Thymosin .alpha.1 promotes tissue repair, angiogenesis and cell migration

The present invention relates to methods for promoting tissue repair, angiogenesis and cell migration. The method of the invention utilizes thymosin a1 (T.alpha.1) peptide to promote tissue repair, angiogenesis and cell migration. The invention further relates to modulating T.alpha.1 activity in tissues.

TECHNICAL FIELD
 The present invention relates generally to tissue repair and more
 specifically to methods for regulating wound healing, angiogenesis and
 cell migration using thymosin .alpha.1 (T.alpha.1).
 BACKGROUND
 Impaired tissue healing is a significant problem in health care. Chronic,
 non-healing wounds are a major cause of prolonged morbidity in the aged
 human population. This is especially the case in bedridden or diabetic
 patients who develop severe, non-healing skin ulcers. In many of these
 cases, the delay in healing is a result of inadequate blood supply either
 as a result of continuous pressure or of vascular blockage. Poor capillary
 circulation due to small artery atherosclerosis or venous stasis
 contribute to the failure to repair damaged tissue. Such tissues are often
 infected with microorganisms that proliferate unchallenged by the innate
 defense systems of the body which require well vascularized tissue to
 effectively eliminate pathogenic organisms. As a result, most therapeutic
 intervention centers on restoring blood flow to ischemic tissues thereby
 allowing nutrients and immunological factors access to the site of the
 wound.
 Wounds (i.e., lacerations or openings) in mammalian tissue result in tissue
 disruption and coagulation of the microvasculature at the wound face.
 Repair of such damage represents an orderly, controlled cellular response
 to injury. All soft tissue wounds, regardless of size, heal in a similar
 manner. Tissue growth and repair are biologic systems wherein cellular
 proliferation and angiogenesis occur.
 Although many of the basic biochemical steps in wound healing have been
 characterized, a number of key regulatory factors have yet to be
 identified. The identification of such factors could lead to improved
 methods for the treatment of disease states associated with ineffectual
 wound healing. In wound healing, lymphoid cells release soluble factors
 that attract fibroblasts and macrophages initiating repair, endothelial
 cell migration, angiogenesis and matrix production.
 An important aspect of wound repair is the revascularization of damaged
 tissue by angiogenesis. The process of angiogenesis involves endothelial
 cell attachment, basement membrane degradation and synthesis, migration
 and proliferation. Mitogenic factors released from lymphoid and
 endothelial cells can induce angiogenesis and promote neovascularization
 of damaged tissue. Regulation of angiogenesis is of considerable
 significance in tissue formation, wound healing and in pathological
 conditions such as cancer, Wegener's granuloma, Takayasu's arteritis,
 systemic lupus erythematosus and other autoimmune diseases.
 Previous studies have used the "scratch" wound closure assay to assess the
 potential effects of an agent on in vitro cell migration. Though
 informative, such a test does not mimic in vivo wound healing conditions
 to the extent that all factors involved in wound closure are present in
 the assay. For this reason, in vivo systems have been developed to assess
 the ability of an agent or factor to modulate wound healing activities.
 T.alpha.1 was initially identified as an immunomodulatory factor which
 affects T-cell maturation, differentiation and function in vitro and in
 vivo. T.alpha.1 can enhance the production of IL-2 and a-IFN and
 upregulate the expression of IL-2 receptors on mitogen-stimulated T-cells.
 In addition, T.alpha.1 has important actions outside the immune system
 related to a role for this peptide and its 113 amino acid parent molecule,
 prothymosin .alpha., in regulating cell proliferation and apoptosis
 (Sburlati et al., Proc. Natl. Acad. Sci. 88:253, 1991).
 SUMMARY OF THE INVENTION
 The present invention is based on the discovery that thymosin .alpha.1
 (T.alpha.1) accelerates wound healing and stimulates angiogenesis. The
 invention is further based on the discovery that T.alpha.1 enhances the
 morphological differentiation of endothelial cells and is a potent
 chemoattractant for endothelial cells and monocytes.
 In a first embodiment, the invention provides a method for accelerating
 wound healing in a subject in need of such treatment including contacting
 the site of the wound with a therapeutically effective amount of a
 composition containing thymosin .alpha.1 peptide.
 In another embodiment, the invention provides a method for modulating
 angiogenesis in a tissue including contacting the tissue with a
 therapeutically effective amount of a composition containing thymosin
 .alpha.1 peptide.
 In another embodiment, the invention provides a method of inhibiting
 angiogenesis in a subject, including administering to the subject a
 composition containing an agent which regulates thymosin .alpha.1
 activity.
 In yet another embodiment, the invention provides a method of diagnosing a
 pathological state in a subject suspected of having pathology
 characterized by a cell proliferative disorder associated with thymosin
 .alpha.1, including obtaining a sample suspected of containing thymosin
 .alpha.1 from the subject, determining the level of thymosin .alpha.1 in
 the sample and comparing the level of thymosin .alpha.1 in the sample to
 the level of thymosin .alpha.1 in a normal standard sample.
 In another embodiment, the invention provides a method for ameliorating a
 cell proliferative disorder associated with thymosin .alpha.1, including
 treating a subject having the disorder, at the site of the disorder, with
 a composition which regulates thymosin .alpha.1 activity.

DETAILED DESCRIPTION OF THE INVENTION
 The present invention arose from an investigation of the effects of
 T.alpha.1 on cell differentiation and on cell migration. In vitro results
 demonstrate that T.alpha.1 enhances the formation of tube-like structures
 by human umbilical vein epithelial cells (HUVECs) cultured on Matrigel.
 Additional experiments demonstrate that T.alpha.1 acts as a
 chemoattractant to stimulate directional HUVEC migration. Boyden chamber
 assays with a variety of cell types demonstrate that migratory stimulation
 occurs specifically with endothelial cells and monocytes. T.alpha.1
 stimulates cell migration in vivo using subcutaneously implanted Matrigel
 and stimulates wound healing when applied topically or injected
 intraperitoneally in a rat wound model.
 The present invention identifies T.alpha.1 as active in promoting
 endothelial cell migration, angiogenesis and wound healing in vivo. In
 vivo plug results indicate that migration and angiogenesis are stimulated
 at or above the levels observed for migration and angiogenesis in response
 to the positive control epithelial cell growth stimulator (ECGS).
 Additionally, T.alpha.1 accelerates wound healing in a punch wound assay.
 Complete reepitheliazation was observed with both intraperitoneal and
 topical applications of T.alpha.1 and neovascularization was more
 extensive in treated wounds. Increased levels of collagen were also
 observed in treated wounds showing that T.alpha.1 treatment can also
 accelerate wound contraction and stimulate the healing process.
 The methods of the present invention results from the identification of the
 effect of thymosin.alpha.1 (T.alpha.1) on wound healing, angiogenesis and
 cell migration. T.alpha.1 enhances the morphological differentiation of
 endothelial cells and is a potent chemoattractant for endothelial cells
 and monocytes. In vivo, T.alpha.1 stimulated angiogenesis in a
 subcutaneous model (see Example 6). When given either topically or
 intraperitoneally, T.alpha.1 accelerated angiogenesis and promoted wound
 healing (see Example 7). Further, the present invention identifies a
 direct effect of T.alpha.1 on monocyte migration (see Table 1).
 Modulation of Tissue Regneration
 In one embodiment, the invention provides a method for accelerating wound
 healing in a subject by applying to the wound a therapeutically effective
 amount of a composition which contains T.alpha.1 peptides. T.alpha.1
 peptide is valuable as a therapeutic in cases in which there is impaired
 healing of skin wounds or there is a need to augment as, normal healing
 mechanisms.
 T.alpha.1 peptide was initially localized in the thymus but is also in
 other tissues. Therefore, agents which stimulate the production of
 T.alpha.1 peptides can be added to a composition that is used to
 accelerate T.alpha.1 production from such cells. In addition, agents which
 promote wound repair can further be included in such compositions to
 augment wound healing. Such agents include members of the family of growth
 factors such as insulin-like growth factor (IGF-I), platelet-derived
 growth factor (PDGF), epidermal growth factor (EGF), transforming growth
 factor beta (TGF-.beta.) and basic fibroblast growth factor (bFGF). More
 preferably, the agent is transforming growth factor beta (TGF-.beta.) or
 other member of the TGF-.beta. superfamily. The T.alpha.1 compositions of
 the invention aid in healing the wound, in part, by promoting the growth
 of connective tissue. The T.alpha.1 compositions are prepared by
 combining, in any pharmaceutically acceptable carrier substance, e.g.,
 inert gels or liquids, the purified T.alpha.1 peptides of the invention.
 As used herein, a "therapeutically effective amount" of a composition
 containing T.alpha.1 for use in tissue repair is defined as that amount
 that is effective in promoting tissue regeneration. Diseases, disorders or
 ailments modulated by T.alpha.1 include tissue repair subsequent to
 traumatic injuries or conditions including arthritis, osteoporosis and
 other skeletal disorders, and bums. Because these problems are due to a
 poor growth response of the fibroblasts, stem cells, chondrocytes,
 osteoblasts or fibroblasts at the site of injury, the addition of an
 active biologic agent that stimulates or induces growth of these cells is
 beneficial. The term "induce" or "induction" as used herein, refers to the
 activation, stimulation, enhancement, initiation and or maintenance of the
 cellular mechanisms or processes necessary for the formation of any of the
 tissue, repair process or development as described herein.
 In another aspect, the invention is useful for revitalizing scar tissue
 resulting from injuries due to surgical procedures, irradiation,
 laceration, toxic chemicals, viral infection bacterial infection or bums.
 The term "scar tissue" means fibrotic or collagenous tissue formed during
 the healing of a wound or other morbid process. For example, T.alpha.1 can
 be included in a controlled release matrix which can be positioned in
 proximity to damaged tissue thereby promoting regeneration and
 revascularization of such tissue. The term "controlled release matrix"
 means any composition which allows the slow release of a bioactive
 substance which is mixed or admixed therein. The matrix can be a solid
 composition, a porous material, or a semi-solid, gel or liquid suspension
 containing bioactive substances. The term "bioactive material" means any
 composition that will modulate tissue repair when used in accordance with
 the method of the present invention. The bioactive materials/matrix can be
 introduced by means of injection, surgery, catheters or any other means
 suitable for modulating tissue repair.
 It is envisioned that the method of the invention can be used to aid wound
 repair in guided tissue regeneration (GTR) procedures. Such procedures are
 currently used by those skilled in the medical arts to accelerate wound
 healing following invasive surgical procedures. Typically, nonresorbable
 or bioabsorbable membranes are used to accelerate wound healing by
 promoting the repopulation of the wound area with cells which form the
 architectural and structural matrix of the tissue. For example, the method
 of the invention can be used in aiding periodontal tissue regeneration in
 a human or lower animal by placing a composition containing a
 bioresorbable polymer, leachable solvent, and T.alpha.1 at a site in need
 of periodontal tissue regeneration in a human or other mammal such that
 the composition is effective for aiding tissue regeneration by releasing a
 therapeutically-effective amount of T.alpha.1 at the site.
 In another aspect, the invention can be useful for the purposes of
 promoting tissue growth during the process of tissue engineering. As used
 herein, "tissue engineering" is defined as the creation, design, and
 fabrication of biological prosthetic devices, in combination with
 synthetic or natural materials, for the augmentation or replacement of
 body tissues and organs. Thus, the present method can be used to augment
 the design and growth of human tissues outside the body for later
 implantation in the repair or replacement of diseased tissues. For
 example, T.alpha.1 may be useful in promoting the growth of skin graft
 replacements which are used as a therapy in the treatment of bums.
 In another aspect of tissue engineering, T.alpha.1 of the present invention
 can be included in cell-containing or cell-free devices which induce the
 regeneration of functional human tissues when implanted at a site which
 requires regeneration. As previously discussed, biomaterial-guided tissue
 regeneration can be used to promote bone regrowth in, for example,
 periodontal disease. Thus, T.alpha.1 can be used to promote the growth of
 reconstituted tissues assembled into three-dimensional configurations at
 the site of a wound or other tissue in need of such repair.
 In another aspect of tissue engineering, T.alpha.1 can be included in
 external or internal devices containing human tissues designed to replace
 the function of diseased internal tissues. This approach involves
 isolating cells from the body, placing them on or within structural
 matrices, and implanting the new system inside the body or using the
 system outside the body. The method of the invention can be included in
 such matrices to promote the growth of tissues contained in the matrices.
 For example, T.alpha.1 can be included in a cell-lined vascular graft to
 promote the growth of the cells contained in the graft. It is envisioned
 that the method of the invention can be used to augment tissue repair,
 regeneration and engineering in products such as cartilage and bone,
 central nervous system tissues, muscle, liver, and pancreatic islet
 (insulin-producing) cells.
 The present invention further provides a method for modulating female
 reproductive tract function. Growth factors have been shown to play a role
 in cyclic mitosis and differentiation of endometrial cellular components,
 recruitment of macrophages in decidualizing the endometrium,
 endometrial-trophoblast interactions, early pregnancy maintenance, and
 endometrial functional regeneration. The term "modulate" as used herein,
 denotes a modification of an existing condition or biologic state.
 Modulation of a condition as defined herein, encompasses both an increase
 or a decrease in the determinants affecting the existing condition. For
 example, administration of T.alpha.1 could be used to augment uterine
 functions in a condition where the promotion of growth is desired. For
 example, the uterus may be treated with T.alpha.1 to promote the growth
 and development of placental membranes or endometrial growth. Furthermore,
 treatment with Tat may be used to promote and maintain a pregnancy by
 facilitating endometrial-trophoblast interaction. Alternatively,
 antagonists to T.alpha.1 could be administered to modulate conditions of
 excessive endometrial growth in which the level of T.alpha.1 is excessive
 in comparison to a normal biologic condition.
 Another therapeutic approach included within the invention involves direct
 administration of reagents or compositions including the T.alpha.1 of the
 invention by any conventional administration technique (for example, but
 not restricted to, local injection, inhalation, or systemic
 administration), to a subject with a fibrotic, a scelortic, or a cell
 proliferative disorder such as, for example, atherosclerosis.
 Administration of T.alpha.1, as described above, can accelerate wound
 healing, induce the formation of tissue repair or regeneration, or promote
 the growth and development of the endometrium. The reagent, formulation or
 composition may also be targeted to specific cells or receptors by any
 method described herein or by any method known in the art of delivering,
 targeting and expressing genes encoding T.alpha.1. The actual dosage of
 reagent, formulation or composition that modulates a fibrotic disorder, a
 scelortic disorder, a cell proliferative disorder, atherosclerosis or
 wound healing depends on many factors, including the size and health of an
 organism. However, one of ordinary skill in the art can use the following
 teachings describing the methods and techniques for determining clinical
 dosages (Spilker B., Guide to Clinical Studies and Developing Protocols,
 Raven Press Books, Ltd., New York, 1984, pp. 7-13, 54-60; Spilker B.,
 Guide to Clinical Trials, Raven Press, Ltd., New York, 1991, pp. 93-101;
 Craig C., and R. Stitzel, eds., Modern Pharmacology, 2d ed., Little, Brown
 and Co., Boston, 1986, pp. 127-33; T. Speight, ed., Avery's Drug
 Treatment: Principles and Practice of Clinical Pharmacology and
 Therapeutics, 3d ed., Williams and Wilkins, Baltimore, 1987, pp. 50-56; R.
 Tallarida, R. Raffa and P. McGonigle, Principles in General Pharmacology,
 Springer-Verlag, New York, 1988, pp. 18-20) or to determine the
 appropriate dosage to use.
 In yet another embodiment, the invention may provide a method of diagnosing
 a pathological state in a subject suspected of having pathology
 characterized by a cell proliferative disorder assoicated with thymosin
 .alpha.1, including obtaining a sample suspected of containing thymosin
 .alpha.1 from the subject, determining the level of thymosin .alpha.1 in
 the sample and comparing the level of thymosin.alpha.1 in the sample to
 the level of thymosin.alpha.1 in a normal standard sample. Such conditions
 include but are not restricted to cell proliferative disorders, various
 fibrotic conditions including scleroderma, arthritis, liver cirrhosis, and
 uterine fibroids. For example, a sample suspected of containing T.alpha.1
 is obtained from a subject, the level of T.alpha.1 peptide is determined
 and compared with the level of T.alpha.1 peptide in a normal tissue
 sample. The level of T.alpha.1 can be determined by immunoassays using
 anti-T.alpha.1 peptide antibodies, for example. Other variations of such
 assays include radioimmunoassay (RIA), ELISA and immunofluorescence.
 Alternatively, nucleic acid probes can be used to detect and quantitate
 T.alpha.1 peptide mRNA for the same purpose.
 In another embodiment, the invention may provide a method for ameliorating
 a cell proliferative disorder associated with T.alpha.1, including
 treating a subject having the disorder, at the site of the disorder, with
 a composition which regulates T.alpha.1 activity. The term "ameliorate"
 denotes a lessening of the detrimental effect of the disease-inducing
 response in the patient receiving therapy. Where the disease is due to an
 overgrowth of cells, an antagonist of T.alpha.1 peptide is effective in
 decreasing the amount of available T.alpha.1. Such an antagonist may be a
 T.alpha.1 specific antibody or functional fragments thereof (e.g., Fab,
 F(ab').sub.2). The treatment requires contacting the site of the disease
 with the antagonist.
 The term "cell proliferative disorder", as used herein, refers to a
 condition characterized by abnormal cell growth. The condition can include
 both hypertrophic (the continual multiplication of cells resulting in an
 overgrowth of a cell population within a tissue) and hypotrophic (a lack
 or deficiency of cells within a tissue) cell growth or an excessive influx
 or migration of cells into an area of a body. The cell populations are not
 necessarily transformed, tumorigenic or malignant cells, but can include
 normal cells as well. For example, T.alpha.1 may be involved in a
 pathological condition by inducing a proliferative lesion in the intimal
 layer of an arterial wall, resulting in atherosclerosis. Instead of trying
 to reduce risk factors for the condition, e.g., lowering blood pressure or
 reducing elevated cholesterol levels, T.alpha.1 peptide inhibitors or
 antagonists of the invention would be useful in interfering with the in
 vivo activity of T.alpha.1 associated with atherosclerosis. T.alpha.1
 peptide antagonists are also useful in treating other disorders associated
 with an overgrowth of connective tissues, such as various fibrotic
 conditions, including scleroderma, arthritis and liver cirrhosis.
 In yet another embodiment, the invention may provide a method of treating a
 subject having a cell proliferative disorder associated with T.alpha.1
 gene expression in a subject. The method includes administering to a
 subject having the disorder a therapeutically effective amount of an agent
 which modulates T.alpha.1 gene expression, thereby treating the disorder.
 The term "modulate" refers to inhibition or suppression of T.alpha.1
 expression when T.alpha.1 is overexpressed, and induction of expression
 when T.alpha.1 is underexpressed. The term "therapeutically effective"
 means that amount of T.alpha.1 agent which is effective in reducing the
 symptoms of the T.alpha.1 associated cell proliferative disorder.
 An agent which modulates T.alpha.1 gene expression may be a polynucleotide
 for example. The polynucleotide may be an antisense, a triplex agent, or a
 ribozyme, as described above. For example, an antisense may be directed to
 the structural gene region or to the promoter region of T.alpha.1.
 T.alpha.1 has been identified as a 28 amino acid peptide which results from
 the cleavage of the N-terminal region of prothymosin .alpha.1. Therefore,
 when a cell proliferative disorder is associated with the expression of
 T.alpha.1, a therapeutic approach which directly interferes with the
 translation of prothymosin .alpha.1 messages into protein is possible. For
 example, antisense nucleic acid or ribozymes could be used to bind to the
 prothymosin .alpha.1 mRNA or to cleave it. Antisense RNA or DNA molecules
 bind specifically with a targeted gene's RNA message, interrupting the
 expression of that gene's protein product. The antisense binds to the
 messenger RNA forming a double stranded molecule which cannot be
 translated by the cell. Antisense oligonucleotides of about 15-25
 nucleotides are preferred since they are easily synthesized and have an
 inhibitory effect just like antisense RNA molecules. In addition,
 chemically reactive groups, such as iron-linked ethylenediaminetetraacetic
 acid (EDTA-Fe) can be attached to an antisense oligonucleotide, causing
 cleavage of the RNA at the site of hybridization. These and other uses of
 antisense methods to inhibit the in vitro translation of genes are well
 known in the art (Marcus-Sakura, Anal., Biochem., 172:289, 1988).
 Antisense nucleic acids are DNA or RNA molecules that are complementary to
 at least a portion of a specific mRNA molecule (Weintraub, Scientific
 American, 262:40, 1990). In the cell, the antisense nucleic acids
 hybridize to the corresponding mRNA, forming a double-stranded molecule.
 The antisense nucleic acids interfere with the translation of the mRNA,
 since the cell will not translate a mRNA that is double-stranded.
 Antisense oligomers of about 15 nucleotides are preferred, since they are
 easily synthesized and are less likely to cause problems than larger
 molecules when introduced into the target prothymosin .alpha.1 producing
 cell. The use of antisense methods to inhibit the in vitro translation of
 genes is well known in the art (Marcus-Sakura, Anal.Biochem., 172:289,
 1988).
 Use of an oligonucleotide to stall transcription is known as the triplex
 strategy since the oligomer winds around double-helical DNA, forming a
 three-strand helix. Therefore, these triplex compounds can be designed to
 recognize a unique site on a chosen gene (Maher, et al., Antisense Res.
 and Dev., 1 (3):227, 1991; Helene, C., Anticancer Drug Design, 6(6):569,
 1991).
 Ribozymes are RNA molecules possessing the ability to specifically cleave
 other single-stranded RNA in a manner analogous to DNA restriction
 endonucleases. Through the modification of nucleotide sequences which
 encode these RNAs, it is possible to engineer molecules that recognize
 specific nucleotide sequences in an RNA molecule and cleave it (Cech,
 J.Amer.Med. Assn., 260:3030, 1988). A major advantage of this approach is
 that, because they are sequence-specific, only mRNAs with particular
 sequences are inactivated.
 There are two basic types of ribozymes namely, tetrahymena-type
 (Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type.
 Tetrahymena-type ribozymes recognize sequences which are four bases in
 length, while "hammerhead"-type ribozymes recognize base sequences 11-18
 bases in length. The longer the recognition sequence, the greater the
 likelihood that the sequence will occur exclusively in the target mRNA
 species. Consequently, hammerhead-type ribozymes are preferable to
 tetrahymena-type ribozymes for inactivating a specific mRNA species and
 18-based recognition sequences are preferable to shorter recognition
 sequences.
 These and other uses of antisense methods to inhibit the in vivo
 translation of genes are well known in the art (e.g., De Mesmaeker, et
 al., 1995. Backbone modifications in oligonucleotides and peptide nucleic
 acid systems. Curr. Opin. Struct. Biol. 5:343-355; Gewirtz, A. M., et al.,
 1996b. Facilitating delivery of antisense -oligodeoxynucleotides: Helping
 antisense deliver on its promise; Proc. Natl. Acad. Sci. U.S.A.
 93:3161-3163; Stein, C. A. A discussion of G-tetrads 1996. Exploiting the
 potential of antisense: beyond phosphorothioate oligodeoxynucleotides.
 Chem. and Biol. 3:319-323).
 Delivery of antisense, triplex agents, ribozymes, competitive inhibitors
 and the like can be achieved using a recombinant expression vector such as
 a chimeric virus or a colloidal dispersion system. Various viral vectors
 which can be utilized for gene therapy as taught herein include
 adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a
 retrovirus. Preferably, the retroviral vector is a derivative of a murine
 or avian retrovirus. Examples of retroviral vectors in which a single
 foreign gene can be inserted include, but are not limited to: Moloney
 murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
 murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number
 of additional retroviral vectors can incorporate multiple genes. All of
 these vectors can transfer or incorporate a gene for a selectable marker
 so that transduced cells can be identified and generated. By inserting a
 polynucleotide sequence of interest into the viral vector, along with
 another gene which encodes the ligand for a receptor on a specific target
 cell, for example, the vector is now target specific. Retroviral vectors
 can be made target specific by inserting, for example, a polynucleotide
 encoding a sugar, a glycolipid, or a protein. Preferred targeting is
 accomplished by using an antibody to target the retroviral vector. Those
 of skill in the art will know of, or can readily ascertain without undue
 experimentation, specific polynucleotide sequences which can be inserted
 into the retroviral genome to allow target specific delivery of the
 retroviral vector containing the antisense polynucleotide.
 Since recombinant retroviruses are defective, they require assistance in
 order to produce infectious vector particles. This assistance can be
 provided, for example, by using helper cell lines that contain plasmids
 encoding all of the structural genes of the retrovirus under the control
 of regulatory sequences within the LTR. These plasmids are missing a
 nucleotide sequence which enables the packaging mechanism to recognize an
 RNA transcript for encapsidation. Helper cell lines which have deletions
 of the packaging signal include but are not limited to .psi.2, 17 and
 2, for example. These cell lines produce empty virions, since no genome
 is packaged. If a retroviral vector is introduced into such cells in which
 the packaging signal is intact, but the structural genes are replaced by
 other genes of interest, the vector can be packaged and vector virion
 produced.
 Alternatively, NIH 3T3 or other tissue culture cells can be directly
 transfected with plasmids encoding the retroviral structural genes gag,
 pol and env, by conventional calcium phosphate transfection. These cells
 are then transfected with the vector plasmid containing the genes of
 interest. The resulting cells release the retroviral vector into the
 culture medium.
 Another targeted delivery system for antisense polynucleotides a colloidal
 dispersion system. Colloidal dispersion systems include macromolecule
 complexes, nanocapsules, microspheres, beads, and lipid-based systems
 including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
 The preferred colloidal system of this invention is a liposome. Liposomes
 are artificial membrane vesicles which are useful as delivery vehicles in
 vitro and in vivo. It has been shown that large unilamellar vesicles
 (LUV), which range in size from 0.2-4.0 um can encapsulate a substantial
 percentage of an aqueous buffer containing large macromolecules. RNA, DNA
 and intact virions can be encapsulated within the aqueous interior and be
 delivered to cells in a biologically active form (Fraley, et al., Trends
 Biochem. Sci., 6:77, 1981). In addition to mammalian cells, liposomes have
 been used for delivery of polynucleotides in plant, yeast and bacterial
 cells. In order for a liposome to be an efficient gene transfer vehicle,
 the following characteristics should be present: (1) encapsulation of the
 genes of interest at high efficiency while not compromising their
 biological activity; (2) preferential and substantial binding to a target
 cell in comparison to non-target cells; (3) delivery of the aqueous
 contents of the vesicle to the target cell cytoplasm at high efficiency;
 and (4) accurate and effective expression of genetic information (Mannino,
 et al., Biotechniques, 6:682, 1988).
 The composition of the liposome is usually a combination of phospholipids,
 particularly high-phase-transition-temperature phospholipids, usually in
 combination with steroids, especially cholesterol. Other phospholipids or
 other lipids may also be used. The physical characteristics of liposomes
 depend on pH, ionic strength, and the presence of divalent cations.
 Examples of lipids useful in liposome production include phosphatidyl
 compounds, such as phosphatidylglycerol, phosphatidylcholine,
 phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides,
 and gangliosides. Particularly useful are diacylphosphatidylglycerols,
 where the lipid moiety contains from 14-18 carbon atoms, particularly from
 16-18 carbon atoms, and is saturated. Illustrative phospholipids include
 egg phosphatidylcholine, dipalmitoylphosphatidylcholine and
 distearoylphosphatidylcholine.
 The targeting of liposomes has been classified based on anatomical and
 mechanistic factors. Anatomical classification is based on the level of
 selectivity, for example, organ-specific, cell-specific, and
 organelle-specific. Mechanistic targeting can be distinguished based upon
 whether it is passive or active. Passive targeting utilizes the natural
 tendency of liposomes to distribute to cells of the reticulo-endothelial
 system (RES) in organs which contain sinusoidal capillaries. Active
 targeting, on the other hand, involves alteration of the liposome by
 coupling the liposome to a specific ligand such as a monoclonal antibody,
 sugar, glycolipid, or protein, or by changing the composition or size of
 the liposome in order to achieve targeting to organs and cell types other
 than the naturally occurring sites of localization.
 The surface of the targeted delivery system may be modified in a variety of
 ways. In the case of a liposomal targeted delivery system, lipid groups
 can be incorporated into the lipid bilayer of the liposome in order to
 maintain the targeting ligand in stable association with the liposomal
 bilayer. Various linking groups can be used for joining the lipid chains
 to the targeting ligand. In general, the compounds bound to the surface of
 the targeted delivery system will be ligands and receptors which will
 allow the targeted delivery system to find and "home in" on the desired
 cells. A ligand may be any compound of interest which will bind to another
 compound, such as a receptor.
 The therapeutic agents useful in the method of the invention can be
 administered parenterally by injection or by gradual perfusion over time.
 Administration may be intravenously, intraperitoneally, intramuscularly,
 subcutaneously, intracavity, or transdermally.
 Preparations for parenteral administration include sterile aqueous or
 non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous
 solvents are propylene glycol, polyethylene glycol, vegetable oils such as
 olive oil, and injectable organic esters such as ethyl oleate. Aqueous
 carriers include water, alcoholic/aqueous solutions, emulsions or
 suspensions, including saline and buffered media. Parenteral vehicles
 include sodium chloride solution, Ringer's dextrose, dextrose and sodium
 chloride, lactated Ringer's intravenous vehicles include fluid and
 nutrient replenishers, electrolyte replenishers (such as those based on
 Ringer's dextrose), and the like. Preservatives and other additives may
 also be present such as, for example, antimicrobials, anti-oxidants,
 chelating agents and inert gases and the like.
 The invention also includes a pharmaceutical composition comprising a
 therapeutically effective amount of T.alpha.1 in a pharmaceutically
 acceptable carrier. Such carriers include those listed above with
 reference to parenteral administration.
 Antibodies that Bind to T.dbd.1
 Antibodies to T.alpha.1 peptide or fragments could be valuable as
 diagnostic tools to aid in the detection of diseases in which T.alpha.1 is
 a pathological factor. Further, antibodies which bind to prothymosin
 .alpha.1 and inhibit or prevent the proteolytic cleavage of T.alpha.1 from
 promothymosin .alpha.1 are included in the present invention.
 Therapeutically, antibodies or fragments of the antibody molecule could
 also be used to neutralize the biological activity of T.alpha.1 in
 diseases where T.alpha.1 is inducing the overgrowth of tissue. Such
 antibodies can recognize an epitope of prothymosin .alpha.1, T.alpha.1, or
 fragments thereof, suitable for antibody recognition and neutralization of
 T.alpha.1 activity. As used in this invention, the term "epitope" refers
 to an antigenic determinant on an antigen, such as a T.alpha.1 peptide, to
 which the paratope of an antibody, such as an T.alpha.1-specific antibody,
 binds. Antigenic determinants usually consist of chemically active surface
 groupings of molecules, such as amino acids or sugar side chains, and can
 have specific three-dimensional structural characteristics, as well as
 specific charge characteristics.
 Preparation of an antibody requires a substantially purified moiety that
 can provide an antigenic determinant. The term "substantially pure" as
 used herein refers to T.alpha.1, or variants thereof, which is
 substantially free of other proteins, lipids, carbohydrates or other
 materials with which it is naturally associated. Substantially purified or
 "isolated" refers to molecules, either nucleic or amino acid sequences,
 that are removed from their natural environment, isolated or separated,
 and are at least 60% free, preferably 75% free, and most preferably 90%
 free from other components with which they are naturally associated. One
 skilled in the art can isolate T.alpha.1 using standard techniques for
 protein purification. The substantially pure peptide will yield a single
 major band on a non-reducing polyacrylamide gel. The purity of the
 T.alpha.1 peptide can also be determined by amino-terminal amino acid
 sequence analysis. T.alpha.1 peptide includes functional fragments of the
 peptide, as long as the activity of T.alpha.1 remains. Smaller peptides
 containing the biological activity of T.alpha.1 are included in the
 invention. As used in the present invention, the term "antibody" includes,
 in addition to conventional antibodies, such protein fragments that have
 the ability to recognize specifically and bind the T.alpha.1 protein or
 variants thereof. Regions of the gene that differ at the protein level are
 well defined. A protein can be raised by expression of the wt gene or of
 the variants, or, preferably, fractions therefore. For example, the
 nucleic acid sequence can be cloned into expression vectors. According to
 this embodiment, the sequence of interest can first be obtained by
 employing PCR, as described above, or from a synthetic gene construction
 with overlapping and ligated synthetic oligonucleotides. Another
 alternative would involve synthesis of a short peptide. All those
 methodologies are well known to one skilled in the art. See, for example,
 Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Volumes 1 and 2
 (1987), with supplements, and Maniatis et al., MOLECULAR CLONING, A
 LABORATORY MANUAL, Cold Spring Harbor Laboratory.
 The genetic sequence discussed above then is expressed in any known,
 commercially available systems. Vectors for subcloning the sequence of
 interest, and subsequent expression into bacterial, yeast, baculovirus,
 insect, or tissue culture are well known to one skilled in the art. The
 subcloning process could, according to one embodiment, produce a fused
 protein with a short N- or C-terminal extension to facilitate subsequent
 purifications on columns or by use of antibodies. Alternatively, the
 protein of interest is purified by standard protein purification
 protocols. See for example PROTEIN PURIFICATION--PRINCIPLES AND PRACTICE,
 Springer Varlag publ., New-York; and PROTEIN BIOTECHNOLOGY, Humana Press,
 Totowa, N.J.
 The preparation of polyclonal antibodies is well-known to those skilled in
 the art.
 See, for example, Green et al., Production of Polyclonal Antisera, in
 IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (Humana Press 1992);
 Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice
 and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992),
 which are hereby incorporated by reference.
 The preparation of monoclonal antibodies likewise is conventional. See, for
 example, Kohler & Milstein, Nature 256:495 (1975); Coligan et al.,
 sections 2.5.1-2.6.7; and Harlow et al., ANTIBODIES: A LABORATORY MANUAL,
 page 726 (Cold Spring Harbor Pub. 1988), which are hereby incorporated by
 reference. Briefly, monoclonal antibodies can be obtained by injecting
 mice with a composition comprising an antigen, verifying the presence of
 antibody production by removing a serum sample, removing the spleen to
 obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to
 produce hybridomas, cloning the hybridomas, selecting positive clones that
 produce antibodies to the antigen, and isolating the antibodies from the
 hybridoma cultures. Monoclonal antibodies can be isolated and purified
 from hybridoma cultures by a variety of well-established techniques. Such
 isolation techniques include affinity chromatography with Protein-A
 Sepharose, size-exclusion chromatography, and ion-exchange chromatography.
 See, e.g., Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;
 Barnes et al., Purification of Immunoglobulin G (IgG), in METHODS IN
 MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (Humana Press 1992). Methods of
 in vitro and in vivo multiplication of monoclonal antibodies are
 well-known to those skilled in the art. Multiplication in vitro may be
 carried out in suitable culture media such as Dulbecco's Modified Eagle
 Medium or RPMI 1640 medium, optionally replenished by a mammalian serum
 such as fetal calf serum or trace elements and growth-sustaining
 supplements such as normal mouse peritoneal exudate cells, spleen cells,
 bone marrow macrophages. Production in vitro provides relatively pure
 antibody preparations and allows scale-up to yield large amounts of the
 desired antibodies. Large scale hybridoma cultivation can be carried out
 by homogenous suspension culture in an airlift reactor, in a continuous
 stirrer reactor, or in immobilized or entrapped cell culture.
 Multiplication in vivo may be carried out by injecting cell clones into
 mammals histocompatible with the parent cells, e.g., syngeneic mice, to
 cause growth of antibody-producing tumors. Optionally, the animals are
 primed with a hydrocarbon, especially oils such as pristane
 (tetramethylpentadecane) prior to injection. After one to three weeks, the
 desired monoclonal antibody is recovered from the body fluid of the
 animal.
 The invention provides a method for detecting T.alpha.1, or variants
 thereof, which includes contacting an anti-T.alpha.1 antibody with a cell
 or protein and detecting binding to the antibody. An antibody which binds
 to T.alpha.1 peptide is labeled with a compound which allows detection of
 binding to T.alpha.1. There are many different labels and methods of
 labeling known to those of ordinary skill in the art. Examples of the
 types of labels which can be used in the present invention include
 enzymes, radioisotopes, fluorescent compounds, colloidal metals,
 chemiluminescent compounds, phosphorescent compounds, and bioluminescent
 compounds. Those of ordinary skill in the art will know of other suitable
 labels for binding to the antibody, or will be able to ascertain such,
 using routine experimentation. For purposes of the invention, an antibody
 specific for T.alpha.1 peptide may be used to detect the level of
 T.alpha.1 in biological fluids and tissues. Any specimen containing a
 detectable amount of antigen can be used. The level of T.alpha.1 in the
 suspect cell can be compared with the level in a normal cell to determine
 whether the subject is predisposed to a T.alpha.1 associated increase in
 angiogenesis.
 The antibodies of the invention are suited for use, for example, in
 immunoassays in which they can be utilized in liquid phase or bound to a
 solid phase carrier. In addition, the antibodies in these immunoassays can
 be detectably labeled in various ways. Examples of types of immunoassays
 which can utilize antibodies of the invention are competitive and
 non-competitive immunoassays in either a direct or indirect format.
 Examples of such immunoassays are the radioimmunoassay (RIA) and the
 sandwich (immunometric) assay. Detection of the antigens using the
 antibodies of the invention can be done utilizing immunoassays which are
 run in either the forward, reverse, or simultaneous modes, including
 immunohistochemical assays on physiological samples. Those of skill in the
 art will know, or can readily discern, other immunoassay formats without
 undue experimentation.
 The antibodies of the invention can be bound to many different carriers and
 used to detect the presence of an antigen comprising the peptide of the
 invention. Examples of well-known carriers include glass, polystyrene,
 polypropylene, polyethylene, dextran, nylon, amylases, natural and
 modified celluloses, polyacrylamides, agaroses and magnetite. The nature
 of the carrier can be either soluble or insoluble for purposes of the
 invention. Those skilled in the art will know of other suitable carriers
 for binding antibodies, or will be able to ascertain such, using routine
 experimentation.
 Another technique which may also result in greater sensitivity consists of
 coupling the antibodies to low molecular weight haptens. These haptens can
 then be specifically detected by means of a second reaction. For example,
 it is common to use such haptens as biotin, which reacts with avidin, or
 dinitrophenyl, puridoxal, and fluorescein, which can react with specific
 antihapten antibodies.
 The invention includes antibodies immunoreactive with T.alpha.1 peptide or
 functional fragments thereof. Antibody which consists essentially of
 pooled monoclonal antibodies with different epitopic specificities, as
 well as distinct monoclonal antibody preparations are provided. Monoclonal
 antibodies are made from antigen containing fragments of the protein by
 methods well known to those skilled in the art (Kohler, et al., Nature,
 256:495, 1975). The term antibody as used in this invention is meant to
 include intact molecules as well as fragments thereof, such as Fab and
 F(ab').sub.2, Fv and SCA fragments which are capable of binding an
 epitopic determinant on T.alpha.1.
 (1) An Fab fragment consists of a monovalent antigen-binding fragment of an
 antibody molecule, and can be produced by digestion of a whole antibody
 molecule with the enzyme papain, to yield a fragment consisting of an
 intact light chain and a portion of a heavy chain.
 (2) An Fab' fragment of an antibody molecule can be obtained by treating a
 whole antibody molecule with pepsin, followed by reduction, to yield a
 molecule consisting of an intact light chain and a portion of a heavy
 chain. Two Fab' fragments are obtained per antibody molecule treated in
 this manner.
 (3) An (Fab').sub.2 fragment of an antibody can be obtained by treating a
 whole antibody molecule with the enzyme pepsin, without subsequent
 reduction. A (Fab').sub.2 fragment is a dimer of two Fab' fragments, held
 together by two disulfide bonds.
 (4) An Fv fragment is defined as a genetically engineered fragment
 containing the variable region of a light chain and the variable region of
 a heavy chain expressed as two chains.
 (5) A single chain antibody ("SCA") is a genetically engineered single
 chain molecule containing the variable region of a light chain and the
 variable region of a heavy chain, linked by a suitable, flexible
 polypeptide linker.
 Alternatively, a therapeutically or diagnostically useful anti-T.alpha.1
 antibody may be derived from a "humanized" monoclonal antibody. Humanized
 monoclonal antibodies are produced by transferring mouse complementary
 determining regions from heavy and light variable chains of the mouse
 immunoglobulin into a human variable domain, and then substituting human
 residues in the framework regions of the murine counterparts. The use of
 antibody components derived from humanized monoclonal antibodies obviates
 potential problems associated with the immunogenicity of murine constant
 regions. General techniques for cloning murine immunoglobulin variable
 domains are described, for example, by Orlandi et al., Proc. Natl. Acad.
 Sci. USA 86: 3833 (1989), which is hereby incorporate din its entirety by
 reference. Techniques for producing humanized monoclonal antibodies are
 described, for example, by Jones et al., Nature 321: 522 (1986); Riechmann
 et al., Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534
 (1988); Carter et al., Proc. Nat'l Acad Sci. USA 89: 4285 (1992); Sandhu,
 Crit. Rev. Biotech. 12: 437 (1992); and Singer et al., J. Immunol. 150:
 2844 (1993), which are hereby incorporated by reference.
 Antibodies of the invention also may be derived from human antibody
 fragments isolated from a combinatorial immunoglobulin library. See, for
 example, Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY,
 VOL. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol. 12: 433 (1994),
 which are hereby incorporated by reference. Cloning and expression vectors
 that are useful for producing a human immunoglobulin phage library can be
 obtained, for example, from STRATAGENE Cloning Systems (La Jolla, Calif.).
 In addition, antibodies of the present invention may be derived from a
 human monoclonal antibody. Such antibodies are obtained from transgenic
 mice that have been "engineered" to produce specific human antibodies in
 response to antigenic challenge. In this technique, elements of the human
 heavy and light chain loci are introduced into strains of mice derived
 from embryonic stem cell lines that contain targeted disruptions of the
 endogenous heavy and light chain loci. The transgenic mice can synthesize
 human antibodies specific for human antigens, and the mice can be sued to
 produce human antibody-secreting hybridomas. Methods for obtaining human
 antibodies from transgenic mice are described by Green et al., Nature
 Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994); and Taylor et
 al., Int. Immunol. 6: 579 (1994), which are hereby incorporated by
 reference.
 As is mentioned above, antigens that can be used in producing
 T.alpha.1-specific antibodies include T.alpha.1 peptides or T.alpha.1
 peptide fragments. The polypeptide or peptide used to immunize an animal
 can be obtained by standard recombinant, chemical synthetic, or
 purification methods. As is well known in the art, in order to increase
 immunogenicity, an antigen can be conjugated to a carrier protein.
 Commonly used carriers include keyhole limpet hemocyanin (KLH),
 thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled
 peptide is then used to immunize the animal (e.g., a mouse, a rat, or a
 rabbit). In addition to such carriers, well known adjuvants can be
 administered with the antigen to facilitate induction of a strong immune
 response.
 Modulation of Angiogenesis
 Angiogenesis, a biological process that includes the proliferation and
 migration of endothelial cells, is defined as the growth of new blood
 vessels, in particular, capillaries and is an essential part of both
 normal and pathological tissue growth. Characteristic elements of
 angiogenesis include endothelial cell proliferation, endothelial cell
 migration, invasion of endothelial cells into tissues, and maturation of
 endothelial cells. Angiogenesis plays a crucial role in such beneficial
 functions as embryogenesis, wound healing and the female reproductive
 cycle, as well as in such abnormal functions as psoriasis, diabetic
 retinopathy, rheumatoid arthritis, hemangiomas, and solid tumor formation.
 In another embodiment, the invention provides a method for modulating
 angiogenesis in a tissue including contacting the tissue with a
 therapeutically effective amount of a composition containing thymosin
 .alpha.1 peptide. It is envisioned that the method of the invention can be
 used to treat tissue damage in a subject after the tissue has been
 deprived of blood supply for a tissue damaging amount of time. The
 treatment can involve administering to the subject an effective amount of
 T.alpha.1 peptide so as to contact the blood deprived tissue, thereby
 promoting angiogenesis in a tissue and treating or preventing tissue
 damage. For example, T.alpha.1 peptide can be administered to prevent or
 treat tissue damage in cardiac tissue resulting from an incomplete or
 complete coronary occlusion by inducing angiogenesis and stimulating
 collateral circulation in the tissue affected by the occlusion. Thus, the
 method of the invention can be used restore blood flow to ischemic tissues
 thereby allowing nutrients and immunological factors access to the site of
 tissue damage.
 In addition, the method of the invention is useful in promoting
 angiogenesis in tissues deprived of adequate blood flow. For example, a
 composition containing T.alpha.1 can promote hair growth in a subject
 desiring such growth. One aspect of hair loss is the loss of
 vascularization in tissues containing hair follicles resulting in
 inadequate blood supplies to the hair follicle. A composition containing
 T.alpha.1 can be applied to the affected area in an amount sufficient to
 cause neovascularization of the tissue resulting in hair growth.
 Pathologically, T.alpha.1 may be involved in diseases in which there is an
 overgrowth of blood vessels, such as cancer, tumor formation and growth,
 diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis and
 psoriasis.
 The ingrowth of capillaries and ancillary blood vessels is essential for
 growth of solid tumors and is thus an unwanted physiological response
 which facilitates the spread of malignant tissue and metastases.
 Inhibition of angiogenesis and the resultant growth of capillaries and
 blood vessels is therefore a component of effective treatment of
 malignancy in use for treatment of cancer patients.
 Thus, in another embodiment, the invention may provide a method of
 inhibiting angiogenesis in a subject, including administering to the
 subject a composition containing an agent which regulates thymosin
 .alpha.1 activity. For example, the inhibition of angiogenesis and
 endothelial cell proliferation can be beneficial in controlling the growth
 of solid tumors. Most, if not all solid tumors, like normal tissue,
 require a steady and sufficient blood supply for optimal growth. Tumors
 are known to make use of angiogenic growth factors to attract new blood
 vessels and ascertain supply with sufficient amounts of nutrients to
 sustain their growth. Many tumors are well vascularized and the inhibition
 of the formation of an adequate blood supply to the tumor by inhibition of
 tumor vascularization, as a result of endothelial cell growth inhibition,
 is beneficial in tumor growth control. Without a strong blood supply,
 rapid and prolonged growth of tumor tissue cannot be sustained. Thus,
 agents which inhibit T.alpha.1 activity may be used to prevent neoplastic
 growth. The T.alpha.1 inhibiting agent may be administered orally,
 parenterally, topically, intravenously, or systemically. In addition, for
 inhibiting tumor cell proliferation and tumor growth, the agent may be
 administered locally directly to the tumor or as a part of a deposited
 slow release formulation. Administration may be on a daily basis for as
 long as needed to inhibit angiogenesis, endothelial cell proliferation,
 tumor cell proliferation or tumor growth. Alternatively, a slow release
 formulation may continue for as long as needed to control tumor growth.
 This dosage regimen may be adjusted to provide the optimum therapeutic
 response. For example, several divided doses may be administered daily or
 the dose may be proportionally reduced as indicated by the exigencies of
 the therapeutic situation.
 In this regard, the compositions of this invention that are useful as
 inhibitors of angiogenesis, endothelial cell proliferation, tumor cell
 proliferation and tumor growth contain a pharmaceutically acceptable
 carrier and an amount of T.alpha.1 modulating agent effective to inhibit
 tumor or endothelial cell proliferation. Such compositions may also
 include preservatives, antioxidants, immunosuppressants and other
 biologically and pharmaceutically effective agents which do not exert a
 detrimental effect on the T.alpha.1 modulating agent. For treatment of
 tumor cells, the composition may include a chemotherapeutic agent, for
 example an anti-cancer agent which selectively kills the faster
 replicating tumor cells, many of which are known and clinically used.
 Exemplary anti-cancer agents include melphalan, cyclophosphamide,
 methotrexate, adriamycin and bleomycin.
 Modulation of Monocyte Chemotaxis
 Monocytes or (i.e., mononuclear phagocytes) are incompletely differentiated
 phagocytic white blood cells which circulate in the blood. Monocytes
 settle in tissues and mature into tissue macrophages. Circulating
 monocytes are involved in clearing antigen/antibody complexes from the
 circulation. Migration of monocytes from blood vessels in to distressed or
 damaged tissues is crucial to the initiation of normal disease fighting
 inflammatory responses. Thus, in another embodiment, the invention
 provides a method of modulating monocyte migration including contacting
 the monocyte with a migration inducing effective amount of thymosin
 .alpha.1 peptide. Thus, T.alpha.1 is useful for attracting monocytes to
 the site of damaged and/or infected tissues for the purpose of preventing
 or ameliorating disease states associated with viral or bacterial
 infections. Previously disclosed methods for applying compositions
 containing T.alpha.1 to a site in need of such treatment can be used to
 promote the prevention or treatment of an infection.
 Monocyte recruitment is also involved in the onset and progression of
 debilitating and life-threatening inflammatory and autoimmune diseases.
 The pathology of these diseases results from the attack of the body's
 immune system defenses on normal tissues. Thus, in another aspect of the
 invention, agents which modulate the activity of T.alpha.1 can be used to
 prevent or ameliorate a disease state associated with inflammatory and
 autoimmune diseases. These disease include, but are not limited to,
 rheumatoid arthritis, psoriasis, contact dermatitis, inflammatory bowel
 disease, multiple sclerosis, atherosclerosis, sarcoidosis, idiopathic
 pulmonary fibrosis, dermatomyosititis, hepatitis, diabetes, allograft
 rejection and graft-vs-host disease.
 Screen for compounds which modulate T.alpha.1 activity
 In another embodiment, the invention provides a method for identifying a
 compound which modulates T.alpha.1 cell migration activity, angiogenesis
 activity or wound healing activity. The method includes: a) incubating
 components comprising the compound and T.alpha.1 under conditions
 sufficient to allow the components to interact; and b) determining the
 effect of the compound on T.alpha.1 activity before and after incubating
 in the presence of the compound. Compounds that affect T.alpha.1 activity
 (e.g., antagonists and agonists) include peptides, peptidomimetics,
 polypeptides, chemical compounds and biologic agents. T.alpha.1 activity
 can be assayed using methodology as described in the present Examples.
 Further, the method includes determining whether a compound has an effect
 on production of T.alpha.1 from prothymosin. Prothymosin can be analyzed
 before and after incubation to determine, for example, if the compound
 inhibits production of T.alpha.1. If a compound inhibits the production of
 T.alpha.1, only prothymosin, or a predominant amount of prothymosin will
 remain in the test sample. Alternatively, if the compound stimulates
 production of T.alpha.1 from prothymosin, less prothymosin and more
 T.alpha.1 will be present in the test sample after incubation. The amino
 acid sequence can be determined by standard N-terminal sequencing methods
 or by contacting the sample with a monoclonal antibody which distinguishes
 between prothymosin and T.alpha.1.
 Incubating includes conditions which allow contact between the test
 compound and the T.alpha.1 or prothymosin. Contacting includes in solution
 and in solid phase, or in a cell. The test compound may optionally be a
 combinatorial library for screening a plurality of compounds. Compounds
 identified in the method of the invention can be further evaluated,
 detected, cloned, sequenced, and the like, either in solution or after
 binding to a solid support, by any method usually applied to the detection
 of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et
 al., Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide
 (ASO) probe analysis (Conner, et al., Proc. Natl. Acad. Sci. USA, 80:278,
 1983), oligonucleotide ligation assays (OLAs) (Landegren, et al., Science,
 241:1077, 1988), and the like. Molecular techniques for DNA analysis have
 been reviewed (Landegren, et al., Science, 242:229-237, 1988).
 Without further elaboration, it is believed that one skilled in the art
 can, using the preceding description, utilize the present invention to its
 fullest extent. The following examples are to be considered illustrative
 and thus are not limiting of the remainder of the disclosure in any way
 whatsoever.
 EXAMPLE 1
 Cell Culture
 Human umbilical vein endothelial cells (HUVECs) were isolated from freshly
 delivered cords according to Jaffe et al. (J. Clin. Invest. 52: 2745,
 1973) and grown on Nunclon dishes (Nunc, Denmark) in RPMI 1640 (Life
 Technologies, Gaithersburg, Md.) supplemented with 20% bovine calf serum
 (Hyclone Laboratories, Logan Utah), 100 U/ml penicillin/streptomycin (Life
 Technologies, Gaithersburg, Md.), 50 .mu.g/ml gentamycin (Life
 Technologies, Gaithersburg, Md.), 2.7 .mu.g/ml amphotericin B
 (fungizone)(Life Technologies, Gaithersburg, Md.), 5 U/ml sodium heparin
 (Fisher Scientific, Pittsburgh, Pa.) and 200 .mu.g/ml endothelial cell
 growth supplement (ECGS) (Collaborative Research, Bedford, Mass.). Cells
 between passage 3 and 5 were used for all experiments. Human coronary
 artery endothelial cells (HCAECs) were grown on Nunclon dishes in the
 HUVEC media. Foreskin fibroblasts (Ffs) were grown in DMEM supplemented
 with 10% fetal bovine serum (Hyclone) on Falcon (Becton Dickinson, N.J.)
 dishes. HT1080 cells (human fibrosarcoma cell line) were grown in Falcon
 flasks in DMEM supplemented with 10% fetal bovine serum. Neutrophils were
 freshly isolated from buffy coats as previously reported (Kibbey et al.,
 J. Cell Phys. 160:185, 1994). Human aortic smooth muscle cells (AOSMCs)
 were grown in smooth muscle cell media (Clonetics, San Diego, Calif.) on
 Nunclon dishes.
 EXAMPLE 2
 Tube formation is enhanced in the presence of T.alpha.1
 Migration and differentiation during angiogenesis can be studied in vitro
 using Matrigel, a basement membrane matrix on which HUVECs undergo
 capillary-like tube formation (Grant et al., J. Cell. Physiol. 153: 614,
 1992; Kubota et al., J. Cell Biol. 107: 1589, 1988). The tube formation
 assay was performed according to Grant et al. (J. Cell. Physiol. 153:614,
 1992) with the following modifications. Twenty-four well Nunclon plates
 were coated with 320 .mu.l of Matrigel (10 mg/ml) and incubated at
 37.degree. C. for 30 min to promote gelling. 30,000 HUVECs were
 resuspended in growth media and added to each well with 1, 10, 100 and
 1000 ng of synthetic T.alpha.1, 20 and 200 .mu.g/ml ECGS (a bovine brain
 extract that contains aFGF and bFGF and acts as a positive control) or
 media alone. The final serum concentrations were 10% or 5%. After 18 h,
 plates were fixed with Diff-Quik (Baxter Healthcare Corporation, McGraw
 Park, Ill.) and the length of the tubes was measured using an Olympus CK2
 microscope with a 2.times. objective connected to a Javelin CCTV camera
 with a 3.3.times. coupler and NIH Image 1.57 software. Three random
 measurements of each of four wells at each culture condition were
 measured. Each experiment was repeated at least three times.
 When 10% serum was used, cells incubated with 100 or 1000 ng/ml T.alpha.1
 formed more tubes than control cells (FIG. 2B). The tubes were
 significantly longer (P.ltoreq.0.0001) with an average length of 316-324
 .mu.m compared to cells in media alone that formed tubes 208 .mu.m long
 (FIG. 1) and had large numbers of unorganized cells (FIG. 2A, arrow). When
 placed in media with 5% serum (FIG. 2C), in media alone or in 1 or 10
 ng/ml T.alpha.1, cells did not form tubes while those incubated with 100
 and 1000 ng/ml T.alpha.1 formed highly organized tubes (FIG. 2D).
 EXAMPLE 3
 Migration effects are cell type specific
 Several cell types were tested to determine if the migratory stimulation
 observed with T.alpha.1 was specific. Foreskin fibroblasts, neutrophils,
 HT1080 fibrosarcoma cells, and human aortic smooth muscle cells did not
 demonstrate any significant migration toward T.alpha.1 (Table 1). HUVEC
 migration assays were carried out in Boyden chambers using 12 ,m pore PVP
 free filters coated with either a 0.1 mg/ml or 0.05 mg/ml solution of
 collagen IV as previously reported (Malinda et al., FASEB J. 11:474,
 1997). Each condition was assayed in triplicate wells at least three times
 unless indicated. HCAECs, AOSMCs, FFs, HT1080 cells and neutrophils were
 cultured or isolated and assayed for migration as previously reported
 (Malinda et al., FASEB J. 11:474, 1997).
 The cells migrated toward their respective positive controls. In contrast,
 human coronary artery endothelial cells migrated to T.alpha.1 with a
 2-fold increase (P&lt;0.0001) in migration over media alone in response to
 1000 ng of T.alpha.1 (Table 1). Significant migration was also observed
 with 1-100 ng doses (P&lt;0.0001) showing that the response to T.alpha.1
 could occur in other endothelial cell types. Monocyte migration was
 stimulated by T.alpha.1 similar to the positive control. Monocyte
 migration increased in response to 1000 ng/ml of T.alpha.1 (P=0.003),
 while migration appeared to be inhibited by 100 ng/ml. Higher levels of
 T.alpha.1 may trigger migration similar to an inflammatory response when
 the cells invade surrounding tissues and the inhibitory response to lower
 levels of T.alpha.1 could be due to cell activation. Therefore, T.alpha.1
 specifically stimulates endothelial cell and monocyte migration.
 EXAMPLE 4
 In vitro "scratch" wound closure is more rapid in the presence of T.alpha.1
 During angiogenesis the cells may be presented with a constant level of
 T.alpha.1. The "scratch" wound closure assay was used to assess the
 potential effects of constant doses of T.alpha.1 on cell migration.
 Confluent monolayers were "scratch" wounded using the tip of a universal
 blue pipette tip and rinsed with PBS. T.alpha.1 (1, 10, 100, and 1000
 ng/ml) or ECGS (200 .mu.g/ml) was added to the wells in fresh media
 lacking ECGS but containing 10 mM thymidine to inhibit cell proliferation
 (Malinda et al., FASEB J. 11: 474, 1997). Antibody inhibition experiments
 were carried out as described above by wounding the cell monolayer and
 then adding either anti-T.alpha.1 polyclonal antibody (1/25, 1/50
 dilution) or pre-immune serum (1/25 dilution), or fresh media to the
 plate.
 Migration of cells into the wounded area was significantly increased in the
 presence of 1000 ng/ml of T.alpha.1 (P&lt;0.0001) over migration in the
 presence of media alone within 2 h after wounding (14% closure vs 1%)
 (FIG. 3A and FIG. 3B). Wound closure was similar to that in the presence
 of the positive control, ECGS, and remained elevated throughout the 10 h
 experiment. Acceleration of wound closure was also observed in response to
 100 ng/ml T.alpha.1 as early as 2 h (7% vs 1%, P&lt;0.0001). When wounded
 monolayers were incubated in the presence of T.alpha.1 antibody, wound
 closure was inhibited 1.6-fold compared to that observed in the presence
 of media alone (P.ltoreq.0.0001). Pre-immune sera had no effect on cell
 migration and there was no indication of altered cell morphology or death
 of the anti-T.alpha.1-treated cells. Antibody reduction of the levels of
 T.alpha.1, therefore, altered HUVEC migration.
 EXAMPLE 5
 In vivo endothelial cell migration and vessel formation are enhanced by
 T.alpha.1
 Since T.alpha.1 enhanced HUVEC migration in vitro, experiments were
 performed to determine if T.alpha.1 promoted cell migration and vessel
 formation in vivo. Endothelial cells invade and form vessels in Matrigel
 plugs containing angiogenic factors (Kibbey et al., J. Cell Phys. 160:185,
 1994; Passaniti et al., Lab. Invest. 67: 519, 1992). Matrigel was mixed
 with T.alpha.1 (5, 50 and 500 .mu.g/ml) or ECGS (10 and 100 ng/ml) and
 injected subcutaneously into three or four C57Bl/6N female mice for each
 condition (Malinda et al., FASEB J. 11:474, 1997). Three random fields
 were collected and analyzed per plug in each of three experiments.
 Matrigel plugs containing 5 or 50 .mu.g/ml of T.alpha.1 showed a
 significant 3- to 3.7-fold increase (P&lt;0.0001, P=0.001 respectively) in
 the number of cells in the plugs compared to plugs containing Matrigel
 alone (FIG. 4). At the lower and higher concentrations, 1 .mu.g/ml and 500
 .mu.g/ml, a decrease in migration was observed. Cells migrate into the
 plug from the area closest to the skin forming vessels (FIG. 5). Plugs
 containing T.alpha.1 (FIGS. 5C and 5D) show many more cells than in the
 Matrigel alone (FIG. 5A). Cell morphology was similar to those in plugs
 containing 10 ng/ml of the positive control ECGS (FIG. 5B). These results
 indicate that T.alpha.1 is a potent cell migration and vessel formation
 factor in vivo.
 EXAMPLE 6
 In vivo wound healing is accelerated by T.alpha.1
 T.alpha.1, whether administered topically or intraperitoneal, significantly
 accelerated wound healing as compared to untreated wounds (FIG. 6A vs 6B,
 and 6C). Six full thickness 8 mm punch biopsy wounds were made on the
 dorsal surface of rats as previously reported (Bhartiya et al., J. Cell.
 Physiol. 150:312, 1992; Sihhu et al., J. Cell. Physiol. 169:108, 1996) and
 T.alpha.1 was given topically at the time of wounding (5 .mu.g in 50
 .mu.l) and again after 48 hr. Controls for the topical treatment received
 identical amounts of saline at the time of wounding and at 48 hr. Three
 rats in each group also received intraperitoneal injections at the time of
 wounding (60 .mu.g in 300 .mu.l) and again every other day. Controls for
 these animals received identical amounts of saline i.p. on the same
 injection schedule. At days 8 and 9 post-wounding, tissue was collected
 and fixed in 10% buffered formalin (i.p. n=18 samples; topical n=9
 samples). The samples were sectioned and stained with H&E and Masson's
 Trichrome (American Histolabs, Gaithersburg, Md.).
 Migration of cells into the granulation tissue and complete
 reepithelization of the epithelium were observed (FIGS. 6B and 6C).
 T.alpha.1 treatment by both methods resulted in considerable capillary
 ingrowth especially, when applied topically (FIG. 6D vs 6E and 6F). These
 results suggest that T.alpha.1 is active in vivo for the formation of
 granulation tissue by promoting cell proliferation, migration and vessel
 formation. Additionally, an increase in the accumulation/biosynthesis of
 collagen by T.alpha.1 treated wounds as compared to the untreated control
 (FIG. 6G vs 6H and 6I) suggests a role for T.alpha.1 in wound contraction
 (Kirsner et al., Dermatologic Clinics 11:629, 1993).
 The invention now being fully described, it will be apparent to one of
 ordinary skill in the art that many changes and modifications can be made
 thereto without departing from the spirit or scope of the appended claims.
 TABLE 1
 Response of Different Cell Types to T.alpha..sub.1
 Thymosin .alpha..sub.1 (ng/ml)
 Positive
 Cell Type 1000 100 10 1 0
 Control
 FF 5.2 .+-. 0.5 4.8 .+-. 0.7 5 .+-. 0.4 5 .+-. 0.4 5 .+-.
 0.3 bFGF (10 ng/ml) =