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WO1996026953A1 - FsF-1 ET LA DETECTION PRECOCE DES FIBROSES - Google Patents

FsF-1 ET LA DETECTION PRECOCE DES FIBROSES Download PDF

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Publication number
WO1996026953A1
WO1996026953A1 PCT/US1996/002726 US9602726W WO9626953A1 WO 1996026953 A1 WO1996026953 A1 WO 1996026953A1 US 9602726 W US9602726 W US 9602726W WO 9626953 A1 WO9626953 A1 WO 9626953A1
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Prior art keywords
fsf
polypeptide
fibrosin
cells
human
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PCT/US1996/002726
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English (en)
Inventor
David J. Wyler
Sadhana Prakash
Xiaoping Zhang
Original Assignee
New England Medical Center Hospitals, Inc.
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Publication date
Application filed by New England Medical Center Hospitals, Inc. filed Critical New England Medical Center Hospitals, Inc.
Priority to AU51772/96A priority Critical patent/AU5177296A/en
Publication of WO1996026953A1 publication Critical patent/WO1996026953A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the field of the invention is lymphokines and fibrosis.
  • Schistosomiasis is one of the most prominent helminthic diseases, estimated to afflict 200 million people in the tropics (Walsh et al., 1979).
  • Two of the schistosome species that infect humans can cause serious morbidity (including portal hypertension and gastrointestinal hemorrhage) as a result of a form of hepatic fibrosis
  • infected individuals may aid the prevention of scarring.
  • anti-inflammatory or immunosuppressive drugs including ethotrexate, cytotoxins, and various corticosteriods may aid the prevention of scarring.
  • these drugs are known to produce a number of relatively severe side effects, treatment of all infected individuals, 94-97% of 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.
  • FsF-1 also referred to as fibrosin
  • 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-1 polypeptide.
  • This polypeptide may be encoded by mammalian DNA, such as that of a mouse or a human.
  • human FsF-1 is meant the polypeptide encoded by the DNA sequence of SEQ ID NO.:11.
  • murine FsF-1 is meant the polypeptide encoded by the DNA sequence of SEQ ID NO. :2.
  • polypeptide is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation) .
  • An FsF-1 polypeptide is preferably a polypeptide having one or more of the biological activities of human FsF-1.
  • 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.
  • 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-l 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.
  • 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 FsF-1 cDNA sequence of SEQ ID NO.:11 beginning a nucleotide 318 (codon AGG) .
  • the 2B3 domain has been further divided into the subdomains, 2B3a and 2B3b.
  • 2B3a subdomain is meant polypeptides 26-45 of SEQ ID N0.:1.
  • 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-1 that function as antagonists of the FsF-1 receptor.
  • the invention features isolated DNA consisting essentially of a DNA sequence encoding an FsF-1 polypeptide, or a fragment of the FsF-1 polypeptide such as the 2B3 domain, the 2B3a subdomain, or the 2B3b subdomain.
  • isolated 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.
  • 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.
  • 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 NO.:3) .
  • 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.
  • 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
  • essentially homogeneous population of cells is meant that at least 99% of the cells contain the vector of the invention (or the isolated DNA) .
  • 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 refers to the subunit sequence similarity between two molecules. When a subunit position in both of the molecules is occupied by the same onomeric 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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.
  • a further feature of the invention is a substantially pure antibody that specifically binds FsF- 1.
  • antibodies according to the invention may be prepared by a variety of methods.
  • the FsF-1 protein, or antigenic fragments thereof can be administered to an animal in order to induce the production of polyclonal antibodies.
  • 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., Eur. J. Immunol . 6:511, 1976; Kohler et al.. Bur J. Immunol .
  • the invention features antibodies which specifically bind a murine FsF-1 polypeptide, a human FsF-1 polypeptide, or another FsF-1 polypeptide.
  • the invention features "neutralizing” antibodies.
  • 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 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.
  • the invention features antibody fragments, including F(ab')2, Fab', Fab, Fv and sFv fragments.
  • substantially pure describes a compound, e.g., a protein, polypeptide, or antibody, that is substantially free from the components that naturally accompany it.
  • 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, polyacrylamide gel electrophoresis, or HPLC analysis.
  • the FsF-1 polypeptide may be used as the active ingredient of 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.
  • 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.
  • 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.
  • a chemical moiety which is capable of being detected, including, without limitation, radioactive isotopes, enzymes, luminescent agents, precipitating agents, and dyes.
  • compositions of the present invention can be assembled in a kit for the detection of FsF-1 polypeptides or mRNA.
  • 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 FsF-1.
  • 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.
  • the polypeptide includes a fragment of a naturally occurring FsF-1 polypeptide.
  • the polypeptide may be full length, may contain the 2B3 domain, or subdo ains 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.
  • the polypeptide includes the amino acid sequence depicted in Fig.
  • 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.
  • 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.
  • 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 that permit immunocomplex formation, and detecting an increase in the relative level of the immunocomplex as an indication of a propensity for pathological fibrosis.
  • 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.
  • 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 l 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.
  • 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) , pleural fluid, cerebral spinal fluid, and the aqueous humor; or from the supernatant of cultured peripheral blood mononuclear cells.
  • the sample is obtained from a mammal, and even more preferably, the mammal is a human.
  • the pathological scarring results from hepatic fibrosis.
  • hepatic fibrosis is the result of the disease schistoso iasis.
  • 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 sarcoidosis.
  • FsF-1 plays an important role in the progression of fibrotic pathogenesis in liver, and tissue fibrosis in other chronic inflammatory disease.
  • 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.
  • 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 who require antifibrotic and/or anthelminthic therapy.
  • 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.
  • 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) .
  • 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.
  • the electropherogram of proteins present in unfractionated granuloma supernatant is shown in lane 1. Note that the migration position of FsF-1 (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.
  • 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 ⁇ l to 5 ⁇ l and then probed with antibodies.
  • Figs. 7A and 7B are a pair of graphs.
  • 7A depicts a heparin-Sepharose eluate (FsF-l:10 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 ⁇ g/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-1: -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 endotheliai 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 IO 4 ; endotheliai cells, 6.1 ⁇ 0.1 x IO 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.
  • 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.
  • 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 ⁇ g/ml, lane 2; 15 ⁇ g/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 FsF-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-l IgG at 2.5 ⁇ g/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.
  • 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.
  • Fig. 17 is a graph that illustrates sib selection in the cloning of 2B3 cDNA that encodes a fibroblast itogen.
  • 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.
  • 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. 17 is a graph that illustrates sib selection in the cloning of 2B3 cDNA that encodes a fibroblast itogen.
  • 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 ⁇ M concentrations.
  • Fig. 20 is a graph depicting the fibroblast mitogenic activity of supernatants from cloned T cell hybridomas stimulated with 10 ⁇ g/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
  • Fig. 28 is a graph of fibroblast proliferative responses to fibrosin. Fibrosin was purified by immunoaffinity 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.
  • FIG. 29 is a bar graph of the results of fibrosin antigen-capture ELISA.
  • Wells were coated with rat anti- fibrosin 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) .
  • pre- immune rabbit IgG 0.5 ⁇ g/ml
  • r ⁇ F IgG 0.5 ⁇ g/ml
  • r ⁇ F IgG 0.5 ⁇ g/ml
  • r ⁇ F IgG 0.5 ⁇ g/ml
  • 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-,91 (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 fibroblasts in response to purified native fibrosin (panel B) ; collagen synthesis, as determined by the incorporation of [ 3 H]- proline into collagenase-sensitive 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) .
  • 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-1; pg/ml) in conditioned medium from cultured rheumatoid synoviocytes that were treated with phytohemaglutin (+PHA) , or were untreated (-PHA) .
  • FsF-1 fibrosin
  • Fig 35 is a bar graph of the concentration of fibrosin (FsF-1; pg/ml) in sera from Brazilian patients with Schistosomiasis mansoni (Schistosomiasis) , and in sera from normal, non-infected Brazilians (Control) .
  • FsF-1 fibrosin
  • 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 ethotrexate.
  • 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 scleroder a cells cultured in MEM Eagle's Medium with 10% FCS and stained with an antibody against type I collagen. The cells were treated with 20 ⁇ g/ml of random IgG (a) , 20 ⁇ g/ml anti-fibrosin antibody (one-day incubation;b) , or 20 ⁇ g/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.
  • conditioned medium from normal fibroblasts is added to the cultured cells prior to staining.
  • conditioned medium from sclerodermal fibroblasts is added to the cultured cells prior to staining.
  • 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
  • conditioned medium from normal fibroblasts is added to the cultured cells prior to staining.
  • conditioned medium from sclerodermal fibroblasts is added to the cultured cells prior to staining.
  • 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 are a series of photomicrographs of cultured sclerodermal cells stained with a monoclonal antibody against fibrosin.
  • scleroderma cells were treated with 20 ⁇ g/ml random IgG.
  • scleroderma cells were treated with 20 ⁇ g/ml anti-fibrosin antibody.
  • TGF£ antibodies (type 1, 2, and 3 and 2.5 ⁇ g/ml each, mixed) .
  • Fig. 42 is a bar graph depicting the response of fibroblasts to immunopurified human serum fibrosin.
  • Fig. 43 is a bar graph depicting the chemotactic response of monocytes to COS cells transfected with a plasmid bearing a 2.8 kb fragment of human fibrosin cDNA
  • mice 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 Guatemalan 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) .
  • HBSS cold Hanks' buffered salt solution
  • 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 ⁇ m diameter; Millipore Corporation, Bedford, MA) .
  • the supernatants were stored in aliquots at -20 or -70 ⁇ C.
  • 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.
  • granuloma cells were dissociated by collagenase treatment using methods as described in Wyler et al. , J, IimJH D l ⁇ 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 ⁇ g/ml of normal rabbit IgG or of rabbit anti-FsF-1 IgG, washed, and then treated with 10 ⁇ g/ml goat anti-rabbit IgG (H and L chain specific) antibody conjugated with fluorescein isothiocyanate (FITC; Fisher Scientific, Pittsburgh, PA) .
  • FITC fluorescein isothiocyanate
  • unstained or FITC-stained cells were treated with phycoerythrin-conjugated rat anti-mouse CD4 antibody (5 ⁇ g/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°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.
  • BSA bovine serum albumin
  • cells were suspended in serum-containing supplemented medium at a density of 5 to 6 x l0 /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 ⁇ l test samples were added to each well. Twenty hours later, 1 ⁇ Ci[ 3 H]-thymidine (specific activity 6.7 Ci/mM, Dupont- NEN Research Products, Boston, MA) was added to each well for 4 h.
  • 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 onolayer by treatment with trypsin-EDTA.
  • Monodispersed cells were counted in a hemocytometer chamber (Cambridge Instruments, Inc., Buffalo, NY).
  • 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 ⁇ m 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 centrifugation and dialyzed before testing in the biologic assays.
  • the heparin-Sepharose purified material was designated FsF-1.
  • FPLC A ion Exchange Chromatographv The fractions eluting from heparin Sepharose with 1.5 M NaCl were dialyzed (6 to 8 kDa cutoff) at 4°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, Anal. Biochem.. 117:307 (1981)).
  • 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) .
  • IFA incomplete Freund's adjuvant
  • 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., J. IfflPMn?”
  • 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 ⁇ l.
  • Bio-Rad nitrocellulose paper
  • 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 l h at room temperature in alkaline phosphatase-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.
  • substrate containing nitroblue tetrazolium chloride and 5-bromo-4 chloro-3imodyl phosphate, p-toluidine salt (Promega)
  • alkaline phosphatase buffer 100 mM Tris-HCl, 100 mM NaCl, 5 mM
  • 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 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., Biotechniques 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.
  • Biosynthetic Labeling of FsF-1 CD4 + cells from granulomas were cultured for 24 h in methionine and cysteine-free RPMI 1640 medium (Select mine»Kit, GIBCO, Grand Island, NY) to which was added 75 ⁇ Ci 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 ⁇ l of the supernatant was first incubated (1-2 hr; 4°C) with normal rabbit IgG (NRIgG; 5 or 15 ⁇ g in 200 ⁇ l) and the mixture was then incubated (1 hr; 4°C) with protein A-Sepharose (50-100 ⁇ l; Sigma) .
  • 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.
  • the heparin affinity of FsF-1 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)).
  • the granuloma-derived mitogen has pi-6.2 (Wyler et al., J. IlfflftV ⁇ Pl T 129:1706 (1982)), thus FsF-1 has a characteristic of class l (acidic) HBGF.
  • Anti-FsF-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.
  • anti-FsF-l 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.
  • HBGF HBGF-like growth factor-1
  • fibroblasts and endotheliai cells proliferate in response to acidic FGF. Inasmuch as the magnitude of the response of endotheliai 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.
  • FsF-1 in concentrations in the range of 1 to 40 ng/ml did not induce endotheliai cell proliferation (Fig. 8B) .
  • 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.
  • Fibroblast proliferate response ( 3 H-thymidine incorporation) to cluture supernatants of CD4 + lymphocytes from schistosomal egg granulomas Additive to 3 H-thymidine fifrrPblagt gu t ⁇ rg incorporation rcPMi 1
  • 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 affinity binding of these factors to heparin is uncharacteristic of most proteins (Lobb et al., supra) .
  • 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.
  • FsF-1 is heparin- binding
  • the granuloma-derived mitogen is a protein with pi -6.2.
  • 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 endotheliai cells.
  • FsF-1 probably is distinct from FGF and sensu stricto is not a class l HBGF. Supporting this conclusion are our observations that antibodies prepared against FsF-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., supra ) 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) .
  • fibroblast mitogenic activity can be detected in extracts of recently isolated egg granulomas (Wyler et al., J. 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., supra) .
  • unfractionated splenocytes and splenic CD4 + lymphocytes isolated by flow cytometry fail to spontaneously secrete fibroblast mitogens (Wyler et al.. Infect. lTHfflu.ni 38:103 (1982); and the present study).
  • 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., filiEEfl).
  • FsF-1 detected by immunoprecipitation corresponded to a prominent 60 kDa protein produced by isolated granuloma CD4 + lymphocytes (Fig. 5) .
  • Our results suggest that FsF-1 might be a major protein produced by this subpopulation of granuloma cells.
  • sensitized splenic lymphocytes from S. mansoni-infected mice when stimulated with an aqueous extract of schistosoma eggs secrete a fibroblast mitogen, presumably FsF-1 (Wyler et al., Infect. ⁇ n ⁇ n. 38:103 (1982)).
  • CD4 + cells are stimulated in vivo to produce FsF-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.
  • FsF-1 not only is 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-1 positive lymphocytes and fibroblasts might stimulate fibroblast growth.
  • cytokines IL-1, TNF, TGF3
  • IL-1, TNF, TGF3 cytokines
  • fibroblast growth factors some of which were purified from other sources and were recognized for this biological property (PDGF [Ross et al., ____! 46:155 (1986)], FGF [Gospodarowicz et al., J. Biol. Chem. 250:2515 (1975); Gospodarowicz et al., J. BiaL,.253:3736 (1978)]), and heparin-binding epidermal growth factor [HB-EGF Higashiyama et al.. Science 251:936 (1991)]) have been found to be produced by macrophages.
  • PDGF Ross et al., _____! 46:155 (1986)
  • FGF Gospodarowicz et al., J. Biol. Chem. 250:2515 (1975); Gospodarowicz et al., J. BiaL,.253:3736 (1978)]
  • FsF-1 is distinct from these fibroblast growth factors.
  • the fact that FsF-1 is a lymphokine is an additional distinguishing characteristic. Indeed, because we have not detected fibroblast mitogenic activity in several purified and recombinant lymphokines, and since avid heparin-binding is not a known characteristic of most lymphokines, we believe that FsF-1 is a previously unidentified lymphokine. DETECTION OF FsF-1 IN GRANULOMA SUPERNATANTS AND
  • FsF-1 can be detected in highly-dilute granuloma supernatant in an antigen-capture ELISA (Nourel, et al.
  • the assay apparently is sensitive to at least pg/ml concentrations, consistent with expectations based on similar antigen-capture assays for other lymphokines (Fig. 12) .
  • An antigen-capture ELISA was also used to detect FsF-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.
  • IgG Antibody Neutralization of Fibrogenic Activity
  • polyclonal IgG was prepared by immunizing rabbits with highly purified murine FsF-1, as 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 ⁇ g/100 ⁇ l) at 37 ⁇ C for 2-3 h in polypropylene culture tubes (Falcon #2063; Becton-Pickinson 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).
  • the beads were removed by centrifugation (lOOOg x 10 min.) The samples were then filter-sterilized (0.22 ⁇ m diameter pore size; Millipore Corp. , Bedford, MA) and tested in a fibroblast proliferation assay.
  • CD4 + lymphocyte clones and cell lines 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 extensively in HBSS and IO 6 cells were cultured in 1 ml serum-free medium supplemented with 0.3 mg/ml BSA and 10 ⁇ g/ml concanavalin A (Con A; Sigma Chemical Co.,St.
  • a murine cDNA library prepared from mRNA isolated from Con A-stimulated cells of the CDC25 line was obtained from DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA.
  • the library was constructed in the vector pcDSR ⁇ 296 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).
  • pcDRa296 allows a high level of expression of full-length cDNA inserts under the control of the simian virus 40 (SV40) early promoter.
  • SV40-derived DNA fragments are arrayed in these vectors to permit transcription, splicing, and polyadenylation of the cloned cDNA.
  • a DNA fragment containing both the SV40 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 transcripts) splicing occurs at the 16s RNA intron junction and places the cDNA initiator ATG codon first in line from the 5' end of the mRNA.
  • E.coli transformants containing the pcDSR ⁇ 296 vectors were expanded in L-broth medium containing ampicillin (50 ⁇ g/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) .
  • DMEM Dulbecco's modified Eagle's medium
  • FBS FBS
  • 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 ⁇ g) in serum-free medium (4 ml per plate) was added, followed by the addition of DEAE dextran (200 ⁇ g/ml; Pharmacia, Piscataway, NJ) .
  • COS-7 cells were washed and treated for 2-3 h with 100 ⁇ M 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.
  • COS-7 cells were transfected with the pcDSR ⁇ 296 plasmid containing a murine IL-4 cDNA insert (DNAX) , and were subsequently 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.
  • DNAX murine IL-4 cDNA insert
  • 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.
  • CDC25 Lvmphocvte Line Produces Fibroblast Mitogen Of the 30 lymphocyte lines and T cell hybridomas we tested, five elaborated detectable fibroblast mitogenic activity in culture supernatants following in vitro stimulation with Con A for 24 h. Con A had no intrinsic fibroblast mitogenic activity in our fibroblast assay.
  • 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 ⁇ g/100 ⁇ l and 5 ⁇ g/ ⁇ l IgG respectively (Fig. 14B) , relative to responses in the presence of the same concentrations of pre-immune IgG (NRIgG) which we previously established does not significantly alter the fibroblast responses to purified 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 rifrrpfrlapt grpwth Fact-pr
  • the CDC25 library was partitioned into pools of IO 3 clones that were seeded into separate wells of microtiter plates containing L-broth and ampicillin. Pools, estimated to contain IO 4 clones, were prepared by combining wells within a row. C0S-7 cells were transfected with plasmid DNA prepared from these pools and the transfectant culture supernatants (tested at various concentrations, to a maximum of 10%) were assayed for their ability to enhance fibroblast [ 3 H]-thymidine uptake at least 2-3 fold above background (positive transfectant) .
  • 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 IO 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.
  • 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.
  • ORF codes 71 amino acids and is followed by 11 untranslated codons that precede the poly(A) tail.
  • 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) .
  • peak mitogenic activity was observed with peptide concentrations between IO" 13 and 10" 11 M; additional activity was detected at concentrations in the 10 ⁇ 6 M range.
  • the dose-response pattern we observed is reminiscent of our experience with crude and purified natural FsF-1 as well as with supernatants of the 2B3 transfectant (Figs. 16A-B) .
  • 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 heterologous 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.
  • 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 cells were fused with thymoma BW 5147 cells using standard methods (Coligan et al., eds.. Current Protocols in Immunology, Vol. 1, John Wiley & Sons, Inc., pp. 3.14.1-3.14.11 (1994)).
  • hybridomas that on stimulation with Con A (in the absence of serum) produced fibroblast mitogenic activity, and confirmed by ELISA, neutralization, and immunopurification that this biological activity derived from FsF-1 in the supernatants. Positive hybridomas were subcloned 2-3 times 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) .
  • 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.
  • SEA (10 ⁇ g/ml; fraction that lacks direct mitogenic activity) stimulates hybridoma ⁇ B12 production of FsF-1. Response to various concentrations of cell culture supernatant are shown relative to CPM in fibroblasts grown in medium alone in Fig. 22.
  • the control was 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 CPM in fibroblast cultured with medium alone. (Fig. 24).
  • T-hybridomas produce the same FsF-1 that we previously purified from granuloma supernatants.
  • Tl C-TCA-CTA-AGC-CAG-AGG-CCA-AAG-TG (SEQ ID NO.
  • Bl A-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)
  • PCR primers T1/B3, T1/B2, and Tl/Bl
  • 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.
  • PBMC peripheral blood mononuclear cells
  • RACE PCR Frohman, in PCJR Protocol . A guide to methods and applications, p. 28- 38. Academic Press, NY 1990
  • RACE NO MORE PCR Weis, Nucleic Acid Res. 22:3427-3428, 1994
  • 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.
  • 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.
  • 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).
  • 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's of its poly-A tail of more than 50 A's.
  • T2 AAG-CCA-GGG-TTG-GAA-GGC-AAA-GGT-CAC-A
  • 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.
  • 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 expression vector.
  • a suitable host cell either prokaryotic or eukaryotic
  • 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.
  • 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-1 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.
  • 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.
  • Prokaryotes most frequently used are various strains E. coli j 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 are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences.
  • fibrosin production appears to be a risk factor for the development of pipe-stem fibrosis.
  • fibrosin from two sources of serum- free conditioned medium: (1) egg granulomas and (2) cloned T cell hybridomas stimulated with concanavalin A (Con A) .
  • 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 vol. I. New York, John Wiley & Sons, Inc. (1994)).
  • T cell hybridomas were initially selected in HAT medium (GIBCO/BRL
  • 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 (HiTrap" Heparin; Pharmacia Biotech, Piscataway, NJ) as described (Prakash et al., _ ⁇ _. Immunol. 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 dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Prakash et al., J. Immunol.
  • 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 ⁇ g protein in Freund's complete adjuvant) , followed by six ip booster injections of fibrosin (2 ⁇ g protein in Freund's incomplete adjuvant) at 2-week intervals.
  • 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.
  • MAR mouse anti-rat K
  • the MAR Model EL 308; Bio-Tek Instruments, 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 set of assays, and adjusted for dilution.
  • mice In studies of murine schistosomiasis, 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 as described above.
  • mice 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).
  • each of three separately prepared rat anti-fibrosin Mabs IP8/C9, IIIG6/C7, and IIIA5/P8 detects a single 60 kP 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 kP protein corresponds to that of fibrosin (Prakash et al. , J. Immunol.
  • mice Serum Levels of Fibrosin Peak at the Same Time the Granulomatous Inflammatory Response Peaks
  • 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 assayed simultaneously, and the results were confirmed in separate additional assays of selected samples.
  • plasma fibrosin concentrations were in the normal range.
  • fibrosin levels were above normal levels in almost all the mice.
  • Fibrosin production by granuloma CP4 + lymphocytes is likely to be stimulated by egg antigens, and CD4 + lymphocyte hypersensitivity to these antigens is most vigorous at week 8 of infection. The hypersensitivity wanes thereafter and is considerably depressed at week 12.
  • 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 described 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 COS cells transfected with human FsF-1 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-sensitive protein in fibroblast culture supernatants using a published microassay method (Diegelmann et al.. Anal. Biochem. (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 fibroblast-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.
  • DMEM Dulbecco's modified Eagle's medium
  • Fibronectin and hyaluronan concentrations in serum-free, fibroblast-conditioned medium were determined by published methods. For these experiments, IO 5 fibroblasts suspended in DMEM-10 were seeded into each 16 mm diameter well of 24-well tissue culture plates and incubated at 37°C in 5% C0 2 -95% air atmosphere 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.
  • Mab recognizes an epitope within the 2B3 peptide that is also expressed in native fibrosin. Fibrosin has Chemotactic Properties that are
  • Purified native fibrosin and 2B3 peptide are both chemotactic agents to fibroblasts when assessed 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.
  • 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 ⁇ M range) .
  • a plasmid bearing a large fragment of human fibrosin cDNA (pBKCMVHFibrosinl) was purified from liquid cultures (QIAGEN plasmid preps) 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 for chemotaxis was carried out in Boyden chambers, which consist of lucite blocks that are separated into two chambers by a polycarbonate filter with 5 ⁇ m pores.
  • Human peripheral monocytes were placed in one chambers 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 (Fig. 43) were expressed as the average number of cells per high power field (HPF) . Cells transfected with the plasmid bearing the human FsF-1 encoding insert stimulated the chemotactic movement of monocytes.
  • Fibronectin and hyaluronan are two matrix components that frequently are produced early in the course of fibrogenesis. Fibrosin stimulates fibroblasts to produce both of these macromolecules (Fig. 32, panel A and Fig. 32, panel B) . Since the addition of cycloheximide blocks the fibrosin-enhanced elaboration of fibronectin, this response depends on de novo protein synthesis rather than simply release of pre-formed fibronectin. Furthermore, the 2B3 peptide stimulates fibronectin synthesis and antifibrosin Mab blocks the stimulatory effect of the truncated peptide and native fibrosin (Fig. 35 panel C) .
  • Fibrosin (optimal concentration 20 ⁇ g/ml) stimulates, in contact-inhibited fibroblast cultures, a net increase in the incorporation of 3 H-proline into collagenase-sensitive secreted protein (Table 3) .
  • fibrosin Although fibrosin also increases total protein synthesis (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) . fibrosin
  • 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.
  • N 3.
  • Hydroxyproline concentration (pM/ ⁇ g DNA) in fibroblast culture supernatants, expressed as percentage of concentration in supernatants of unstimulated (medium only) cultures.
  • Ratio of hydroxyproline to proline concentration in supernatants of stimulated fibroblasts expressed as a percent of this concentration ratio of unstimulated cultures.
  • Ratio of hydroxyproline to leucine concentration in supernatants of stimulated fibroblasts expressed as a percent of this concentration ratio for umstimulated cultures.
  • 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 schistosomiasis.
  • 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 described above, were incorporated into hydron pellets and implanted into the corneal stroma of rats. Induction of neovacsularization from the limbal vasculature was monitored for 7 days following implantation. Corneas were examined histologically for non-specific inflammation. Purified native fibrosin induced an angiogenic response in rat corneas without inciting an inflammatory reaction. The minimum amount of fibrosin required to elicit an angiogenic response (5 ng/implant) is comparable to the amount of bFGF, VEGF, or TNF ⁇ required to induce similar responses.
  • the 2B3 peptide (maximum amount tested 2.5 ng) also elicited a distinct, inflammation-free angiogenic response.
  • 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 responses, as revealed by experiemnts with the 2B3 peptide.
  • the fibrogenic and angiogenic properties of fibrosin could account for the histopathy of liver fibrosis in experimental schistosomiasis.
  • fibrosin could also play a role in formation of granulation tissue during would healing.
  • FIBROSIN IS OVERPRODUCED BY SMOOTH MUSCLE CELLS IN THE BOWEL OF A CROHN'S DISEASE PATIENT
  • FIBROSIN IS ABUNDANTLY PRODUCED BY RHEUMATOID SYNOVIOCYTE CULTURES
  • 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
  • Fig. 38 is a series of photomicrographs of scleroderma cells cultured in MEM Eagle's Medium with 10% FCS. These experiments examined the effect of an antifibrosin antibody on the production of collagen by scleroderma cells.
  • the cultured scleroderma cells were treated with 20 ⁇ g/ml of random IgG, or 20 ⁇ g/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.
  • 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 similar 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • a pharmaceutically acceptable carrier substance e.g., physiological saline
  • 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 dosage range would be 1 ng to 10 mg of the polypeptide, and treatment can be repeated as deemed necessary to promote healing.
  • the nucleic acids of the invention can be used therapeutically.
  • Oligonucleotides which are antisense to FsF-1 mRNA can be utilized as an antifibrotic therapy.
  • the method would involve introduction of the antisense 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 cells are described, for example, in Weinberg et al., U.S. Patent No. 4,740,463. OTHER EMBODIMENTS
  • 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 hybridizes 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 FsF-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 sequences 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.
  • glycosylation e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to glycosylating enzymes from cells that normally provide such processing, e.g., mammalian glycosylation enzymes.
  • phosphorylated amino acid residues e.g., phosphotyrosine, phosphoserine, or phosphothreonine
  • 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., ⁇ or ⁇ 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.
  • plasmids pCR3MFibrosin; in TOPIOF'
  • plasmid pcDSR ⁇ 29b/2B3; in DH5 ⁇
  • plasmid pBKCMVHFibrosinl; in XLOLR
  • the deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited plasmid, and in any case, for a period of at least 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.

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Abstract

Procédé permettant de repérer les sujets ayant une propension à la fibrose pathologique. Dans ce procédé, on prélève un échantillon chez un sujet présentant une maladie inflammatoire chronique et on met l'échantillon en contact avec un anticorps spécifique du facteur de stimulation des fibroblastes 1 (FsF-1), dans des conditions permettant la formation d'un immunocomplexe; toute augmentation dans le niveau relatif de l'immunocomplexe indique une propension à la fibrose pathologique. L'invention porte également sur les polypeptides FsF-1 et les anticorps spécifiques de ces FsF-1, ainsi que sur les séquences d'ADN codant ces polypeptides FsF-1.
PCT/US1996/002726 1995-02-28 1996-02-28 FsF-1 ET LA DETECTION PRECOCE DES FIBROSES WO1996026953A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2302359A1 (fr) 2009-09-24 2011-03-30 Université De Reims Champagne-Ardenne Spectroscopie de sérum en infrarouge pour l'évaluation non invasive de fibrose hépatique chez des patients souffrant d'une maladie chronique du foie
WO2011036267A2 (fr) 2009-09-24 2011-03-31 Universite De Reims Champagne Ardenne (U.R.C.A.) Spectroscopie infrarouge de sérum pour évaluation non invasive de fibrose hépatique chez des patients atteints de maladies chroniques du foie

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