Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/02—Nasal agents, e.g. decongestants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
  • C12Y301/01047—1-Alkyl-2-acetylglycerophosphocholine esterase (3.1.1.47), i.e. platelet-activating factor acetylhydrolase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates generally to platelet-activating factor acetylhydrolase and more specifically to novel purified and isolated polynucleotides encoding human plasma platelet-activating factor acetylhydrolase, to the platelet- activating factor acetylhydrolase products encoded by the polynucleotides, to materials and methods for the recombinant production of platelet-activating factor acetylhydrolase products and to antibody substances specific for platelet-activating factor acetylhydrolase.
• Platelet-activating factor is a biologically active phospholipid synthesized by various cell types. In vivo and at normal concentrations of 10 "1 to 10 M, PAF activates target cells such as platelets and neutrophils by binding to specific G protein-coupled cell surface receptors [Venable et al. , J. Lipid Res. , 34: 691-701 (1993)]. PAF has the structure l-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine. For optimal biological activity, the sn-1 position of the PAF glycerol backbone must be in an ether linkage with a fatty alcohol and the sn-3 position must have a phosphocholine head group.
• PAF functions in normal physiological processes (e.g. , inflammation, hemostasis and parturition) and is implicated in pathological inflammatory responses
• PAF- AH PAF acetylhydrolase
• PAF- AH PAF acetylhydrolase
• PAF- AH also inactivates oxidatively fragmented phospholipids such as products of the arachidonic acid cascade that mediate inflammation. See, Stremler et al, J. Biol. Chem. , 266(11): 11095-
• PAF-AH The inactivation of PAF by PAF- AH occurs primarily by hydrolysis of the PAF sn-2 acetyl group and PAF-AH metabolizes oxidatively fragmented phospholipids by removing sn-2 acyl groups.
• Two types of PAF-AH have been identified: cytoplasmic forms found in a variety of cell types and tissues such as endothelial cells and erythrocytes, and an extracellular form found in plasma and serum.
• Plasma PAF-AH does not hydrolyze intact phospholipids except for PAF and this substrate specificity allows the enzyme to circulate in vivo in a fully active state without adverse effects.
• the plasma PAF-AH appears to account for all of the PAF degradation in human blood ex vivo [Stafforini et al , J. Biol. Chem. , 262(9): 4223- 4230 (1987)].
• plasma PAF-AH has biochemical characteristics which distinguish it from cytoplasmic PAF-AH and from other characterized lipases. Specifically, plasma PAF-AH is associated with lipoprotein particles, is inhibited by diisopropyl fluorophosphate, is not affected by calcium ions, is relatively insensitive to proteolysis, and has an apparent molecular weight of 43,000 daltons. See, Stafforini et al (1987), supra. The same Stafforini et al. article describes a procedure for partial purification of PAF-AH from human plasma and the amino acid composition of the plasma material obtained by use of the procedure.
• Cytoplasmic PAF-AH has been purified from erythrocytes as reported in Stafforini et al. , J. Biol. Chem., 268(6): 3857-3865 (1993) and ten amino terminal residues of cytoplasmic PAF-AH are also described in the article. Hattori et al, J. Biol Chem. , 268(25):
• 18748-18753 (1993) describes the purification of cytoplasmic PAF-AH from bovine brain. Subsequent to filing of the parent application hereto the nucleotide sequence of bovine brain cytoplasmic PAF-AH was published in Hattori et al , J. Biol. Chem. , 269(231): 23150-23155 (1994). On January 5, 1995, three months after the filing date of the parent application hereto, a nucleotide sequence for a lipoprotein associated phospholipase A2 (Lp-PlJV)) was published in Smithkline Beecham PLC Patent Cooperation Treaty (PCT) International Publication No. WO 95/00649.
• Lp-PlJV lipoprotein associated phospholipase A2
• the nucleotide sequence of the Lp-PLA2 differs at one position when compared to the nucleotide sequence of the PAF-AH of the present invention.
• the nucleotide difference results in an amino acid difference between the enzymes encoded by the polynucleotides.
• the amino acid at position 379 of SEQ ID NO: 8 is a valine while the amino acid at the corresponding position in Lp-PLA2 is an alanine.
• the nucleotide sequence of the PAF- AH of the present invention includes 124 bases at the 5' end and twenty bases at the 3' end not present in the Lp-PLA2 sequence.
• Lp-PLA2 sequence was deposited in GenBank under Accession No. U24577 which differs at eleven positions when compared to the nucleotide sequence of the PAF-AH of the present invention.
• the nucleotide differences results in four amino acid differences between the enzymes encoded by the polynucleotides.
• the amino acids at positions 249, 250, 274 and 389 of SEQ ID NO: 8 are lysine, aspartic acid, phenylalanine and leucine, respectively, while the respective amino acid at the corresponding positions in the GenBank sequence are isoleucine, arginine, leucine and serine.
• the recombinant production of PAF-AH would make possible the use of exogenous PAF-AH to mimic or augment normal processes of resolution of inflammation in vivo.
• the administration of PAF-AH would provide a physiological advantage over administration of PAF receptor antagonists because PAF-AH is a product normally found in plasma.
• PAF receptor antagonists which are structurally related to PAF inhibit native PAF-AH activity, the desirable metabolism of PAF and of oxidatively fragmented phospholipids is thereby prevented.
• the inhibition of PAF-AH activity by PAF receptor antagonists counteracts the competitive blockade of the PAF receptor by the antagonists. See, Stremler et al. , supra.
• the release of oxidants results in inactivation of the native PAF-AH enzyme in turn resulting in elevated local levels of PAF and PAF-like compounds which would compete with any exogenously administed PAF receptor antagonist for binding to the PAF receptor.
• treatment with recombinant PAF-AH would augment endogenous PAF- AH activity and compensate for any inactivated endogenous enzyme.
• the present invention provides novel purified and isolated polynucleotides (i.e. , DNA and RNA both sense and antisense strands) encoding human plasma PAF-AH or enzymatically active fragments thereof.
• Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences.
• the DNA sequence encoding PAF-AH that is set out in SEQ ID NO: 7 and DNA sequences which hybridize to the noncoding strand thereof under standard stringent conditions or which would hybridize but for the redundancy of the genetic code, are contemplated by the invention.
• Autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating PAF-AH sequences and especially vectors wherein DNA encoding PAF-AH is operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided.
• procaryotic or eucaryotic host cells are stably transformed with DNA sequences of the invention in a manner allowing the desired PAF-AH to be expressed therein.
• Host cells expressing PAF- AH products can serve a variety of useful purposes.
• Host cells of the invention are conspicuously useful in methods for the large scale production of PAF-AH wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by, for example, immunoaffinity purification.
• a non-immunological method contemplated by the invention for purifying PAF-AH from plasma includes the following steps: (a) isolating low density lipoprotein particles; (b) solubilizing said low density lipoprotein particles in a buffer comprising lOmM CHAPS to generate a first PAF-AH enzyme solution; (c) applying said first PAF-AH enzyme solution to a DEAE anion exchange column; (d) washing said DEAE anion exchange column using an approximately pH 7.5 buffer comprising ImM CHAPS; (e) eluting PAF-AH enzyme from said DEAE anion exchange column in fractions using approximately pH 7.5 buffers comprising a gradient of 0 to 0.5 M NaCl; (f) pooling fractions eluted from said DEAE anion exchange column having PAF-AH enzymatic activity; (g) adjusting said pooled, active fractions from said DEAE anion exchange column to lOmM CHAPS to generate a second PAF-AH enzyme solution; (h) applying said second PAF-
• the buffer of step (b) is 25 mM Tris-HCl, lOmM CHAPS, pH 7.5; the buffer of step (d) is 25 mM Tris-HCl, ImM CHAPS; the column of step (h) is a Blue Sepharose Fast Flow column; the buffer of step (i) is 25mM Tris-HCl, lOmM CHAPS, 0.5M KSCN, pH 7.5; the column of step (j) is a Cu Chelating Sepharose column; and the buffer of step (k) is 25 mM Tris-HCl, lOmM CHAPS, 0.5M NaCl, 50mM imidazole at a pH in a range of about pH 7.5-8.0.
• a method contemplated by the invention for purifying enzymatically- active PAF-AH from E. coli producing PAF-AH includes the steps of: (a) preparing a centrifugation supernatant from lysed E. coli producing PAF-AH enzyme; (b) applying said centrifugation supernatant to a blue dye ligand affinity column; (c) eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer comprising lOmM CHAPS and a chaotropic salt; (d) applying said eluate from said blue dye ligand affinity column to a Cu ligand affinity column; and (e) eluting PAF-
• the column of step (b) is a Blue Sepharose Fast Flow column
• the buffer of step (c) is 25mM Tris-HCl, lOmM CHAPS, 0.5M KSCN, pH 7.5
• the column of step (d) is a Cu Chelating Sepharose column
• the buffer of step (e) is 25mM Tris-HCl, lOmM CHAPS, 0.5M NaCl, lOOmM imidazole, pH 7.5.
• Another method contemplated by the invention for purifying enzymatically-active PAF-AH from E. coli producing PAF-AH includes the steps of: (a) preparing a centrifugation supernatant from lysed E. coli producing PAF-AH enzyme; (b) diluting said centrifugation supernatant in a low pH buffer comprising lOmM CHAPS; (c) applying said diluted centrifugation supernatant to a cation exchange column equilibrated at about pH 7.5; (d) eluting PAF-AH enzyme from said cation exchange column using 1M salt; (e) raising the pH of said eluate from said cation exhange column and adjusting the salt concentration of said eluate to about 0.5M salt; (f) applying said adjusted eluate from said cation exchange column to a blue dye ligand affinity column; (g) eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer comprising about 2M to about 3
• the buffer of step (b) is 25mM MES, lOmM CHAPS, ImM EDTA, pH 4.9;
• the column of step (c) is an S sepharose column equilibrated in 25mM MES, lOmM CHAPS, ImM EDTA, 50mM NaCl, pH 5.5;
• PAF-AH is eluted in step (d) using ImM NaCl;
• the pH of the eluate in step (e) is adjusted to pH 7.5 using 2M Tris base;
• the column in step (f) is a sepharose column;
• the buffer in step (g) is 25mM Tris, lOmM CHAPS, 3M NaCl, ImM EDTA, pH 7.5;
• the buffer in step (h) is 25mM Tris, 0.5M NaCl, 0.1 % Tween 80, pH 7.5.
• Still another method contemplated by the invention for purifying enzymatically-active PAF-AH from E.coli includes the steps of: (a) preparing an E.coli extract which yields solubilized PAF-AH supernatant after lysis in a buffer containing CHAPS; (b) dilution of said supernatant and application to a anion exchange column equilibrated at about pH 8.0; (c) eluting PAF-AH enzyme from said anion exchange column; (d) applying said adjusted eluate from said anion exchange column to a blue dye ligand affinity column; (e) eluting the said blue dye ligand affinity column using a buffer comprising 3.0M salt; (f) dilution of the blue dye eluate into a suitable buffer for performing hydroxylapatite chromatography; (g) performing hydroxylapatite chromatography where washing and elution is accomplished using buffers (with or without CHAPS); (h) diluting said hydroxylapatite eluate to
• the lysis buffer is 25 mM Tris, lOOmM NaCl, ImM EDTA, 20mM CHAPS, pH 8.0; in step (b) the dilution of the supernatant for anion exchange chromatography is 3-4 fold into 25 mM Tris, ImM EDTA, lOmM CHAPS, pH 8.0 and the column is a Q-Sepharose column equilibrated with 25mM Tris, ImM EDTA, 50mM NaCl, lOmM CHAPS, pH 8.0; in step (c) the anion exchange column is eluted using 25mM Tris, ImM EDTA, 350mM NaCl, lOmM CHAPS, pH 8.0; in step (d) the eluate from step (c) is applied directly onto a blue dye affinity column; in step (e) the column is eluted with 3M NaCl, lOmM CHAPS, 25mM Tris, pH 8.0; in step (b) the
• suitable formulation buffers for use in step (1) which stabilize PAF-AH include 50mM potassium phosphate, 12.5mM Aspartic acid, 125mM NaCl pH 7.4 (approximately, with and without the addition of Tween-80 and or Pluronic F68) or 25mM potassium phosphate buffer containing (at least) 125mM NaCl, 25mM arginine and 0.01 % Tween-80 (with or without Pluronic F68 at approximately 0.1 and 0.5 %).
• Yet another method contemplated by the invention for purifying enzymatically active rPAF-AH products from E. coli includes the steps of: (a) preparing an E. coli extract which yields solubilized rPAF-AH product supernatant after lysis in a buffer containing Triton X-100, (b) dilution of said supernatant and application to an immobilized metal affinity exchange column equilibrated at about pH 8.0; (c) eluting rPAF-AH product from said immobilized metal affimty exchange column with a buffer comprising imidazole; (d) adjusting the salt concentration and applying said eluate from said immobilized metal affinity column to an hydrophobic interaction column (HIC#1); (e) eluting said HIC#1 by reducing the salt concentration and/or increasing the detergent concentration; (f) titrating said HIC#1 eluate to a pH of about 6.4; (g) applying said adjusted HIC#1 eluate to
• step (a) adjusting said CEX#1 eluate with sodium chloride to a concentration of about 2.0M; (j) applying said adjusted CEX#1 eluate to a hydrophobic interaction column (HIC#2) equilibrated at about pH 8.0 and about 2.0M sodium chloride; (k) eluting said HIC#2 hy reducing the salt concentration and/or increasing the detergent concentration; (1) diluting said HIC#2 eluate and adjusting to a pH of about 6.0; (m) applying said adjusted HIC#2 eluate to a cation exchange column (CEX#2) equilibrated at about pH 6.0; (n) eluting the rPAF-AH product from said CEX#2 with a suitable formulation buffer.
• the lysis buffer is 90mM TRIS, 0.125 %
• Triton X-100, 0.6M NaCl, pH 8.0, and lysis is carried out in a high pressure homogenizer; in step (b) the supernatant is diluted into equilibration buffer (20mM TRIS, 0.5M NaCl, 0.1 % Triton X-100, pH 8.0), a zinc chelate column (Chelating Sepharose Fast Flow, Pharmacia, Uppsala, Sweden) is charged, equilibrated with equilibration buffer, loaded with the diluted supernatant, and washed with 20mM
• TRIS 0.5M NaCl, 4M urea, 0.1 % Triton X-100, pH 8.0, followed by washing with 20mM TRIS, 0.5M NaCl, 0.02% Triton X-100, pH 8.0; in step (c) elution is accomplished with 20mM Tris, 50mM imidazole, 0.02% Triton X-100, pH 8.0; in step (d) the eluate is adjusted to ImM EDTA and 2M NaCl, a Phenyl Sepharose 6 Fast Flow (Pharmacia) is equilibrated with equilibration buffer (2.0M NaCl, 25mM
• Tris 0.02% Triton X-100, pH 8.0
• step (e) elution is accomplished with 25mM NaPO ⁇ 3 % Triton X-100, pH 6.5
• step (g) a Macro- Prep High S Column (Bio-Rad Labs, Richmond, CA) is equilibrated with equilibration buffer (20mM NaPO 4 , 0.02 % Triton X-100, pH 6.4), loaded with the adjusted eluate from step (f), washed with equilibration buffer, and washed with 25mM Tris, 0.02% Triton X-100, pH 8.0; in step (h) elution is accomplished with 25mM Tris, 0.02% Triton X-
• step (k) elution is accomplished with lOmM Tris, 3.0% Triton X-100, pH 8.0; in step (1) dilution is into equilibration buffer (20mM succinate, 0.1 % PLURONIC F68, pH 6.0); in step (m) a SP Sepharose Fast Flow (Pharmacia) column is equilibrated with the equilibration buffer of step (1), loaded with eluate from step (1), and washed with equilibration buffer; and in step (n) elution is accomplished with 50mM NaPO 4 , 0.7M NaCl, 0.1 % PLURONIC F68,
• PAF-AH products may be obtained as isolates from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving procaryotic or eucaryotic host cells of the invention.
• PAF-AH products having part or all of the amino acid sequence set out in SEQ ID NO: 8 are contemplated. Specifically contemplated are fragments lacking up to the first twelve N-terminal amino acids of the mature human PAF-AH amino acid sequence set out in SEQ ID NO: 8, particularly those having Met 46 , Ala 47 or Ala 4 g of SEQ ID NO: 8 as the initial N-terminal amino acid.
• fragments thereof lacking up to thirty C-terminal amino acids of the amino acid sequence of SEQ ID NO: 8, particularly those having He 4 29 and Leu ⁇ j as the C-terminal residue.
• polynucleotides including DNA encoding such fragments or variant fragments are provided by the invention, as well as methods of recombinantly producing such fragments or variants by growing host cells comprising such DNA.
• Presently preferred PAF-AH products include the prokaryotic polypeptide expression products of DNA encoding amino acid residues et 4 g through Asn 44j of SEQ ID NO: 8, designated rPH.2, and the prokaryotic polypeptide expression products of DNA encoding amino acid residues Met 4 g through He 4 29 °f SEQ ID NO: 8, designated rPH.9.
• Both the rPH.2 and rPH.9 products display less amino-terminal heterogeneity than, for example, the corresponding prokaryotic expression products of DNA encoding the full mature sequence of PAF-AH preceded by a translation initiation codon.
• the rPH.9 product displays greater carboxy terminal homogeneity (consistency).
• PAF-AH products of the invention may be full length polypeptides, fragments or variants.
• Variants may comprise PAF-AH analogs wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced or wherein one or more nonspecified amino acids are added: (1) without loss of one or more of the enzymatic activities or immunological characteristics specific to PAF-AH; or (2) with specific disablement of a particular biological activity of PAF-AH. Proteins or other molecules that bind to PAF-AH may be used to modulate its activity.
• antibody substances e.g. , monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like
• other binding proteins specific for PAF-AH are also comprehended by the present invention.
• binding proteins of the invention are the monoclonal antibodies produced by hybridomas 90G11D and 90F2D which were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on September 30, 1994 and were respectively assigned Accession Nos. HB 11724 and HB 11725.
• monoclonal antibody produced by hybridoma 143 A which was deposited with the ATCC on June 1 , 1995 and assigned Accession No. HB 11900.
• Proteins or other molecules which specifically bind to PAF-AH can be identified using PAF-AH isolated from plasma, recombinant PAF- AH, PAF-AH variants or cells expressing such products. Binding proteins are useful, in turn, in compositions for immunization as well as for purifying PAF-AH, and are useful for detection or quantification of PAF-AH in fluid and tissue samples by known immunological procedures. Anti-idiotypic antibodies specific for PAF-AH- specific antibody substances are also contemplated. The scientific value of the information contributed through the disclosures of DNA and amino acid sequences of the present invention is manifest.
• DNA sequence information provided by the present invention also makes possible the development, by homologous recombination or "knockout" strategies [see, e.g. , Kapecchi, Science, 244: 1288-1292
• Polynucleotides of the invention when suitably labelled are useful in hybridization assays to detect the capacity of cells to synthesize PAF- AH. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in the PAF-AH locus that underlies a disease state or states. Also made available by the invention are anti-sense polynucleotides relevant to regulating expression of PAF-AH by those cells which ordinarily express the same.
• PAF-AH preparations of the invention to mammalian subjects, especially humans, for the purpose of ameliorating pathological inflammatory conditions is contemplated. Based on implication of the involvement of PAF in pathological inflammatory conditions, the administration of PAF-AH is indicated, for example, in treatment of asthma [Miwa et al , J. Clin. Invest., 82: 1983-1991 (1988); Hsieh et al , J. Allergy Clin.
• mice for many of the foregoing pathological conditions have been described in the art.
• a mouse model for asthma and rhinitis is described in Example 16 herein;
• a rabbit model for arthritis is described in Zarco et at., supra;
• rat models for ischemic bowel necrosis/necrotizing enterocolitis are described in Furukawa et al, Ped.
• a rabbit model for stroke is described in Lindsberg et al , (1990), supra;
• a mouse model for lupus is described in Matsuzaki et al , supra;
• a rat model for acute pancreatitis is described in Kald et al, supra:
• a rat model for pulmonary edema resulting from IL-2 therapy is described in Rabinovici et al. , supra;
• a rat model of allergic inflammation is described in Watanabe et al. , supra);
• a canine model of renal allograft is described in Watson et al.
• PAF-AH compositions for use in methods for treating a mammal susceptible to or suffering from PAF- mediated pathological conditions comprising administering PAF-AH to the mammal in an amount sufficient to supplement endogenous PAF-AH activity and to inactivate pathological amounts of PAF in the mammal.
• Therapeutic/pharmaceutical compositions contemplated by the invention include PAF-AH products and a physiologically acceptable diluent or carrier and may also include other agents having anti-inflammatory effects. Dosage amounts indicated would be sufficient to supplement endogenous PAF-AH activity and to inactivate pathological amounts of PAF. For general dosage considerations see Remmington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA (1990). Dosages will vary between about 0.1 to about 1000 ⁇ g PAF-AH/kg body weight.
• compositions of the invention may be administered by various routes depending on the pathological condition to be treated.
• administration may be by intraveneous, subcutaneous, oral, suppository, and/or pulmonary routes.
• administration of PAF-AH by the pulmonary route is particularly indicated.
• Contemplated for use in pulmonary administration are a wide range of delivery devices including, for example, nebulizers, metered dose inhalers, and powder inhalers, which are standard in the art. Delivery of various proteins to the lungs and circulatory system by inhalation of aerosol formulations has been described in Adjei et al , Pharm. Res. , 7(6): 565-569 (1990) (leuprolide acetate); Braquet et al. , J. Cardio. Pharm., 73(Supp. 5): s. 143-
• FIGURE 1 is a photograph of a PVDF membrane containing PAF-AH purified from human plasma
• FIGURE 2 is a graph showing the enzymatic activity of recombinant human plasma PAF-AH
• FIGURE 3 is a schematic drawing depicting recombinant PAF-AH fragments and their catalytic activity
• FIGURE 4 depicts mass spectroscopy results for a recombinant PAF- AH product, rPH.2.
• FIGURE 5 depicts mass spectroscopy results for a recombinant PAF- AH product, rPH.9.
• FIGURE 6 is a bar graph illustrating blockage of PAF-induced rat foot edema by locally administered recombinant PAF-AH of the invention;
• FIGURE 7 is a bar graph illustrating blockage of PAF-induced rat foot edema by intravenously administered PAF-AH;
• FIGURE 8 is a bar graph showing that PAF-AH blocks PAF-induced edema but not zymosan A-induced edema;
• FIGURES 9A and 9B present dose response results of PAF-AH anti- inflammatory activity in rat food edema
• FIGURES 10A and 10B present results indicating the in vivo efficacy of a single dose of PAF-AH over time;
• FIGURE 11 is a line graph representing the pharmacokinetics of PAF-
• FIGURE 12 is a bar graph showing the anti-inflammatory effects of PAF-AH in comparison to the lesser effects of PAF antagonists in rat foot edema.
• FIGURE 13 presents results indicating that PAF-AH neutralizes the apoptotic effects of conditioned media from HIV- 1 -infected and activated monocytes.
• Example 1 presents a novel method for the purification of PAF-AH from human plasma.
• Example 2 describes amino acid microsequencing of the purified human plasma PAF-AH. The cloning of a full length cDNA encoding human plasma PAF-AH is described in
• Example 3 Identification of a putative splice variant of the human plasma PAF-AH gene is described in Example 4. The cloning of genomic sequences encoding human plasma PAF-AH is described in Example 5.
• Example 6 desribes the cloning of canine, murine, bovine, chicken, rodent and macaque cDNAs homologous to the human plasma PAF-AH cDNA.
• Example 7 presents the results of an assay evidencing the enzymatic activity of recombinant PAF-AH transiently expressed in COS 7 cells.
• Example 8 describes the expression of full length, truncated and chimeric human PAF-AH DNAs in E. coli, S. cerevisiae and mammalian cells.
• Example 9 presents protocols for purification of recombinant PAF-AH from E. coli and assays confirming its enzymatic activity.
• Example 10 describes various recombinant PAF-AH products including amino acid substitution analogs and amino and carboxy-truncated products, and describes experiments demonstrating that native PAF-AH isolated from plasma is glycosylated. Results of a Northern blot assay for expression of human plasma PAF-AH RNA in various tissues and cell lines are presented in Example 11 while results of in situ hybridization are presented in Example 12.
• Example 13 describes the development of monoclonal and polyclonal antibodies specific for human plasma PAF-AH.
• Examples 14, 15, 16, 17, 18 and 19 respectively describe the in vivo therapeutic effect of administration of recombinant PAF-AH products of the invention on acute inflammation, pleurisy, asthma, necrotizing enterocolitis, adult respiratory distress syndrome and pancreatitis in animal models.
• Example 20 describes the in vitro effect of recombinant PAF-AH product on neurotoxicity associated with HIV infection.
• Example 21 presents the results of immunoassays of serum of human patients exhibiting a deficiency in PAF- AH activity and describes the identification of a genetic lesion in the patients which is apparently responsible for the deficiency.
• PAF-AH was purified from human plasma in order to provide material for amino acid sequencing.
• LDL low density lipoprotein
• Tween 20 low density lipoprotein (LDL) particles were precipitated from plasma with phosphotungstate and solubilized in 0.1 % Tween 20 and subjected to chromatography on a DEAE column (Pharmacia, Uppsala, Sweden) according to the method of Stafforini et al. (1987), supra, but inconsistent elution of PAF-AH activity from the DEAE column required reevaluation of the solubilization and subsequent purification conditions.
• Tween 20, CHAPS (Pierce Chemical Co., Rockford, IL) and octyl glucoside were evaluated by centrifugation and gel filtration chromatography for their ability to solubilize LDL particles.
• CHAPS provided 25% greater recovery of solubilized activity than Tween 20 and 300% greater recovery than octyl glucoside.
• LDL precipitate solubilized with lOmM CHAPS was then fractionated on a DEAE
• Sepharose Fast Flow column an anion exchange column; Pharmacia
• buffer containing ImM CHAPS to provide a large pool of partially purified PAF-AH ("the DEAE pool") for evaluation of additional columns.
• the DEAE pool was used as starting material to test a variety of chromatography columns for utility in further purifying the PAF-AH activity.
• the columns tested included: Blue Sepharose Fast Flow (Pharmacia), a dye ligand affinity column; S-Sepharose Fast Flow (Pharmacia), a cation exchange column; Cu Chelating Sepharose (Pharmacia), a metal ligand affinity column; Fractogel S (EM Separations, Gibbstown, NJ), a cation exchange column; and Sephacryl-200 (Pharmacia), a gel filtration column. These chromatographic procedures all yielded low, unsatisfactory levels of purification when operated in ImM CHAPS.
• the novel protocol utilized to purify PAF-AH for amino acid sequencing therefore comprised the following steps which were performed at 4°C.
• Human plasma was divided into 900 ml aliquots in 1 liter Nalgene bottles and adjusted to pH 8.6.
• LDL particles were then precipitated by adding 90 ml of 3.85 % sodium phosphotungstate followed by 23 ml of 2M MgC ⁇ .
• the plasma was then centrifuged for 15 minutes at 3600 g.
• Pellets were resuspended in 800 ml of 0.2% sodium citrate.
• LDL was precipitated again by adding 10 g NaCl and 24 ml of 2M M C ⁇ .
• LDL particles were pelleted by centrifugation for 15 minutes at 3600 g. This wash was repeated twice.
• Protein was eluted with an 8 L, 0 - 0.5M NaCl gradient and 480 ml fractions were collected. This step was necessary to obtain binding to the Blue Sepharose Fast Flow column below. Fractions were assayed for acetylhydrolase activity essentially by the method described in Example 4.
• Active fractions were pooled, and the pool was adjusted to pH 8.0 with 1M Tris-HCl pH 8.0.
• the active pool from Blue Sepharose Fast Flow chromatography was loaded onto a Cu Chelating Sepharose column (2.5 cm x 2 cm;
• the Cu Chelating Sepharose pool was reduced in 50 mM DTT for 15 minutes at 37°C and loaded onto a 0.75 mm, 7.5 % polyacrylamide gel.
• Gel slices were cut every 0.5 cm and placed in disposable microfuge tubes containing 200 l 25mM Tris-HCl, lOmM CHAPS, 150mM NaCl. Slices were ground up and allowed to incubate overnight at 4°C. The supernatant of each gel slice was then assayed for PAF-AH activity to determine which protein band on SDS-PAGE contained PAF-AH activity. PAF-AH activity was found in an approximately 44 kDa band.
• Protein from a duplicate gel was electrotransf erred to a PVDF membrane (Immobilon-P, Millipore) and stained with Coomassie Blue.
• a photograph of the PVDF membrane is presented in FIGURE 1.
• approximately 200 ⁇ g PAF-AH was purified 2 x 10 -fold from 5 L human plasma.
• a 3 x 10 -fold purification of PAF-AH activity is described in Stafforini et al. (1987), supra.
• the approximately 44 kDa protein band from the PAF-AH- containing PVDF membrane described in Example 1 was excised and sequenced using an Applied Biosystems 473A Protein sequencer. N-terminal sequence analysis of the approximately 44 kDa protein band corresponding to the
• FKDLGEENFKALVLIAF A search of protein databases revealed this sequence to be a fragment of human serum albumin.
• the upper half of the same PVDF membrane was also sequenced and the N-terminal amino acid sequence determined was:
• Example 3 A full length clone encoding human plasma PAF-AH was isolated from a macrophage cDNA library.
• RNA was harvested from peripheral blood monocyte-derived macrophages. Double-stranded, blunt-ended cDNA was generated using the Invitrogen Copy Kit (San Diego, CA) and BstXl adapters were ligated to the cDNA prior to insertion into the mammalian expression vector, pRc/CMV (Invitrogen). The resulting plasmids were introduced into E. coli strain XL-1 Blue by electroporation. Transformed bacteria were plated at a density of approximately 3000 colonies per agarose plate on a total of 978 plates. Plasmid DNA prepared separately from each plate was retained in individual pools and was also combined into larger pools representing 300,000 clones each.
• the codon choice tables of Wada et al Nuc. Acids Res. , 19S: 1981-1986 (1991) were used to select nucleotides at the third position of each codon of the primer.
• the primer was used in combination with a primer specific for either the SP6 or T7 promoter sequences, both of which flank the cloning site of pRc/CMV, to screen the macrophage library pools of 300,000 clones. All PCR reactions contained 100 ng of template cDNA, 1 ⁇ g of each primer, 0.125mM of each dNTP, lOmM Tris-HCl pH 8.4, 50mM MgC ⁇ and 2.5 units of Taq polymerase.
• PCR primers set out below which are specific for the cloned PCR fragment described above, were then designed for identifying a full length clone.
• Antisense Primer (SEQ ID NO: 6)
• DNA from the transformed bacteria was subsequently screened by hybridization using the original cloned PCR fragment as a probe.
• Colonies were blotted onto nitrocellulose and prehybridized and hybridized in 50% formamide, 0.75M sodium chloride, 0.075M sodium citrate, 0.05M sodium phosphate pH 6.5, 1 % polyvinyl pyrolidine, 1 % Ficoll, 1 % bovine serum albumin and 50 ng/ml sonicated salmon sperm DNA.
• the hybridization probe was labeled by random hexamer priming. After overnight hybridization at 42 °C, blots were washed extensively in 0.03M sodium chloride, 3mM sodium citrate, 0.1 % SDS at 42°C.
• the nucleotide sequence of 10 hybridizing clones was determined.
• One of the clones, clone sAH 406-3 contained the sequence predicted by the original peptide sequence of the PAF-AH activity purified from human plasma.
• the DNA and deduced amino acid sequences of the human plasma PAF-AH are set out in SEQ ID NOs: 7 and 8, respectively.
• Clone sAH 406-3 contains a 1.52 kb insert with an open reading frame that encodes a predicted protein of 441 amino acids. At the amino terminus, a relatively hydrophobic segment of 41 residues precedes the N-terminal amino acid (the isoleucine at position 42 of SEQ ID NO: 8) identified by protein microsequencing.
• the encoded protein may thus have either a long signal sequence or a signal sequence plus an additional peptide that is cleaved to yield the mature functional enzyme. The presence of a signal sequence is one characteristic of secreted proteins.
• the protein encoded by clone sAH 406-3 includes the consensus GxSxG motif (amino acids 271-275 of SEQ ID NO: 8) that is believed to contain the active site serine of all known mammalian Upases, microbial Upases and serine proteases. See Chapus et al, Biochimie, 70: 1223-1224 (1988) and Brenner, Nature, 334: 528-530 (1988).
• Table 2 is a comparison of the amino acid composition of the human plasma PAF-AH of the invention as predicted from SEQ ID NO: 8 and the amino acid composition of the purportedly purified material described by Stafforini et al (1987), supra.
• amino acid composition of the mature form of the human plasma PAF-AH of the invention and the amino acid composition of the previously purified material that was purportedly the human plasma PAF-AH are clearly distinct.
• a putative spUce variant of the human PAF-AH gene was detected when PCR was performed on macrophage and stimulated PBMC cDNA using primers that hybridized to the 5' untranslated region (nucleotides 31 to 52 of SEQ ID NO: 7) and the region spanning the translation termination codon at the 3 1 end of the PAF-
• AH cDNA nucleotides 1465 to 1487 of SEQ ID NO: 7
• the PCR reactions yielded two bands on a gel, one corresponding to the expected size of the PAF-AH cDNA of Example 3 and the other was about 100 bp shorter. Sequencing of both bands revealed that the larger band was the PAF-AH cDNA of Example 3 while the shorter band lacked exon 2 (Example 5 below) of the PAF-AH sequence which encodes the putative signal and pro-peptide sequences of plasma PAF-AH.
• the predicted catalytic triad and aU cysteines were present in the shorter clone, therefore the biochemical activity of the protein encoded by the clone is likely to match that of the plasma enzyme.
• Genomic human plasma PAF-AH sequences were also isolated.
• the structure of the PAF-AH gene was determined by isolating lambda and PI phage clones containing human genomic DNA by DNA hybridization under conditions of high stringency. Fragments of the phage clones were subcloned and sequenced using primers designed to anneal at regular intervals throughout the cDNA clone sAH 406- 3. In addition, new sequencing primers designed to anneal to the intron regions flanking the exons were used to sequence back across the exon-intron boundaries to confirm the sequences. Exon/intron boundaries were defined as the points where the genomic and cDNA sequences diverged. These analyses revealed that the human PAF-AH gene is comprised of 12 exons.
• Exons 1, 2, 3, 4, 5, 6, and part of 7 were isolated from a male fetal placental Ubrary constructed in lamda FIX (Stratagene). Phage plaques were blotted onto nitroceUulose and prehybridized and hybridized in 50% formamide, 0.75M sodium chloride, 75mM sodium citrate, 50mM sodium phosphate (pH 6.5), 1 % poly vinyl pyroUdine, 1 % FicoU, 1 % bovine serum albumin, and 50 ng/ml sonicated salmon sperm DNA. The hybridization probe used to identify a phage clone containing exons 2-6 and part of 7 consisted of the entire cDNA clone sAH 406-3.
• a clone containing exon 1 was identified using a fragment derived from the 5' end of the cDNA clone (nucleotides 1 to 312 of SEQ ID NO: 7). Both probes were labeUed with - ⁇ P by hexamer random priming. After overnight hybridization at 42 °C, blots were washed extensively in 30mM sodium chloride, 3mM sodium citrate, 0.1 % SDS at 42°C. The DNA sequences of exons 1, 2, 3, 4, 5, and 6 along with partial surrounding intron sequences are set out in SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively.
• exon 7 as weU was subcloned from a PI clone isolated from a human PI genomic Ubrary.
• PI phage plaques were blotted onto nitrocellulose and prehybridized and hybridized in 0.75M sodium chloride, 50mM sodium phosphate (pH 7.4), 5mM EDTA, 1 % polyvinyl pyroUdine, 1 % FicoU, 1 % bovine serum albumin, 0.5 % SDS, and 0.1 mg/ml total human DNA.
• the hybridization probe labeled with ⁇ 2 P by hexamer random priming, consisted of a 2.6 kb EcoRl fragment of genomic DNA derived from the 3 ' end of a lambda clone isolated above. This fragment contained exon 6 and the part of exon 7 present on the phage clone. After overnight hybridization at 65 °C, blots were washed as described above. The DNA sequences of exons 7, 8, 9, 10, 11, and 12 along with partial surrounding intron sequences are set out in SEQ ID NOs: 15, 16, 17, 18, 19, and 20, respectively.
• FuU length plasma PAF-AH cDNA clones were isolated from mouse, canine, bovine and chicken spleen cDNA Ubraries and a partial rodent clone was isolated from a rat thymus cDNA Ubrary.
• the clones were identified by low stringency hybridization to the human cDNA (hybridization conditions were the same as described for exons 1 through 6 in Example 5 above except that 20 % formamide instead of 50% formamide was used).
• a 1 kb Hindi ⁇ fragment of the human PAF- AH sAH 406-3 cDNA clone (nucleotides 309 to 1322 of SEQ ID NO: 7) was used as a probe.
• a partial monkey clone was isolated from macaque brain cDNA by PCR using primers based on nucleotides 285 to 303 and 851 to 867 of SEQ
• nucleotide and deduced amino acid sequences of the mouse, canine, bovine, chicken, rat, and macaque cDNA clones are set out in SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively.
• human plasma PAF-AH is expected to have a region that mediates its specific interaction with the low density and high density Upoprotein particles of plasma. Interaction with these particles may be mediated by the N- terminal half of the molecule which has large stretches of amino acids highly conserved among species but does not contain the catalytic triad of the enzyme.
• Example 7 To determine whether human plasma PAF-AH cDNA clone sAH 406-3 (Example 3) encodes a protein having PAF-AH activity, the pRc/CMV expression construct was transiently expressed in COS 7 ceUs. Three days foUowing transfection by a DEAE Dextran method, COS ceU media was assayed for PAF-AH activity.
• CeUs were seeded at a density of 300,000 ceUs per 60 mm tissue culture dish. The foUowing day, the ceUs were incubated in DMEM containing 0.5 mg/ml DEAE dextran, 0. ImM chloroquine and 5-10 ⁇ g of plasmid DNA for 2 hours. Cells were then treated with 10% DMSO in phosphate-buffered saline for 1 minute, washed with media and incubated in DMEM containing 10% fetal calf serum previously treated with dusopropyl fluorophosphate (DFP) to inactivate endogenous bovine serum PAF-AH. After 3 days of incubation, media from transfected ceUs were assayed for PAF-AH activity.
• DFP dusopropyl fluorophosphate
• PAF-AH activity was determined by measuring the hydrolysis of • ⁇ -acetate from [acetyl- H] PAF (New England Nuclear, Boston, MA). The aqueous free - ⁇ -acetate was separated from labeled substrate by reversed-phase column chromatography over octadecylsiUca gel cartridges (Baker Research Products, PhilUpsburg, PA). Assays were carried out using 10 ⁇ l transfectent supernatant in 0.1 M Hepes buffer, pH 7.2, in a reaction volume of 50 ⁇ l. A total of 50 pmoles of substrate were used per reaction with a ratio of 1:5 labeled: cold PAF.
• media from ceUs transfected with sAH 406-3 contained PAF-AH activity at levels 4-fold greater than background. This activity was unaffected by the presence of EDTA but was aboUshed by ImM DFP.
• FuU length and various truncated human plasma PAF-AH DNAs and a chimeric mouse-human PAF-AH DNA were expressed in E. coli and yeast and stably expressed in mammaUan cells by recombinant methods.
• PCR was used to generate a protein coding fragment of human plasma PAF-AH cDNA from clone sAH 406-3 which was readUy amenable to subcloning into an E. coli expression vector.
• the subcloned segment began at the 5' end of the human gene with the codon that encodes Ile 4 2 (SEQ ID NO: 8), the N-terminal residue of the enzyme purified from human plasma. The remainder of the gene through the native termination codon was included in the construct.
• the 5' sense PCR primer utilized was: SEQ ID NO: 28
• Transformants from overnight cultures were peUeted and resuspended in lysis buffer containing 50mM Tris-HCl pH 7.5, 50mM NaCl, lOmM CHAPS, ImM EDTA, 100 ⁇ g/ml lysozyme, and 0.05 trypsin-inhibiting units (TIU)/ml
• the N-terminus of natural plasma PAF-AH was identified as Ile 4 2 by amino acid sequencing (Example 2). However, the sequence immediately upstream of He 4 2 does not conform to amino acids found at signal sequence cleavage sites [i.e. , the "-3-1-rule" is not foUowed, as lysine is not found at position -1; see von Heijne, Nuc. Acids Res. , 14: 4683-4690 (1986)]. Presumably a more classical signal sequence is recognized by the ceUular secretion system, foUowed by endoproteolytic cleavage.
• Truncated recombinant human PAF-AH products were also produced in E. coli using a low copy number plasmid and a promoter that can be induced by the addition of arabinose to the culture.
• One such N-terminaUy truncated PAF-AH product is the recombinant expression product of DNA encoding amino acid residues Met 4 g through Asn 441 of the polypeptide encoded by fuU length PAF-AH cDNA (SEQ ID NO: 8), and is designated rPH.2.
• the plasmid used for production of rPH.2 in bacterial cells was pBAR2/PH.2, a pBR322-based plasmid that carries (1) nucleotides 297 to 1487 of SEQ ID NO: 7 encoding human PAF-AH beginning with the methionine codon at position 46, (2) the araB-C promoters and araC gene from the arabinose operon of Salmonella typhimurium, (3) a transcription termination sequence from the bacteriophage T7, and (4) a repUcation origin from bacteriophage fl.
• SpecificaUy, pBAR2/PH.2 included the following segments of DNA:
• PAF-AH product is the recombinant expression product of DNA encoding amino acid residues Met 4 g through He 4 29 of the polypeptide encoded by fuU length PAF-AH cDNA (SEQ ID NO: 8).
• the DNA encoding rPH.9 was inserted into the same vector used for production of rPH.2 in bacterial ceUs.
• This plasmid was designated pBAR2/PH.9 and SpecificaUy included the foUowing segments of DNA: (1) from the destroyed AatQ site at position 1958 to the EcoRI site at nucleotide 6239 of the vector sequence containing an origin of repUcation and genes encoding resistance to either ampicillin or tetracycUne derived from the bacterial plasmid pBR322; (2) from the EcoRI site at position 6239 to the Xbal site at position 131, DNA from the Salmonella typhimurium arabinose operon (Genbank accession numbers Ml 1045, Ml 1046, Ml 1047, J01797); (3) from the Xbal site at position 131 to the Ncol site at position 170, DNA containing a ribosome binding site from pET-21b (Novagen, Madison, WI); (4) from the Ncol site at position 170 to the Xhol site at position 1328, human PAF-AH DNA sequence; (5) from the Xhol site at position 1328
• PAF-AH products in pBAR2/PH.2 and pBAR2/PH.9 is under the control of the araB promoter, which is tightly repressed in the presence of glucose and absence of arabinose, but functions as a strong promoter when L- arabinose is added to cultures depleted of glucose. Selection for ceUs containing the plasmid can be accompUshed through the addition of either ampicillin (or related antibiotics) or tetracycUne to the culture medium.
• a variety of E. coli strains can be used as a host for recombinant expression of PAF-AH products, including but not
• AH is not depleted from the medium during the induction period, resulting in higher levels of PAF-AH compared to that obtained with strains that are capable of metabolizing arabinose.
• Any suitable media and culturing conditions may be used to express active PAF-AH products in various E. coli strains.
• rich media formulations such as LB, EDM295 (a M9 based minimum medium supplemented with yeast extract and acid hydrolysed casein), or "defined” media such as A675, an A based minimal medium set at pH 6.75 employing glycerol as a carbon source and supplemented with trace elements and vitamins, permit substantial production of rPAF-AH products.
• TetracycUne is included in the media to maintain selection of the plasmid.
• the plasmid pBAR2/PH.2 was transformed into the E. coli strain MC1061 (ATCC 53338), which carries a deletion of the arabinose operon and thereby cannot metabolize arabinose.
• MCI 061 is also a leucine auxotroph and was cultivated by batch-fed process using a defined media containing casamino acids that complement the leucine mutation.
• the E. coli M1061 cells transformed with pBAR2/PH.2 were grown at 30° C in batch media containing 2 gm/L glucose.
• Glucose serves the dual purpose of carbon source for cell growth, and repressor of the arabinose promoter.
• batch glucose levels were depleted ( ⁇ 50 mg/L)
• a nutrient feed (containing 300 gm/L glucose) was started.
• the feed was increased linearly for 16 hours at a rate which limited acid bi-product formation.
• the nutrient feed was switched to media containing glycerol instead of glucose.
• 500 gm/L L-arabinose was added to a final concentration of 5 gm/L.
• the glycerol feed was kept at a constant feed rate for 22 hours.
• CeUs were harvested using hoUow-fiber filtration to concentrate the suspension approximately 10-fold.
• CeU paste was stored at -70° C.
• the final culture volume of about 75 liters contained 50-60 gm PAF-AH.
• High level production of rPAF-AH products can be achieved when pBAR2/PH.2 or PH.9 is expressed by strains SB7219 or MC1061. Other strains deficient in arabinose degradation are suitable for high ceU density production.
• the cells are cultured under the following conditions. ExponentiaUy growing SB7219;pBAR2/PH.2 and SB7219;pBAR2/PH.9 strains are seeded into fermentors containing batch medium containing 2 g/L glucose. Once glucose is consumed, the tanks are fed with a glycerol solution containing trace elements, vitamins, magnesium and ammonium salt to maintain healthy exponential growth.
• the tanks are maintained at 30 C, provided air to supply oxygen and agitated to maintain the dissolved oxygen level above about 15 % saturation.
• ceU density of the culture is above 110 g/L (wet ceU mass)
• constant feed rate is imposed and a bolus addition of L-arabinose is added to the culture (about 0.5% final).
• Product formation is observed for 16-22 hours.
• the cultures typicaUy achieve 40-50 g/L (dry ceU weight). CeUs are harvested by centrifugation, stored at -70° C, and rPAF-AH product purified for analysis. Specific productivities in excess of 150 units/ml/OD ⁇ Q ⁇ are routinely obtained.
• Plasmids constructed for expression of PAF-AH employ a strong viral promoter from cytomegalovirus, a polyadenylation site from the bovine growth hormone gene, and the SV40 origin of replication to permit high copy number repUcation of the plasmid in COS ceUs.
• Plasmids were electroporated into cells.
• a first set of plasmids was constructed in which the 5' flanking sequence (pDCl/PAFAH.l) or both the 5' or 3' flanking sequences (PDC1/PAFAH.2) of the human PAF-AH cDNA were replaced with flanking sequences from other genes known to be expressed at high levels in mammaUan ceUs.
• a construct (pRc/MS9) containing the cDNA encoding mouse PAF-AH in the mammaUan expression vector pRc/CMV resulted in production of secreted PAF-AH at the level of 5-10 units/ml (1000 fold above background) after transfection into COS ceUs. Assuming that the specific activity of the mouse PAF-AH is about the same as that of the human enzyme, the mouse cDNA is therefore expressed at a 500-1000 fold higher level than is the human PAF-AH cDNA.
• pRc/PH.MHCl contains the coding sequence for the N-terminal 97 amino acids of the mouse PAF- AH polypeptide (SEQ ID NO: 21) fused to the C-terminal 343 amino acids of human PAF-AH in the expression vector pRc/CMV (Invitrogen, San Diego, CA).
• the second chimeric gene in plasmid pRc/PH.MHC2, contains the coding sequence for the N-terminal 40 amino acids of the mouse PAF-AH polypeptide fused to the C- terminal 400 residues of human PAF-AH in pRc/CMV.
• Transfection of COS ceUs with pRc/PH.MHCl led to accumulation of 1-2 units/ml of PAF-AH activity in the media.
• Conditioned media derived from cells transfected with pRc/PH.MHC2 was found to contain only 0.01 units/ml of PAF-AH activity.
• the approximately 290 bp Asp718/BamHI fragment was derived from a PCR fragment that was made using the dual asymmetric PCR approach for construction of synthetic genes described in Sandhu et al , Biotechniques , 12: 14-16 (1992).
• the synthetic Asp718/BamHI fragment was ligated with DNA fragments encoding the remainder of the human PAF-AH molecule beginning with nucleotide 453 of SEQ ID NO: 7 such that a sequence encoding authentic human PAF-AH enzyme was inserted into the mammaUan expression vector pRc/CMV (Invitrogen, San Diego) to create plasmid pRc/HPH.4.
• the complete sequence of the recoded gene is set out in SEQ
• the 5' flanking sequence adjacent to the human PAF-AH coding sequence in pRc/HPH.4 is from that of a mouse cDNA encoding PAF-AH in pRc/MS9 (nucleotides 1 to 116 of SEQ ID NO: 21).
• the recoded PAF-AH gene from pRc/HPH.4 wiU be inserted into a mammaUan expression vector containing the dihydrofolate reductase (DHFR) gene and DHFR-negative Chinese hamster ovary cells wUl be transfected with the vector.
• the transfected cells wiU be subjected to methotrexate selection to obtain clones making high levels of human PAF-AH due to gene amplification.
• Recombinant human plasma PAF-AH (beginning at He 4 2) expressed in E. coli was purified to a single Coomassie-stained SDS-PAGE band by various methods and assayed for activities exhibited by the native PAF-AH enzyme.
• Example 1 for native PAF-AH The following steps were performed at 4°C. PeUets from 50 ml PAF-AH producing E. coli (transformed with expression construct trp AH) were lysed as described in Example 8. Solids were removed by centrifugation at 10,000 g for 20 minutes. The supernatant was loaded at 0.8 ml/minute onto a Blue Sepharose Fast Flow column (2.5 cm x 4 cm; 20 ml bed volume) equiUbrated in buffer D (25mM Tris-HCl, lOmM CHAPS, 0.5M NaCl, pH 7.5).
• the column was washed with 100 ml buffer D and eluted with 100 ml buffer A containing 0.5M KSCN at 3.2 ml/minute. A 15 ml active fraction was loaded onto a 1 ml Cu Chelating Sepharose column equiUbrated in buffer D. The column was washed with 5 ml buffer D foUowed by elution with 5 ml of buffer D containing lOOmM imidazole with gravity flow. Fractions containing PAF-AH activity were analyzed by SDS- PAGE.
• PeUets 100 g of PAF-AH-producing E. coli (transformed with the expression construct pUC trp AH) were resuspended in 200 ml of lysis buffer (25mM Tris, 20mM CHAPS, 50mM NaCl, ImM EDTA, 50 ⁇ g/ml benzamidine, pH 7.5) and lysed by passing three times through a microfluidizer at 15,000 psi. SoUds were removed by centrifugation at 14,300 x g for 1 hour. The supernatant was dUuted 10- fold in dUution buffer [25mM MES (2-[N-morpholino] ethanesulfonic acid), lOmM
• the S pool was loaded at 1 ml/minute onto a Blue Sepharose Fast Flow column (2.5 cm x 4 cm; 20 ml) equiUbrated in Buffer F (25mM Tris, lOmM CHAPS, 0.5M NaCl, ImM EDTA, pH 7.5).
• Buffer F 25mM Tris, lOmM CHAPS, 0.5M NaCl, ImM EDTA, pH 7.5
• the column washed with 100 ml Buffer F and eluted with 100 ml Buffer F containing 3M NaCl at 4 ml/minute.
• the Blue Sepharose Fast Flow chromatography step was then repeated to reduce endotoxin levels in the sample. Fractions containing PAF-AH activity were pooled and dialyzed against Buffer G (25mM Tris pH 7.5, 0.5M NaCl, 0.1 % Tween 80, ImM EDTA).
• the purification product obtained appeared on SDS-PAGE as a single intense band below the 43 kDa marker with some diffuse staining directly above and below it.
• the recombinant material is significantly more pure and exhibits greater specific activity when compared with PAF-AH preparations from plasma as described in Example 1.
• CeUs are dUuted 1: 1 in lysis buffer (25mM Tris pH 7.5, 150mM NaCl, 1 % Tween 80, 2mM EDTA). Lysis is performed in a chiUed microfluidizer at 15,000-20,000 psi with three passes of the material to yield > 99 % ceU breakage. The lysate is diluted
• Still another method contemplated by the invention for purifying enzymaticaUy-active PAF-AH from E.coli includes the steps of: (a) preparing an E.coli extract which yields solubilized PAF-AH supernatant after lysis in a buffer containing CHAPS; (b) dilution of the said supernatant and appUcation to a anion exchange column equUibrated at about pH 8.0; (c) eluting PAF-AH enzyme from said anion exchange column; (d) applying said adjusted eluate from said anion exchange column to a blue dye Ugand affinity column; (e) eluting the said blue dye Ugand affinity column using a buffer comprising 3.0M salt; (f) dUution of the blue dye eluate into a suitable buffer for performing hydroxylapatite chromatography; (g) performing hydroxylapatite chromatography where washing and elution is accompUshed using buffers (with or without CHAPS); (h) d
• the lysis buffer is 25mM Tris, lOOmM NaCl, ImM EDTA, 20mM CHAPS, pH 8.0; in step (b) the dUution of the supernatant for anion exchange chromatography is 3-4 fold into 25mM Tris, ImM EDTA, lOmM CHAPS, pH 8.0 and the column is a Q-Sepharose column equiUbrated with 25mM Tris, ImM EDTA, 50mM NaCl, lOmM CHAPS, pH 8.0; in step (c) the anion exchange column is eluted using 25mM Tris, ImM EDTA, 350mM NaCl, lOmM CHAPS, pH 8.0; in step (d) the eluate from step (c) is appUed directly onto a blue dye affinity column; in step (e) the column is eluted with 3M NaCl, lOmM CHAPS, 25mM Tris,
• step (h) dUution of said hydroxylapatite eluate for cation exchange chromatography is accompUshed by dilution into a buffer ranging in pH from approximately 6.0 to 7.0 comprising sodium phosphate (with or without CHAPS); in step (i) a S Sepharose column is equiUbrated with 50mM sodium phosphate, (with or without) lOmM CHAPS, pH 6.8; in step (j) elution is accompUshed with a suitable formulation buffer such as potassium phosphate 50mM, 12.5mM aspartic acid, 125mM NaCl, pH 7.5 containing 0.01 % Tween-80; and in step (k) cation exchange chromatrography is accompUshed at 2-8 ° C .
• a suitable formulation buffer such as potassium phosphate 50mM, 12.5mM aspartic acid, 125mM NaCl, pH 7.5 containing 0.01 % Tween-80.
• suitable formulation buffers for use in step (1) which stabilize PAF-AH include 50mM potassium phosphate, 12.5mM Aspartic acid, 125mM NaCl pH 7.4 (approximately, with and without the addition of Tween-80 and or Pluronic F68) or 25mM potassium phosphate buffer containing (at least) 125mM NaCl, 25mM arginine and 0.01 % Tween-80 (with or without Pluronic F68 at approximately 0.1 and 0.5 %).
• recombinant PAF-AH enzyme rapidly degraded an oxidized phosphoUpid (glutaroylPC) which had undergone oxidative cleavage of the sn-2 fatty acid.
• Native plasma PAF-AH has several other properties that distinguish it from other phosphoUpases including calcium-independence and resistance to compounds that modify sulfhydryl groups or disrupt disulfides.
• Both the native and recombinant plasma PAF-AH enzymes are sensitive to DFP, indicating that a serine comprises part of their active sites.
• An unusual feature of the native plasma PAF acetylhydrolase is that it is tightly associated with
• Upoproteins in circulation and its catalytic efficiency is influenced by the Upoprotein environment.
• PAF-AH of the invention When recombinant PAF-AH of the invention was incubated with human plasma (previously treated with DFP to aboUsh the endogenous enzyme activity), it associated with low and high density Upoproteins in the same manner as the native activity. This result is significant because there is substantial evidence that modification of low density Upoproteins is essential for the cholesterol deposition observed in atheromas, and that oxidation of Upids is an initiating factor in this process.
• PAF-AH protects low density Upoproteins from modification under oxidizing conditions in vitro and may have such a role in vivo. Administration of PAF-AH is thus indicated for the suppression of the oxidation of Upoproteins in atherosclerotic plaques as well as to resolve inflammation.
• Example 10 Various other recombinant PAF-AH products were expressed in E. coli.
• the products included PAF-AH analogs having single amino acid mutations and PAF-AH fragments.
• PAF-AH is a Upase because it hydrolyses the phosphoUpid PAF. Wh e no obvious overall similarity exists between PAF-AH and other characterized Upases, there are conserved residues found in comparisons of structuraUy characterized Upases.
• a serine has been identified as a member of the active site. The serine, along with an aspartate residue and a histidine residue, form a catalytic triad which represents the active site of the Upase.
• the three residues are not adjacent in the primary protein sequence, but structural studies have demonstrated that the three residues are adjacent in three dimensional space.
• PAF-AH coding sequence were modified to encode alanine residues and were expressed in E. coli. As shown in Table 8 below wherein, for example, the abbreviation "S108A" indicates that the serine residue at position 108 was changed to an alanine, point mutations of Ser273, ASP29 , or His351 com Pl ete ty destroy PAF- AH activity.
• the distances between active site residues is simUar for PAF-AH (Ser to Asp, 23 amino acids; Ser to His, 78 amino acids) and other Upases.
• Cysteines are often critical for the functional integrity of proteins because of their capacity to form disulfide bonds.
• the plasma PAF-AH enzyme contains five cysteines. To determine whether any of the five is critical for enzyme actvity, each cysteine was mutated individuaUy to a serine and the resulting mutants were expressed in E. coli. PreUminary activity results using partiaUy purified preparations of these recombinantly produced mutants are shown below in the second column of Table 8, while results using more purified preparations are shown below in the third column of Table 8. The data show that aU of the cysteine mutants had largely equivalent activity, so that none of the cysteines appears to be necessary for PAF-AH activity.
• C-terminal deletions were prepared by digesting the 3 ' end of the PAF- AH coding sequence with exonuclease IH for various amounts of time and then Ugating the shortened coding sequence to plasmid DNA encoding stop codons in aU three reading frames.
• Ten different deletion constructs were characterized by DNA sequence analysis, protein expression, and PAF-AH activity. Removal of twenty-one to thirty C-terminal amino acids greatly reduced catalytic activity and removal of fifty-two residues completely destroyed activity. See FIGURE 3.
• the purified material described above was also subject to analysis for glycosylation.
• Purified native PAF-AH was incubated in the presence or absence of N-Glycanase, an enzyme that removes N-linked carbohydrates from glycoproteins.
• the treated PAF-AH samples were electrophoresed through a 12% SDS polyacrylamide gel then visualized by Western blotting using rabbit polyclonal antisera. Protein not treated with N-Glycanase migrated as a diffuse band of 45-50 kDa whereas the protein treated with the glycanase migrated as a tight band of about 44 kDa, demonstrating that native PAF-AH is glycosylated.
• N-terminal heterogeneity was also observed in purified preparations of recombinant PAF-AH (Ile 4 2 N-terminus). These preparations were a mixmre of polypeptides with N-termini beginning at Ala 7, Ile 4 2, or the artificial initiating Met . j adjacent to Ile 4 2-
• PH.2 vector minus the translation initiating methionine which is expected to be post- translationaUy removed.
• the lower mass peak was approximately 1200 atomic mass units less.
• rPH.2 the expression product of DNA encoding Met ⁇ -Asn ⁇ j
• rPH.9 the expression product of DNA encoding Met 4 6-Ile 4 29 preparations were purified for further comparison with purified rPAF- AH (expression product of DNA encoding Ile ⁇ -Asn ⁇ j ).
• rPH.9 was produced by E. coli strain SB7219 and purified generaUy according to the zinc chelate purification procedure described above, while rPH.2 was produced by E. coli strain MC1061 and purified as described below.
• the transformed ceUs were lysed by dUution of the ceU paste with lysis buffer (100 mM succinate, 100 mM NaCl, 20 mM CHAPS, pH 6.0). The slurry was mixed and lysed by high pressure disruption. The lysed ceUs were centrifuged and the supernatant containing rPH.2 was retained. The clarified supernatant was dUuted 5-fold in 25 mM sodium phosphate buffer containing, 1 mM EDTA, 10 mM CHAPS, pH 7.0. The dUuted supernatant was then appUed to the Q Sepharose column.
• lysis buffer 100 mM succinate, 100 mM NaCl, 20 mM CHAPS, pH 6.0.
• the slurry was mixed and lysed by high pressure disruption.
• the lysed ceUs were centrifuged and the supernatant containing rPH.2 was retained.
• the clarified supernatant was d
• the column was washed first with 3 column volumes of 25 mM sodium phosphate buffer containing 1 mM EDTA, 50 mM NaCl, 10 mM CHAPS, pH 7.0 (Wash 1), then washed with 10 column volumes of 25 mM Tris buffer containing 1 mM EDTA, 10 mM CHAPS, pH 8.0 (Wash 2) and with 10 column volumes of 25 mM Tris buffer containing 1 mM EDTA, 100 mM NaCl, 10 mM CHAPS, pH 8.0 (Wash 3). Elution was accompUshed with 25 mM Tris buffer containing 1 mM EDTA, 350 mM NaCl, 10 mM CHAPS, pH 8.0.
• the Q Sepharose eluate was dUuted 3-fold in 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 then appUed to a Blue Sepharose column.
• the column was washed first with 10 column volumes of 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0.
• the column was then washed with 3 column volumes of 25 mM Tris, 0.5 M NaCl, 10 mM CHAPS, pH 8.0. Elution was accompUshed with 25 mM Tris, 3.0 M NaCl, 10 mM CHAPs, pH 8.0.
• the Blue Sepharose eluate was dUuted 5-fold in 10 mM sodium phosphate, 10 mM CHAPS, pH 6.2 then appUed to the chromatography column.
• the column was washed with 10 column volumes of 10 mM sodium phosphate, 100 mM NaCl, 0.1 % Pluromc F68, pH 6.2. rPH.2 was eluted with 120 mM sodium phosphate, 100 mM NaCl, 0.1 % Pluronic F-68, pH 7.5.
• the hydroxyapatite eluate was dUuted 6- fold with 10 mM sodium phosphate, 0.1 % Pluronic F68, pH 6.8.
• the dUuted hydroxyapatite eluate was adjusted to pH 6.8 using 0.5 N succinic acid and then appUed to a SP Sepharose column.
• the SP Sepharose column was washed with 10 column volumes 50 mM sodium phosphate, 0.1 % Fluronic F68, pH 6.8 and eluted with 50 mM sodium phosphate, 125 mM NaCl, 0.1 % Pluronic F68, pH 7.5.
• the eluted rPH.2 was formulated by dUuting to a final concentration of 4 mg/ml in 50 mM sodium phosphate, 125 mM NaCl, 0.15 % Pluronic F68, pH 7.5, and Tween 80 was added to a final concentration of 0.02 % Tween 80.
• the formulated product was then filtered through a 0.2 ⁇ membrane and stored prior to use.
• the rPH.2 preparation had less N-terminal heterogeneity compared to rPAF-AH.
• the N-terminus analysis of the rPH.9 preparation was simUar to that of rPH.2, but less C-terminal heterogeneity was observed for the rPH.9 preparation relative to rPH.2.
• the purified rPH.2 preparation contained a major sequence with an N- terminus of Ala 47 (about 86-89 %) and a minor sequence with an N-terminus of Ala g (about 11-14%), with the ratio of the two N-termini being fairly consistent under different fermentation conditions.
• the purified rPH.9 preparation also contained a major sequence with an N-terminus of Ala 47 (about 83-90%) and a minor sequence with an N-terminus of Ala 4 g (about 10-17%).
• the rPH.2 spectrum exhibited two peaks in the spectrum at a mass value expected for the rPAF-AH product (see FIGURE 4), simUar to the pattern observed with the partiaUy purified protein in section B. l. above.
• the secondary, lower molecular weight peak was typicaUy present at approximately 20% to 30% of the total.
• the rPH.9 spectrum showed a predominant peak at a mass consistent with that expected for the fuU length translation product for the PH.9 vector, minus the translation initiating methionine (see FIGURE 5).
• a small sUghtly lower molecular weight shoulder peak was also observed for rPH.9 that represented approximately 5 % of the total.
• Sodium dodecyl sulfate-poiyacrylamide electrophoresis was performed on purified rPAF-AH, rPH.2 and rPH.9 preparations.
• a compUcated banding pattern was observed for rPH.2 around the electrophoretic migration range expected for the rPAF-AH product, based on protein molecular weight standards.
• One, or in some gels, two predominant bands were seen, with readUy observed secondary bands above and below the primary band.
• Purified rPH.2 and rPH.9 have enzymatic activity indistinguishable from that of endogenous PAF-AH purified from serum, and rPH.2 and rPH.9 bind to lipropotein in a simUar manner as purified endogenous PAF-AH.
• Example 11 A preliminary analysis of expression patterns of human plasma PAF-
• AH mRNA in human tissues was conducted by Northern blot hybridization.
• RNA was prepared from human cerebral cortex, heart, kidney, placenta, thymus and tonsil using RNA Stat 60 (Tel-Test "B", Friendswood, TX). Additionally, RNA was prepared from the human hematopoietic precursor-like ceU line, THP-1 (ATCC TIB 202), which was induced to differentiate to a macrophage- like phenotype using the phorbol ester phorbolmyristylacetate (PMA). Tissue RNA and RNA prepared from the premyelocytic THP-1 ceU line prior to and 1 to 3 days after induction were electrophoresed through a 1.2% agarose formaldehyde gel and subsequently transferred to a nitrocellulose membrane.
• RNA Stat 60 Tel-Test "B", Friendswood, TX.
• PMA phorbol ester phorbolmyristylacetate
• PAF is synthesized in the brain under normal physiological as weU as pathophysiological conditions. Given the known pro-inflammatory and potential neurotoxic properties of the molecule, a mechanism for localization of PAF synthesis or for its rapid cataboUsm would be expected to be critical for the health of neural tissue. The presence of PAF acetylhydrolase in neural tissues is consistent with it playing such a protective role. Interestingly, both a bovine heterotrimeric intraceUular PAF-AH [the cloning of which is described in Hattori et al. , J. Biol. Chem., 269(31): 23150-23155 (1994)] and PAF-AH of the invention have been identified in the brain.
• the human homologue of the bovine brain intraceUular PAF-AH cDNA was cloned, and its mRNA expression pattern in the brain was compared by Northern blotting to the mRNA expression pattern of the
• PAF-AH of the invention by essentiaUy the same methods as described in the foregoing paragraph.
• the regions of the brain examined by Northern blotting were the cerebeUum, meduUa, spinal cord, putamen, amygdala, caudate nucleus, thalamus, and the occipital pole, frontal lobe and temporal lobe of the cerebral cortex. Message of both enzymes was detected in each of these tissues although the heterotrimeric intraceUular form appeared in greater abundance than the secreted form.
• PAF-AH RNA The expression of PAF-AH RNA in monocytes isolated from human blood and during their spontaneous differentiation into macrophages in culture was also examined. Little or no RNA was detected in fresh monocytes, but expression was induced and maintained during differentiation into macrophages. There was a concomitant accumulation of PAF-AH activity in the culture medium of the differentiating ceUs. Expression of the human plasma PAF-AH transcript was also observed in the THP-1 ceU RNA at 1 day but not 3 days foUowing induction. THP-1 cells did not express mRNA for PAF-AH in the basal state.
• Human tissues were obtained from National Disease Research Interchange and the Cooperative Human Tissue Network. Normal mouse brain and spinal cord, and EAE stage 3 mouse spinal cords were harvested from S/JLJ mice. Normal S/JLJ mouse embryos were harvested from eleven to eighteen days after fertilization.
• tissue sections were placed in Tissue Tek ⁇ cryomolds (MUes Laboratories, Inc. , NaperviUe, IL) with a smaU amount of OCT compound (MUes, Inc. , Elkhart, IN). They were centered in the cryomold, the cryomold fiUed with
• the tissues were hybridized in situ with radiolabeled single-stranded mRNA generated from DNA derived from an internal 1 Kb HindQI fragment of the PAF-AH gene (nucleotides 308 to 1323 of SEQ ID NO: 7) by in vitro RNA transcription incorporation 35 S-UTP (Amersham) or from DNA derived from the heterotrimeric intraceUular PAF-AH cDNA identified by Hattori et al.
• the probes were used at varying lengths from 250-500 bp.
• Hybridization was carried out overnight (12-16 hours) at 50°C; the ""S-labeled riboprobes (6 x IQr cpm/section), tRNA (0.5 ⁇ g/section) and diethylpyrocarbonate (depc)-treated water were added to hybridization buffer to bring it a final concentration of 50% formamide, 0.3M NaCl, 20 mM Tris pH 7.5, 10% dextran sulfate, IX Denhardt's solution, 100 mM dithiothretol (DTT) and 5 mM EDTA.
• DTT dithiothretol
• sections were washed for 1 hour at room temperature in 4X SSC/10 mM DTT, then for 40 minutes at 60 °C in 50% formamide/lX SSC/10 mM DTT, 30 minutes at room temperature in 2X SSC, and 30 minutes at room temperature in 0.1X SSC.
• the sections were dehydrated, air dried for 2 hours, coated with Kodak NTB2 photographic emulsion, air dried for 2 hours, developed (after storage at 4°C in complete darkness) and counterstained with hematoxyUn eosin .
• Cerebellum In both the mouse and the human brains, strong signal was seen in the Purkinje ceU layer of the cerebeUum, in basket ceUs, and individual neuronal ceU bodies in the dentate nucleus (one of the four deep nuclei in the cerebellum). Message for the heterotrimeric intraceUular PAF-AH was also observed in these ceU types. AdditionaUy, plasma PAF-AH signal was seen on individual ceUs in the granular and molecular layers of the grey matter. Hippocampus. In the human hippocampus section, individual ceUs throughout the section, which appear to be neuronal cell bodies, showed strong signal. These were identified as polymorphic cell bodies and granule ceUs. Message for the heterotrimeric intraceUular PAF-AH was also observed in hippocampus.
• Both normal and Crohn's disease colons displayed signal in the lymphatic aggregations present in the mucosa of the sections, with the level of signal being sUghtly higher in the section from the Crohn's disease patient.
• the Crohn's disease colon also had strong signal in the lamina propria. SimUarly, a high level of signal was observed in a diseased appendix section wl ⁇ le the normal appendix exhibited a lower but stiU detectable signal.
• the sections from the ulcerative coUtis patient showed no evident signal in either the lymphatic aggregations or the lamina limbal.
• the expression pattern had differentiated into signal in the cortex, hindbrain (cerebeUum and brain stem), nerves leaving the lumbar region of the spinal cord, the posterior portion of the mouth/ throat, the liver, the kidney, and possible weak signal in the lung and gut.
• PAF-AH mRNA expression in the tonsil, thymus, lymph node, Peyer's patches, appendix, and colon lymphatic aggregates is consistent with the conclusions that the probable predominant in vivo source of PAF-AH is the macrophage because these tisues aU are populated with tissue macrophages that serve as phagocytic and antigen-processing ceUs.
• PAF-AH inflamed tissues would be consistent with the hypothesis that a role of monocyte-derived macrophages is to resolve inflammation. PAF-AH would be expected to inactivate PAF and the pro- inflammatory phosphoUpids, thus down-regulating the inflammatory cascade of events initiated by these mediators.
• PAF has been detected in whole brain tissue and is secreted by rat cerebeUar granule cells in culture.
• PAF binds a specific receptor in neural tissues and induces functional and phenotypic changes such as calcium mobilization, upregulation of transcription activating genes, and differentiation of the neural precursor ceU line,
• Monoclonal antibodies specific for recombinant human plasma PAF-AH were generated using E. coli produced PAF-AH as an immunogen.
• Mouse #1342 was injected on day 0, day 19, and day 40 with recombinant PAF-AH.
• the mouse was injected with the immunogen in PBS, four days later the mouse was sacrificed and its spleen removed sterilely and placed in 10ml serum free RPMI 1640.
• a single-ceU suspension was formed by grinding the spleen between the frosted ends of two glass microscope sUdes submerged in serum free RPMI 1640, supplemented with 2 mM L-glutamine,
• NS-1 myeloma ceUs kept in log phase in RPMI with 11 % fetal bovine serum (FBS) (Hyclone Laboratories, Inc. , Logan, Utah) for three days prior to fusion, were centrifuged at 200 g for 5 minutes, and the peUet was washed twice as described in the foregoing paragraph.
• FBS fetal bovine serum
• the ceU peUet was dislodged by tapping the tube and 1 ml of 37° C PEG 1500 (50% in 75mM Hepes, pH 8.0) (Boehringer Mannheim) was added with stirring over the course of 1 minute, foUowed by adding 7 ml of serum free RPMI over 7 minutes. An additional 8 ml RPMI was added and the ceUs were centrifuged at 200 g for 10 minutes.
• the peUet was resuspended in 200 ml RPMI containing 15% FBS, 100 ⁇ M sodium hypoxanthine, 0.4 ⁇ M aminopterin, 16 ⁇ M thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 x 10 6 thymocytes/ml and plated into 10 Corning flat bottom 96 weU tissue culture plates (Corning, Corning New York). On days 2, 4, and 6, after the fusion, 100 ⁇ l of medium was removed from the wells of the fusion plates and replaced with fresh medium.
• the fusion was screened by ELISA, testing for the presence of mouse IgG binding to recombinant PAF-AH.
• Immulon 4 plates (Dynatech, Cambridge, MA) were coated for 2 hours at 37 °C with 100 ng/well recombinant PAF-AH dUuted in 25mM TRIS, pH 7.5. The coating solution was aspirated and 200ul/weU of blocking solution
• Hybridomas cloned were 90D1E, 90E3A, 90E6C, 90G11D (ATCC HB 11724), and 90F2D (ATCC HB 11725).
• the monoclonal antibodies produced by hybridomas were isotyped using the Isostrip system (Boehringer Mannheim, IndianapoUs, IN). Results showed that the monoclonal antibodies produced by hybridomas from fusion 90 were aU
• Hybridomas were generated as described for fusion 90 but were screened by Western blotting rather than ELISA to identify Western-competent clones.
• recombinant PAF-AH was mixed with an equal volume of sample buffer containing 125mM Tris, pH 6.8, 4% SDS, lOOmM dithiothreitol and 0.05 % bromphenol blue and boUed for five minutes prior to loading onto a 12% SDS polyacrylamide gel (Novex).
• sample buffer containing 125mM Tris, pH 6.8, 4% SDS, lOOmM dithiothreitol and 0.05 % bromphenol blue and boUed for five minutes prior to loading onto a 12% SDS polyacrylamide gel (Novex).
• FoUowing electrophoresis at 40 mAmps proteins were electrotransferred onto a polyvinyUdene fluoride membrane (Pierce) for 1 hour at 125 V in 192mM glycine, 25mM Tris base, 20% methanol, and 0.01 % SDS.
• the membrane was incubated in 20mM Tris, lOOmM NaCl (TBS) containing 5 % bovine serum albumin (BSA, Sigma) overnight at 4°C.
• TBS Tris, lOOmM NaCl
• BSA bovine serum albumin
• the blot was incubated 1 hour at room temperature with rabbit polyclonal antisera dUuted 1/8000 in TBS containing 5 % BSA, and then washed with TBS and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG in TBS containing 5 % BSA for 1 hour at room temperature.
• the blot was again washed with TBS then incubated with 0.02% 5-bromo-4-chloro-3-indolyl phosphate and 0.03% nitroblue tetrazoUum in lOOmM Tris-HCl, pH 9.5, lOOmM NaCl, and 5mM MgCl 2 .
• the reaction was stopped with repeated water rinses.
• Hybridoma 143 A reacted with PAF-AH in Western blots and was cloned (ATCC HB 11900).
• Polyclonal antisera specific for human plasma PAF-AH was raised in rabbits by three monthly immunizations with 100 ⁇ g of purified recombinant enzyme in Fruend's adjuvant.
• Example 14 Experimental studies were performed to evaluate the in vivo therapeutic effects of recombinant PAF-AH of the invention on acute inflammation using a rat foot edema model [Henriques et al, Br. J. Pharmacol , 106: 579-582 (1992)]. The results of these studies demonstrated that rPAF-AH blocks PAF-induced edema. ParaUel studies were done to compare the effectiveness of PAF-AH with two commerciaUy avaUable PAF antagonists. A. Preparation of PAF-AH
• E. coli transformed with the PAF-AH expression vector pue trp AH were lysed in a microfluidizer, soUds were centrifuged out and the ceU supernatants were loaded onto a S -Sepharose column (Pharmacia). The column was washed extensively with buffer consisting of 50mM NaCl, lOmM CHAPS, 25mM MES and
• Purity of PAF-AH isolated in this manner was generaUy 95 % as assessed by SDS-PAGE with activity in the range of 5000-10,000 U/ml. Additional quality controls done on each PAF-AH preparation included determining endotoxin levels and hemolysis activity on freshly obtained rat erythrocytes.
• a buffer containing 25mM Tris, lOmM CHAPS, 0.5M NaCl, pH 7.5 functioned as storage media of the enzyme as weU as carrier for administration. Dosages used in experiments were based on enzyme activity assays conducted immediately prior to experiments.
• Edema was quantitated by measuring the foot volume immediately prior to administration of PAF or zymosan and at indicated time point post-chaUenge with
• Edema is expressed as the increase in foot volume in milliUters. Volume displacement measurements were made on anesthetized animals using a plethysmometer (UGO BasUe, model #7150) which measures the displaced water volume of the immersed foot. In order to insure that foot immersion was comparable from one time point to the next, the hind feet were marked in indeUble ink where the hairline meets the heel. Repeated measurements of the same foot using this technique indicate the precision to be within 5 % .
• PAF-AH was injected locaUy between the foot pads, or systematicaUy by IV injection in the tail vein.
• rats received 100 ⁇ l PAF-AH
• AH (4000-6000 U/ml) deUvered subcutaneously between the right hind foot pads. Left feet served as controls by administration of 100 ⁇ l carrier (buffered salt solution).
• carrier buffer (buffered salt solution).
• rats received the indicated units of PAF-AH in 300 ⁇ l of carrier administered IV in the tail vein.
• Controls received the appropriate volume of carrier IV in the taU vein.
• FIGURE 6 wherein edema is expressed as average increase in foot volume (ml) +. SEM for each treatment group, Ulustrates that PAF-induced foot edema is blocked by local administration of PAF-AH. The group which received local PAF-AH treatment prior to PAF chaUenge showed reduced inflammation compared to the control injected group.
• the group which received 2000 U of PAF-AH given by the IV route showed a reduction in inflammation over the two hour time course.
• Mean volume increase for the PAF-AH treated group at two hours was 0.10 ml ⁇ _ 0.08 (SEM), versus 0.56 ml + . 0.11 for carrier treated controls.
• Control rats were injected IV with a 300 ⁇ l volume of carrier.
• the PAF antagonists were administered IP because they are solubilized in ethanol.
• Rats injected with either CV3988 or Alprazolam were challenged with PAF 30 minutes after administration of the PAF antagonist to allow the PAF antagonist to enter circulation, while PAF-AH and carrier-treated rats were chaUenged 15 minutes after enzyme administration.
• Rats injected with PAF-AH exhibited a reduction in PAF- induced edema beyond that afforded by the established PAF antagonists CV3988 and Alprazolam. See FIGURE 12 wherein edema is expressed as average increase in volume (ml) ⁇ SEM for each treatment group.
• rPAF-AH is effective in blocking edema mediated by PAF in vivo.
• Administration of PAF-AH products can be either local or systemic by
• PAF induced inflammation and may be of therapeutic value in diseases where PAF is the primary mediator.
• the degree of vascular leak was determined by the quantity of Evans blue dye in the pleural space which was quantitated by absorbance at 620 nm. Rats pretreated with PAF-AH were found to have much less vascular leakage than control animals
• Example 16 Recombinant PAF-AH enzyme of the invention was also tested for efficacy in a model of antigen-induced eosinophil recruitment.
• the accumulation of eosinophUs in the airway is a characteristic feature of late phase immune responses which occur in asthma, rhinitis and eczema.
• BALB/c mice (Charles River) were sensitized by two intraperitoneal injections consisting of 1 ⁇ g of ovalbumin (OVA) in 4 mg of aluminum hydroxide (Imject alum, Pierce Laboratories, Rockford, IL) given at a 2 week interval.
• OVA ovalbumin
• Imject alum Pierce Laboratories, Rockford, IL
• mice Fourteen days foUowing the second immunization, the sensitized mice were chaUenged with either aerosolized OVA or saline as a control. Prior to chaUenge mice were randomly placed into four groups, with four mice/group. Mice in groups 1 and 3 were pretreated with 140 ⁇ l of control buffer consisting of 25mM tris, 0.5M NaCl, ImM EDTA and 0.1 % Tween 80 given by intravenous injection. Mice in groups 2 and 4 were pretreated with 750 units of PAF-AH (activity of 5,500 units/ml given in 140 ⁇ l of PAF-AH buffer).
• control buffer consisting of 25mM tris, 0.5M NaCl, ImM EDTA and 0.1 % Tween 80 given by intravenous injection.
• mice in groups 2 and 4 were pretreated with 750 units of PAF-AH (activity of 5,500 units/ml given in 140 ⁇ l of PAF-AH buffer).
• mice in groups 1 and 2 were exposed to aerosoUzed PBS as described below, whUe mice in groups 3 and 4 were exposed to aerosoUzed OVA. Twenty-four hours later mice were treated a second time with either 140 ⁇ l of buffer (groups 1 and 3) or 750 units of PAF-AH in 140 ⁇ l of buffer (groups 2 and 4) given by intravenous injection.
• EosinophU infiltration of the trachea was induced in the sensitized mice by exposing the animals to aerosolized OVA.
• Sensitized mice were placed in 50 ml conical centrifuge tubes (Corning) and forced to breath aerosoUzed OVA (50 mg/ml) dissolved in 0.9% saline for 20 minutes using a nebulizer (Model 646, DeVUbiss Corp., Somerset, PA). Control mice were treated in a simUar manner with the exception that 0.9 % saline was used in the nebulizer.
• Forty-eight hours foUowing the exposure to aerosoUzed OVA or saline mice were sacrificed and the tracheas were excised. Tracheas from each group were inbeded in OCT and stored at -70° untU sections were cut.
• tissue sections from the four groups of mice were stained with either Luna solution and hematoxylin-eosin solution or with peroxidase. Twelve 6 ⁇ thick sections were cut from each group of mice and numbered accordingly. Odd numbered sections were stained with Luna stain as foUows. Sections were fixed in formal-alcohol for 5 minutes at room temperamre, rinsed across three changes of tap water for 2 minutes at room temperamre then rinsed in two changed of H ⁇ O for 1 minute at room temperature. Tissue sections were stained with Luna stain 5 minutes at room temperamre (Luna stain consisting of 90 ml Weigert's Iron hematoxylin and 10 ml of 1 % Biebrich
• SUdes were rinsed in tap water for 5 minutes at room temperamre and 2 drops of 1 % osmic acid was appUed to each section for 3-5 seconds. SUdes were rinsed in tap water for 5 minutes at room temperamre and counterstained with Mayers hematoxyUn at 25 °C at room temperamre. SUdes were then rinsed in running tap water for 5 minutes and dehydrated across 70%-95 %-100% ethanol 1 minute each at room temperamre. Slides were cleared through two changes of xylene for 1 minute each at room temperamre and mounted in Cytoseal 60. The number of eosinophils in the submucosal tissue of the trachea was evaluated.
• mice from groups 1 and 2 were found to have very few eosinophUs scattered throughout the submucosal tissue.
• tracheas from mice in group 3 which were pretreated with buffer and exposed to nebulized OVA, were found to have large numbers of eosinophUs throughout the submucosal tissue.
• the tracheas from mice in group 4 which were pretreated with PAF-AH and exposed to nebulized OVA were found to have very few eosinophUs in the submucosal tissue comparable to what was seen in the two control groups, groups 1 and 2.
• a PAF-AH product of the invention was also tested in two different rat models for treatment of necrotizing enterocoUtis (NEC), an acute hemorrhagic necrosis of the bowel which occurs in low birth weight infants and causes a significant morbidity and mortality.
• NEC necrotizing enterocoUtis
• Previous experiments have demonstrated that treatment with glucocorticoids decreases the incidence of NEC in animals and in premature infants, and the activity of glucocorticoids has been suggested to occur via an increase in the activity of plasma PAF-AH.
• BSA (0.25%)-saUne (groups 1 and 2) or PAF (0.2 ⁇ g/100 gm) suspended in BSA saline (groups 3 and 4) was injected into the abdominal aorta at the level of the superior mesenteric artery 15 minutes after rPH.2 or vehicle injection as previously described by Furukawa, et al. [J.Pediatr.Res. 34:231 -2A1 (1993)].
• the smaU intestines were removed after 2 hours from the Ugament of Trietz to the cecum, thoroughly washed with cold saUne and examined grossly. Samples were obtained from microscopic examination from the upper, middle and lower portions of the small intestine. The tissues were fixed in buffered formalin and the sample processed for microscopic examination by staining with hematoxylin and eosin. The experiment was repeated three times.
• the intestine obtained from groups 1, 2 and 4 demonstrated a normal villous architecture and a normal population of ceUs within the lamina propria.
• the group treated with PAF alone showed a fuU thickness necrosis and hemorrhage throughout the entire mucosa.
• the plasma PAF-AH activities were also determined in the rats utilized in the experiment described above. PAF-AH activity was determined as foUows.
• blood samples Prior to the tail vein injection, blood samples were obtained. Subsequently blood samples were obtained from the vena cava just prior to the injection of PAF and at the time of sacrifice. Approximately 50 ⁇ l of blood was coUected in heparinized capiUary tubes. The plasma was obtained foUowing centrifugation (980 x g for 5 minutes). The enzyme was assayed as previously described by Yasuda and Johnston, Endocrinology, 130: 708-716 (1992).
• the mean plasma PAF-AH activity of aU rats prior to injection was found to be 75.5 jh 2.5 units (1 unit equals 1 nmoles x min "1 x ml "1 plasma).
• the mean plasma PAF-AH activities 15 minutes foUowing the injection of the vehicle were 75.2 +_ 2.6 units for group 1 and 76.7 + . 3.5 units for group 3.
• the plasma PAF-AH activity of the animals injected with 25,500 units rPH.2 was 2249 ⁇ 341 units for group 2 and 2494 +_ 623 units for group 4.
• the activity of groups 2 and 4 remained elevated (1855 + . 257 units) untU the time of sacrifice
• rPH.2 In order to determine if the protection against NEC in rats was dose dependent, animals were treated with increasing doses of rPH.2 15 minutes prior to PAF administration. Initially, rPH.2, ranging from 25.5 to 25,500 units were administered into the tail vein of rats. PAF (0.4 ⁇ g in 0.2 ml of BSA-saline) was subsequently injected into the abdominal aorta 15 minutes after the administration of rPH.2. The small intestine was removed and examined for NEC development 2 hours after PAF administration. Plasma PAF-AH activity was determined prior to the exogenous administration of the enzyme, and 15 minutes and 2 1/4 hours after rPH.2 administration. The results are the mean of 2-5 animals in each group.
• rats were injected once with a fixed amount of the enzyme via the taU vein and subsequently chaUenged with PAF at various time points.
• rPH.2 (8,500 units in 0.3 ml) or vehicle alone was administered into the taU vein of rats, and PAF (0.36 ⁇ g in 0.2 ml of BSA-saline) was injected into the abdominal aorta at various times after the enzyme administration.
• the small intestines were removed 2 hours after the PAF injection for gross and histological examinations in order to evaluate for NEC development.
• Plasma PAF-AH activities were determined at various times after enzyme administration and two hours after PAF administration. The mean value ⁇ _ standard error for enzyme activity was determined for each group.
• rPH.2 To assess the efficacy of rPH.2, three different groups of rats were treated with the compound via enteral deUvery, intraperitoneal deUvery or both.
• the rPH.2 preparation had 0.8 mg/ml protein and approximately 4000 Units/mg PAF-AH activity, with a ⁇ 0.5 EU/mg endotoxin/protein ratio.
• EnteraUy dosed animals were given 25 ⁇ (80 U) of rPH.2 via the orogastric tube dUuted into each feeding (every three hours).
• IntraperitoneaUy dosed animals were given 75 ⁇ by intraperitoneal injection twice daUy. Control animals received appropriate volumes of buffer (20 mM NaPO 4 , pH 7.4) without the rPH.2 and were studied simultaneously with each experimental group. MortaUty and signs of NEC were evaluated for each treatment group, and differences were analyzed statisticaUy using Fischer's Exact test. A p- value of ⁇ 0.05 was considered significant. Results are shown in Table
• Control i.p. admin. 7/10 8/10 rPH.2 (240 U i.p. twice daUy) 6/11 8/11 Control (enteral admin.) 19/26 21/26 rPH.2 (80 U enterally every 3 hours s)) 6/26 7/26
• Control i.p. + enteral admin. 10/17 12/17 rPH.2 (240 U i.p. twice daily and 3/14 7/14 80 U enterally every 3 hours)
• Data represent cumulative results from four different experiments for i.p. dosing, four experiments for enteral dosing, and three experiments for i.p. + enteral dosing.
• the onset of symptoms was simUar between this group and controls (40 + 5 hours in controls vs 36 ⁇ 7 hours in rPH.2-treated rats) and the degree of NEC in both groups was simUar (median score 2.6 in controls vs. 2.5 in rPH.2-treated rats).
• Platelet-activating factor injected intravenously into guinea pigs produces a profound lung inflammation reminiscent of early ARDS in humans.
• a cannula is placed into the jugular vein of anaesthetized male Hartly guinea pigs (approximately 350-400 grams) and PAF dUuted in a 500 ⁇ l volume of phosphate buffered saline with 0.25 % bovine serum albumin as a carrier (PBS-BSA) is infused over a 15 minute period of time at a total dosage ranging from 100-400 ng/kg.
• PBS-BSA bovine serum albumin as a carrier
• NeutrophUs and red blood cells are present in the alveolar spaces of PAF treated guinea pigs but absent in control or sham infused animals.
• Evidence of epithelial cell damage is also evident and pronounced of hyaline membrane formation in human ARDS patients. Protein determinations done on bronchoalveolar lavage
• BAL fluid was collected by lavaging the lungs 2X with 10ml of saline containing 2 ⁇ /ml heparin to prevent clotting.
• samples were dUuted 1:10 in saline and the OD 280 was determined.
• BAL fluid from sham guinea pigs was found to have a protein concentration of 2.10 + 1.3 mg/ml.
• BAL fluid from animals infused with PAF was found to have a protein concentration of 12.55 ⁇ 1.65 mg/ml.
• BAL fluid was found to have a protein concentration of 1.13 + 0.25 mg/ml which is comparable to the sham controls and demonstrates that PAF-AH product completely blocks lung edema in response to
• Rats Male Wistar rats (200-250 g) were purchased from Charles River Laboratories (Wilmington, MA). They were housed in a climate controUed room at 23+2 °C with a 12 hour Ught/dark cycle and fed standard laboratory chow with water ad libitum. Animals were randomly assigned to either control or experimental groups. Rats were anesthetized with 50 mg/kg pentobarbital sodium intraperitoneaUy, and a polyvinyl catheter (size V3, Biolab products, Lake Havasu, AZ) was placed by cutdown into the jugular vein. The catheter was tunneled subcutaneously to exit in the dorsal cervical area, and the animals were allowed to recover from anesthesia.
• a polyvinyl catheter size V3, Biolab products, Lake Havasu, AZ
• the rats were given free access to water but were fasted overnight. Experiments were performed the next day on conscious animals. During the interim, catheter patency was maintained by constant infusion of saline (0.2 ml/h). On the day of the experiment, the animals were intravenously injected with rPH.2 or vehicle control, foUowed by an infusion of either (1) 5 ⁇ g/kg per hour of caerulein for 3.5 hours, or (2) 10 ⁇ g/kg per hour of caerulein for 5 hours, (Research Plus, Bayonne, NJ). Immediately after completion of the infusion, the animals were anesthetized with pentobarbital sodium, their abdomens were opened, and 5 ml of blood aspirated from the inferior vena cava for subsequent assays.
• pancreas was harvested. Pieces of pancreas were either fixed in a 4% phosphate buffered formaldehyde solution for histological examination or immediately deep frozen at -80° C for measurements of myeloperoxidase activity. Additional pieces of pancreas were assessed for pancreatic water content and pancreatic amylase and trypsin as described below. Myeloperoxidase activity, a measure of neutrophU sequestration, was assessed in the pancreas and lung as described below. Pulmonary vascular permeabUity was also assessed as described below. Statistical analysis of the data was accompUshed using unpaired Student's t-test. The data reported represent means + S.E.M. of at least three different experiments. Differences in the results were considered significant when p ⁇ 0.05.
• pancreatic water content Pancreas pieces were blotted dry and weighed (wet weight), and were then desiccated for 34 hrs at 120°C and reweighed (dry weight). Pancreatic water content was calculated as the difference between wet and dry weight and expressed as a percentage of the pancreatic wet weight. A rise in pancreatic water content was considered to indicate the development of edema.
• Amylase activity in serum was measured using 4,6-ethyUdene (G7)-p- nitrophenyl (G ⁇ -c D-maltoplaside (ET-G7PNP) (Sigma Chemical Co., St. Louis, MO) as substrate according to Pierre et al. , Clin. Chem. , 22:1219 (1976).
• Trypsin activity was measured fluorimetricaUy using Boc-Gin- Ala- Arg- MCA as the substrate. Briefly, 200 ⁇ l of the sample and 2.7ml of 50 mM Tris-buffer (pH 8.0) containing 150 mM NaCl, ImM CaC ⁇ and 0.1 % bovine serum albumin were mixed in a cuvette. One hundred ⁇ l of substrate was added to the sample after
• Acinar ceU injury/necrosis was defined as either (a) the presence of acinar ceU ghosts or (b) vacuoUzation and swelling of acinar cells and destmction of the histo-architecmre of whole or parts of the acini, both of which had to be associated with an inflammatory reaction.
• the amount of acinar ceU injury/necrosis and the total area occupied by acinar tissue were each quantitated mo ⁇ hometricaUy using computerized planimetric image analysis video unit (model CCD-72, Dage-MTl, Michigan city, IN) equipped with NIH-1200 image analysis software. Ten randomly chosen microscopic fields (125x) were examined for each tissue sample. The extent of acinar ceU injury/necrosis was expressed as the percent of total acinar tissue which was occupied by areas which met the criteria for injury/ necrosis.
• Obstruction of the common bUiopancreatic duct also typicaUy results in severe pancreatitis-associated lung injury quantifiable by lung vascular permeabiUty and histological examination.
• Two hours before the animals were kiUed an intravenous bolus injection of 5 mg/kg fluorescein isothiocyanate albumin (FITC-albumin, Sigma Chemical Co., St. Louis, MO) was given.
• Pulmonary microvascular permeabiUty was evaluated by quantifying the leakage of FITC-albumin from the vascular compartment into the bronchoalveolar space. Briefly, just after sacrifice, the right bronchus was blocked using a clamp and the trachea exposed.
• the right lung was lavaged by using a cannula inserted into the trachea.
• Three washes of saline (60 ml lavage) were pooled and the FITC fluorescence in serum and lavage was measured at excitation 494 nm and emission 520 nm.
• the fluorescence ratio of lavage fluid to blood was calculated and taken as a measure of microvascular permeabiUty in the lung.
• the lung was also stained with H&E and examined histologicaUy.
• pancreatic edema water content
• histology that were induced by infusion of caerulein alone.
• Administration of rPH.2 also had no effect on caerulein-induced activation of pancreatic trypsinogen or amylase content.
• pancreatitis Infusion of a higher dose of caerulein, 10 ⁇ g/kg/h for 5 hours, to rats resulted in a more severe pancreatitis, characterized relative to the controls by a more pronounced increase in serum amylase activity and pancreatic edema, a marked increase in pancreatic MPO activity, and a significant increase in trypsinogen activation and amylase activity in the pancreas.
• Pancreatic histology indicated not only pancreatic edema and acinar cell vacuoUzation but also some patchy necrosis and a few infiltrating ceUs.
• rPH.2 (5 or 10 mg/kg intravenously) 30 min. before the start of caemlein (10 ⁇ g/kg/h) infusion ameUorated the magnitude of many of the pancreatic changes induced by the infusion of caemlein alone. Results are shown in Table 10 below.
• rPH.2 treatment at a dose of 5mg/kg resulted in decrease of serum amylase activity (from 10984+ 1412 to 6763 + 1256). The higher 10 mg/kg dose of rPH.2 did not result in further improvement of hyperamylasemia.
• pancreatitis associated lung injury has been observed both cUnicaUy and in several models of pancreatitis. Infusion of caemlein at 5 ⁇ g/kg/h for 3.5 h, which resulted in a mUd form of pancreatitis, did not result in significant injury to the lungs. However, infusion of caemlein at 10 ⁇ g/kg/h for 5 hours, which resulted in more severe pancreatitis, also resulted in lung injury quantified by increased lung vascular permeabiUty (0.31 +0.04 to 0.79+0.09), lung MPO activity (indicating neutroplul sequestration) and neutrophU infiltration on histological examination.
• rPH.2 at a dose of 5 mg/kg 30 min prior to caemlein infusion significantly ameliorated the rise in lung MPO activity induced by the infusion of caemlein alone (3.55 + 0.93 for caemlein alone vs. 1.51 ⁇ 0.26 for caemlein with rPH.2).
• rPH.2 treatment significantly decreased the severity of microscopic changes observed in the lung tissue after caemlein infusion.
• the caerulein-induced increase in lung vascular permeabiUty was reduced by rPH.2 treatment, although not statistically significant.
• the higher 10 mg/kg dose of rPH.2 was no more effective than the lower dose in decreasing the severity of caerulein- induced lung injury.
• CER 5 mg/kg 10 mg/kg (no CER) lO ⁇ g/kg/h rPH.2 rPH.2
• a ceUotomy was performed through a midline incision under sterile conditions and the common bUe pancreatic duct was Ugated in aU animals to induce acute necrotizing pancreatitis. Additionally, the cystic duct was Ugated to prevent the gallbladder from serving as a bUe reservoir.
• the animals were randomly assigned to either control or experimental groups. Starting at Day 2 after Ugation of the pancreatic duct, the experimental group received 5 mg/kg body weight per day of rPH.2 (suppUed in a 4mg/ml solution) intravenously via the taU vein, while the control group received an intravenous injection of the same volume of placebo vehicle only. After 1 and 2 days of treatment (at Day 3 and Day 4 after Ugation of the pancreatic duct) the animals were euthanized by a sodium-pentobarbital overdose. Blood samples were drawn from the heart for measurements of serum amylase, serum
• pancreas Upase and serum bUirubin, and the pancreas was harvested. Pieces of pancreas were either fixed in a 4% phosphate buffered formaldehyde solution for histological examination or immediately deep frozen at -80°C for measurements of myeloperoxidase activity. Additional pieces of pancreas were assessed for pancreatic water content and pancreatic amylase as described above in section A of this example.
• Myeloperoxidase activity was assessed in the pancreas as described above. Pulmonary vascular permeabiUty was also assessed as described above.
• results reported represent mean + standard error of the mean (SEM) values obtained from multiple determinations in 3 or more separate experiments.
• rPH.2 Intravenous administration of rPH.2 (5 mg/kg/day) starting at Day 2 after Ugation of the pancreatic duct ameUorated the magnitude of many of the pancreatic changes induced by duct obstruction and placebo treatment alone.
• One day of rPH.2 treatment reduced semm amylase levels in comparison to placebo treated animals, although the difference was not statisticaUy significant, and two days of rPH.2 treatment (at Day 4 after Ugation of the pancreatic duct) significantly reduced se m amylase levels compared to placebo.
• rPH.2 One or two days of rPH.2 treatment reduced semm Upase levels relative to controls, although the difference was not statistically significant. Two days of rPH.2 treatment reduced pancreatic amylase content relative to controls, although one day of treatment resulted in an increase in pancreatic amylase. Treatment with rPH.2 was not observed to affect semm bilirubin levels, pancreas myeloperoxidase activity or pancreas water content.
• the major characteristic histological changes induced by obstruction of the bUiopancreatic duct included marked necrosis, infiltration of inflammatory ceUs, acinar ceU vacuoUzation, and marked distention of the acinar lumina.
• Mo ⁇ hometrical examination of the pancreas for acinar ceU injury showed a major protective effect of rPH.2 on the pancreas after one and two days of rPH.2 treatment. After one day of rPH.2 treatment, the acinar cell injury was reduced to about 23 % of total acinar cell tissue, compared to 48 % injury for the placebo-treated animals. This reduction of acinar ceU injury was even more pronounced after two days of treatment, at which time rPH.2 treatment resulted in about 35% injury of the total acinar ceU tissue, compared to about 60% injury for the placebo-treated animals.
• Lung vascular permeabUity quantified by FITC injection showed a highly significant difference after one and two days of rPH.2 treatment compared to placebo group. Histological examination of the lung showed severe lung injury in aU placebo-treated animals. Lung mjury was characterized by an extensive inflammatory response with interstitial and intraalveolar infiltration of mainly macrophages, lymphocytes and neutrophUs, and by a patchy but marked interstitial edema and thickening of the alveolar membranes. Administration of rPH.2 resulted in a marked decrease of infiltration of inflammatory cells and a reduction of interstitial edema at aU times.
• HIV-1 Human immunodeficiency virus type 1
• HIV- 1 -infected monocytes activated by a variety of antigenic stimuU, including contact with neural cells, secrete high levels of neurotoxic pro-inflammatory cytokines, including PAF.
• the effect of rPH.2 on the neurotoxicity of conditioned media from HIV-infected and activated monocytes was assessed.
• Monocytes were infected with HIV and activated as foUows. Monocytes were recovered from peripheral bone marrow ceUs (PBMC) of HIV- and hepatitis B-seronegative donors after leukopheresis and purified (> 98%) by countercurrent centrifugal elutriation as described in Genis et al , J. Exp. Med. , 176: 1703-1718 (1992). CeUs were cultured as adherent monolayers (1 x 10 4 ceUs/ml in T-75 culmre flasks) in DMEM (Sigma, St. Louis, MO) with recombinant human macrophage colony stimulatory factor (MSCF) (Genetics Institute, Inc.
• Human cerebral cortical neuron cell cultures were estabUshed as follows.
• Human fetal brain tissue was obtained from the telencephalon of second trimester (13-16 weeks gestation) human fetal brain tissue according to a modified procedure of Banker and Cowan, Brain Res. , 126:391-A25 (1977). Briefly, brain tissue was coUected, washed in 30 ml of cold Hank's BSS (containing Ca and Mg - " 1 1 - ⁇ + 25 mM HEPES, and 5X gentamicin), separated from adherent meninges
• Nitex bag and gently triturated through a flame-poUshed Pasteur pipet 10-15 times.
• the tissue was centrifuged at 550 rpm, 5 minutes, 4°C, and the peUet was resuspended in 5-10 ml of MEM-hipp (D-glucose, 5 grams/Uter; L-glutamine, 2 mM;
• HEPES 10 mM; Na pyruvate, 1 mM; KC1, 20 mM) containing NI components
• CeUs were tently triturated 5 times with a 10 ml pipet and plated at a density of 10 5 ceUs/12 mm glass coversUp pre-coated with poly-L-lysine (70K-150K MW, Sigma, St. Louis, MO) placed in 24 weU culmre dishes. One ml of media was pipetted into each culmre weU. CeUs were cultured for 10-28 days at 37°C in a humidified atmosphere of 5% CO2/95% air, changing media every 3 days. Under these conditions, cultures were > 60-70% homogeneous for neurons, with 20-30% astrocytes, ⁇ 1 % microglia and ⁇ 10% macrophage and microgUa staining.
• neuronal cultures express sufficient levels of N-methyl-D-aspartate (NMDA) or non-NMDA receptors to die after excitotoxic doses of NMDA or alpha-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid (AMP A).
• NMDA N-methyl-D-aspartate
• AMP A alpha-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid
• the neurotoxicity assay was conducted as foUows.
• the test samples which were (a) conditioned media from LPS-stimulated HIV-1 infected monocytes, (b) control media, (c) conditioned media with added rPH.2 at 51 ⁇ g/ml or (d) conditioned media with added vehicle for rPH.2, were appUed to the neuronal ceU cultures at a 1:10 v/v concentration for 24 hours.
• Neurotoxicity was measured by identifying apoptotic nuclei in situ on neuronal coversUps fixed in 4% paraformaldehyde, employing a commercial kit (Apop Tag; ONCOR, Gaithersburg, MD) that uses terminal deoxynucleotidyl transferase (TdT) to bind digoxigenin-dUPT to free 3' -OH ends of newly cleaved DNA (TUNEL staining). Digitized images of TUNEL-stained neurons in _>_15 randomly selected microscopic fields were analyzed for number of TUNEL-stained nuclei/number of total neurons per 50X field using computerized mo ⁇ hometry (MCID, Imaging Research, St. Catherine, Ontario, Canada).
• Apop Tag ONCOR, Gaithersburg, MD
• TdT terminal deoxynucleotidyl transferase
• TUNEL staining Digitized images of TUNEL-stained neurons in _>_15 randomly selected microscopic fields were
• the plates were blocked for 1 hour at room temperature with 0.5 % fish skin gelatin (Sigma) dUuted in CMF-PBS and then washed three times.
• Patient plasma was dUuted in PBS containing 15mM CHAPS and added to each weU of the plates (50 ⁇ l/weU).
• the plates were incubated for 1 hour at room temperature and washed four times.
• Fifty ⁇ l of 5 ⁇ g/ml monoclonal antibody 90F2D which was biotinylated by standard methods and dUuted in PBST, was added to each well, and the plates were incubated for 1 hour at room temperature and then washed three times.
• E. coli expression construct containing the mutation was generated by methods simUar to that described in Example 10.
• the expression constmct generated no PAF-AH activity whUe a control constmct lacking the mutation was fully active.
• This amino acid substitution presumably results in a structural modification which causes the observed deficiency of activity and lack of immunoreactivity with the PAF-AH antibodies of the invention.
• PAF-AH specific antibodies of the invention may thus be used in diagnostic methods to detect abnormal levels of PAF-AH in semm (normal levels are about 1 to 5 U/ml) and to foUow the progression of treatment of pathological conditions with PAF-AH. Moreover, identification of a genetic lesion in the PAF- AH gene aUows for genetic screening for the PAF-AH deficiency exhibited by the Japanese patients. The mutation causes the gain of a restriction endonuclease site (Mae II) and thus allows for the simple method of Restriction Fragment Length
• CAA ACT AAA ATC CCC CGG GGA AAT GGG CCT TAT TCC GTT GGT TGT ACA 365 Gin Thr Lys lie Pro Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr 55 60 65
• GCA ACG GTT ATT CAG ACT CTT AGT GAA GAT CAG AGA TTC AGA TGT GGT 103 Ala Thr Val lie Gin Thr Leu Ser Glu Asp Gin Arg Phe Arg Cys Gly 280 285 290
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• ACCCTCCAAA ACCCCTACAC AGTGTTTCAA ACAGAGAGAC CCTCAATAAT TGCATATCTT 120
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• CTGGAGGAGT TGGGGTTCCT CAATAATTGG CTGTGGGTCT ATTGATCAGT CCTAGACCTG 420
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• CAGAACTCTT CAGGAATAGA GAAATACAAT TAGGATTAAA ATAGGTTTTT TAAAAGTCTT 300 GTTTCAAAAC TGTCTAAAAT TATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGAGTGAGAGAG 360
• ATC CCA AAC AAA GAA TAT TTT TTG GGT CTT AGT ATA TTT CTT GGA ACA 452 He Pro Asn Lys Glu Tyr Phe Leu Gly Leu Ser He Phe Leu Gly Thr 100 105 110
• GAA CAC AGA GAC AGA TCT GCA TCG
• GCA ACT TAC TTT TTT GAA
• GAC CAG 692 Glu His Arg Asp Arg Ser Ala Ser Ala Thr Tyr Phe Phe Glu Asp Gin 180 185 190

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