The field of the invention is lymphokines and fibrosis.

Schistosomiasiβ is one of the most prominent helminthic diseases, estimated to afflict 200 million people in the tropics (Nalsh et al., 1979). Two of the schistosome species that infect humans (Schiεtosoma ansoni and S. japonicum) can cause serious morbidity (including portal hypertension and gastrointestinal hemorrhage) as a result of a form of hepatic fibrosis

(Cheever et al., 1967). However, only a relatively small subpopulation (3-6%) of infected individuals develop this scarring; the others remain generally healthy (Chen et al., 1988). Traditional forms of anthelminthic therapy have a number of undesirable side effects, and treatment with the relatively new drug, praziquantel, is very expensive, thus making anthelminthic treatment of all infected individuals impractical. In addition, while early anthelminthic therapy may aid in preventing liver scarring, it has not been established that this is an invariable outcome of treatment (Homeida et al., 1988).

Alternatively, early aggressive treatment of infected individuals with anti-inflammatory or ixomunosuppressive drugs including methotrexate, cytotoxins, and various corticosteriods may aid the prevention of scarring. However, given that these drugs are known to produce a number of relatively severe side effects, treatment of all infected individuals, 94-97% of

- 2 - which would never develop the progressive fibrotic form of the disease, is both undesirable and impractical. Several other chronic inflammatory diseases, including pulmonary fibrosis, scleroderma/progressive systemic sclerosis, sarcoidosis, sclerosing cholangitis, primary biliary cirrhosis, and inflammatory bowel disease also can result in organ dysfunction due to pathological fibrosis. As in schistosomiasis, only a subpopulation of individuals with these diseases develop debilitating organ scarring. Thus, methods are needed that would make it possible to predict which patients will develop the progressive fibrotic forms of these inflammatory diseases so that more aggressive, antifibrotic courses of treatment can be limited to only those individuals who would benefit from these treatments.

PTOHMTC QF THE IFVBPHgB FsF-1, also referred to as fibrosin, is a novel lymphokine that is a heparin-binding growth factor. It stimulates fibroblast proliferation, collagen and hyaluronan synthesis, and acts as a chemoattractant for fibroblasts. FsF-1/fibrosin is distinct from other previously characterized heparin-binding growth factors.

The invention features a substantially pure FsF-l polypeptide. This polypeptide may be encoded by mammalian DNA, such as that of a mouse or a human. By "human FsF-l" is meant the polypeptide encoded by the DNA sequence of SEQ ID NO.ill. By "murine FsF-1" is meant the polypeptide encoded by the DNA sequence of SEQ ID NO.:2. By "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation) . An FsF-l polypeptide is preferably a polypeptide having one or more of the biological activities of human FsF-1. As described

herein, these activities include stimulation of fibroblast proliferation, stimulation of fibronectin, collagen, and hyaluronan synthesis, and chemoattraction. The invention also includes biologically active polypeptide fragments or analogs of the FsF-1 of the invention.

By "biologically active" is meant possessing any in vivo or in vitro activity that is characteristic of the FsF-1 of the invention as assayed by the methods described herein. A biologically active FsF-1 polypeptide or polypeptide fragment generally possesses at least 40%, more preferably at least 70%, and most preferably at least 90% of the activity of human FsF-1 described herein. A biologically active FsF-1 polypeptide may also have activity that is 30%, 50%, 70%, 80%, or 90% of the activity of the human or murine FsF-1 2B3 domain. A biologically active FsF-1 polypeptide may also have activity that is 30%, 50%, 70%, 80%, or 90% of the activity of a subdomain of the human or murine FsF-1 2B3 domain, such as 2B3a or 2B3b. By "2B3 domain of FsF- 1" is meant that portion of FsF-1 corresponding to amino acids 26 (Arg encoded by AGG) to 96 (Leu encoded by CTA) of murine FSF-1 (SEQ ID N0.:1). The "2B3 domain of human FsF-1" corresponds to the 71 amino acid polypeptide of the murine 2B3 domain of FsF-1 shown in SEQ ID N0.:1. The 2B3 domain of human FsF-1 is encoded by nucleotides within the human fibrosin cDNA sequence of SEQ ID NO.:11, beginning at nucleotide 318 (codon AGG) . The 2B3 domain has been further divided into the subdomains, 2B3a and 2B3b. By "2B3a subdomain" is meant polypeptides 26-45 of SEQ ID NO.:l. By "2B3b subdomain" is meant polypeptides 46-96 of SEQ ID N0.:1. The 2B3 domain exhibits comparable activity to full-length fibrosin in a cellular proliferation assay, such as the fibroblast proliferation assay described herein.

The polypeptide of the invention may be immuno- purified from the supernatant of granuloma cultures, or from the serum of a mammal, such a mouse or a human. The polypeptide of the invention may be a synthetic polypeptide.

The invention also includes an FsF-1 polypeptide, FsF-1 polypeptide fragments, or analogs of FsF-l that function as antagonists of the FsF-1 receptor.

In another aspect, the invention features isolated DNA consisting essentially of a DNA sequence encoding an FsF-l polypeptide, or a fragment of the FsF-1 polypeptide such as the 2B3 domain, the 2B3a subdomain, or the 2B3b subdomain.

By "isolated", as used herein in reference to DNA, is meant a DNA that is not immediately contiguous with (i.e., covalently linked to) both of the coding sequences with which it is immediately contiguous in the naturally occurring genome of the organism from which the DNA of the invention is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector (e.g., an autonomously replicating virus or plasmid) , or into the genomic DNA of a prokaryote or eukaryote; DNA which exists as a separate molecule independent of other DNA sequences such as a cDNA or genomic DNA fragment produced by chemical means (e.g., polymerase chain reaction, ligase chain reaction) , or by restriction endonuclease treatment; and recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence(s). Also included in the isolated DNAs of the invention are single-stranded DNAs that are generally at least 10 nucleotides long, preferably at least 18 nucleotides long, more preferably at least 30 nucleotides long, and ranging up to full-length of the gene or cDNA encoding an FsF-1 polypeptide. The single-

stranded DNAs can also be detectably labelled for use as hybridization probes, and can be antisense.

Preferably, the isolated DNA hybridizes under conditions of high stringency to a nucleic acid having the sequence of Fig. 25 (SEQ ID N0.:2) or Fig. 26 (SEQ ID N0.:3).

By "high stringency" is meant, for example, conditions such as those described herein below for the isolation of human FsF-1 cDNA (also see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989).

The DNA of the invention can be incorporated into a vector [which may be provided as a purified preparation (e.g., a vector separated from the mixture of vectors which make up a library) ] containing a DNA sequence encoding an FsF-1 polypeptide of the invention or a fragment of the FsF-1 polypeptide, and a cell or essentially homogeneous population of cells (e.g. prokaryotic cells, or eukaryotic cells such as mammalian cells) which contain the vector or the isolated DNA described above.

By "essentially homogenous population of cells" is meant that at least 99% of the cells contain the vector of the invention (or the isolated DNA) . Preferably, the vector is capable of directing expression of an FsF-1 polypeptide (for example, in a cell transfected or transformed with the vector) .

A nucleic acid "consisting essentially of" a particular sequence of nucleotides as used herein refers to that particular sequence and other sequences that are the same as the first sequence but for the addition to or removal from the sequence of a few nucleotides (e.g. , 2 to 10) which does not alter the amino acid sequence encoded by the nucleic acid.

"Similarity", as used herein in reference to DNA or polypeptide sequences, refers to the subunit sequence similarity between two molecules. When a subunit position in both of the molecules is occupied by the same monomeric subunit (i.e., the same nucleotide or amino acid, respectively) , then the molecules are the same at that position. The similarity between two nucleotide or two amino acid sequences is a direct function of the number of matching or homologous positions, e.g., if half the positions in two DNA or two amino acid sequences are the same, then the sequences are 50% similar.

By "substantially similar" is meant a polypeptide or nucleic acid that is at least 60%, preferably 75%, more preferably 80%, and most preferably 95% the same as a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.

Sequence similarity, can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) .

In the case of amino acid sequences which are less than 100% the same as a reference sequence the non- similar positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,

glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine and tyrosine.

Where a particular polypeptide is said to have a specific percent similarity to a reference polypeptide of a defined length, the percent similarity is relative to the reference peptide. Thus, a peptide that is 50% the same as a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% the same as the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. A further feature of the invention is a substantially pure antibody that specifically binds FβF- 1. By "specifically binds" is meant an antibody that binds to FsF-1 and that does not substantially recognize and bind to other antigenically-unrelated molecules. Antibodies according to the invention may be prepared by a variety of methods. For example, the FsF-1 protein, or antigenic fragments thereof, can be administered to an animal in order to induce the production of polyclonal antibodies. Alternatively, antibodies according to the invention may be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Bur. J. Immunol . 6:511, 1976; Kohler et al., Bur J. Immunol . 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981) . The invention features antibodies which specifically bind a murine FsF-1 polypeptide, a human FsF-1 polypeptide, or another FsF-1 polypeptide. In particular the invention features "neutralizing" antibodies. By "neutralizing" antibodies is meant antibodies that interfere with any of the biological activities of FsF-1. The activities

described herein include stimulation of fibroblast proliferation, stimulation of fibronectin, hyaluronan, and collagen synthesis, and stimulation of fibroblast chemotaxis. The neutralizing antibody may reduce the fibroblast proliferation activity of FsF-1, preferably full length naturally occurring human FsF-1, preferably by 50%, more preferably by 70%, and most preferably by 90% or more. Any standard fibroblast proliferation assay, including the assay described herein, may be used to measure fibroblast proliferation.

In addition to intact monoclonal and polyclonal anti-FsF-1 antibodies, the invention features various genetically engineered antibodies, humanized antibodies, and antibody fragments, including F(ab')2, Fab', Fab, Fv and sFv fragments. Antibodies can be humanized by methods known in the art, e.g., monoclonal antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA) . Fully human antibodies, such as those expressed in transgenic animals, are also features of the invention (Green et al., Nature Genetics 7:13-21, 1994).

Ladner (U.S. Patent 4,946,778 and 4,704,692) describes methods for preparing single polypeptide chain antibodies. Ward et al., (Nature 341:544-546, 1989) describe the preparation of heavy chain variable domains, which they term "single domain antibodies," which have high antigen-binding affinities. McCafferty et al., (Nature 348:552-554, 1990) show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen, and that rare phage (one in a million) can be isolated after affinity chromatography. Boss et al. (U.S. Patent 4,816,397) describes various methods for producing immunoglobulins, and immunologically functional fragments thereof, which include at least the variable

domains of the heavy and light chain in a single host cell. Cabilly et al. (U.S. Patent 4,816,567) describe methods for preparing chin-eric antibodies.

As used herein, the term "substantially pure" describes a compound, e.g., a protein, polypeptide, or antibody, that is substantially free from the components that naturally accompany it. Typically, a compound is substantially pure when at least 60%, preferably at least 75%, more preferably at least 90%, and most preferably at least 99%, of the total material (by weight) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, polyaerylamide gel electrophoresis, or HPLC analysis. The FsF-1 polypeptide, according to the invention, may be used as the active ingredient of therapeutic compositions. In such therapeutic compositions, the active ingredient may be formulated with a physiologically-acceptable carrier or anchored in the membrane of a cell. .'Such therapeutic compositions are used to stimulate fibroblast proliferation and extracellular matrix synthesis, e.g., to promote wound healing. The method involves applying the therapeutic composition, preferably topically, to a wound of a mammal in a dosage effective to stimulate fibroblast proliferation and thereby accelerate wound closure.

Another aspect of the invention is a therapeutic composition that contains a substantially pure antibody as the active ingredient of the composition. The antibody of the therapeutic composition may bind human FβF-1, murine FsF-1, the 2B3 domain of human or murine FsF-1, or the subdomains, 2B3a and 2B3b, of human or murine 2B3. The antibody of the therapeutic composition may be conjugated to an immunotoxin. The antibody would be formulated with a physiologically-acceptable carrier,

such as physiological saline, for administration to a patient in a dose sufficient to suppress fibrosis, the chemotactic movement of fibroblaβts, angiogeneβis, or inflammation. Antibodies of the invention can be administered by any standard route including intraperitoneally, intramuscularly, subcutaneously, or intravenously. It is expected that the preferred route of administration will be intravenous. As is well known in the medical arts, dosages for any one patient depends on many factors, including the general health, sex, size, body surface area, and age of the patient, as well as the particular compound to be administered, time and route of administration, and other drugs being administered concurrently. Dosages for the antibodies of the invention will vary, but a preferred dosage for intravenous administration is approximately 0.01 mg to 100 mg/ml/blood volume. Determination of correct dosage for a given application is well within the abilities of one of ordinary skill in the art of pharmacology. Skilled artisans will be aided in their determination of adequate dosage by previous studies. For example, Abraham et al., (1995, J. Amer. Med. Assoc. 273:934-941) administered TNF-α monoclonal antibody (TNF-α-MAb) at doses of 1 to 15 mg/kg. TNF-α-MAb was well tolerated by all patients; despite the development of human antimurine antibodies in these patients, no serum sickness-like reactions, skin reactions, or systemic allergic reactions developed. Similarly, Rankin et al., (1995, Br. J. Rheumatol. 34:334-342) administered a single intravenous dose of 0.1, 1.0 or 10 mg/kg of an engineered human antibody, CDP571 that neutralizes human TNF-α. Both studies also detail the criteria used to select patients and their subsequent physical evaluation.

The patient in need of such treatment may have been diagnosed as having pulmonary fibrosis, adult

respiratory distress syndrome, cystic fibrosis, asthma, emphysema, scleroderma/progressive systemic sclerosis, sarcoidosis, sclerosing cholangitis, primary biliary cirrhosis, glomerulonephritis, renal failure, inflammatory bowel disease, gastrointestinal fibrosis, Crohn's disease, ulcerative colitis, intestinal occlusion, a fibrotic cancer, optic fibrosis, dermal fibrosis, scleroderma, marrow fibrosis, joint fibrosis, and vascular fibrosis which could have been caused by a chronic inflammatory disease.

The isolated DNA of the invention can be used to detect the level of mRNA encoding FsF-1 in a sample. The method involves contacting the sample with all or a portion of a single-stranded nucleic acid of the invention under hybridization conditions which allow the formation of nucleic acid duplexes between the nucleic acid and mRNA in the sample, and then determining the amount of duplexes in the sample as an indication of a propensity for tissue fibrosis. The detection of the duplexes can involve any standard techniques for identifying duplex molecules. Preferably, either the nucleic acid of the invention, or the mRNA from the sample are labeled with a chemical moiety which is capable of being detected, including, without limitation, radioactive isotopes, enzymes, luminescent agents, precipitating agents, and dyes.

Individuals skilled in the art will readily recognize that the compositions of the present invention can be assembled in a kit for the detection of FsF-1 polypeptides or mRNA. Typically, such kits include reagents containing the nucleic acids or antibodies of the present invention with instructions and suitable packaging for their use as part of an assay for FβF-1.

In another aspect, the invention features an FsF-1 polypeptide (or a substantially pure preparation

thereof) , produced by the expression of a recombinant DNA molecule encoding the FsF-1 polypeptide. Preferably, the polypeptide includes a fragment of a naturally occurring FsF-1 polypeptide. The polypeptide may be full length, may contain the 2B3 domain, or subdomains of 2B3, such as 2B3a and 2B3b, and is capable of stimulating the growth, mitogenesis and chemotaxis of fibroblasts according to the assays described herein. More preferably, the polypeptide also stimulates fibroblast extracellular matrix synthesis. Most preferably, the polypeptide includes the amino acid sequence depicted in Fig. 18 (SEQ ID NO.:l).

Another feature of the invention is a therapeutic method designed to mitigate pathological scarring by inhibiting production (via transcriptional or translational mechanisms) of the FsF-1 protein or DNA of the invention. The method entails administering to a patient suffering from a chronic inflammatory disease antisense molecules to the DNA of the invention. By "antisense" is meant a molecule that is 10 or more nucleotides long and that is the reverse complement of a portion of the coding strand of the double stranded DNA of the invention.

Pathological fibrosis in schistosomiasis is preceded by a chronic granulomatous inflammatory reaction to helminth eggs deposited in the liver. In patients, there is a well-established association between the presence of severe liver fibrosis and relatively high levels of T cell responsiveness to schistosomal antigens during chronic infection. This is a striking finding, because such brisk T cell responsiveness —which typically is present in the early stages of the infection— is markedly reduced in many (or most) chronically-infected patients without severe liver fibrosis. These observations indicate that spontaneous

dσtrn-regulation of anti-schistosomal, especially anti- schistosomal egg antigen (anti-SEA) T cell responses typically occur in chronic infection in patients, whereas persistently high responsiveness contributes to the development of fibrosis.

The invention also features methods for identifying individuals with a propensity for pathological fibrosis. The methods include providing a sample from an individual with a chronic inflammatory disease, contacting the sample with an antibody specific for FsF-1 under conditions which permit immunocomplex formation, and detecting an increase in the relative level of the immunocomplex as an indication of a propensity for pathological fibrosis. By "relative level" is meant the relative amount of immunocomplex detected when compared to the level in a sample from a normal individual.

In an individual at a chronic stage of the disease, an increased level of the immunocomplex in a single sample is indicative that they are at risk of serious fibrosis. In an individual at an early stage of the disease, the method further involves providing a first and a second sample from the individual over a period of time (e.g., every 3 to 6 months over a period of 1 to 3 years) , and detecting a persistent increase in the relative level of the immunocomplex as an indication of a propensity for pathological fibrosis.

The sample may be any biological sample. Preferably, the sample is a blood, serum or plasma sample, but may also be a urine sample; a tissue sample (e.g., biopsy); an effusion obtained from a joint, the abdominal cavity (e.g., ascites) , pleura1 fluid, cerebral spinal fluid, and the aqueous humor; or from the supernatant of cultured peripheral blood mononuclear

cells. Also preferably, the sample is obtained from a mammal, and even more preferably, the mammal is a human.

In one preferred embodiment, the pathological scarring results from hepatic fibrosis. In another related embodiment, hepatic fibrosis is the result of the disease schistosomiasis. in other embodiments, the pathological fibrosis is a result of various chronic inflammatory diseases including pulmonary fibrosis, adult respiratory distress syndrome, cystic fibrosis, asthma, emphysema, scleroderma/progressive systemic sclerosis, sarcoidosis, sclerosing cholangitis, primary biliary cirrhosis, glomerulonephritis, renal failure, inflammatory bowel disease, gastrointestinal fibrosis, Crohn's disease, ulcerative colitis, intestinal occlusion, a fibrotic cancer, optic fibrosis, dermal fibrosis, scleroderma, marrow fibrosis, joint fibrosis, and vascular fibrosis.

We have purified a potent fibroblast mitogen, FsF- 1, and have demonstrated that this polypeptide is overproduced by the CD4 cells contained within these egg granulomas. Thus, we have concluded that FsF-l plays an important role in the progression of fibrotic pathogenesis in liver, and tissue fibrosis in other chronic inflammatory disease. In addition, FsF-1 is overproduced in lymphocytes of the organ wherein scarring develops, and in relatively high amounts (i.e., compared to animals which do not develop fibrosis) in serum, plasma, blood, urine and serous effusions. Thus, detection of FsF-1 in the lymphocytes from patients with chronic inflammatory diseases using the method of the present invention provides a relatively simple and rapid means to detect those individuals with a propensity to develop the progressive fibrotic forms of these diseases. This provides the advantage of allowing clinicians to limit

treatment to only the subpopulation of infected individuals which require antifibrotic and/or anthelminthic therapy.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications mentioned herein are incorporated by reference. Examples of the preferred methods and materials will now be described. These examples are illustrative only, and not intended to be limiting as those skilled in the art will understand that methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. DETAILED PEgCRIPTIQN

The drawings will first briefly be described. Fig. 1 is a gel filtration chromatograph (Biogel P-30) of unconcentrated egg granuloma culture supernatant. Samples of each fraction were assayed at a final concentration of 1/20 for their ability to stimulate fibroblast proliferation (uptake of [ ³ H]- thymidine) . Each point is a mean of three determinations (SEM ≤ 10% of the mean) . Elution positions of relevant molecular weight standards are indicated. Shown is a representative experiment of more than six experiments. Fig. 2 is a heparin-Sepharose affinity chromatograph of a pool of biologically active fractions obtained by gel filtration chromatography (Biogel P-30) of crude granuloma culture supernatant. Thirty 1 ml fractions were collected. Each fraction was tested for its ability to stimulate fibroblast proliferation. The mean cpm of triplicate determinations is indicated (SEM ≤ 10%) . Shown are the results of a representative experiment of two performed.

- 16 -

Fig. 3 is a FPLC anion exchange chromatograph of purified FsF-1. The eluted material from heparin- Sepharose was applied to a Mono Q column and eluted at a rate of 1 ml/min with a gradient of NaCl (0 to 2.2 M NaCl) . Arrow marks the elution position of commercial heparin. Each point represents the mean of three determination: SEM ≤ 10%. Shown is a representative experiment of two performed.

Fig. 4 is a photograph of an SDS-polyacrylamide gel (10% acrylamide; silver stained) . FsF-1 (lane 2) was prepared from granuloma culture supernatants by our published methods and used for immunizing rabbits to prepare anti-FsF-1 IgG described in this report. For comparison, the electropherogram of proteins present in unfractionated granuloma supernatant is shown in lane 1. Note that the migration position of FsF-l (lane 2) corresponds to that of a major protein (MW 60 kD) present in the starting material (lane 1).

Fig. 5 is a Western blot of cell-free supernatants from egg granuloma cultures (granuloma supernatant) probed with anti-FsF-1 antibody. Granuloma supernatant was subjected to SDS-PAGE, stained with Coomassie Blue (lane a) , electrophoretically transferred to Immobilon-P Transfer Membranes (Millipore) and then treated with anti-FsF-1 IgG (lane b) , or pre-immune IgG (lane c) , followed by alkaline phosphatase-conjugated goat anti-rabbit IgG (Promega Corp., Madison, WI) and developed with substrate as described. The migration position of standard molecular weight markers is shown. Figs. 6A and 6B are dot blot ELISA of FsF-1 (10 ng) , plasma fibronectin (FN; 20 ng) , acidic fibroblast growth factor (aFGF; 100 ng) , and platelet derived growth factor (PDGF; 40 ng) applied to nitrocellulose paper in a volume of 1 μl to 5 μl and then probed with antibodies.

Figs. 7A and 7B are a pair of graphs. 7A depicts a heparin-Sepharose eluate (FsF-l:lθ ng/ml) or FGF (5 ng/ml) incubated with either normal rabbit IgG (open bar) or with anti-FsF-1 (closed bar) at a final concentration of 2.5 μg/ml. Remaining fibroblast-mitogenic activity is represented as percent cpm obtained with untreated mitogens. 7B depicts PDGF (10 ng/ml) , biologically active peak from P-30 chromatography, or heparin- Sepharose eluate (FsF-l: -10 ng/ml) treated with either normal rabbit IgG (open bar) or anti-PDGF IgG (closed bar; 50 mg/ml) . The supernatants were then tested for fibroblast mitogenic activity. Shown is a representative experiment of three performed.

Figs. 8A and 8B are graphs showing growth of bovine aortic endothelial cells (open symbols) and human fibroblasts (solid symbols) in response to FGF (A) or to heparin-Sepharose purified FsF-1 (B) . Growth response was determined by counting numbers of cells per culture after a 96 h incubation. Baseline counts (medium alone) were: fibroblasts, 6.5 ± 0.1 x 10 ⁴ ; endothelial cells, 6.1 ± 0.1 x 10 ⁴ . The first two points in B represent responses to FsF-1 at concentrations of 0.01 and 0.1 vol %. Shown is a representative experiment of two performed. Each point represents a mean of four determinations (SEM ≤ 10%) .

Figs. 9A and 9B are curvilinear representations of flow cytometry analysis of dissociated granuloma cells stained with NRS plus FITC-conjugated goat anti-rabbit IgG (a) or anti-FsF-1 IgG followed by FITC-conjugated goat anti-rabbit IgG (b) .

Figs. 10A-10D are contour plots of flow cytometry analysis of monodispersed cells obtained from isolated hepatic egg granulomas. Cells enzymatically dissociated from intact granulomas were stained in the following manner and then analyzed with a FACScan: unstained cells

(a) ; phycoerythrin-conjugated rat anti-mouse CD4 antibody (b) ; anti-FsF-1 IgG followed by FITC-conjugated goat anti-rabbit IgG (c) ; anti-FsF-1 IgG, followed by FITC-conjugated anti-rabbit IgG, followed by phycoerythrin conjugated rat anti-CD4 (d) .

Fig. 11 is an autoradiograph of metabolically- labeled proteins produced by granuloma CD4* lymphocytes following SDS-PAGE. CD4 ⁺ lymphocytes were purified by FACS from suspensions of enzymatically-dissociated granuloma cells and incubated for 24 h in the presence of ³⁵ S-methionine- ³⁵ S-cysteine. Cell-free supernatants were then either subjected to 10% SDS-PAGE directly (lane 1) or were precleared by incubation with Sepharose- conjugated normal rabbit IgG and then treated with anti-FsF-1 IgG using two different antibody concentration (5 μg/ml, lane 2; 15 μg/ml, lane 3) prior to electrophoresis. The migration position of standard molecular weight markers is indicated.

Fig. 12 is a line graph depicting the results of an ELISA antigen-capture assay of FsF-1 in granuloma supernatants.

Fig. 13 is a line graph that depicts the levels of FβF-1 in sera of uninfected or infected mice in an ELISA assay. Fig. 14A is a line graph that depicts fibroblast [ ³ H]-thymidine incorporation in response to culture supernatants of CDC25 cells stimulated with concanavalin A (con A) for 24 h. Mean ± SEM of triplicate determinations is shown for each concentration of culture supernatant tested.

Fig 14B is a bar graph that demonstrates the partial neutralization of the fibroblast mitogenic activity in CDC25 culture supernatant. Supernatants were incubated for 1 h with either pre-immune, normal rabbit IgG (NRIgG), or rabbit anti-FsF-1 IgG at 2.5 μg/ml. The

mixture was tested at 5% vol/vol concentration. NRIgG did not affect the response to culture supernatant. Mean ± SEM of triplicate determinations is shown.

Fig. 15 is a graph that depicts fibroblast [ ³ H]~ thymidine incorporation in response to culture supernatants from COS-7 cells transfected with plasmid DNA representing the whole CDC25 cDNA library (closed symbols) and from sham-transfected COS-7 cells (open symbols) . Mean ± SEM of triplicate determinations is shown for each concentration of culture supernatant shown.

Fig. 16a is a graph that depicts the growth of fibroblasts in response to culture supernatants from COS- 7 cells transfected with plasmid DNA containing the clone 2B3 cDNA insert. The mean + SEM of fibroblast cell number determined after 96 h of incubation is shown for each concentration of transfectant culture supernatant tested (indicated on logarithmic scale) . Cultures of fibroblasts maintained for 96 h in medium alone contained 6.7 ± 0.3 x 10 ⁴ cells.

Fig. 16b is a bar graph that depicts the neutralization of fibroblast stimulating activity in culture supernatants of COS-7 cells transfected with clone 2B3-containing plasmid DNA. Supernatants were incubated for 1 h with 7.5 μg/ml of either normal rabbit IgG (NRIgG) or rabbit anti-FsF-1 IgG followed by adsorption with protein A-Sepharose and tested at a final concentration of 0.01% vol/vol for the ability to stimulate fibroblast [ ³ H]-thymidine incorporation. The effect of treatments is shown relative to the response of fibroblasts to untreated transfectant culture supernatant tested at 0.01 vol%.

Fig. 17 is a graph that illustrates sib selection in the cloning of 2B3 cDNA that encodes a fibroblast mitogen. COS-7 cells were transfected with plasmid DNA

and the transfectant supernatants were tested at various concentrations for their ability to stimulate fibroblast [ ³ H] thymidine incorporation. Following transfection with the entire library cDNA (10 ⁶ clones), pools of clones were sequentially screened and selected for their ability to encode biologically active macromolecules. The inverse correlation between the number of clones per screen and the concentration of the corresponding transfectant culture supernatant that maximally stimulated fibroblast responses reflects the progressive enrichment of cDNA that encodes fibrogenic activity. Fig. 18 is an illustration of the nucleotide sequence and predicted amino acid sequence of the 2B3 cDNA insert (SEQ ID NO.:l). Codons 1-25 are derived from the vector; the insert begins with codon AGG (asterisk) and ends with the termination codon TAA (codon 97) .

Fig. 19 is a bar graph that depicts fibroblast growth in response to 72 h incubation with various doses of the synthetic 2B3 peptide in the absence of serum. The mean (±SEM) responses of triplicate determinations in a representative experiment are shown. The horizontal line depicts the mean fibroblast number grown in medium alone (baseline) . In all, twelve experiments were performed; cells were enumerated at 72 h in 6 experiments, and at 96 h in 6 experiments. Maximum responses at pM concentrations were 3-4-fold baseline, and 2-3-fold baseline at μM concentrations.

Fig. 20 is a graph depicting the fibroblast mitogenic activity of supernatants from cloned T cell hybridomas stimulated with 10 μg/ml Con A.

Fig. 21 is a bar graph of the elution profile of the hybridoma-derived mitogen from T hybridoma B12.

Fig. 22 is a graph depicting the SEA stimulation of FsF-1 production in hybridoma B12.

Fig. 23 is a photograph of a Western Blot of crude supernatants from con A-stimulated T cell hybridomas using anti-FsFl Mabs.

Fig. 24 is a bar graph depicting the immunopurification of mitogenic activity with anti-FsF-1 Mab from T hybridoma B12.

Fig. 25 is the nucleotide sequence of mouse fibrosin cDNA (SEQ ID NO. : 2). Underlined are the PCR primers, T2 and B4. Fig. 26 is the nucleotide sequence of human fibrosin cDNA (SEQ ID NO. : 3).

Fig. 27 is a photograph of a Western blot stained with rat monoclonal antibodies (Mab) . Crude egg granuloma culture supernatants (lane 1) and T cell hybridoma culture supernatants (lane 2) were subjected to SDS-PAGE and electrotransferred to nitrocellulose paper. The presence of fibrosin (60 kD) was revealed by staining with three separate anti-fibrosin Mabs ("D6"-D8/CD9; "G1"-IIIG6/C7; "A1"«IIIA5/D8) . No bands were revealed following incubation with normal rat IgG. Fig. 28 is a graph of fibroblast proliferative responses to fibrosin. Fibrosin was purified by im unoaffinity chromatography from two representative sources: T cell hybridomas (closed circles) and normal mouse serum (closed squares) and applied to fibrobasts in culture. The addition of homologous anti-fibrosin Mab to cultures abrogated the responses to T cell-derived fibrosin (open circles) . Mean ± SEM of triplicate determinations are shown for a representative experiment. Fig. 29 is a bar graph of the results of fibrosin antigen-capture ELISA. Wells were coated with rat anti- ibrosin monoclonal IgG-containing hybridoma culture supernatants, the wells were then blocked with gelatin- containing buffer, and 1 pg/ml purified fibrosin was added for 18 h (room temperature) . After washing, pre-

immune rabbit IgG (0.5 μg/ml), rαF IgG (0.5 μg/ml), or rαF IgG (0.5 μg/ml) previously incubated (18 h, room temperature) with purified fibrosin (1 ng/ml) (""blocked" IgG) was added. The plates were washed and developed with alkaline phosphatase-conjugated goat anti-rabbit IgG. Mean of duplicate determinations is shown.

Fig. 30 is a bar graph of the production of fibronectin by fibroblasts in response to fibrosin and 2B3 synthetic peptide. The graph also shows that these responses are neutralized by anti-fibrosin monoclonal antibody (Mab) .

Fig. 31A is a graph of fibroblast chemotactic response to purified native murine fibrosin. Mean ± SEM of triplicate determinations. Fig. 3IB is a graph of the fibroblast chemotactic response to synthetic 2B3 peptide.

Fig. 31C is a bar graph of the fibroblast . chemotactic response to fibrosin (40 ng/ml) and TGF-01 (86 pg/ml) and the selective neutralization of the response by anti-fibrosin monoclonal antibody (Mab; 7.5 ng/ml) .

Figs. 32A-32D are a series of bar graphs depicting the production of fibronectin by fibroblasts in response to purified native fibrosin after 6 h (open bars) and 24 h (striped bars) of stimulation (panel A) ; the production of hyaluronan by ibroblasts in response to purified native fibrosin (panel B); collagen synthesis, as determined by the incorporation of [ ³ H]-proline into collagenase-βensitive protein (panel C; dummy control is a 1.5 M NaCl eluate of a heparin affinity column to which no material had been adsorbed) ; and neutralization of collagen synthesis induction by fibrosin (panel D) . In D, fibrosin and TGF-9 were incubated with anti-fibrosin monoclonal antibody and the collagen synthetic response

to this mixture was compared to the response of untreated agonists.

Fig. 33 is a bar graph of spontaneous fibrosin production by smooth muscle cells isolated from the muscularis mucosae of normal human bowel, or bowel from a patient with Crohn's disease.

Fig. 34 is a bar graph of the concentration of fibrosin (FsF-l; pg/ml) in conditioned medium from cultured rheumatoid synoviocytes that were treated with phytohemaglutin (+PHA) , or were untreated (-PHA) .

Fig 35 is a bar graph of the concentration of fibrosin (FsF-1; pg/ml) in sera from Brazilian patients infected with Schistosoma mansoni (Schistosomiasis) , and in sera from normal, non-infected Brazilians (Control) . Fig. 36 is a pair of line graphs depicting the concentration of fibrosin in patients with biliary cirrhosis during the course of treatment with colchicine or methotrexate.

Fig. 37 is the nucleotide sequence of human fibrosin cDNA (SEQ ID NO.:11). This sequence includes the sequence that encodes the 2B3 domain.

Figs. 38A-38C are a series of photomicrographs of scleroderma cells cultured in MEM Eagle's Medium with 10% PCS and stained with an antibody against type I collagen. The cells were treated with 20 μg/ml of random IgG (a) , 20 μg/ml anti-fibrosin antibody (one-day incubation;b) , or 20 μg/ml anti-fibrosin antibody (two-day incubation;c) .

Figs. 39A-39C are a series of photomicrographs of cultured fibroblasts stained with an antibody against type I collagen. In (a) conditioned medium from normal fibroblasts is added to the cultured cells prior to staining. In (b) conditioned medium from sclerodermal fibroblasts is added to the cultured cells prior to staining. In (c) conditioned medium from sclerodermal

fibroblasts that has been depleted with anti-fibrosin antibodies is added to the cultured cells prior to staining.

Figs. 40A-40C are a series of photomicrographs In (a) conditioned medium from normal fibroblasts is added to the cultured cells prior to staining. In (b) conditioned medium from sclerodermal fibroblasts is added to the cultured cells prior to staining. In (c) conditioned medium from sclerodermal fibroblasts that has been depleted with anti-fibrosin antibodies is added to the cultured cells prior to staining.

Figs. 41A-41C is a series of photomicrographs of cultured sclerodermal cells stained with a monoclonal antibody against fibrosin. In (a) scleroderma cells were treated with 20 μg/ml random IgG. In (b) scleroderma cells were treated with 20 μg/ml anti-fibrosin antibody. In (c) scleroderma cells were treated with 20 μg/ml anti- TGF£ antibodies (type l, 2, and 3 and 2.5 μg/ml each, mixed) . Fig. 42 is a bar graph depicting the response of fibroblasts to immunopurifled human serum fibrosin.

Fig. 43 is a bar graph depicting the chemotactic response of monocytes to the supernatant of COS cells transfected with a plasmid bearing a 2.8 kb fragment of human fibrosin cDNA (pBKCMVHFibrosinl) . The assay was carried out in Boyden chambers as described herein (see also Wyler et al., J, TUT-Tfli-nPl- . 188:478, 1977). REAGENTS AND PROCEDURES Animals and Schistosomiasis Infection C57BL/6NcrLBr female mice (18 to 20 g; Taconic Farms, Inc. , Germantown, NY) were infected by the intraperitoneal injection of 35-50 cercariae of S. mansoni (Puerto Rican strain) suspended in 0.5 ml sterile saline. The mice were euthanized eight weeks later by

inducing C0 ₂ narcosis and their livers were placed in cold Hanks' buffered salt solution (HBSS) .

Isolation of Granulomas and Preparation of Granuloma Supernatant Isolation of granulomas was performed as described previously (Wyler et al., Science. 202:438 (1978),

Pellegrino et al., J. Parasitol. 42:564 (1956)).

Briefly, livers were homogenized in cold HBSS using a

Waring Blender (New Hartford, CT) . Granulomas were isolated from hepatic parenchymal debris by three to five cycles of serial sedimentation at 1 x g in HBSS. Granulomas were suspended at 10% (v/v) in serum-free culture medium RPMI 1640 supplemented with antibiotics and L-glutamine and cultured for 20 to 24 h at 37°C in an atmosphere containing 5% C0 ₂ -95% air. Cell-free supernatant from the granuloma cultures was retrieved by centrifugation (1000 x g 20 min at 4°C) and filter sterilized (0.22 μm diameter; Millipore Corporation, Bedford, MA) . The supernatants were stored in aliquots at -20 or -70 ^β C.

In some experiments cell-free supernatants were retrieved by centrifugation (200 g x 10 min) and used to purify FsF-1 for immunization of rabbits, or to conduct Western blot analysis. In other experiments, granuloma cells were dissociated by collagenase treatment using methods as described in Wyler et al., 132:3142 (1984). The granuloma cell suspension was first washed with HBSS and then with a solution of phosphate- buffered 0.15 M NaCl containing NaN ₃ (0.015 M) and goat serum (1% v/v; Sigma Chemical Co., St. Louis, MO). The washed cells were pelleted and incubated at 4°C for 0.5 h each in the presence of 10-15 μg/ml of normal rabbit IgG or of rabbit anti-FsF-1 IgG, washed, and then treated with 10 μg/ml goat anti-rabbit IgG (H and L chain specific) antibody conjugated with fluorescein isothiocyanate (FITC; Fisher Scientific, Pittsburgh, PA) .

In some experiments, unstained or FITC-stained cells were treated with phycoerythrin-conjugated rat anti-mouse CD4 antibody (5 μg/ml; Becton-Dickinson, Mountain View, CA) .

Antibody-treated cells were sorted by FACStar Plus flow cytometry and analyzed by a FACScan (Becton- Dickinson) or a .Coulter Epics 541 flow cytometer (Coulter Electronics Inc. , Hialeah, FL) . The fields were gated to exclude autofluorescent and dead cells. The CD4 ⁺ sorted cells were washed and suspended (0.6-1.0 x 10 ⁶ /ml) in serum-free medium RPMI 1640 supplemented with 0.3 mg/ml bovine serum albumin (BSA; Sigma, St. Louis, MO) and incubated for 24 h at 30 ^β C in 5% C0 ₂ -95% air humid atmosphere. Cell-free supernatants were retrieved by centrifugation (200 g x 10 min; 4°C) and analyzed for fibroblast mitogenic activity.

Cell Culture and Cell Proliferation Assays Human diploid fibroblast cultures were established from newborn foreskin as described previously (Wyler et al., .τ _t T mn r-1 , 130:1371 (1983)). Primary cultures of bovine aortic endothelial cells (BAEC) were prepared by and were a kind gift of Dr. Michael Gimbrone (Harvard Medical School, Boston, MA; Gimbrone et al., J. Cell. Biology 60:673 (1974)). All cells were grown to confluency in 75 cm ² polystyrene tissue culture flasks (Nuncalon, Rochester, NY) in supplemented medium RPMI 1640 containing 10% inactivated FCS (GIBCO Laboratories, Grand Island, NY) . When the cultures reached confluency (approximately every 4 d) , cells were passaged by treatment with 0.2% trypsin-0.1% sodium EDTA (trypsin- EDTA) .

For the proliferation assay, cells were suspended in serum-containing supplemented medium at a density of 5 to 6 x 10 /ml. One ml of the cell suspension was seeded in each well of a 24-well polystyrene tissue culture plate (Nuncalon) and incubated overnight. Cells were

then washed twice with warm (37°C) HBSS and replenished with serum-free medium. On the next day, 100 μl test samples were added to each well. Twenty hours later, 1 μCi[ ³ H]-thymidine (specific activity 6.7 Ci/mM, Dupont- NEN Research Products, Boston, MA) was added to each well for 4 h. Cells were then trypsinized and harvested onto glass fiber filters with a cell harvester (Titertek, Flow Laboratories, Rockville, MD) . The magnitude of incorporation of [ ³ H]-thymidine was estimated by scintillation spectrometry.

In selected experiments, cell growth was also assessed by direct quantitation. The cells were cultured as described above. Test samples were added for 96 h, after which the cultures were washed and detached from the monolayer by treatment with trypsin-EDTA.

Monodispersed cells were counted in a hemocytometer chamber (Cambridge Instruments, Inc., Buffalo, NY).

Gel Filtration Chromatoαraphv and Heparin Affinity Chromatoαraphv A 1 x 40 cm column of Bio-Gel P-30 (Bio-Rad

Laboratories, Rockville Center, NY) was equilibrated with PBS (0.15 M; pH 7.4) and calibrated with the following molecular weight markers: blue dextran, OVA, chymotrypsin A, and myoglobin (gel filtration markers; Sigma Chemical Co., St. Louis, M0). Approximately 1 ml of unconcentrated exude granuloma culture supernatant was loaded onto the column, which was then run at 4°C with PBS at a rate of 5 to 6 ml/h. One ml fractions were collected, 0.3 mg/ml BSA (Sigma) was added as a carrier, and the fractions were then dialyzed (Nominal exclusion, 6 to 8 kDa), first against HBSS, followed by RPMI 1640. The dialyzed material was filter-sterilized before testing in the proliferation assay. The two or three fractions eluting from the P-30 column with peak fibroblast-stimulating activity (Fig. 1) were pooled and mixed with an equal volume of washed-heparin-Sepharose

CL-6B (Pharmacia LKB, Uppsala, Sweden) . The mixture was rocked gently overnight at 37°C in polypropylene tubes (Corning, Glassworks, Corning, NY) that had been pretreated by incubation with BSA (1 mg/ml) followed by washing with PBS. The slurry was poured into a column (1 x 15 cm. Bio-Rad) . Material that was not adsorbed (fall- through fraction) was reapplied to the column. The column was then washed extensively with PBS (0.15 M NaCl) and elution was carried out over a 2-3 h period with a 30 ml continuous salt gradient (0 to 2.5 M NaCl) and 1 ml fractions were collected. The conductance (ohms ^-1 ) of each fraction was determined (model CDM, Radiometer, Copenhagen, Denmark) . Fractions were dialyzed against medium RPMI 1640 and filter sterilized before testing in the biologic assay.

After determining the concentration of NaCl with which the fibroblast proliferative activity eluted from heparin-Sepharose, a "batch elution" procedure was used for preparing biologically active material from heparin- Sepharose beads. The biologically active fractions prepared by initial gel filtration chromatography were adsorbed to heparin-Sepharose as described above. The tubes were centrifuged (1000 x g for 10 min) and the supernatant was recovered and filter-sterilized (0.22 μm diameter pore) . The beads were then washed three times with PBS. After the last wash the beads, suspended in an equal volume of 3.0 M NaCl, were gently mixed at 37°C for 1 h. The supernatant was removed by centrif gation and dialyzed before testing in the biologic assays. The heparin-Sepharose purified material was designated FsF-1. V T C Anion Exchange Chro atoσraphv The fractions eluting from heparin Sepharose with 1.5 M NaCl were dialyzed (6 to 8 kDa cutoff) at 4 ^β C against two to three changes of PBS, followed by two changes of 20 mM Tris-HCl, pH 8.0, and applied to a Mono

Q column (HR 5/5, Pharmacia, Uppsala, Sweden) that had been equilibrated in the starting buffer (20 mM Tris-HCl, pH 8.0). The fast-performance liquid chromatography apparatus (Pharmacia) was operated at 4°C. Elution was achieved with a gradient of 0 to 2.2 M NaCl in 20 mM

Tris-HCl, pH 8.0, at a flow rate of 1 ml/min. Forty 1 ml fractions were collected. Absorbance (280 nm) and the conductance of each fraction was monitored and 0.3 mg/ml of BSA was added as a carrier protein before each fraction was dialyzed against medium and tested in the proliferation assays.

SDS-PAGE

Samples were combined with an equal volume of buffer consisting of 10 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 2% dithiothreitol, and 0.01% bromophenol blue. Samples were analyzed by SDS-PAGE as described by Laemmli (Laemmli et al., Nature 227:680 (1970)). Separating gels of 10% acrylamide and stacking gels of 7% were used. Gels were stained with silver nitrate (Morrissey, Anfl. t. Biocheffi,, 117:307 (1981)).

Preparation and Analysis of anti-FsF-1 Antibodies Initial (preimmune) serum samples were obtained from two female NZW rabbits (3 to 4 kg, Buckshire Corp., Perkasie, PA) which were then immunized with purified FsF-1 by repeated intradermal injections on the back. Ten ml of unfractionated granuloma supernatant (approximately 10 mg of protein by Bradford assay; Bradford Anal. Biochem. 72:248, 1976)) was processed by gel filtration and heparin affinity chromatography (batch elution procedure) as described above. Purified FsF-1 was concentrated to a volume of 1 to 1.5 ml (approximately 1 to 2 μg; fluorescamine protein assay (Bohlen et al., Blophvs. 155:213 (1973)) by ultrafiltration using a 6-8 kDa nominal exclusion cellophane dialysis bag suspended in a slurry of

polyethylene glycol (molecular mass * 8 kDa, Sigma) . The concentrate was emulsified in an equal volume of CFA (Sigma) . The rabbits were boosted at 4- to 6-wk intervals by intradermal injections of the same amount of purified FsF-1 in incomplete Freund's adjuvant (IFA) (Sigma) . Aliquots of serum obtained routinely 6 to 8 days after each booster injection were stored at either - 20 or -70°C. IgG from preimmune or immune serum was prepared by protein A-Sepharose (Sigma) chromatography (Goding, J. Immunol. Methods 20:241 (1978)).

The specificity of the anti-FsF-1 antibody was assessed by dot-blot ELISA as described in Hawkes et al., Anal. Biochem. 119:142 (1981). Briefly, purified FsF-1 (10 to 15 ng) ; purified acidic FGF (100 ng, 200 ng) ; PDGF (40 ng, 100 ng) ; purified human plasma fibronectin (2, 20, 100 ng (prepared as described in Wyler et al., ___ Immunol. 138:1581 (1987)); rhIL-2 (2 ng, 12 ng, 20 ng; Genzyme, Boston, MA); rmIL-3 (2.5 ng, 7.5 ng; Genzyme) rmIL-4 (0.7 ng, 4 ng, 7 ng; Collaborative Research, Bedford, MA); rmIL-6 (2.5 ng, 15 ng, 25 ng; Biosource International, Westlake Village, CA) ; rh IL-7 (0.75 ng, 4.5 ng, 7.5 ng; Biosource International); and rm GM-CSF (0.25 ng, 1.5 ng; 2.5 ng; Genzyme), were adsorbed to nitrocellulose paper (Bio-Rad) in a volume of 1 to 5 μl. The nitrocellulose was then washed overnight in blocking buffer (PBS containing 5% w/v Carnation nonfat powdered milk) . The nitrocellulose was then incubated at room temperature for 1 h with anti-FsF-1 IgG antibody (1:50 final dilution) . After extensive washing with blocking buffer, the nitrocellulose was incubated for 0.5 to 1 h at room temperature in alkaline phoβphatase-conjugated goat anti-rabbit IgG (Promega Corporation Madison, WI) . The development of the blot was carried out by incubation with substrate (containing nitroblue tetrazolium chloride and 5-bromo-4 chloro-3imodyl phosphate, p-toluidine salt

(Promega)) dissolved in alkaline phosphatase buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl ₂ , pH 9.5): 33 ml nitroblue tetrazolium chloride and 16.5 ml 5-bromo-4 chloro-3-imodyl phosphate p-toluidine salt were used for every 5 ml of the buffer. The reaction was stopped with deionized water.

Antibody Neutralization of Biologic Activity Purified acidic FGF, PDGF or heparin-Sepharose purified FsF-1 (approximately 10-20 ng in 100 μl) or culture supernatant of granuloma-derived CD4 ⁺ lymphocytes) were combined with preimmune IgG (2.5 μg in 100 μl) or anti-FsF-1 rabbit IgG (2.5 μg in 100 μl) and the mixture was incubated at 37°C for 3 to 4 h in polypropylene culture tubes that had been pretreated with BSA. The samples were then filter sterilized and tested in the fibroblast proliferation assay. Alternatively, the samples were incubated with immobilized protein A- Sepharose to remove Ag-antibody complexes; the samples were centrifuged at 1000 x g for 10 min and the supernatant was tested for fibrogenic activity. Amino Acid Analysis of FsF-1

Amino acid composition of FsF-1 was determined by analysis of the 1.5 M NaCl eluate from the heparin- Sepharose bound to polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA (LeGendre et al.,

Biotechnicmes 6:154 (1988)). The amino acid composition was determined by the standard method of Waters PICO-TAG. Interleukins Murine rIL-3, IL-4, and recombinant mouse granulocyte-macrophage CSF were obtained from Genzyme, Boston, MA. Murine rIL-6, human rIL-7, and human rIL-8 were obtained from Bio-Source International, Westlake Village, CA. Human PDGF, a-endothelial cell growth factor, and goat anti-human PDGF (IgG) were purchased from Collaborative Research. Bovine acidic FGF used for

the dot blot ELISA assay and neutralization studies and rabbit anti-bovine acidic FGF fragment (Leu 60-Leu 98) , polyclonal IgG were obtained from UBI, Lake Placid, NY. Biosvnthetic Labeling of FsF-1 CD4 ⁺ cells from granulomas were cultured for 24 h in methionine and cysteine-free RPMI 1640 medium (Selectamine«Kit, GIBCO, Grand Island, NY) to which was added 75 μCi Tran ³⁵ S-Label» ( ³⁵ S E. coli hydrolysate labeling reagent containing 70% L-methionine [ ³⁵ S] and 15% L-cysteine [ ³⁵ S]; sp. act. 1181 Ci/mmole; ICN Biomedical, Irvine, CA) . The cell-free culture supernatants were collected at 24 h by centrifugation (200 g x 10 min) and 200 μl of the supernatant was first incubated (1-2 hr; 4*C) with normal rabbit IgG (NRIgG; 5 or 15 μg in 200 μl) and the mixture was then incubated (1 hr; 4*C) with protein A-Sepharose (50-100 μl; Sigma). Two hundred microliters of supernatant of this mixture was retrieved following centrifugation (12,000 g x 5 min) and re¬ treated in a similar manner, this time with anti-FsF-1 IgG (5 or 15 μg in 200 μl) and protein A-Sepharose. The beads were pelleted (12,000 g x 5 min), washed twice in phosphate-buffered 0.15 M NaCl and then boiled for 5 min in sample buffer (10 mM Tris-HCl; 2% sodium dodecyl sulfate; 5% glycerol; 2% dithiothreitol; 0.05% pyronine Y; pH 6.8). Supernatant from these treated beads was subjected to electrophoresis in a 70 x 70 x 0.5 mm slab gel of 10% acrylamide (BioRad Labs, Richmond, CA) using standard method (Laemmli, Nature 227:680 (1970)). Following electrophoresis, proteins were transferred electrophoretically (1-2 h; 70 volts) onto nitrocellulose paper (BioRad) using standard procedures. The nitrocellulose paper was then exposed to X-ray film (X- OMAT, AR; Kodak, Rochester, NY) for 72 h at -70 ^» C. PURIFICATION OF FsF-1

Gel filtration chromatography of granuloma culture supernatant resolves the fibroblast growth factor in fractions with apparent molecular mass 25 to 28 kDa (Fig. 1) . Each fraction was tested at different dilutions (from 1/10 to 1/50); maximum stimulation of [ ³ H]- thymidine incozporation was obtained with a dilution of 1/20 of the most active fraction.

A pool of two or three consecutive active fractions obtained by gel filtration was subjected to heparin-affinity chromatography. When the elution was performed with a linear gradient of NaCl (0 to 2.5 M) , peak mitogenic activity (containing 85 to 90% of the total activity) eluted from the affinity column with 1.25 to 1.5 M NaCl (Fig. 2). Accordingly, we used 1.5 M NaCl to elute the fibroblast mitogen in subsequent experiments. By this procedure, most of the biologic activity was retrieved in the adsorbed fraction; negligible activity was present in the unadsorbed fraction. Maximum fibroblast stimulation was achieved when the active fraction was tested to a dilution of

1/100 and 1/1000. Nonspecific binding of the fibroblast mitogen to heparin-free Sepharose 4B beads was not detected in control experiments.

Heparin-Sepharose fractionated material was subjected to anion-exchange FPLC chromatography (Fig. 3) . Fibroblast mitogenic activity eluted from the column with 1.2 to 1.5 M NaCl in a single active peak; maximum activity was present in fractions eluting with 1.5 M NaCl. These fractions were active at a concentration of 1/100 to 1/1000. Only fractions 17 and 18 detectably adsorbed uv light (280 nm) . Commercial heparin (from porcine intestinal mucosa, Sigma, cat. no. H-3125) when applied to a mono Q column under the same conditions also eluted in these two factors.

The biologically active fraction that eluted from heparin-Sepharose was analyzed by silver stain of SDS- PAGE (Fig. 4) and under reducing conditions revealed a single band with molecular mass - 60 kDa. This band corresponds to the migration position of one of the major proteins detected in electropherograms of crude granuloma supernatant.

Rabbit IgG produced in response to immunization with heparin-purified FsF-1 reacts by dot-blot ELISA with heparin-purified FsF-1 and neutralizes its biological activity. Anti FsF-l IgG but not pre-immune IgG also identifies in Western blot of crude granuloma supernatant FsF-1 (MW 60 kD) , and two of its degradation products (Fig. 5). Identity of FsF-1

The heparin affinity of FsF-1, as revealed in the above experiments, suggested that the mitogen might be the same as a previously-defined HBGF. Such factors are classified in part according to their anodic or cathodic migratory behavior during IEF (Lobb et al., J. Biol. Chem. 261:1924 (1986)). We established that the granuloma-derived mitogen has pi-6.2 (Wyler et al., J. Immunol. 129:1706 (1982)), thus FsF-1 has a characteristic of class 1 (acidic) HBGF. We determined the amino acid composition of FsF-1 and compared it with that of bovine acidic FGF. At least 6 of the 15 amino acids analyzed different (by - 50%) in their mole percent content (Table 1) . The possibility that the FsF-1 preparation might have been contaminated with heparin places into question the accuracy of the mole-percent determination of serine and glycine (Folkman et al., Science 235:442 (1987)). Nonetheless, the extent of the differences in amino acid composition of FsF-1 and aFGF indicates their molecular distinctiveness inasmuch

as the structure of aFGF is highly conserved in evolution

(Lobb et al., Anal. Biochem. 154:1 (1986)).

TABLE 1

Comparison of the amino acid composition of heparin-purified FSF-1 (two separate preparations) and bovine acidic FGF

Amino Acid Mol Percent*

FSF-1 FSF-1 FGF

Asp and Asn 2.93 1.85 9.63

Glu and Gin 14.22 9.85 11.00 Ser 12.31 14.33 6.88 Gly 22.87 24.16 9.70 His .08 .26 3.40 Arg 5.05 3.97 4.13

Thr 6.15 6.23 6.18 Ala 8.85 7.62 3.40 Pro 5.89 5.46 4.82 Tyr 2.08 3.58 4.80 Val 5.46 5.94 3.40

Met 0.72 1.85 0.68 He 3.75 4.02 4.13 Leu 7.55 7.69 13.06 Phe 1.96 2.50 4.82 Lys 0.13 0.72 8.94

* Mol percent - mole of amino acid over mole of total protein X 100%

We also conducted a series of experiments to assess the potential similarity of FsF-1 to acidic FGF, the prototype class 1 HBGF molecule (Lobb et al., supra) . We also compared FsF-1 with PDGF, a group of closely related mesenchymal cell mitogens (LeGendre et al., supra) .

Anti-FβF-1 antibody detected FsF-1 in both purified form and in crude granuloma supernatant in a dot-blot ELISA assay (Fig. 6) . The antibody did not detect acidic FGF or PDGF, mitogens that were detected with the appropriate homologous antibodies. Furthermore, anti-FsF-1 did not detect plasma fibronectin or acidic FGF in a dot-blot ELISA; nor did anti-fibronectin antibody react with FsF-1. Crude granuloma supernatant

had no biological activity characteristic of TNF in an L929 cytotoxicity assay and purified TNF was not mitogenic in our assay which utilizes serum-free conditions. We detected no IL-2 activity in granuloma supernatants (Wyler et al., J, ^τ TMBUπo ι 129:1706 (1982)), and detected no fibroblast mitogenic activity in rIL-2. Anti-FsF-1 antibody inhibited the proliferative responses of fibroblasts to FsF-1 (Fig. 7A; p < 0.005; comparison by Student's t-test of mean fibroblast responses to FsF-l in the presence of normal rabbit IgG or anti-FsF-1 IgG in four separate experiments) . Under identical conditions, the antibody preparation did not significantly (p > 0.4) affect the mitogenic activity of acidic FGF. Furthermore, anti-PDGF, which abrogated the mitogenic effects of PDGF, had no effect on FsF-1 (Fig. 7B) . A characteristic feature of class 1 HBGF is their ability to stimulate endothelial cell proliferation (Folkman et al., silB-ca) • As shown in Figure 8A, fibroblasts and endothelial cells proliferate in response to acidic FGF. Inasmuch as the magnitude of the response of endothelial cells to the lower (<2 ng/ml) concentration of acidic FGF is greater than that of fibroblasts, these cells appear to be the more sensitive to this mitogen. In contrast, FsF-1 in concentrations in the range of 1 to 40 ng/ml, did not induce endothelial cell proliferation (Fig. 8B) .

Finally, we detected no biological activity in the following commercially-prepared cytokines; rIL-3 (0.2-200 U/ml), rIL-4 (0.05-20 U/ml) rhIL-5 (1-1000 U/ml) , rIL-6 (0.002-100 U/ml) , IL-7 (0.02-100 U/ml) rIL-8 (0.02-100 U/ml) or GM-CSF (0.3-20 U/ml). The lack of relevant biological activity in these cytokines implies that FsF-1 is unique.

Flow Cvtometrv of Granuloma Cells We analyzed granuloma cells by flow cytometry after they were treated with anti- FsF-1 IgG (or preimmune IgG) and FITC-conjugated anti-IgG with or without subsequent treatment with phycoerythrin- conjugated anti-CD4 antibody (Figures 9 and 10) . In the mouse, CD4 is expressed on a subpopulation of lymphocytes but not on macrophages (Crocker et al., J. EXP. Med. 166:613 (1987)). Using one- and two-color flow cytometric analysis, we determined that approximately 20- 25% of the CD4 ⁺ lymphocytes also stained specifically

We next employed FACS to obtain a highly-enriched (99% pure) population of CD4 ⁺ granuloma cells that we then incubated at a density of 0.5-1.0 x 10 ⁶ cells/ml for 24 h in serum-free medium. The conditioned medium from these cultures stimulated fibroblast proliferation (Table 2) . In contrast, culture supernatants of CD4 ⁺ lymphocytes isolated from spleen cell suspensions prepared from uninfected mice contained no such biological activity. This indicates that the treatment of cells with anti-CD4 antibody in the course of their purification did not trigger the secretion of FsF-1.

TABLE 2

Fibroblast proliferate response ( ³ H-thymidine incorporation) to cluture supernatants of CD4 ⁺ lymphocytes from schistosomal egg granulomas Additive to ³ H-thymidine fibroblast culture incorporation fCPMV

Medium alone 5282 ± 822

FBS, 10% 40,765 ± 6099 granuloma CD4 ⁺ cells l:5 ² 10,411 ± 411

1:15 7305 ± 36

1:50 6330 ± 356 spleen CD4 ⁺ cells

1:5 5063 ± 852

1. Mean ± SEM of triplicate determinations in representative experiment of three performed. 2. Final dilution of CD4 ⁺ cell supernatant tested in fibroblast cultures.

Biosynthetic Labeling of FsF-1 in CD4 ⁺ lymphocytes The foregoing results suggested that egg granuloma-derived CD4 ⁺ lymphocytes produce FsF-l. To confirm this conclusion more precisely, and exclude the possibility that FsF-1 was merely bound to the CD4 ⁺ lymphocytes and subsequently released, we incubated these isolated granuloma cells with [ ³⁵ S]-methionine/cysteine for 24 h and processed the culture supernatants by immunoprecipitation with anti-FsF-1. Autoradiographs of the electroblots of SDS-PAGE preparations of untreated culture supernatant disclosed in excess of 10-15 distinct bands (Fig. 11) . In contrast, immuno-precipitation of the CD4 ⁺ lymphocyte supernatant with anti-FsF-1 IgG revealed a single 60 kDa band (Figure 11) . On the basis of its Rf, this band corresponds to the major labelled product of the isolated lymphocytes and to that of heparin-affinity purified FsF-1 from granuloma

supernatant (Prakash et al. , supra) . As noted by Western analysis, preimmune IgG does not react with this protein (Fig. 5).

Heparin affinity chromatography has proven to be a valuable purification procedure in the isolation of certain mesenchymal growth factors and angiogenic factors (Lobb et al., supra). An advantage of this technique is that the relatively high af inity binding of these factors to heparin is uncharacteristic of most proteins (Lobb et al., SSSBΣΛ) - The simplicity of the scheme we were able to devise for purifying FsF-1 from culture supernatants is a consequence of its heparin-binding property. Early in our studies, we noted that an initial gel iltration step enhances the efficiency of the subsequent affinity chromatography step, perhaps by removing fibronectin, which is another heparin-binding protein that is a constituent of these supernatants (Wyler, Rev. Infect. Pis. 9 Suppl:5391 (1987)), from the crude granuloma supernatants. The final anion exchange FPLC (Fig. 3) confirmed the homogeneity of biologic activity in the purified fractions, and the single band (or occasionally a doublet) detected on silver-stained SDS-PAGE gels (Fig. 4) supports the conclusion that purification was achieved. Furthermore, we estimate that the purification scheme resulted in approximately 10,000- to 50,000-fold enrichment in specific activity. We base this estimate on our measurement of the total protein concentration of crude granuloma culture supernatant (-1 mg/ml; Bradford assay (Bradford, Anal. Bioche . 72:248 (1976)), our detection of protein in the heparin- Sepharose eluate near the lower limit of sensitivity of the fluorescamine assay (-200 ng/ml (Bohlen et al., supra)), and the dilution of material yielding maximum proliferative responses being 1:10 to 1:20 for crude material and 1:100 for purified material.

The apparent molecular mass -60 kDa of FβF-1 revealed by SDS-PAGE conflicts with our estimates of molecular mass -25 to 28 kDa by gel filtration chromatography (Fig. 1) . By Western blot analysis of crude granuloma supernatants probed with polyclonal anti-FsF-1 antibody, we detected a single band in the range of 55 to 58 kDa and at times also a doublet band at 30 kDa. The basis for this apparent discrepancy in m.w. determination by gel filtration and by SDS-PAGE remains to be elucidated. However, a number of factors are known to affect migration in gel filtration. In contrast, only the 60 kDa protein was identified by immunoprecipitation of metabolically-labeled CD4* lymphocyte-derived proteins (Fig. 11) . This indicates that the minor bands are apparently products of degradation of FsF-1 most likely generated by granuloma-derived proteases present in culture supernatants (but not in CD4 ⁺ lymphocyte culture supernatants) . One possibility is that FsF-l forms aggregates, and that such aggregates formed during heparin-affinity chromatography are resistant to dissociation under the conditions we used in conducting SDS-PAGE.

In addition to providing for a convenient purification method, the fact that FsF-1 is heparin- binding has important implications in establishing its molecular identity. We previously determined that the granuloma-derived mitogen is a protein with pi -6.2. These properties suggest that FsF-1 might be a member of the acidic heparin-binding growth factor class of proteins (class 1 HBGF) , mitogenic proteins of diverse cellular origin that are structurally the same or closely related, and highly conserved between mammalian species (Harper et al.. Biochemistry 25:4097 (1986)). The class 1 HBGF, exemplified by acidic FGF, are all potent mitogens for fibroblasts as well as endothelial cells.

Our indicator endothelial cells from bovine aorta responded in a characteristic manner to bovine FGF but not FsF-l (Fig. 8) . It is unlikely that the lack of response to FsF-1 is due to species differences, because the HBGFs are structurally and functionally conserved (Burgess et al., Annu. Rev. Biochem 58:575 (1989)), and because we found that another cytokine from egg granulomas with molecular characteristics distinct from FsF-l could stimulate proliferation of bovine aortic endothelial cells, but not fibroblasts (Wyler et al., ___ Infect. Pis. 155:728 (1987)). This suggests that FβF-1 probably is distinct from FGF and sensu stricto is not a class 1 HBGF. Supporting this conclusion are our observations that antibodies prepared against FβF-1 neither react with FGF in a dot-blot ELISA nor neutralize its biologic activity, whereas they do both to FsF-1 (Figs. 6 and 7) . Finally, the amino acid content of FsF- 1 and FGF (acidic and basic) reveal differences indicating that these molecules are not the same (Table 1) , and the amino acid sequence of the peptide derived from FsF-1 is distinct from other known proteins. The antibody preparations permitted us to distinguish FsF-1 from other heparin-binding growth factors (Prakash et al., SME-Cfi) and to determine by flow cytometry that a subpopulation (20-25%) of CD4 ⁺ lymphocytes in granuloma cell suspensions apparently express FsF-1 on their surface (Figs. 9 and 10). The fact that culture supernatants of CD4 ⁺ lymphocytes isolated from egg granulomas contained fibroblast mitogenic activity that was neutralized with anti-FsF-1 IgG indicated that these cells secrete the mitogen (Table 2) . The definitive evidence that the CD4 ⁺ lymphocytes produce and secrete FsF-1 was obtained in experiments involving biosynthetic labelling and immunoprecipitation of CD4 ⁺ proteins (Fig. 4). Although the results of the

present studies do not exclude the possibility that other granuloma cells also might be potential sources of FsF-1, our prior studies indicate that FβF-1 is not secreted by granuloma macrophages (Wyler et al., supra) . Furthermore, since S. mansoni egg granulomas from mice treated with anti-IL-5 antibodies lack eosinophils (Sher et al., Proc. Natl. Acad Sci. USA 87:61 (1990)) but nonetheless secrete fibrogenic activity, eosinophils are an unlikely source of FsF-1. On the other hand, egg granulomas from s. mansoni-infected, congenitally athymic mice (which lack mature T lymphocytes and do not develop hepatic fibrosis) produce no fibroblast mitogen (Prakash et al., J. Immunol. 144:317 (1990)). This observation is consistent with our conclusion that FsF-1 is a lymphokine.

We conclude that the granuloma CD4 ⁺ lymphocytes are stimulated in vivo to produce FsF-l, and that this production is not induced artificially during isolation of the lymphocytes. Several points support this conclusion. First, in contrast to many of the studies that have examined production of fibrogenic proteins by chronic inflammatory cells (lymphocytes and macrophages; for example, see Wahl et al., j, T^Tnuηpj 121:942 (1978); Wahl et al., Lvmphokines 2:179 (1981)), we do not add antigens, mitogens, or other nonspecific stimuli to our granuloma or cell cultures. Second, the cell sorting methods we used did not trigger lymphocytes to produce a fibroblast mitogen (see above) . Third, fibroblast mitogenic activity can be detected in extracts of recently isolated egg granulomas (Wyler et al., _[__ Infect. Pis. 144:254 (1981)) and is detectable in the cell-free supernatants of isolated egg granulomas within a few hours of their in vitro incubation (Wyler et al., aunra.. Fourth, unfractionated splenocytes and splenic CD4 ⁺ lymphocytes isolated by flow cytometry fail to

spontaneously secrete fibroblast mitogens (Wyler et al., infect. Immun. 38:103 (1982); and the present study). On the other hand, sensitized splenic lymphocytes stimulated with an aqueous extract of schistosomal eggs (concanavalin-binding fraction of soluble egg antigen) do secrete a fibroblast mitogen, presumably FsF-1 (Wyler et al., fiUEtfi).

It is noteworthy that FsF-l detected by immunoprecipitation corresponded to a prominent 60 kDa protein produced by isolated granuloma CD4 ⁺ lymphocytes (Fig. 5) . Our results suggest that FsF-l might be a major protein produced by this subpopulation of granuloma cells. We have observed that sensitized splenic lymphocytes from 5. mansoni-infected mice, when stimulated with an aqueous extract of schistosoma eggs secrete a fibroblast mitogen, presumably FβF-1 (Wyler et al., Infect. Ti.mi.-n- 38:103 (1982)). It therefore seems likely that CD4* cells are stimulated in vivo to produce FβF-1 in response to egg antigens. Based on our current observation, such production may continue at least briefly when the lymphocytes are dissociated from the eggs and antigen-presenting cells. The notable finding that FsF-l not only iβ secreted but also can remain associated with the surface of CD4 ⁺ lymphocytes suggests that in addition to the action of the secreted cytokine, direct contact between membrane-associated FsF-l positive lymphocytes and fibroblasts might stimulate fibroblast growth.

A number of cytokines (IL-1, TNF, TGF0) , some of which were originally identified on the basis of the other biological activities they possess, also have been shown to exhibit fibrogenic activity in vitro (tor example, see Schmidt et al., J. Immunol. 128:2177 (1982); Vilcek et al., J. EXP. Med. 163:632 (1986); Sugarman et al., Science 230:943 (1985); Masβague, J. Biol. Chem.

260:7059 (1984); Leof et al., Proc. Natl. Acad. Sci. USA. 83:2453 (1986)). In addition, fibroblast growth factors, some of which were purified from other sources and were recognized for this biological property (PDGF [Ross et al., Cell 46:155 (1986)], FGF [Gospodarowicz et al., J. Biol. Chem. 250:2515 (1975); Gospodarowicz et al., J. Biol. 253:3736 (1978)]), and heparin-binding epidermal growth factor [HB-EGF Higaβhiyama et al., Science 251:936 (1991)]) have been found to be produced by macrophages. However, based on its biochemical composition, physicochemical properties, and antigenicity, FsF-1 is distinct from these fibroblast growth factors. Moreover, the fact that FsF-1 iβ a lymphokine iβ an additional distinguishing characteristic. Indeed, becauβe we have not detected fibroblaβt mitogenic activity in βeveral purified and recombinant lymphokineβ, and since avid heparin-binding is not a known characteristic of most lymphokineβ, we believe that FsF-1 iβ a previouβly unidentified lymphokine. DETECTION OF FβF-1 IN GRANULOMA SUPERNATANTS AND

SERUM OF MICE INFECTED WITH S. mansoni

FsF-1 can be detected in highly-dilute granuloma supernatant in an antigen-capture ELISA (Nourel, et al. (1994) Am. J. Troo. Med. Hvg. 50:585-594). Wells of ELISA plates were coated with one of the Mabs shown above (approximately 100 ng/well) . After blocking with gelatin, crude granuloma supernatants (that contain approximately 4 μg/ml FsF-l) were added at the dilutions shown. After removing the unbound material, the captured FsF-1 was detected with monospecific, polyclonal rabbit anti-FsF-1 IgG followed by alkaline phoβphataβe- conjugated goat anti-rabbit IgG. Pre-immune rabbit IgG did not detect the captured FβF-1. The readingβ were blanked against O.D. of wells in which buffer instead of granuloma supernatant was added. The asβay apparently iβ sensitive to at least pg/ml concentrations, consistent

with expectations based on βimilar antigen-capture assayβ for other lymphokineβ (Fig. 12) .

An antigen-capture ELISA was also used to detect FβF-1 in sera of uninfected C57BL/6 mice or mice infected with Schistosoma mansoni for 8 or 20 weeks. Sera from two sets of mice were compared simultaneously in the same assay. A rat anti-murine FsF-1 monoclonal antibody (IgG) bound to Nunculon plates (which were then blocked with a gelatin-containing blocking buffer) was used to capture FβF-1 in sera diluted 1:100. After washing the plates with blocking buffer, rabbit IgG anti-murine FsF-1 (diluted 1:200) was added. After incubation and washing, a goat anti-rabbit IgG conjugated with alkaline phosphatase was added. After washing, the substrate was added and the optical density read in an ELISA reader. These readings were blanked against wells in which the reagents from all steps of the assay were included with the exception of the mouse serum. A control assay which was also conducted simultaneously, was the same in all respects to the experimental assays but for the exclusion of the monospecific rabbit anti-FsF-1 detector.

The results of this assay (Fig. 13) indicate that normal mouse serum contains very little, if any, detectable FsF-1, but sera from infected mice contain elevated levels that continue to rise during mid-chronic stage. The control assayβ performed further confirm the specificity of the assay; the FsF-1 levels observed are not due to either the reaction of the rabbit anti-FsF-1 IgG with the monoclonal antibody, or the reaction of the captured antigen with the goat anti-rabbit IgG.

Antibody Neutralization of Fibrogenic Activity Specific polyclonal IgG waβ prepared by immunizing rabbits with highly purified murine FsF-1, aβ previously described. This antibody preparation did not react (by dot blot ELISA) with a variety of recombinant murine

lymphokines. IgG purified from pooled sera of rabbits prior to immunization (NRIgG) , and which did not react with FsF-1, served as a control. Cell-free supernatants (undiluted) from lymphocytes or transfected COS-7 cells were incubated with IgG (final concentration, 2.5 to 7.5 μg/100 μl) at 37°C for 2-3 h in polypropylene culture tubes (Falcon #2063; Becton-Dickinson Labware, Lincoln Park, NJ) that had been previously treated with BSA (1 mg/ml) and washed, to reduce non-specific adsorption of proteins. Immune complexes were removed from selected mixtures with protein A coupled to Sepharose beads (Sigma). Following incubation for 1 h at 37°c, the beads were removed by centrifugation (lOOOg x 10 min.) The samples were then filter-sterilized (0.22 μm diameter pore size; Millipore Corp., Bedford, MA) and tested in a fibroblast proliferation asβay.

C QNING AND ANALYSIS OF MURINE cDNA Our efforte to obtain amino acid βequence data from the purified FsF-1 polypeptide (also designated fibrosin) were hindered by the fact that FsF-1 is blocked to Edman degradation and is also strikingly resistant to enzymatic proteolysiβ. Accordingly, as an alternative approach to establish its molecular identity, we sought to clone FβF-1 by heterologouβ expression of a CD4 ⁺ lymphocyte-derived cDNA library in COS-7 cells.

Several CD4 ⁺ lymphocyte clones and cell lines (provided as gifts from D.C. Parker, University of Oregon, Portland, OR, and L. Glimcher, Harvard School of Public Health, Boston, MA) were screened for their ability to secrete fibroblast mitogenic activity into culture supernatants following in vitro stimulation. Cells were propagated in supplemented medium RPMI 1640 containing 10% FBS. Cells at mid-log growth were washed extenβively in HBSS and 10 ⁶ cells were cultured in 1 ml serum-free medium supplemented with 0.3 mg/ml BSA and 10

μg/ml concanavalin A (Con A; Sigma Chemical Co.,St. Louie, MO) for 24 h at 37°C in a humidified atmosphere of 5% C0 ₂ -95% air. Cell-free supernatants were retrieved by centrifugation (200g x 10 min) and stored at -20°C until tested in a fibroblast proliferation aββay, aβ described above. Five of the 30 cell lines tested were positive in the fibroblast proliferation aββay, and a CDC25 cell line (CD4 ⁺ Th2 lymphocyte line; Tony et al., J. Exp. Med. 162:1695-1708, 1986) was selected for further analysis. This cell line was propagated biweekly as described (Tony et al., supra) and used 2-3 weeks later for preparation of culture supernatants. cDNA Library and Expression Cloning

A murine cDNA library prepared from mRNA isolated from Con A-stimulated cells of the CDC25 line was obtained from DNAX Reβearch Institute of Molecular and Cellular Biology, Palo Alto, CA. The library waβ constructed in the vector pcDSRα296 which contains a unique promoter that permits high-level, transient expression of the cDNA insert in COS-7 cells (Takebe et al., Mol. cell Bio. 8:466-472, 1988). Like the parental pcD vector originally designed by Okayama and Berg (supra) , pcDRa296 allows a high level of expression of full-length cDNA inserts under the control of the simian virus 40 (SV40) early promoter. SV 0-derived DNA fragments are arrayed in these vectors to permit transcription, splicing, and polyadenylation of the cloned cDNA. A DNA fragment containing both the SV 0 early region promoter and two introns normally used to splice the virus 16s and 19s late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. This DNA fragment can promote two alternate kinds of splicing. Most often (60-70% of transcriptβ) splicing occurs at the 16β RNA intron junction and placeβ the cDNA initiator ATG codon

first in line from the 5' end of the mRNA. When splicing occurs at the 19s RNA intron, it retains an ATG codon upstream of the cDNA in the processed mRNA. Therefore, if the clone contains an incomplete cDNA, translation from the upstream ATG codon may yield a fusion protein. ∑.coli transformants containing the pcDSRα296 vectors were expanded in L-broth medium containing ampicillin (50 μg/ml) and plasmids were isolated on Qiagen columns (Qiagen Inc. , Chatsworth, CA) . For transfection, 5 x 10 ⁵ COS-7 cells (Okayama et al. Meth. Enzvm. 154:3-28, 1987) were seeded on 60 mm tissue culture dishes (Falcon) in supplemented Dulbecco's modified Eagle's medium (DMEM; GIBCO) containing 10% FBS (GIBCO) . Cells were grown overnight to a density visually estimated to be 60-70% confluency. Cells were then washed with serum-free DMEM buffered with 5 mM Tris (pH 7.4) and plasmid DNA (1-5 μg) in serum-free medium (4 ml per plate) was added, followed by the addition of DEAE dextran (200 μg/ml; Pharmacia, Piscataway, NJ) . After co-incubation with p asmid DNA for 4-5 h, COS-7 cells were washed and treated for 2-3 h with 100 μM chloroquine (Sigma) in the presence of 2% FBS. Cells were then washed and grown overnight in DMEM with 4% FBS. The following day, cells were washed and replenished with DMEM containing 0.3 mg/ml BSA. Forty-eight to seventy-two hours later, culture supernatants were collected and tested for their ability to stimulate fibroblast proliferation.

For controls, COS-7 cells were tranβfected with the pcDSRα296 plasmid containing a murine IL-4 cDNA insert (DNAX) , and were subβequently maintained under the above conditions except that the cells were cultured in 2% FBS. Supernatants of these cultures were harvested after 48 to 72 hours and tested for IL-4 activity on an indicator cell line (HT-2; Rennick et al. J. Immunol.

134:910-914, 1985) and in the fibroblast proliferation assay.

DNA Nucleotide Sequence Analysis

Nucleotide sequences on both positive and negative cDNA strands were determined by the dideoxy-chain termination protocol with supercoiled DNA templates (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977) . Areas which were rich with G-C residues were sequenced by PCR using a cycle sequencing kit from Epicentre Technologies. The nucleotide sequence was compared by FASTA and BLAST programs to sequences archived in GenBank.

Peptide synthesis A 71 amino acid residue oligopeptide (MW 7620, as confirmed by mass spectrome ry) with sequence corresponding to that of the cDNA (amino acid arginine at position 26 to leucine at position 96, Figure 18; SEQ ID NO.: 1) was syntheβized by the methods of t- boc NMP chemistry using Perkin-Elmer Applied Biosystemβ 430A Peptide Synthesizer (Clark-Lewis, Science 231:134- 139, 1986). The peptide was purified by HPLC using Vydac CA column and eluted in a single fraction with trifluoroacetic acid/acetonitrile gradient.

CDC25 Lv phocvte Line Produces Fibroblast Mitogen Of the 30 lymphocyte lines and T cell hybridomas we tested, five elaborated detectable fibroblast mitogenic activity in culture supernatantβ following in vitro stimulation with Con A for 24 h. Con A had no intrinsic fibroblast mitogenic activity in our fibroblast assay. We chose line CDC25, a Th2 line for which a cDNA library was available for further analysis, culture supernatants of CDC25 cells stimulated fibroblast [ ³ H]- thymidine incorporation in a concentration-dependent manner; peak responses were detected at a concentration of 10% (the maximum tested; Fig. 14A) . Anti-FsF-1 IgG reduced the activity of the CDC25 culture supernatant by

21% and 47% with 2.5 μg/100 μl and 5 μg/μl IgG respectively (Fig. 14B) , relative to responseβ in the presence of the same concentrations of pre-immune IgG

(NRIgG) which we previously established does not significantly alter the fibroblast responβeβ to puri ied

FsF-1. Furthermore, in Western blot anti-FsF-1 IgG recognizes a single protein band (apparent MW 50-60 kD) in CDC25 culture supernatants subjected to SDS-PAGE;

NRIgG does not recognize this protein. CDC25 Library Contains cDNA that Encodes a Fibroblast Growth Factor

Culture supernatants of COS-7 cells transfected with the entire CDC25 library (containing approximately 10 ⁶ clones) stimulated fibroblast [ ³ H]-thymidine incorporation in a concentration-dependent manner (Fig. 15) . Because culture supernatants from IL-4 cDNA - transfectants (which contained 200-250 U/ml IL-4 based on results in the HT-2 proliferation asβay) had minimal effects on fibroblast proliferation, we concluded that the CDC25 library contained cDNA that specifically encoded a fibroblast mitogen that is not IL-4. We uβed sib-selection as a strategy to clone this cDNA (Yokota et al., Proc. Natl. Acad. Sci. USA 82:68-72, 1985; Lee et al. Proc. Natl. Acad. Sci. USA 83:2061-2065, 1986). The CDC25 library was partitioned into pools of 10 ³ clones that were seeded into separate wells of microtiter plates containing L-broth and ampicillin. Poolβ, estimated to contain 10* clones, were prepared by combining wellβ within a row. COS-7 cellβ were tranβfected with plaβmid DNA prepared from theβe poolβ and the tranβfectant culture βupernatantβ (tested at various concentrations, to a maximum of 10%) were aβsayed for their ability to enhance fibroblast [ ³ H]-thymidine uptake at least 2-3 fold above background (positive transfectant) .

Of the 20 pools initially prepared and tested, 3 (15%) were positive. One of the three pools (pool B) was selected because the biological activity in the supernatant of pool B transfectants could be neutralized with anti-FsF-1 IgG. Pool B was subdivided into 8 separate pools (each containing approximately 10 ³ clones) and plasmid DNA prepared from each pool was used to transfect COS-7 cells. Conditioned medium from four of the transfectants (50%) had significant mitogenic activity. Based on antibody neutralization of bioactivity, one of the pools (2B) was selected. Pool 2B was then subdivided into 16 pools, each estimated to contain 100 clones. Five of the pools (31%) produced positive transfectants. Two of the positive pools were plated on solid agar, and ten colonies were screened; 8 of these colonies produced positive transfectants.

One of the clones (2B3) was subcloned twice on solid agar and analyzed in detail. The supernatant of 2B3 transfectant was active in a fibroblast growth assay over a wide concentration range; the log-linear dose-response relationship was biphasic (Fig. 16A) . Anti-FsF-1 IgG virtually abolished the fibrogenic activity of 2B3 transfectant supernatant (Fig. 16B) . Notably, with each round of sib selection, the potency of the positive transfectant culture supernatants (dilution producing maximum fibroblast stimulation) increased (Fig. 17) . The potency increased approximately 2000-fold from transfection with the whole library to transfection with 2B3 clone. Nucleotide Sequence of Clone 2B3

Clone 2B3 contains a cDNA insert of 216 bp and a single open reading frame (ORF) starting with arginine (nucleotide position 76-78; Fig. 18; SEQ ID NO.: 1) and terminating with leucine (nucleotide position 289-291) followed by the stop codon TAA. Thus, the ORF codeβ 71

amino acids and is followed by 11 untranslated codons that precede the poly(A) tail. Since this ORF does not contain an internal initiating ATG, polypeptide synthesis iβ initiated at the first in-phase ATG codon present in the vector's 16s splice junction area, 72 base pairs upstream from the 5' end of the insert, and the total open reading frame of 288 nucleotides encodes a fusion protein. No significant homology of this sequence with sequences archived in GenBank could be identified. Analysis of the deduced amino acid sequence (Fig. 18; SEQ ID NO.: 1)) indicates that the NH ₂ terminal 15 to 20 deduced amino acid residues of the fusion protein are hydrophobic, and therefore predicted to serve as a secretion signal sequence (Perlman et al., J. Mol. Biol. 167:391-409). If it does βerve thiβ function it would be expected that cleavage occurβ following the alanine residue at position 15 or 20 of the fusion protein, resulting in a mature peptide containing either 81 or 76 amino acids (predicted MW 9-10 kD.) Alternative cleavage sites might exist. Since gel filtration chromatography of the transfectant culture βupernatantβ diβplayβ fibroblaβt mitogenic activity in fractions with MW 30 kD, the protein is also modified in vivo by glycosylation.

Fibroblast Growth Stimulation bv Synthetic Peptide A 71-mer peptide which was synthesized from the deduced amino acid sequence of 2B3 stimulated fibroblast proliferation (based on cell counting) in a concentration-dependent manner (Fig. 19) . Notably, in 12 separate assays (6 in which cells were assayed after 72 h incubation and 6 after 96 h incubation) , peak mitogenic activity was observed with peptide concentrations between 10" ¹³ and 10" ¹¹ M; additional activity was detected at concentrations in the 10" ⁶ M range. The dose-response pattern we observed iβ reminiβcent of our experience with

crude and purified natural FsF-1 as well as with supernatants of the 2B3 transfectant (Figs. 16A-B) .

Preparation and Analysis of Murine τ-σeiι

Hybridomas that Produce FsF-1/Fibrosin We prepared and cloned T-hybridoma cells that secrete FsF-1 upon Con A stimulation because this relatively homogeneous source: (1) provides a "renewable" in vitro source from which we can purify natural FsF-1, (2) provided mRNA from which -full length fibrosin cDNA could be prepared for analysis and heterologouε expression and, (3) allows standardization of in situ hybridization methods to detect fibrosin mRNA, and in the future can be used to study regulation of fibrosin gene expression. Individual (cloned) FsF-l ⁺ lymphocytes were selected by FACS (using rabbit anti-FsF-1 IgG and FITC- goat anti- rabbit IgG as described previously) from a population of splenocytes obtained from a mouse with 8 week S. mansoni infection and stimulated overnight with Con A. Individual cellβ were fuβed with thymoma BW 5147 cells using standard methods (Coligan et al., edβ.. Current Protocols in Immunology, Vol. 1, John Wiley 6 Sons, Inc., pp. 3.14.1-3.14.11 (1994)). We selected hybridomas that on stimulation with Con A (in the absence of serum) produced fibroblast mitogenic activity, and confirmed by ELISA, neutralization, and immunopurification that thiβ biological activity derived from FβF-1 in the supernatants. Positive hybridomaβ were subcloned 2-3 timeβ by limiting dilution. Cloned hybridoma cells were adapted to growth in GIBCO SFM (serum-free medium) to avoid serum contamination of culture supernatants. Representative results are shown in Fig. 20.

Analysis of these T cell hybridomas indicate T hybridomas produce fibroblast mitogenic activity (Fig. 20) . Second, the hybridoma-derived mitogen is

heparin-binding and elutes with high NaCl concentration (two characteristic properties of granuloma-derived FsF- 1) (Fig. 21) : the non-binding fraction (fall-through) had no activity, and the 1.0 and 1.5 M NaCl eluate contained only 60 kD protein (SDS-PAGE) that was detected by

Western blot using anti-FsF-1 antibodies. Third, SEA (10 μg/ml; fraction that lacks direct mitogenic activity) stimulates hybridoma # B12 production of FsF-1. Reβponβe to variouβ concentrations of cell culture supernatant are shown relative to CFM in fibroblastβ grown in medium alone in Fig. 22. Fourth, Western blot analysis of crude hybridoma supernatant using anti-FsF-1 Mabs (D6, Gl, Al) that had been prepared to granuloma-derived FβF-1 identified a 60 kD band in the same manner that they identified FsF-1 in crude granuloma culture supernatants. (Fig. 23) (Lane 1, hybridoma supernatant; lane 2, granuloma supernatant) . Noteworthy iβ that the Mabs do not react with albumin (ELISA) and partially neutralize bioactivity. In addition, crude culture supernatant from Con λ- stimulated T hybridoma (B12) was incubated with Mab IgG (#111A5/D8, 5 μg/ml) and immune complexeβ were precipitated with Sepharoβe-conjugated mouβe anti-rat kappa Mab. Beadβ were retrieved by centrifugation, washed with PBS (pH 7.2), placed in Centricon-100 filtration units and treated with 0.5 M Na-acetate (pH 4.3), then subjected to ultrafiltration of the proteins < 100 kD. The filtrate was neutralized with Tris buffer and tested in the bioassay. The control waβ identical treatment of T-hybridoma supernatant, but excluding incubation with anti-FsF-1 Mab. Bioactivity was compared with heparin- purified T-hybridoma-derived mitogen. Medium control is CFM in fibroblast cultured with medium alone. (Fig. 24) . These resultβ indicate that a Mab againβt FsF-1 immunopurified the mitogenic activity produced by the Con

A stimulated T cell hybridoma B12. Taken together, these data provide strong evidence that T-hybridomas produce the same FsF-1 that we previously purified from granuloma supernatants. Cloning of Full-Lenqtn Fibrosin ς NAs from Mouse

T-Cell Hvbrido a Cells and Human Peripheral Blood Lymphocytes

We first established (by RT PCR) that Con A- stimulated murine T-hybridoma cells expresβ 2B3 mRNA and confirmed this conclusion by in situ hybridization of Con

A-activated T-T hybridoma cells using a 194 bp ds 2B3- derived oligonucleotide probe having the sequence:

C-TCA-CTA-AGC-CAG-AGG-CCA-AAG-TGC-CCC-CCT-CCC-TTT-CGC- CTA-CCA-CCC-AAG-TTC-TCA-TGC-CCT-CCG-AGG-GCT-GAG-GAA-GGA- GGA-ACT- AAA-GGA-ATA-GGG-GTT-TCA-TGT-ACA-TAT-TTA-TCA-CCC- CTT-CCA-CAA- ATC-CCC-CAG-ACC-TTT-TGT-ACA-TTT-TTA-CAG-GGG- TGC-CCC-TCC-CTA- TAA-TTC-CCT-TCC-TGG-T. (SEQ ID NO.: 4)

Next, we designed four gene-specific DNA oligomerβ based on the 2B3 nucleotide sequence; we uβed them for additional (confirmatory) RT PCR, and subsequently also for PCR cloning. The four PCR primers are named Tl, B3, B2, and Bl (the "T" stands for top βtrand βequence and the "B" stands for bottom strand sequence; the order of Tl, B3, B2, and Bl reflects their order in 2B3 as oriented from 5' end to 3' end of the top strand).

Tl: C-TCA-CTA-AGC-CAG-AGG-CCA-AAG-TG (SEQ ID NO. : 5) Bl: λ-CCA-GGA-AGG-GAA-TTA-TAG-GGA-GG (SEQ ID NO.: 6) B2: CC-TTT-AGT-TCC-TCC-TTC-CTC-AGC-C (SEQ ID NO.: 7) B3: CA-TGA-GAA-CTT-GGG-TGG-TAG-GCG-A (SEQ ID NO.: 8) With three pairs of PCR primers (T1/B3, T1/B2, and Tl/Bl) and total RNA prepared from mouse T-hybridoma cells, we obtained RT PCR products with the expected sizes of 60 bp, 93 bp, and 194 bp, respectively. When we examined total RNA prepared from PHA-stimulated human peripheral blood mononuclear cells (PBMC) with these

primer pairs, we obtained only one RT PCR product of 60 bp.

To clone the 5' segment of fibrosin cDNA, we first tried two PCR cloning methods, "RACE PCR" (Frohman, in PCR Protocol. A guide to methods and applications, p. 28- 38. Academic Press, NY 1990) and "RACE NO MORE PCR" (Weis, Nucleic Acid Res. 22:3427-3428, 1994) with the three specific PCR primers Bl, B2, and B3. The human (PBMC) and murine (T hybridoma) RNAs were used to prepare total cDNAs as templates. Because the RACE NO MORE was successful with mouse cDNA, we pursued this method. RACE NO MORE method is based on the rare event of 5' end PCR priming in a linear PCR with only one reverse primer annealed to a random hexamer primed cDNA whose 5' end sequence is unknown. A subsequent second PCR with the first primer and a second reverse primer located 5' end to the first primer specifically amplifies the 5' end unknown region plus a stretch of known sequence at the 3' end. From the mouse source, we obtained in the second PCR a 470 bp product with primer pair B2/B1, which followed the first PCR that was carried out with the Bl primer. When primer pair B3/B1 was used in the second PCR, we obtained a 435 bp product, as expected. Both 470 bp and 435 bp products were loaded on separate gels, and after electrophoresis the bands were excised and purified (Wang and Rossman, Nucleic Acid Reβ. 22:2862-2863, 1994). Confirmatory experiments provided the expected reβultβ. Specifically, by regular PCR, the purified 470 bp yielded a 93 bp product with primer pair T1/B2, and a 60 bp product with primer pair T1/B3. The 435 bp product yielded only the 60 bp product.

The 470 bp and 435 bp products were then cloned into pCR3 vector of Invitrogen's Eukaryotic TA Cloning Kit following the manufacturer's protocol. DNA sequencing of several resulting plasmid clones of each

product revealed that the 470 bp and 435 bp products all contained the expected portions of mouse 2B3 sequence at the 3' end. The 5' end was composed of the Bl sequence, followed by a 9 bp portion of the 2B3 segment (an artifact—not unexpected— created by this new PCR approach) , which in turn was followed downstream by a new 311 bp sequence adjacent to the complete 2B3 sequence. These sequences indicated that both the 470 bp and the 435 bp products were derived from a common RACE NO MORE PCR event. Notably, the 311 bp sequence contained three in-frame ATG triplets; the first one was located at bp 76, the second 15 bp downstream from the first. The sequences surrounding the first and the second ATGs conform with Kozak's criteria for translation initiation (Kozak, J. Cell Biol. 108:229-241, 1989), and thus, could be sites for translational initiation.

The mouse cDNA has a total length of 571 bp with a 75 bp untranslated leader sequence and encodes a polypeptide chain of about 149 residues (including 78 encoded by the upstream 311 bp sequence and 71 encoded by 2B3) (Fig. 25; SEQ ID NO.: 2).

To confirm that the new 311 bp mouse sequence created by RACE NO MORE was not an aberration of the method, and to obtain the complete apparently full-length fibrosin cDNAs from both mouse and human sources, we designed two PCR primers. Primer T2 is a 28-mer forward primer derived from the mouse sequence in the 5' end untranslated leader sequence, upstream from the first ATG; primer B4 is a 35-mer reverse primer, derived from the mouse 2B3 sequence at its 3' end untranslated region, including 10 A'β of its poly-A tail of more than 50 A'β.

T2 : AAG-CCA-GGG-TTG-GAA-GGC-AAA-GGT-CAC-A

(SEQ ID NO. : 9) B4 : TTT-TTT-TTT-TCC-AGT-CTG-AGG-ATT-TAA-TTA-ACC-AG

(SEQ ID NO. : 10)

Using these primers, we obtained a 524 bp PCR product from the cDNA prepared using total RNA from the mouse T cell hybridoma, and a 500 bp PCR product from the cDNA prepared using total RNA from human PBMC.

Both PCR products were gel purified, and then used as templates in separate PCRs with a panel of seven pairs of mouse primers: T2/B1, T2/B2, T2/B3, Tl/Bl, T1/B2, T1/B3, and T1/B4. The mouse 524 bp PCR product generated seven bands of the expected sizes, and the human 500 bp PCR product yielded six bands of the expected sized. DNA sequencing of both PCR products have

^' demonstrated that human (Fig. 26; SEQ ID NO.: 3) and mouse (Fig. 25; SEQ ID NO.: 2) fibrosin cDNAs are comprised of two portions: (1) the 311 bp segment in the 5' region; and, (2) the 2B3 segment in the 3' region. Sequence analysis indicates that there are considerable interspecies differences in the 5' portion of fibrosin cDNA, including some sequence deletions. On the other hand, the 2B3 segment is conserved. The conservation of the 2B3 segment is not surprising since this contains the biologically-active domain that can stimulate growth of both murine and human fibroblastβ in vitro.

EXPRESSION OF FsF-1 POLYPEPTIDE

Polypeptides according to the invention can be produced by expression from a recombinant nucleic acid having a sequence encoding part or all of an FsF-1 polypeptide of the invention, using any appropriate expression system: e.g., transformation of a suitable host cell (either prokaryotic or eukaryotic) with the recombinant nucleic acid in a suitable expresβion vector.

The precise host cell used is not critical to the invention. The method of transfection and the choice of expression vector will depend on the host system selected. Mammalian cell transfection methods are described, e.g., in Ausubel et al. (Current Protocols in Molecular Biology, supra) ; expression vectors may be chosen from those discussed, e.g., in Cloning Vectors: A Laboratory Manual (P.H. Pouwels et al., Supp. 1987). Stably transfected cells can be produced by integration of FsF-1 DNA into host cell chromosomes.

DNA sequences encoding the polypeptides of the invention can also be expressed in a prokaryotic host cell. DNA encoding an FsF-l polypeptide of the invention or a fragment thereof is carried on a vector operably linked to control signals capable of effecting expression in the prokaryotic host. If desired, the coding sequence can contain, at its 5' end, a sequence encoding any of the known signal sequences capable of effecting secretion of the expressed protein into the periplasmic space of the host cell, thereby facilitating recovery of the protein and subsequent purification. Prokaryoteβ most frequently used are various strains Σ. coli; however, other microbials can also be used. Plasmid vectors are used which contain replication origins, selectable markers, and control sequences derived from a species compatible with the microbial host. Commonly used prokaryotic control sequences (also referred to as "regulatory elements") are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences. DETECTION OF CIRCULATING FsF-1/FIBROSIN ANP

PRODUCTION 9F MONOCLONAL ANTIBODIES

To pursue investigations of the role of FsF-1 in liver fibrosis in schistosomiaβiβ, we developed the antigen-capture immunoasβay described above for quantifying circulating FsF-1. Given below we review

various features of thiβ immunoassay, including and its ability to detect marked, transient elevations in plasma FsF-1 levels in infected mice. Because the alterations in plasma fibrosin concentration parallel the time course of fibrosin production in the hepatic granulomas, it is likely that circulating fibrosin levelβ reflect intrahepatic production. Accordingly, fibrogeneeie in schistosomiasis may be monitored by quantifying circulating fibrosin levels in infected patients. Moreover, persistent fibrosin production appears to be a risk factor for the development of pipe-stem fibrosis. We purified fibrosin from two βourceβ of βerum- free conditioned medium: (1) egg granulomas and (2) cloned T cell hybridomas stimulated with concanavalin A (Con A) . As described above, egg granulomas were isolated from the livers of mice infected for 8 weeks with S. mansoni and incubated overnight in medium RPMI 1640. T cell hybridomas were prepared by published methods (Kruisbeck et al., Current Protocols in Im-Ou-LQ-LSSQ-:* vol. I. New York, John Wiley k Sons, Inc. (1994)). Dispersed βplenocyte from an 8-week old infected mouse were fused with BW5147.G.1.4 thyoma cells (TIB #48; originally obtained from the American Type Culture Collection [ATCC]). Viable T cell hybridomas were initially selected in HAT medium (GIBCO/BRL

Products, Gaithersburg, MD) and βubβequently expanded in non-selective medium. Individual fibrosin-positive cells were cloned from the hybridoma cultures by fluorescence- activated cell sorting (FACS) . Cells were treated with rabbit anti-fibrosin IgG followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma Chemical Co., St. Louie, MO), and then cloned using a FACStar Plus flow cytometry (Becton-Dickinson, Mountain View, CA) . Individual fibrosin-positive cells were dispensed into 96-microwell culture plates (Nunculon,

Nunc, Roskilde, Denmark) and cultures were expanded. Two million washed hybridoma cells per ml were cultured overnight in serum-free RPMI 1640 in the presence of Con A (10 μg/ml; Sigma) and cell-free supernatants were screened for the presence of immunoreactive fibrosin in ELISA (Hombeck et al.. Current Protocols in Immunology, vol. I. New York, John Wiley & Sons, Inc. (1994)) using rabbit anti-fibrosin IgG. Fibrosin-secreting hybridomas were then subcloned at least twice by limiting dilution. The presence of fibrosin was confirmed by the ability of conditioned media of Con A-stimulated cloned hybridomas to stimulate human fibroblast proliferation (Prakash et al., J. Immunol. 61:3985-3987 (1993)), and the ability of rabbit anti-fibrosin IgG to specifically neutralize this biological activity (reduction of response >50%) .

Fibrosin was purified from the two sources by a slight modification of published methods (Prakash et al., J. Immunol. 146:1679-1684 (1991)). Conditioned media were first subjected to affinity chromatography on immobilized gelatin to remove fibronectin (Engvall et al.. Int. J. cancer 20:1-5 (1977)). Nonadsorbed material (fall-through fraction) was then subjected to heparin- affinity chromatography (Hi'Trap* Heparin; Pharmacia Biotech, Piscataway, NJ) as described (Prakash et al., it. supra) . The loaded column was first washed with 0.5 M NaCl and then the 1.5 M NaCl eluate was collected. This fraction contains a single detectable protein (60 kD) when it is subjected to sodium dodecylβulfatβ polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Prakash et al.,

146:1679-1684 (1991)). The biological activity of the purified material was confirmed in the fibroblast proliferation assay described above (Wyler et al., ___ Infect. Pis. 144:254-262 (1981)). We determined that our purification method provides 95-98% pure fibrosin at a

rate of approximately 4 μg fibrosin protein/ml starting material (granuloma- or T cell hybridoma-conditioned medium) .

Monoclonal antibodies to murine fibrosin were produced by B cell hybridomas that were prepared by fusing rat splenocytes with myeloma line SP2/0-Agl4 (TIB# 1581, originally obtained from ATCC; Yokoyama, Current Protocols in Immunology, vol. I. New York, John Wiley & Sons, Inc. (1994)). Female Lewis rats received a primary immunization by intraperitoneal (ip) injection of heparin-purified fibrosin (from granuloma-conditioned medium; 2 μg protein in Freund'β complete adjuvant), followed by six ip booster injections of fibrosin (2 μg protein in Freund'β incomplete adjuvant) at 2-week intervals. When serum anti-fibrosin antibody was detected in the rats (by ELISA) , a final ip injection of 4 μg fibrosin without adjuvant was administered, and cell fusions were performed 5 days later. Hybridoma supernatants were initially screened by ELISA for reaction with unfractionated serum-free granuloma- conditioned medium, and positive supernatants were then tested for the presence of antibodies to purified fibrosin. The selected hybridomas were cloned and subcloned by limiting dilution. IgG was purified from serum-free hybridoma conditioned medium (Hybridoma SFM; cat.# 125045; GIBCO/BRL) by immunoaffinity chromatography with immobilized mouse anti-rat K (MAR) monoclonal IgG. The MAR Model EL 308; Bio-Tek Instrumentβ, Inc., Burlington, VT) . Serum and plasma samples were tested at 1:100 and 1:1000 dilution to achieve fibrosin concentrations in the linear portion of the standard curve. Plasma and serum fibrosin concentrations were calculated using the formula that relates O.D. ₄₀₅ ("x") to pg protein/ml ("y"), using the log-linear plot of the

standard curve generated for each βet of assays, and adjusted for dilution.

In studies of murine βchiβtoβomiasis, C57BL/6NcrLBr female mice (18 to 20 g; Taconic Farms, Inc., Germantown, NY) were infected by the intraperitoneal injection of 35-50 cercariae of S. mansoni (Puerto Rican βtrain) βuβpended in 0.5 ml sterile saline as described above. For serum and plasma sampling, mice were individually ear tagged for identification, anesthetized by inhalation of methoxyflurane (Metofane, Pitman-Moore, Inc., Mundelein, IL) , and retro-orbital sinus blood was obtained into plain or heparinized hematocrit tubes (Arthur H. Thomas Co., Philadelphia, PA). The serum or plasma was retrieved after hematocrit centrifugation and was stored at -20*C in 0.5 ml polypropylene vials (Brinkmann Instruments, Inc. , Westbury, NY) .

We confirmed the specificity of the anti-fibrosin Mabs and polyclonal rabbit antifibrosin IgG employed in the antigen-capture ELISA. In Western blot analysis, each of three separately prepared rat anti-fibrosin Mabs (ID8/C9, IIIG6/C7, and IIIA5/D8) detects a single 60 kD protein in serum-free conditioned medium from egg granuloma cultures and T cell hybridoma cultures (Fig. 27). Normal rat IgG reveals no protein bands by this method (results not shown) . The electrophoretic migration of the 60 kD protein corresponds to that of fibrosin (Prakash et al., J. Immunol. 146:1679-1684

(1991)). To further validate that the 60 kD protein recognized by the Mabs is fibrosin, we subjected a variety of fibrosin-containing fluids to immunoaffinity chromatography. Immobilized IgG Mab ID8/C9 was employed to adsorb fibrosin from four separate sources: (1) conditioned medium from egg granuloma cultures, (2) T

cell hybridoma cultures, (3) aqueous extracts of T cell hybridomas, and (4) normal mouse serum. Acid (pH 4.3) eluates from the immunoaffinity column stimulated fibroblast proliferation (Fig. 28) . The fibrogenic activity of the immunopurified fibrosin as well as that of a synthetic truncated fibrosin peptide (2B3 peptide) was abrogated by the addition of the homologous Mab to fibroblast cultures (Fig. 28) .

We previously established the specificity of rabbit anti-fibrosin polyclonal IgG and, in additional experiments determined that the only detectable contaminating IgG has anti-albumin specificity. Accordingly, we subjected the anti-fibrosin IgG preparation to three cycles of immunoadsorption on immobilized bovine serum albumin (Sigma) , and confirmed by ELISA that this procedure successfully depleted the preparation of detectable anti-albumin IgG. To validate that this preparation was appropriately specific for use as the antigen detector in the antigen-capture ELISA, we compared results using pre-immune rabbit IgG, anti- fibrosin rabbit IgG, and anti-fibrosin IgG preincubated with purified fibrosin ("blocked") . Only the anti- fibrosin IgG (not blocked) could detect fibrosin in the antigen-capture assay (Fig. 29) . We performed experiments in order to determine the preferable form and concentration of anti-fibrosin Mab, detector anti-fibrosin polyclonal IgG alkaline phosphatase-conjugated goat and rabbit IgG, and substrate that provided for the most senβitive antigen-capture assay. Based on these studies, we routinely coat the wells of ELISA plates with Mab ID8/C9-containing culture supernatants aβ the antigen-capture reagent. All O.D. ₄₀₅ readings are blanked against wells to which all reagents except fibrosin are added. Based on the reβultβ of more than 12 assays, which were compared with a standard curve

that was generated by asβaying known concentrations of heparin-purified fibrosin, we established that this ELISA is sensitive to at least 0.005 pg/ml and retains a log linear relationship of fibrosin concentration to O.D. ₄₀₅ up to l pg/ml; correlation coefficients (r ² ) ranged between 0.916 and 0.996 in these assays. In view of the high sensitivity of the assays, we routinely test samples at ten-fold dilutions (typically 1:100 and greater) to confirm that our calculations are based on measurements in the linear portion of the standard curve.

Serum Levels of Fibrosin in Uninfected and S. mansoni Infected Mice

We detected fibrosin in concentrations ranging from 2-9 pg/ml in serum of uninfected mice, and substantially elevated levels (75-2500 pg/ml) in mice infected for 8 weeks with S. mansoni . In view of these observations, we subsequently monitored fibrosin levels in individual mice during the course of their infection. We first established that there was no significant difference in the concentration of fibrosin detected in plasma and serum samples that were obtained simultaneously from 5 infected mice (data not shown) . Furthermore, by assaying fibrosin concentrations on multiple occasions in the same plasma samples stored at different temperatures, we determined that fibrosin antigenicity was most stable at -20°C for several months but labile at room temperature (data not shown) .

Serum Levels of Fibrosin Peak at the Same Time the Granulomatous Inflammatory Response Peaks On two βeparate occaβionβ, two monthβ apart, groups of 5 mice were infected with S. mansoni and were bled at regular intervals thereafter. Ten uninfected strain-, age- and sex-matched mice served as controls. All the plasma sample (stored at -20°C) were asβayed simultaneously, and the results were confirmed in separate additional assays of selected samples. At week

4 of infection, before schiβtomiasis eggs were depoβited in tissue, plasma fibrosin concentrations were in the normal range. At week 6, when ovipoβition had begun, ibrosin levels were above normal levels in almost all the mice. At week 8, when the granulomatous inflammatory response in the liver to newly deposited eggs reachea a peak, fibrosin levels reached their peak in all but one mouse. Finally, at week 12, when the granulomatous inflammatory response was undergoing down-regulation, fibrosin levels fell to control levels.

In related experiments, we additionally determined in 10 mice that at day 5 and week 4 of infection the concentration of plasma fibrosin was indistinguishable from control levels. Furthermore, after declining by week 12, fibrosin levels remained in the control range at week 16 (n-16) , 18 (n-4) , and 20 (n«3) in surviving mice (data not shown) .

In this study, we sought to detect circulating fibrosin in S. mansoni-infected mice in whom this fibrogenic cytokine appears to play a role in the pathogenesis of liver fibrosis. Using the antigen- capture ELISA for fibrosin that we describe here for the first time, we quantified circulating fibrosin concentrations in uninfected mice and in mice at different times during schistosomiasis infection. This assay employs as the antigen-capture reagent a rat monoclonal antibody (Mab ID8/C9) prepared against native murine fibrosin. Our other anti-fibrosin monoclonal antibodies could not be used as the detector since all the Mabs apparently recognize the same or vicinal epitopes. With an oligospecific, polyclonal anti- fibrosin rabbit IgG as the detector, the asβay iβ able to detect as little as 0.05 pg/ml fibrosin.

The time course in the elevation of plasma fibrosin concentrations suggests that egg deposition in

the liver and egg granuloma formation (which occurs after week 5 of infection) are prerequisites to fibrosin production. We believe that the hepatic egg granulomas are the likely source of elevated circulating levels of fibrosin, although intestinal granulomas might be additional sources. It is particularly noteworthy that plasma fibrosin levels peak at week 8 of infection, since this is when granuloma formation and granuloma fibrogenic cytokine production are most vigorous (Colley, Parasitic diseases. vol. I New York: Marcel Dekker, Inc. pp 1-83 (1981); Prakash et al., Hepatholocry 13:970-976 (1991)). Furthermore, the decline of these levels to the normal range at week 12 corresponds to the time at which fibrosin production by hepatic egg granulomas appears to cease (Prakash et al., Hepatholoov 13:970-976 (1991)). Hepatic fibrogenesis (as measured by extracellular matrix synthesis and deposition) during murine schistosomiasis follows a similar biphasic pattern: significant increase after granuloma formation, and reduction during the chronic stage (El Menza et al., Hepatology 9:50-56

(1989)). Accordingly, it seems likely that fibrosin and other granuloma-derived fibrogenic cytokines exert regulatory influences on hepatic fibrogenesis, and that fluctuations in plasma fibrosin concentrationβ are appropriate surrogate markers for monitoring hepatic fibrogenesiβ.

The fluctuationa in circulating fibroβin levelβ might represent a manifestation of antischistoβomal hypersensitivity. Fibrosin production by granuloma CD4 ⁺ lymphocytes is likely to be stimulated by egg antigens, and CD4 ⁺ lymphocyte hyperβensitivity to theβe antigens iβ most vigorous at week 8 of infection. The hypersensitivity wanes thereafter and is considerably depreββed at week 12. The likelihood that fibroβin production is down-regulated by an immunological

- 68 - mechanism is suggested by the observations that adoptive transfer of spleen cells from chronically-infected donor mice (week 23) to acutely-infected recipientβ (4 week) prevents production of fibrogeneic factors (biological assayβ) by egg granulomas present in recipients' liver at 8 weeks of infection (Prakash et al., Hepatoloov 13:970- 976 (1991)). Although these experimental results are reminiscent of ones observed in βtudies of the regulation of the granulomatous inflammatory response (Colley, Parasitic diseases, vol. I. New York: Marcel Dekker, Inc., pp. 1-83 (1981)), the specific immunological pathways that might regulate the elaboration of fibrogenic mediators, including fibrosin, might be distinct from those that regulate the magnitude of the granulomatous inflammation (Cheever et al., J. Immunol. 153:753-759 (1994)).

The down-regulated antischistoβomal lymphocyte responsiveness that is characteristic of chronic infection in inbred mice as well as in patients who do not develop severe liver fibrosiβ contrasts with the exuberant antischistosomal T cell hyperβensitivity observed in patients with pipe-stem fibrosiβ (Colley, Parasitic diseases, vol. I. New York: Marcel Dekker, Inc., pp. 1-83 (1981); Ottesen et al., Clln. EXP. Immunol. 33:38-47 (1978); Colley et al.. Am. J. Troo. Med. Hyg. 35:793-802 (1986); Hafez et al.. Am. J. Trop. Med. Hvo. 44:424-433 (1991)). Whereas most patients during chronic infection experience substantial down- regulation of the vigorous antischistosomal hypersensitivity that they initially develop during the acute infection, it appears that a subpopulation of patients, specifically those at risk for pipe-stem fibrosis, fails to do so. It appears that transient production of fibrosin by hepatic egg granulomas results in minimal, largely perigranulomatous scarring in

infected mice and most patients, whereas chronic, unmodified production of fibrosin in selected patients is likely a critical aspect in their development of extensive liver fibrosin. FIBROGENIC PROPERTIES OF FIBROSIN

In this series of experiments we used fibrosin that was purified from egg granuloma conditioned medium, as described above.

These studies also utilized the 71 amino acid- residue peptide 2B3 (Mr 7620) , which was synthesized as described above. 2B3 is a biologically active, truncated fibrosin peptide that is based on the deduced amino acid sequence of a single open reading frame in a truncated fibrosin cDNA (clone 2B3) that we cloned from a murine cDNA library, also as described above.

Monoclonal antibody was prepared, as described above, to heparin-purified murine fibrosin. Assays of Biological Activity Primary human dermal fibroblast cultures were prepared and propagated as deβcribed above, and assessed between passage 4 and 12. Fibroblast chemotaxis was assessed in modified Boyden chambers by published methods (see Wyler et al., J. Immunol. 188:478, 1977). Experiments using Boyden chambers were also performed, as described below, to assess the ability of supernatant from COS cells transfected with human FsF-l to stimulate the chemotaxis of human peripheral monocytes.

Fibroblast collagen synthesis was measured by quantifying the incorporation of ³ H-proline into trichloroacetic acid-precipitated, collagenase-βenβitive protein in fibroblast culture supernatants using a published microassay method (Diegelmann et al.. Anal. Blochem. (1990)). Collagen synthesis results were subsequently confirmed and extended by using HPLC to analyze the relative concentration of hydroxyproline and

reference amino acids in hydrolysates of serum-free fibroblaβt-conditioned medium. For this purpose, fibroblasts suspended in antibiotic-containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (DMEM-10) were grown to confluency in 100 mm diameter tissue culture dishes. Cultures were extensively washed with serum-free DMEM, replenished with DMEM supplemented with 0.1% FCS (DMEM-0.1) and incubated overnight, at which time agonists were added. Six hours later, the conditioned medium was harvested and combined with supernatants from several DMEM-0.1 washed off the monolayer. Protein in the cell-free supernatant was precipitated with cold ethanol and the precipitate was lyophilized, hydrolyzed, and subjected to HPLC. The DNA content of the cell monolayer was determined by standard methods (Ausubel et al. Current Protocols in Molecular Biology, supra) . The hydroxyproline concentration per ng of cellular DNA of the ratio of hydroxyproline to proline or to leu-cine was compared for fibroblast cultures maintained with or without addition of purified fibroβin or TGF/J (Collaborative Reβearch) .

Fibronectin and hyaluronan concentrations in serum-free, fibroblast-conditioned medium were determined by publiβhed methodβ. For theβe experiments, 10 ^s fibroblastβ βuβpended in DMEM-10 were βeeded into each 16 mm diameter well of 24-well tissue culture plates and incubated at 37*C in 5% C0 ₂ -95% air atmoβphere until the cell layer appeared confluent. The cultures were extensively washed, replenished with DMEM-0, and incubated for an additional 18-24 h, at which time stimuli were added to the cultures. Aliquots of conditioned medium were retrieved and stored at -20°C until tested.

An anti-FβF-1 Monoclonal Antibody Neutralizes the Mitogenic Effect of Fibrosin

The rat monoclonal antifibrosin IgG (Mab) we prepared against native fibrosin partially neutralizes the mitogenic effect of native fibrosin and the synthetic

2B3 peptide when added to fibroblast culture (Fig. 30) .

Immunoadsorption with immobilized Mab entirely depletes the biological activity in solutions of purified fibrosin

(data not shown) . These observations indicate that the Mab recognizes an epitope within the 2B3 peptide that is also expressed in native fibrosin.

Fibrosin has Chemotactic Properties that are Neutralized bv an antl-FsF-l Monoclonal Antibody

Purified native fibrosin and 2B3 peptide are both chemotactic agents to fibroblasts when aββeββed in modified Boyden chambers (Fig. 31 panels A-C) . The addition of anti-fibrosin Mab markedly reduces this biological effect of purified native fibrosin (Fig. 31, panel C) . It therefore appears that the chemotactic domain, like the mitogenic domain, is contained in the

2B3 region. It is noteworthy that the optimal chemotactic concentration of the 2B3 peptide (10 nM range) is at least an order of magnitude higher than the optimal concentration of the native molecule (100 μM range) .

Biological Activity in Transfected COS Cell Supernatants: Monocvte Chemotaxis

A plasmid bearing a large fragment (2.8 kb) of human fibrosin cDNA (pBKCMVHFibrosinl) was purified from liquid cultures (QIAGEN purification kit) and transfected by lipofection into COS cells. The efficiency of the transfection was determined by a chromogenic reaction. As a negative control, COS cells were also transfected with plasmid lacking the fibrosin encoding insert. The supernatant was collected from the cells after 24 hours in culture under serum-free conditions. The assay fro

chemotaxis was carried out in Boyden chambers, which consist of lucite blocks that are separated into two chambers by a polycarbonate filter with 5 μm pores. Human peripheral monocytes were placed in one chamber and the supernatant collected from transfected COS cells was placed in the second chamber. The cells remained in the chamber for 90 minutes before the filters were harvested and stained. Cells were counted under high power magnification and the results were expressed as the average number of cells per high power field (HPF; Fig. 43). COS cells transfected with the plasmid bearing the human FsF-l encoding insert stimulated the chemotactic movement of monocytes to a much greater extent than did the supernatant collected from cells transfected with the control plasmid.

The Stimulation of Extracellular Matrix Deposition is Blocked bv an anti-FβF-1 Monoclonal Antibody

Excessive extracellular matrix deposition is a distinctive pathological hallmark of tissue fibrosiβ. Fibronectin and hyaluronan are two matrix components that frequently are produced early in the course of fibrogeneβiβ. Fibrosin stimulates fibroblastβ to produce both of theβe macromolecules (Fig. 32, panel A and Fig. 32, panel B) . Since the addition of cycloheximide blocks the fibrosin-enhanced elaboration of fibronectin, thiβ reβponβe dependβ on de novo protein βyntheβiβ rather than simply release of pre-formed fibronectin. Furthermore, the 2B3 peptide stimulates fibronectin βyntheβiβ and antifibroβin Mab blocks the stimulatory effect of the truncated peptide and native fibrosin (Fig. 35 panel C) . Fibrosin (optimal concentration 20 μg/ml) stimulates, in contact-inhibited fibroblast cultures, a net increase in the incorporation of ³ H-proline into collagenase-βenβitive secreted protein (Table 3). Although fibrosin also increaseβ total protein βyntheβiβ (collagenase-sensitive plus insensitive protein) , it

stimulates a 40% increase above basal levels in the ratio of collagen-to-total protein synthesis when assessed with this assay (Table 3).

Table 3: Relative collagen synthesis in response to fibrosin

Additive Relative collagen βyntheβiβ % to culture EXPt I ¹ Exoer. 2 ²

CP.TP hY rPrPCP Hv ^p ro:leu

Medium 6.2 ± 1 1.0 ± 0.7 0.42 ± 0.16

Fibrosin:

4 ng/ml NT 2.6 ± 0.2 0.72 ± 0.05 40 nα/ml 8.7 t P«4 2.8 ± 0.6 0.66 t 0.14

1. The incorporation of ³ H-proline into collagenous protein (CP) is expressed as a percent on incorporation into total protein (TP) . N≥6.

2. The molar concentration in fibroblast culture supernatants of hydroxyproline (hypro) that reflect collagen synthesis is expressed as a percentage of molar concentration of proline (pro) or leucine (leu) that reflect total protein synthesis.

We confirmed these observations by using another method to assess collagen synthesis, i.e., determining by HPLC the concentration of hydroxyproline relative to selected other amino acids in hydrolysate of fibroblaat culture βupernatantβ. In response to fibrosin, the concentration of hydroxyproline increased in the supernatant and the ratio of hydroxyproline to proline increased by 180% above the basal ratio (medium alone; Table 3) . Furthermore, the addition of anti-fibroβin Mab specifically reduced the collagen synthetic response of fibroblastβ to fibrosin (Fig. 32) . Limited supplies permitted us to conduct only one set of experiments with the 2B3 peptide. We observed a dose-dependent increaβe in hydroxyproline (229% above unstimulated control) and an increase in the ratio of hydroxyproline-to-proline (234% above control) in the βupernatantβ of fibroblastβ stimulated with μM concentrations of 2B3 peptide (Table 4).

Table 4: Collagen synthesis in response to 2B3 peptide

Resource; % control h prø ¹ hYPro?PrP ² hvnrotleu ³

5 X 10" ⁶ 158 234 200 5 X 10 ^*5 229 200 159

5 X 10" ⁴ 116 75 73

1. Hydroxyproline concentration (pM/μg DNA) in fibroblast culture supernatants, expressed aβ percentage of concentration in supernatants of unstimulated (medium only) cultures.

2. Ratio of hydroxyproline to proline concentration in supernatants of stimulated fibroblastβ expressed as a percent of this concentration ratio of unstimulated cultures.

3. Ratio of hydroxyproline to leucine concentration in supernatants of stimulated fibroblasts expresβed aβ a percent of thiβ concentration ratio for umstimulated cultureβ.

These results clearly demonstrate that fibrosin has multiple fibrogenic properties demonstrable in vitro. Furthermore, results of experiments with the 2B3 peptide and anti-fibrosin Mab (that reacts with this peptide) strongly suggest that the biological activities reside within the portions of anti-fibrosin that contains the 2B3 peptide sequence. Fine structure-function analysis should reveal whether the same domain (within the 2B3 stretch) is responsible for all these observed biological effects or whether subregionβ therein promote different effects. In any case, fibrosin differs from some of the other fibrogenic factors that either have mitogenic activity or induce ECM production, but not both. Based on these biological properties it can be expected that in vivo fibrosin plays a role in the fibroblast recruitment, proliferation, and enhanced ECM production involved in

scar formation, such as is observed in the setting of certain chronic inflammatory disorders, including schistoβomiaβiβ.

FSF-1/FIBROSIN IS ANGIOGENIC in vivo Since the hepatic granulomas that form following S. mansoni infection also undergo neovascularization, we investigated whether fibrosin is also anhiogenic.

Fibrosin, purified as described above, or the 2B3 peptide, synthesized as deβcribed above, were incorporated into hydron pellets and implanted into the coroeal βtroma of rate. Induction of neovacβularization from the limbal vaβculature was monitored for 7 dayβ following implantation. Corneas were examined histologically for non-specific inflammation. Purified native fibrosin induced an angiogenic reβponse in rat corneas without inciting an inflammatory reaction. The minimum amount of fibroβin required to elicit an angiogenic response (5 ng/implant) is comparable to the amount of bFGF, VEGF, or TNFα required to induce similar responses. The 2B3 peptide (maximum amount tested 2.5 ng) also elicited a distinct, inflammation-free angiogenic response. In vitro effects of fibrosin on endothelial cells are being evaluated. We conclude that fibrosin has angiogenic properties in vivo with a potency similar to that of other well established angiogenic factors. The angiogenic activity of fibrosin derives from the same domain in the protein that signals fibrogenic responβeβ, as revealed by experiemnts with the 2B3 peptide. The fibrogenic and angiogenic properties of fibrosin could account for the histopathy of liver fibrosiβ in experimental schistosomiasis. Moreover, fibrosin could also play a role in formation of granulation tissue during would healing.

FIBROSIN IS OVERPRODUCED BY SMOOTH MUSCLE Hfr,^ TW THE BOWEL OF A CROHN'S DISEASE PATIENT

We examined the production of fibrosin by smooth muscle cells that were isolated from the muscularis mucosae of a normal bowel and of a Crohn's disease patient. The tissue from the patient with Crohn's disease produced over ten-fold more fibrosin than the normal bowel (Fig. 33). The normal jejunal-derived cells made little basal fibrosin, whereas those from the small bowel of a Crohn's disease patient overexpressed the lymphokine. Particularly noteworthy is that the margin of the resected abnormal bowel, which itself is histologically normal (Margin; Fig. 33) yields smooth muscle cells that produce less fibrosin than the Crohn's ileum, but more than the normal bowel. These results indicate that fibrosin overproduction by smooth muscle - cells in Crohn's disease could be an important part of the fibrotic complications in this inflammatory disorder.

FIBROSIN IS ABUNDANTLY PRODUCEP BY RHEUMATQIP SYNOVIOCyTE CULTURES

We assayed the concentration of fibrosin in the conditioned medium of rheumatoid synoviocyte cultures.

The two donors (patient J and patient W) had rheumatoid arthritis and cells were cultured from removed pannus. The results indicate that these mesenchymal cells make fibroβin, and can be induced to produce increaβed amounts with phytohemaglutinin (PHA; Fig. 34) . Studies are currently underway to compare fibrosin production in rheumatoid and oβteoarthritic synovial cells in culture. Rheumatoid arthritis may have fibrosin as a part of its pathogenesis and these studies indicate that investigation of this poββibility iβ feasible given the assays we have established for fibrosin.

FIBROSIN LEVELS ARE ELEVATED IN SERA OF PATIENTS WITH SCHISTOSOMIASIS AND INFLAMMATORY BOWEL uι_u_____

We determined the level of ibrosin in stored sera that was obtained from Brazilian patients that were infected with Schistosoma mansoni and in similarly stored sera from normal (control) individuals. We have assayed more than 100 samples and obβerved significantly elevated levelβ in most (Fig. 35) . This observation indicates that the assays described herein could be applied to studies designed to determine whether or not a given individual is at risk for liver fibrosis in this infection.

We have also shown that FsF-1/Fibrosin can be immunopurified from the sera of patients, as described above (see "Gel Filtration Chromatography and Heparin Affinity Chromatography"). Furthermore, this immunopurified human serum fibrosin is biologically active in the fibroblast proliferation assay, as described above (see also Fig. 42) .

The concentration of serum fibrosin is also elevated in patients with inflammatory bowel disease (IBD) and in glomeruli from rats with ATS-induced glomerulonephritis in vitro. THE PRODUCTION OF FIBROSIN PROVIDES A RELIABLE

INDEX OF TREATMENT OUTCOME

We monitored fibrosin production in patients with primary biliary cirrhosis that were elevated above upper 95% C.I. of normal controls (Fig. 36; horizontal line). When the levels were monitored during treatment with colchicine or methotrexate, we found that two of three patients with a poor outcome had levels of fibroβin that continued to riβe over the course of the treatment regime. In contrast, in three of three cases where the patients responded well to treatment, the level of fibrosin declined into the normal range.

ANALYSIS OF CULTURED SCLERODERMA CELLS AND

NORMAL FIBROBLASTS

A series of experiments were conducted on scleroderma cells and normal fibroblastβ that were Fig. 38 iβ a βerieβ of photomicrographβ of βcleroderma cellβ cultured in MEM Eagle's Medium with 10% FCS. These experiments examined the effect of an antifibroβin antibody on the production of collagen by βcleroderma cells. The cultured scleroderma cells were treated with 20 μg/ml of random IgG, or 20 μg/ml anti-fibrosin antibody for either one or two days, and stained with an antibody against type I collagen (Fig. 38) . Treatment with the anti-fibrosin antibody diminished the expression of type I collagen. The effect of conditioned medium on collagen expression was also investigated. Normal fibroblasts - were cultured as described and exposed to either conditioned medium from normal fibroblasts, conditioned medium from sclerodermal fibroblasts, or conditioned medium from sclerodermal fibroblasts that had been depleted with anti-fibrosin antibodies. The cells were then stained with an antibody against type I collagen. Conditioned medium from scleroderma cells stimulated collagen production, which was blocked by addition of anti-fibrosin antibodies to the conditioned medium (Fig. 39) . A βimilar observation was made for the production of fibronectin: conditioned medium from sclerodermal cells stimulated fibronectin production, which was blocked by addition of anti-fibrosin antibodies to the conditioned medium (Fig. 40) .

The expression of fibrosin by βcleroderma cells was demonstrated directly by staining cultured scleroderma cells with a monoclonal antibody against fibrosin (Fig. 41).

USE

Using the methods described herein, overexpression of the nucleic acids of the invention can be used to isolate large quantities of FsF-1 polypeptides that are capable of efficiently stimulating fibroblast growth, proliferation and chemotaxis. Alternatively, the methods described herein can be used to generate synthetic or recombinant polypeptides that are capable of inhibiting one or more of the biological activities of the naturally occurring FsF-1 polypeptide. For example, such antagonistic peptides can be produced and tested in any of the assays described herein for the ability to neutraize the mitogenic and/or chemotactic activity of the naturally occurring polypeptide. In one application, the FsF-1 polypeptides of the invention will be admixed with a pharmaceutically acceptable carrier substance, e.g., physiological saline, and administered to a mammal, e.g., a human, suffering from a wound or a burn. The particular mode of administration is preferably topical. The dosage of polypeptide will vary, depending on such factors as the type and severity of the lesion, but will generally be at a dosage sufficient to stimulate adequate fibroblast proliferation and chemotaxis to the lesion site. A typical doβage range would be 1 ng to 10 mg of the polypeptide, and treatment can be repeated aβ deemed necessary to promote healing.

In another application, the antibodies of the invention which have been characterized, according to the methods described herein, as being capable of neutralizing the activity of an FsF-1 polypeptide will be used to treat dieorders in which the reduction of FβF-1 levels are desirable, e.g., chronic inflammatory diseaseβ. In one example, the antibodies of the invention can be conjugated to any immunotoxin well known

to those skilled in the art, and used to target FsF-l producing cells. These antibodies will be formulated in a pharmaceutically acceptable carrier substance and administered, e.g. , intravenously, intramuscularly, orally, parenterally, transdermally, or topically. The particular mode will depend upon the condition being treated and the general status of the animal, and will be apparent to those skilled in the art. The dosage of the antibody will also vary, depending on such factors as the type and severity of the disease, but will generally be at a dosage sufficient to inhibit the formation of a serious fibrotic conditions. A typical dosage range would be l ng to 10 mg of the antibody per kg body weight, and can be repeated weekly or daily as deemed necessary.

The nucleic acids of the invention can also be used therapeutically. Oligonucleotides which are antisense to FβF-1 mRNA (or nucleic acid constructs which express RNA that iβ antiβenβe to FβF-1 mRNA) can be utilized aβ an antifibrotic therapy. The method would involve introduction of the antiβenβe oligonucleotide into lymphocytes in vivo. The antisense hybridizes with endogenous FsF-1 mRNA, interfering with translation of the protein, thereby reducing production of the polypeptide. Methods of antisense design and introduction into host cellβ are deβcribed, for example, in Weinberg et al., U.S. Patent No. 4,740,463. OTHER EMBODIMENTS Other embodiments are within the following claims. For example, the invention includes any protein which is substantially homologous to murine or human FsF-1 as well as other naturally occurring FsF-1 polypeptides. Also included are: allelic variations; natural mutants; induced mutants; chimeric polypeptides that include a FsF-1 polypeptide; proteins encoded by DNA that

hybrid!zee under high stringency conditions (e.g., see Current Protocols in Molecular Biology, supra to a naturally occurring nucleic acid encoding FsF-1; and polypeptides or proteins specifically bound by antisera to FβF-1, especially by antisera specific to the 2B3 fragment of FsF-1.

Preferred analogs of the invention include FsF-1 polypeptides (or biologically active fragments thereof) whose sequenceβ differ from the naturally occurring FsF-1 polypeptide by amino acid sequence differences or by modifications that do not affect sequence, or by both. Analogs of the invention will generally exhibit at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% or even 99%, similarity with all or part of a naturally occurring FsF- 1 polypeptide. The length of comparison sequences will generally be at least 20 amino acids residues, and preferably, more than 40 amino acid residues. Differences in amino acid sequence can be by conservative amino acid substitutions, for example substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more nonconservative amino acid substitutions, deletions, or insertions which do not destroy the analog's biological activity as measured by the assays described herein. Modifications include in vivo or in vitro chemical derivatization of polypeptides, e.g., acetylation or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its βyntheβiβ and processing or in further processing steps, e.g., by exposing the polypeptide to glycosylating enzymes from cells that normally provide such procesβing, e.g., mammalian glycosylation enzymes. Also embraced are versions of the same primary amino acid sequence that

have phosphorylated amino acid residues, e.g., phosphotyrosine, phoβphoserine, or phosphothreonine; and analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., β or γ amino acids. Preferred analogs also include FsF-1 (or biologically active fragments thereof) which are modified for the purpose of increasing peptide stability, e.g., one or more desaturated peptide bonds, or non-peptide bonds. Alternatively, increased stability can be conferred by cyclizing the peptide molecule.

While preferred embodiments have been illustrated and described, it is understood that the present invention is capable of variation and modification and, therefore, should not be limited to the precise details set forth, but should include such changes and alterations that fall within the purview of the following claims.

__________ Under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, deposit of has been with the American Type Culture Collection (ATCC) of Rockville, MD, USA, where the deposits were given Accession Numbers 69993, 69995 and 69994, deposited on February 21, 1996. These deposits included, respectively: (1) a plasmid (pCR3MFibrosin; in TOPIOF') containing an insert of approximately 0.5 kb of mouse fibrosin cDNA, (2) a plasmid (pcDSRα29b/2B3; in DH5α) containing an insert encoding the mouse 2B3 domain, and (3) a plasmid (pBKCMVHFibroβinl; in XLOLR) containing approximately 2.8 kb of human fibroβin cDNA.

Applicants' assignee. New England Medical Center Hospital, Inc. , represent that the ATCC is a depository

affording permanence of the deposit and ready accessibility thereto by the public if a patent iβ granted. ^' All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. §122. The deposited material will be maintained with all the care necesβary to keep it viable and uncontaminated for a period of at leaβt five years after the most recent request for the furnishing of a sample of the deposited plaβmid, and in any caβe, for a period of at leaβt thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicants' assignees acknowledge their duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.