SK47399A3 - Truncated platelet-activating factor acetylhydrolase - Google Patents

Abstract

The present invention provides purified and isolated polynucleotide sequences encoding human plasma platelet-activating factor acetylhydrolase. Also provided are materials and methods for the recombinant production of platelet-activating factor acetylhydrolase products which are expected to be useful in regulating pathological inflammatory events.

Description

Truncated PAF acetyl hydrolase

-activating factor ^

Technical field

The present invention relates to

CAH) Platelet Activating Factor hydrolyzing hydrolyzes) and more specifically the newly purified and isolated polynucleotides encoding human plasma PAF acetyl hydrolase and PAF-AH products encoded by polynucleotides. Further materials and methods of producing recombinant PAF-AH products and antibodies against PAF-AH.

BACKGROUND OF THE INVENTION

PAF is a biologically active phospholipid synthesized by various cell types. In vivo, and at normal concentrations of 10 ^-10 to 10 ^-9 M PAF, activates target cells (such as platelets and neutrophils) by binding to specific surface receptors associated with G proteins CVenable et al., J. Lipid Res., 34: 691- 701 (C1993)).

PAF has the structure 1-Cl-alkyl-2-acetyl-sn-glycero-3-phosphocholine. For optimal biological activity, a fatty alcohol must be attached at the s-1 position of the PAF backbone and a s-3 position must have a phosphocholoryl leader group.

PAF works in the normal physiological processes of Cnapr. inflammation, haemostasis and delivery) and its role is assumed in the case of pathological inflammatory processes Cnapr. asthma, anaphylaxis. septic shock and arthritis) CVeriable et al., supra, Lindsberg et al., Ann. Neurol., 30: 117-129 (1998). The probability of the role of PAF in pathological responses has led to attempts to modulate PAF activity. Most of these experiments have focused on the development of PAF activity antagonists that would interfere with the binding of PAF to cell surface receptors. See e.g. Heuer et al., Clin. Exp. Allergy. 22: 980-983 (1992).

Synthesis and secretion of PAF, its degradation and purification are apparently highly controlled, to the extent that the pathological action of PAF- may result from the breakdown of PAF regulatory mechanisms. This leads to its overproduction, inappropriate production or inability to degrade. An alternative source of modulating PAF activity would include mimicking or augmenting the natural processes that distinguish inflammation. Macrophages (Stafforini et al., J. Biol. Chem. 265 (17), 9682-9687 (1990). Hepatocytes and HepG2 Human Hepatum Cell Lines (Saton et al., J. Clin. Invest., 87, 476-). 481 (1991) and Tarbet et al., J. Biol. Chem. 266 (25) = 16667-16673 (1991) described the release of the enzymatic activity, PAF acetyl hydrolase (PAF-AH), which inactivates PAF. inactivates PAF, PAF-AH also inactivates oxidatively fragmented phospholipids such as the arachidory cascade products that transmit inflammation (see Streinler et al., J. Biol. Chem., 266 (17) < 11095-11103 (1991)). PAF PAF-AH is mainly due to the hydrolysis of PAF to the sn-2 acetylation group and PAF-AH metabolizes oxidatively fragmented phospholipids by removing the sn-2 acyl scapil Two types of PAF-AH have been identified cytoplasmic form found in different cell and tissue types as are endothelial and erythrocytes and extracellular form found in plasma and serum. hydrolyzes intact phospholipids with the exception of PAF, and this substrate specificity allows the enzyme to circulate in vivo in a fully active state without adversely affecting it. Plasma PAF-AH appears to be responsible for all degradations of PAF in human blood ex vivo (Stafforini et al., J. Biol. Chem., 162 (9), 4223-430 (1987)).

It appears. whereas, while the cytoplasmic and plasma forms of PAF-AH have identical substrate specificity, plasma PAF-AH has biochemical properties. which differ from cytoplasmic PAF-AH and other 11pases. In particular, plasma PAF-AH is associated with lipoprotein particles, is digested with disopropyl fluorophosphate. it is relatively insensitive to proteolysis and has a molecular weight of 43,000 Da (see Stafforini et al. (1987), supra). The same Stafforini paper describes a process for partial purification of PAF-AH from human plasma and the amino acid composition of the material obtained using this process. Cytoplasmic PAF-AH was purified from erythrocytes (Stafforini et al., J. Biol. Chem., 268 (6) t 3857-3865 (1993)) and ten amino acid residues from the amino-terminus of cytoplasmic PAF-AH were also described in the article . Hattori et al., J. Biol. Chem. 268 (25) with 18748-18753 described purification of cytoplasmic PAF-AH from bovine brain. Following a patent application, the nucleotide sequence of bovine brain cytoplasmic PAF-AH was published in Hattori et al., J. Biol. Chem., 269 (237) with 23150-23155 (1994). On January 5, 1995, three months after filing a patent proposal for this document, the nucleotide sequence of the lipoprotein-associated phospholipase As (Lp-PLA-a) with the Smithkline Beecham PLC Patent Cooperation Treaty (PTC) was published. U095 / 00,469th The nucleotide sequence of Lp-PLA _α is different in one position compared to the nucleotide sequence of PAF-AH of the present invention. The nucleotide difference (corresponding to position 1297 of SEQ ID NO: 7) resulted in an amino acid difference between the enzymes encoded by these polynucleotides. The amino acid at position 379 of SEQ ID NO: 8 is valier, while the amino acid at the corresponding position. Lp-PLAa is alanine. In addition, the nucleotide sequence of the PAF-AH of the present invention comprises 124 bases at the 5 'end and 20 bases at the 3' end not present in the Lp-PLA- sequence. U24577 deposited Lp-PLA _α sequence, which differs by 11 positions compared to the nucleotide sequence of PAF-AH of the present invention. Nucleotide differences (corresponding to positions 79, 81, 84, 85, 86, 121, 122, 904, 905, 911, 983 and 1327 of SEQ ID NOs 7) resulted in four amino acid differences for the enzymes encoded by these polynucleotides. The amino acids at positions 249, 250, 274 and 369 of SEQ ID NO: 8 are lysine, aspartic acid, phenylalanine, and leucine, while the corresponding amino acids at the appropriate positions in the GenBank are isoleucine, arginine. leucine and serine.

Recombinant production of PAF-AH would allow the use of exogenous PAF-AH to mimic or augment normal processes to recognize inflammation in vivo. Administration of PAF-AH would provide a physiological advantage over the administration of PAF receptor antagonists, since PAF-AH is a substance normally found in plasma. PAF receptor antagonists that are structurally related to PAF also inhibit native PAF-AH activity. Thus, administration of PAF-AH would protect the desired metabolism of PAF and the metabolism of oxidatively fragmented phospholipids. In fact, inhibition of PAF-AH activity by PAF-AH receptor antagonists obstructs competitive blockade of PAF receptor by antagonists (Cpozri Stremler et al., Supra). In addition, at the site of acute inflammation, for example, oxidans are released which lead to inactivation of the native PAF-AH enzyme, resulting in increased local levels of PAF and PAF-like wells (PAF-like). These agents may further compete with any exogenously administered PAF receptor antagonist for binding to the PAF receptor. In contrast, treatment with recombinant PAF-AH would increase the endogenous activity of PAF-AH, thereby compensating for any inactivation of the endogenous enzyme. Thus, there is a need in the art for the identification and isolation of a polynucleotide sequence encoding human plasma PAF-AH, and for the development of material and mebodes useful for recombinant production of PAF-AH and production of substances for detecting PAF-AH in plasma.

SUMMARY OF THE INVENTION

The present invention provides novelly purified and isolated polynucleotides (e.g., DNA and RNA - both 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 partially or totally chemically synthesized DNA sequences. The present invention contemplates DNA sequences encoding PAF-AH that are described in SEQ ID NO: 7 and DNA sequences that hybridize under stringent conditions to the coding strand of the sequence or that would hybridize for redundancy of the genetic code. Also contemplated by the invention are biological replicas (e.g., copies of isolated DNA sequences generated in vivo or in vitro) of the DNA sequences of the invention. Autonomously replicating recombinant constructs are also provided. such as plasmid and viral vectors incorporating PAF-AH sequences, and in particular vectors wherein the DNA encoding PAF-AH is operably linked to endogenous or exogenous expression control DNA sequences and transcriptional terminators.

According to another aspect of the invention, prokaryotic and eukaryotic host cells are stably transformed with DNA sequences of the invention in a manner allowing the desired expression of PAF-AH within these cells. Host cells expressing PAF-AH products can serve a variety of useful targets. Such cells create a valuable source of immunogens for the development of antibody substances specifically immunoreacting with PAF-AH. The host cells of the invention are clearly useful in large-scale PAF-AH production methods, wherein the cells are grown in suitable culture media and the desired polypeptide products are isolated from these cells or from the medium. in which the cells were grown, for example by immunoaffinity purification.

The non-immunological methods contemplated by the invention for plasma PAF-Ah purification include the steps of a) isolating low density lipoprotein particles; b) solubilizing the above low density lipoprotein particles in a buffer containing 10 mM CHAPS. thus forming a first solution of the PAF-AH enzyme; c) loading the aforementioned first PAF-AH enzyme solution onto a DFAF anion-1 n-nex column; d) washing the above DEAE anion-exchange column with a pH of about 7.5 containing 3 ml of CHAPS; e) eluting the PAF-AH enzyme. the aforementioned DEAE anion-1 non-exchanging column of dn fractions with a pH buffer of about 7.5 containing a NaCl gradient of 0 to 0.51 * 1; f) pooling fractions having PAF-AH enzymatic activity eluted from the above-mentioned DFAF anion-1-exchanger column; g) transferring the aforementioned pooled active fractions from the aforementioned DEAE anion-innex clone dn 10ml * 1 CHAPS. thus, a second solution of the PAF-AH enzyme was generated; h) loading the aforementioned second enzyme solution onto a blue dye Igg affinity column; 1) eluting the PAF-AH enzyme from the above affinity column with blue dye ligandoin buffer containing 10 mM CHAPS and chaotropic salts; j) loading the eluate from the above blue dye ligand affinity column onto a Cu 11gand affinity column; k) eluting the enzyme PAF-AH from the above affinity column with a Cu ligand buffer containing 10 mM CHAPS and Imidazole; 1) loading the eluate from the above affinity Cu11gand column on SDS-PAGE; and m) isolating the approximately 44 kDa PAF-AH enzyme from the SDS polyalkrylamide gel. Preferably, the buffer in step b) is 25 ml * 1 Tris-HCl, pH CHAPS, pH 7.5; the buffer in step d) is 25 mM Tris-HCl, 1 mM CHAPS; the column of step h) is a Blue Sepharose Fast Flu column; the buffer in step i) is 25 mM Tris-HCl, 10 ml * 1 CHAPS, 0.5 KSCN, pH 7.5; the column in step j) is a Cu Chelating Sepharose column and the buffer of step k) is 25ml * 1 Tris-HCl, 10mM CHAPS, 0.51 * 1 NaCl. 50ml * I imidazole at a pH in the range of about 7.5-8.0.

The method contemplated by the invention for purifying enzymatically active PAF-AH from f. a PAF-AH producing coli comprising the steps of: b) loading the above centrifugation supernatant onto a blue dye 11gandom affinity column; c) eluting the PAF-AH enzyme from the above-described blue dye ligand affinity column containing 10 mM CHAPS and chaotropic salts; d) loading the eluate from the above blue dye ligand affinity column onto a Cu ligand affinity column; e) eluting the enzyme PAF-AH from the above affinity column with a Cu ligand buffer containing 10 mM CHAPS and imidazole. Preferably, the column of step b) is a Blue Sepharose Fast Floui column; the buffer of step c) is 25 ml * 1 Tris-HCl, 10 mM CHAPS, 0.5 M KSCN. pH 7.5; the column in step d) is a Cu Chelating Sepharose column and the buffer of step e) is 25 mM Tris-HCl, 10 mM CHAPS, 0.5 M NaCl. 50mM Imidazole, pH 7.5.

Another method of purifying the enzymatically active PAF-AH from E. coli producing PAF-AH is contemplated in the invention. This method includes the following steps; a) preparing a centrifugation supernatant from lysed E. coli producing PAF-AH 'enzyme; b) diluting the above centrifugation supernatant in a low pH buffer containing 10 mM CHAPS; c) loading the above diluted centrifugation supernatant onto a cation exchange column equilibrated to a pH of about 7.5; d) eluting the PAF-AH enzyme from the above cation-exchange IM column with salt; e) raising the pH of said eluate from said cation exchange column and adjusting the salt concentration of said eluate to a salt concentration of about 0.51 * 1; f) loading the above eluate from the above cation-exchange column onto a blue dye 11gandom affinity column; g) eluting the enzyme PAF-AH from the above affinity column with a blue dye ligandoin buffer containing salts at a concentration of about 2 µl to 31 µl; h) dialyzing the above eluate from the above blue dye 11gandom affinity column using a buffer containing about 0. 1X Tween. Preferably, the buffer in step b) is 25ml * 1 MES. 10mM CHAPS. lml * l EDTA. pH 4.9; the column in step c) is a S Sepharose column equilibrated in 25 µl * 1 MES, 10 ml * 1 CHAPS, 1 mM EDTA, 50 ml * 1 NaCl. pH 5.5; PAF-AH is eluted in step d) with 1 ml * 1 NaCl; The pH of the eluate in step e) is adjusted to pH 7.5 using 21 * 1 Tris-base; the column in step f) is a sepharose column; the buffer in step g) is 25 ml * 1 Tris. 10ml * 1 CHAPS. 31 * 1 NaCl. lml * l EDTA. pH 7.5 and the buffer in step h) is 25 ml * 1 Tris, 0.5 µl NaCl, 0.1X Tween 8D, pH 7.5.

Another method of purifying enzymatically active PAF-AH from E. coli is contemplated in the invention. This method comprises the following steps of a) preparing an E. coli extract which, after lysis in a buffer containing CHAPS, results in a supernatant containing solubilizable PAF-AH; b) diluting the above supernatant and applying it to an anion exchange column equilibrated to a pH of about 8.0; c) eluting the PAF-Ah enzyme from the above anion-exchange column; d) applying said adjusted eluate from said anion-exchange column to a blue dye ligand containing affinity column: d) eluting said blue dye ligand column with a buffer containing 3, D1 * 1 salt; f) diluting the eluate from the column containing the blue dye ligand to a buffer suitable for performing hydroxylapatite chromatography; g) performing hydroxylapatite chromatography, wherein the washing and elution is carried out using Cs buffers containing Ci without CHAPS); h) not diluting the hydroxylapatta eluate to a cation exchange resin; i) applying a hydroxylapatta eluate to the aforementioned salts suitable for the aforementioned diluted cation-ion exchange column at a pH in the range of about 6. D to 7.0; j) eluting PAF-AH from the above cation-exchange column with a suitable buffer of a suitable composition; k) performing cation-exchange chromatography in the cold; and 1) the composition of a liquid or frozen form of PAF-AH in the absence of CHAPS.

Preferably, in the above step: a) the lysis buffer is a different composition. 25ml * l Tris. 100mL NaCl. lml * l EDTA. 20inch CHAPS. pH 8.0; in step b), the dilution of the supernatant for anion-exchange chromatography is 3-4 times to 25 ml of Tris. 1µl EDTA, 10ml * 1 CHAPS. pH 8. Q and column is O-Sepharose column equilibrated with 25 µM Tris. 1 µl EDTA, 50 µl NaCl. 10mL CHAPS, pH 8.0; in step c), the anion exchange column is eluted using 25 ml Tris. 1 µL EDTA, 350 µL NaCl, 10 µL CHAPS, pH 8.0; in step d), the eluate of step c) is applied directly to the blue dye 11gandom affinity column; in step e) the column is eluted with a buffer containing 3 µl of NaCl. 10ml CHAPS, 25ml Tris. pH 8.0; in step f), the blue dye eluate is diluted for hydroxylapatite chromatography. The eluate is diluted with 10 ml sodium phosphate, 10 ml ml NaCl. 10mL CHAPS, pH 6.2; in step g), hydroxylapatite chromatography is performed using a hydroxylapatite column equilibrated with 10mL sodium phosphate, 100mL NaCl, 10mL CHAPS. Elution is performed using 50 ml of sodium phosphate. 100mL NaCl C with Ci without 10mL CHAPS), pH 7.5; in step h) the dilution of the above hydroxylap bit eluate for cation-exchange chromatography is carried out with a buffer having a pH in the range of about 6.0 to

7. 0 and containing sodium phosphate Cs (without CHAPS); in step i) the S-Sepharose column is equilibrated with SOmil 1 sodium phosphate (with Ci without 10mil CHAPS), pH 6.8; in step j), the elution is carried out with a buffer of a suitable composition, such as, for example, SO1, potassium phosphate. 12, 5ml acid.

and containing 0.012 Tween 80;

cation-ion exchange chromatography at 2-8 ^e C. An example for a suitable composition of the buffer usable in step 1) that stabilizes PAF-AH is SO1 / ml potassium phosphate, 12.5. aspartic, 125mL NaCl, pH about 7.4 Cs Ci without the addition of Tween 80 and / or Plurlnic F68) or 25mL phosphate buffer (C *) containing C at least 125'mL NaCl. 25 ml of arglin and 0. 012 Tween-80 C with Ci without Pluronlck aspartic, 125 ml of NaCl. The pH of 7.5 in step k) is carried out

F68 w at a concentration of approximately 0.1 and 0.5%).

said HIC # 1 by reducing the concentration of detergents; HIC # 1 to a pH of about 6.4; g)

Yet another method of purifying the enzymatically active rPAF-AH product δ. coli is contemplated in the invention. The method comprises the following steps * a) preparing an E. coli extract which, after lysis in a buffer containing Triton X-100, results in a solublized supernatant of the rPAF-AH product; b) diluting the above superriatant and loading it onto an immobilized metal affinity exchange column equilibrated to a pH of about 8.0; c) eluting the rPAF-AH product from the above affinity exchange column with an immobilized metal buffer containing imidazole; d) adjusting the salt concentration and loading the above eluate from the immobilized metal affinity exchange column onto a hydrophobic column (Chydrophobic interaction column (CHICtt1)); e) eluting a higher salt concentration and / or increasing f) titrating the aforesaid eluate from loading the aforesaid conditioned eluate from HCl (CCl 1) onto a cation-ion exchange column (equilibrated to a pH of about 8.4; h) eluting the above CEXtt1 using sodium chloride concentration; 1) adjusting the above eluate from CEX # 1 with sodium chloride to a concentration of about 2.01 * 1; j) loading the above eluate from CEXtt1 onto a hydrophobic column (Chydrophobic iriteractiori column CHIC # 2) equilibrated to a pH of about 8.0 and a sodium chloride concentration of about 2.01 * 1; k) eluting the above HIC # 2 by reducing the salt concentration and / or increasing the detergent concentration; 1) diluting the above eluate from HIC # 2 and adjusting the pH to about 6.0; m) loading the above treated eluate from HIC82 onto a cation-ion exchange column (CCEX # 2) equilibrated to about pH 6.0; n) eluting the rPAF-AH product from the above CEX # 2 buffer with a suitable composition.

It is best to use lysis buffer 90ml * I TRIS, 0.125X Triton X-100 in step a) above. 0. 61 * 1 NaCl, pH 8. 0. The lysis is carried out in a high-pressure homogenizer; in step b), the supernatant is diluted with 20 ml * 1 TRIS equilibration buffer. 0.51 * 1 NaCl. 0, 1X Triton X-100. pH 8.0). zinc chelate column COhelatlng Sepharose Fast Flou, Pharmacia. Uppsala. Sweden) is charged and equilibrated with equilibration buffer. The undiluted supernatant is applied to the column and the column is washed with 20 ml of TRIS buffer. 0. 51 * 1 NaCl. 41 * 1 urea, 0. IX Triton X-100. pH 8.0 and further washed with 20ml * 1 TRIS. 0.51 * 1 NaCl, 0.02% Triton X-100. pH 8.0; in step c) elution is accomplished with a 20mL Tris solution, 50ml * 1 Imidazole, 0.02% Triton X-100, pH 8.0; in step d) the eluate is adjusted to 1 ml * 1 EDTA and 21 * 1 NaCl, a Phenyl Sepharose 6 Fast Flow (Pharmacia) column is equilibrated with Equilibration Buffer C 2. 01 * 1 NaCl, 25 ml * 1 Tris, 0.02% Triton X-100 pH 8.0) and the eluate of step c) is applied at room temperature. The column is washed with equilibration buffer and further washed with 25 ml * 1 NaPO ₄ , 0.20% Triton X-100, pH 6.5 at a rate of 30 cm / hr. ; in step

e) elution is accomplished with 25 ml * 1 NaPCLi solution. 3% Triton X-100, pH 6.5; in step g), the illo-prep High S Column (Bio-Rad Labs, Richmond, CA, USA) is equilibrated with equilibration buffer (20ml * 1 NaPCLi, 0.02% Triton X-100, pH 6.4). The conditioned eluate from step f) is applied to the column, washed with equilibration buffer and further washed with a buffer of 25 ml * 1 NaPCL2, 0.02% Triton X-100, pH 8.0; in step h) elution is accomplished with a 25ml * 1 NaPO _{4 solution} . 0.02% Triton X-100, 1.31 * 1 NaCl, pH 8.0; in step j), Bakerbond Wide Pore Hi-Propyl C ₃ (Baker, Philipsburg, NJ) is equilibrated with equilibration buffer (0.02% Triton X-100, 21 * 1 NaCl, 25ml * 1 Tris, pH 8.0). The treated eluate of step i) is applied to this column at room temperature, the column is washed with equilibration buffer and further washed with a solution of 0.20% Triton X-100, 25 ml Tris, pH 8.0 at a rate of 30 cm / h; in step k), elution was accomplished using a 10ml * 1 Tris, 3.0% Triton X-100 solution. pH 8.0; in step 1) is not diluted in equilibration buffer (20mL * succinate, 0.1% PLURONIC F68 pH 6.0); in step m) the SP Sepharose Fast Flow column (Pharmacia) is equilibrated with the equilibration buffer of step 1) and the eluate from step 1) further loaded onto it is washed with equilibration buffer; and in step n) elution is accomplished with a 50ml * 1 NaPCl * solution. 0. 71 * 1 NaCl. 0.1% PLURONIC F68. 0.02% TUEEN 80, pH 7.5.

PAF-AH products may be obtained as isolates from natural cell sources or may be chemically synthesized.

Preferably, however, they are produced by recombinant procedures involving the host eukaryotic and prokaryotic cells of the invention. PAF-AH products having part or all of the amino acid sequence described in SEQ ID NO: 8 are contemplated in the present invention. In particular, fragments that lack up to the first twelve N-terminal amino acids of the human PAF-AH amino acid sequences described in SEO ID NO: 8 are contemplated. Preferably, those having the Initial N-terminal amino acid 1 * let ₄ > Ala ₄₇ or Ala _4a SEO ID NO: 8. Also contemplated are fragments of this lacking up to thirty C-terminal amino acids of the amino acid sequence of SEO ID NO: 8, particularly those having a Leu ₄₃₁ as the C-terminal residue. Also contemplated are variants of PAF-AH or PAF-AH, or those having an amino group replaced in the sequence of SEO ID NO: 8 and selected from the group consisting of S108A, S273A.

D 286 N. D 296 A, D 304 A, D 338 A, H 351 A, H 395 A. H 399 A. C 67 S, C 229 S, C 291 S, C 334 S. C 407 S, D 286 A, D 286 N and D 304 A. As noted above, polynucleotides (including DNA) encoding such fragments or fragment variations are provided by the invention. Also provided by the invention are methods of recombinantly producing these fragments or variations by growing host cells containing such DNA. At the same time preferred PAF-AH products include the prokaryotic polypeptldové expression products of DNA encoding residues amiriokyselinové L * _{4 a} to let Asn _{44 tons} SEQ ID NO: 8, and rPH.2 called polypeptldové prokaryotic expression products of DNA encoding of amino acid residues L * ₄ years _<*, and Ile ₄₂ 9 SEO ID NO: 8 RPH.9. Both rPH.2 and rPH.9) exhibit lower amino terminal heterogeneity than. for example, the corresponding prokaryotic polypeptide expression products of DNA encoding the entire sequence of PAF-AH with the preceding translational initiation codon. In addition, rPH.9 exhibits higher carboxy-terminal homogeneity (consistency). The use of mammalian host cells is contemplated to provide for such posttranslational modifications (e.g., myristal, glycosylation, truncation, human and tyrosine-, serine- and threonine phosphorylation) that may be required to confer optimal biological activity of the recombinant expression products of the invention. The PAF-AH products described herein may be full length, may be fragments or variations. Variants may include PAF-AH analogues where one or more Criapr is specified. naturally encoded) amino acids selected or replaced or wherein one or more unspecified amino acids are added 1) without losing one or more PAF-AH-specific enzymatic activities or immunological characteristics, or 2) with a specific inability to a particular PAF-AH biological activity. Proteins or other molecules that bind to PAF-AH can be used to modulate its activity.

Also contemplated by the present invention are antibody substances (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR grafted antibodies, and the like) and other PAF-AH-specific binding proteins. In particular, the demonstrative binding proteins of the invention are monoclonal antibodies produced by the hybridomas 90G11D and 90F2D, which have been deposited in the American Type Culture Collection (CCCC), 12301. Parklawn Drive, Rockville, IN ID 20852, Sep. 30, 1994 and assigned Accession Nos. HB 11724 and HB 11725. Another demonstrative binding protein of the invention is a monoclonal antibody produced by hybridoma 143A, which has been deposited in the ATCC

1 June 1995 and assigned to him by Accession No. is HB 11900. Proteins or other molecules of Cnapr. lipids or small molecules) that specifically bind to PAF-AH can be identified using plasma-isolated PAF-AH, recombinant PAF-AH, variants of PAF-AH, or cells expressing such products. The binding proteins are subsequently useful in compositions for immunization as well as for purification of PAF-AH. They are also useful for detecting or quantifying PAF-AH in fluids and tissue samples using known immunological techniques. Antlidlotypic antibodies specific for PAF-AH specific antibody substances are also contemplated.

The scientific value of the information provided through the disclosure of the DNA and amino acid sequence of the present invention is apparent. One of a series of examples of knowledge of the cDNA sequence for PAF-AH allows the use of DNA / DNA hybridizations to isolate genomic DNA sequences encoding PAF-AH and to specify PAF-AH expression control regulatory sequences such as promoters, operators, and the like. DNA / DNA hybridization procedures performed with the DNA sequences of the invention under standard stringency conditions also seem to permit isolation of DNA encoding allelic variants of PAF-AH, other structurally related proteins. which share one or more of the biochemical and / or immunological properties of PAF-AH as well as protein homologues of PAF-AH from non-human sources. The DNA sequence information of the present invention also permits using or knockout strategies (see, e.g., Kapecchi,

1288-1292 (1989)), the development of rodents that are unable to express a functional PAF-AH enzyme or express a variant of the PAF-AH enzyme. The suitably labeled polyriucleotides described herein are useful in hybridization assays to detect the capacity of cells to synthesize PAF-AH. The polynucleotides described in the invention may also form the basis of diagnostic methods useful in the identification and genetic engineering of a PAF-AH center that highlights a disease state or conditions. The invention also makes available antisense polynucleotides important in regulating the expression of PAF-AH by those cells that usually epxrlmme the same.

provided by homologous recombinations Science. 244

Administration of the PAF-AH preparations of the invention to mammalian subjects, particularly humans, for the purpose of ameliorating pathological inflammatory conditions is contemplated. Based on evidence of PAF involvement in pathological inflammatory conditions, administration of PAF-AH is indicated e.g. in the treatment of asthma (Liwa et al., J. Clin Invest. 82, 1983-1991 (1988) with Hsieh et al., J. Allergy Clin., Immunol., 91, 650-657 (1993), Yamashida et al. Allergy., 49, 60-63 (1994)), anaphylaxis (Venable et al., Supra), shock (Venable et al. Supra), reperfusion injury and central nervous system ischemia (Lindsberg et al. (1991) , supra), antigen-induced arthritis (Zarco et al., Clin. Exp. Immunol. 88: 318-323 (1992)). atherogenesis (Handley et al., Drug Dev. Res. 7: 361-375 (1986)) Crohn's Disease (Denlzot et al., Dgestive Diseases and Sciences, 37 (3), 432-437 (1982)). ischemic intestinal necrosis / necrotizing enterocolitis (Denizot et al., supra and Caplan et al., Acta Paediatr., Suppl. 396, 11-17 (1994)), ulcerative colitis (Denizot et al., supra), ischemic cerebral episodes (Satori et al., Stroke 23: 1090-1092 (1992)), ischemic brain injury (Llndsberg et al., Stroke, 21, 1452-1457 (1990) and Llndsberg et al. (1991), supra) systemic lupus erythematosus (Matsizaki et al., Clinica Chimica Acta, 210: 139-144 (1992)), acute paricreatitis (Kald et al., Pancreas, 8 (4): 440-442 (1993)), blood poisoning ( Kald et al., Supra), acute post-streptococcal gloinerulonephritis (Mezzario et al., J. Am. Soc. Nephrol., 4: 235-242 (1993)), pulmonary edema in response to IL-2 treatment (Rablriovici et al. , J. Clin. InvesL., 89 (1669-1673 (1992)), allergic inflammation (Watanabe et al., Br. J. Pharmacol., 111: 123-130 (1994)), ischemic renal failure (Grino ela. , Annals of Internal! Leddclrie, 121 ( 5): 345-347 (1994)), premature labor (Hoffman et. al .. AM. J. Obstet. Gynecol. 162 (2): 525-528 (1990) and Makl. et al., Proc. Natl. Acad. Sci. USA, 85: 728-732 (1988)), adult respiratory insufficiency syndrome (Rabinovici et al., J. Appl. Physiol., 74 (4): 1791-1802 (1993). Matsuinoto et al. Clin. Exp. Pharmacol. Physiol., 19: 509-515 (1992) and Rodriguez-Roisin et al., J. Clin Invest., 93: 188-194 (1994) .The use of PAF-AH formulations for the treatment of HIV central nervous system infection is also The treatment as used herein includes both prophylactic and therapeutic treatments.

For many of the aforementioned pathological conditions, an experimental animal model has been described. For example, a mouse model for asthma and rhinitis is described in Example 16 of this document; a rabbit model for arthritis is described in Zarco et al., supra; rat models for intestinal ischemic necrosis / necrotizing enterocollosis are described in Furukaua et al., Ped. Res., 34 (2): 237-241 (1993) and Caplan et al., Supra; the rabbit stroke model is described in Lndsberg et al., (1990), supra; the mouse model for lupus is described in Matsuzaki et al., supra; the rat model for acute pancreatitis is described in Kald et al., supra; rat! the model for pulmonary edema as a result of IL-2 therapy is described in Rabinovici et al., supra; rat! the allergic inflammatory model is described in Uatanable et al., supra; a canine model for kidney allograft is described in Uatson et al. Transplantatiori, 56 (4) «1047-1049 (1993) and rat and guinea pig models of adult respiratory distress syndrome are separately described in Rabinovici et al., supra and Lellouch-Tubiana. , Am. Rev. Respir. Dis. 137 (948-954, C1988).

Particularly contemplated in the invention are PAF-AH compositions useful in a method of treating a mammal which is readily subject to or suffering from a pathological condition caused by PAF. Such treatment comprises administering PAF-AH to the mammal in amounts sufficient to complement the endogenous activity of PAF-AH and to inactivate pathological levels of PAF in the mammal.

The therapeutic-pharmacological composition contemplated by the invention includes PAF-AH products and physiologically acceptable diluents or carriers. It may also include other substances having anti-inflammatory effects. The indicated dose amount would be sufficient to complement the endogenous activity of PAF-AH and to inactivate pathological amounts of PAF. For general consideration of dosages, see Remmingtori's Pharmaceutical Sciences, 18 ^tH Editlon. Plack Publishing Co., Easton, PA (1990). Dosages will range between 0.1 to about 100 µg PAF-AH / kg body weight. The therapeutic compositions of the invention may be administered by various routes depending on the pathological conditions to be treated. Administration can be, for example, intravenously, subcutaneously, orally. in the form of suppositories and / or pulmonary pathways.

In the case of pathological lung conditions. administration of PAF-AH by the pulmonary route is preferably indicated. For pulmonary administration, a wide variety of delivery devices are contemplated for these purposes. e.g. nebulizers, measuring inhalers and powder inhalers. Delivery of Various Proteins to the Lung and Circulatory System Inhalation of aerosol mixtures has been described in Adjel et al., Pharm. Res. 7 (6): 565-569 (1990) (leuprolide acetate); Braquet et al., J. Cardio. Pharm., 13 (Supp.5) <p. 143-146 (1989) (endothelin-1); Hubbard et al., Annals of Internal Medicine. ΙΓΓ (3). 206-212 (1989) (? -Antitrypsin) with Smith et al., J. Clin Invest., 84s 1145-1146 (1989) (? -1-proteinase Inhibitor); Debs et al., J. Immunol., 140, pp. 3482-3488 (1993) (recombinant gamain-terferin and tumor necrosis factor alpha); Patent Cooperation Treaty (PCT) US 94/20069 published September 15, 1994 (recombinant pegylated granulocyte colony stimulating factor).

Describe the figures in the drawings

A number of other aspects and advantages of the present invention will be apparent from the following detailed explanations in which reference will be made to the drawings in which:

Fig. 1s A photograph of a PVDF membrane containing PAF-AH purified from human plasma.

Fig. 2s Graph showing the erizymatic activity of recombinant human plasma PAF-AH.

Fig. 3s Schematic drawing showing recombinant fragments of PAF-AH and their catalytic activities.

Fig. 4 - Mass spectroscopy of rPH.2 of PAF-AH.

Fig. 5 &apos; RPH.9 spectroscopy of PAF-AH.

recombinant ombinant recombinant

Fig. 6: Bar graph illustrating blockade of PAF-induced rat foot edema by locally administered recombinant PAF-AH of the invention.

Fig. 7 Bar graph illustrating blockade of PAF-induced rat foot edema by intravenously administered recombinant PAF-AH.

Fig. 8s Bar graph showing that PAF-AH blocks PAF-induced edema, but not zymosan A-induced edema.

Fig. 9A and 9Bs Dose-dependent arithi-inflammatory activity of PAF-AH in rat foot edema.

Fig. 10A and 10B: In vivo Time Dependence of Single Dose PAF-AH.

Fig. 11: Line graph showing the pharmacokinetics of PAF-AH in rat bloodstream.

Fig. 12: Bar graph showing the inflammatory effects of PAF-AH compared to the lower effect of PAF antagonists in rat foot edema.

Fig. 13: Representation of results indicating that PAF-AH neutralizes the apoptotic effects of medium from HIV-1 infected and activated monocytes.

DETAILED DESCRIPTION OF THE INVENTION

The following examples serve to illustrate the invention.

Example 1 discloses a new method for purifying PAF-AH from human plasma. Example 2 describes the microsquencing of amino acid residues of PAF-AH purified from human plasma. Example 3 describes the cloning of the cDNA of the entire Cfull lenth gene (cDNA) encoding human plasma PAF-AH. In Example 4, various variants of the composition of the gene encoding human plasma PAF-AH are described. Cloning of gene sequences encoding human plasma PAF-AH is described in Example 5. Example 6 describes cloning of cDNA homologs for human plasma PAF-AH for canine. mouse, bovine, chicken, rodent and cynomolgus cDNAs. Example 7 describes the results of a test demonstrating recombinant enzyme activity

PAF-AH transiently expressed in COS 7 cells. Example 8 describes the expression of DNA representing a full-length DNA strand, truncated DNA, and and chimeric DNA for human PAF-AH in E. coli, S. cerevisiae, and mammalian cells. Example 9 sets forth a protocol for the purification of recombinant PAF-AH from E. coli and assays confirming its enzymatic activity. Example 10 describes various recombinant products for PAF-AH including amino acid residue analogues and amino and carboxykone-derived products. Further, there is described an experiment that native PAF-AH isolated from plasma is the result of Northern truncation analyzes to prove glycosylated.

blots to demonstrate the expression of human plasma PAF-AH RNA in various tissues and cell lines are presented in Example 11. whereas the 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 describe in vivo animal models the therapeutic effects of administration of recombinant PAF-AH products of the invention on acute inflammation; pleural inflammation, asthma, necrotizing enterocolitis (intestinal catarrh, adult respiratory distress syndrome, and paricreatitis. Example 20 describes the in vitro effect of recombinant PAF-AH product on HIV-associated neurotoxicity. Example 21 shows the results of immunoassay examinations in human sera deficiency patients -AH and describes the identification of genetic disorders in patients that are obviously responsible for this deficiency.

Example 1

Purification was performed to obtain material for the amino acid sequence of human plasma PAF-AH.

A. Optimization of Purification Conditions

First, low-density lipoprotein particles (LDL5 lipoprotein-LDL5) were removed from the plasma by preciprocating plasma phosphovate, followed by solubilization in 0, 1X Tween 20, followed by DEAE CPharmacia column chromatography, Upforala, Suffen, Sueden and others. C1987), see above, but inconsistent elution of PAF-AH activity from the DEAE column required re-evaluation of solubilization and subsequent purification conditions.

Tween 20, CHAPS (Pierce Chemical Co., Rockford, IL) and octyl glucoside were evaluated in centrifugation methods and gel filtration chromatography used for their ability to solublize LDL particles. CHAPS showed a 25X greater activity recovery in the soluble fraction than Tween 20 and a 300X greater activity yield than octylglucoside. The LDL precipate solubilized with 10 mM CHAPS was then separated on a DEAE Sepharose Fast Flcw column (an anion exchange column; Pharmacia) using a buffer containing linol.

CHAPS. This gave a large PAF-AH CDEAE pool). which columns.

the amount of partially uncleaned was used to evaluate others

The DEAE pool was used as a starting material to test other different chromatographic columns that were used to further purify the PAF-AH activity. Among the columns tested were x Blue Sepharose Fast Flou (coloring), an affinity chromatography column with color 11gand; S-Sepharose Fast Flou (Pharmacia); Cu Chelatirig Sepharose (Pharmacia), affinity chromatography column using metal Uganda; Fractogel S (EM Separations, Gibbstoun. NJ), a cation exchanger and Sephaacryl -200 (Pharmacia), gel filtration column. However, all these chromatographic procedures yielded only a low degree of dilution when Sephacryle 1mM CHAPS chromatography was used. Subsequent gel

S-200 in 1mM CHAPS gave an enzymatically active fraction, but eluted from the column over a larger size range than predicted size of approximately 44 kDa. This finding suggests that LDL proteins are aggregated in solution.

Different LDL samples were therefore evaluated by analytical gel filtration, where the degree of aggregation and the effect on PAF-AH activity were evaluated. Samples from the DEAE pool and Freshly Dissolved LDL Precipitates were analyzed on a Superose 12 (Pharmacia) column equilibrated with 1 ml of CHAPS. Both samples were eluted from the column over a wide molecular weight range. The high activity was found in a fraction with a molecular weight of 150 kDa. When samples were analyzed on a Superose 12 column equilibrated with 10 ml CHAPS. The greatest amount of activity was found in a fraction with a molecular weight of approximately 44 kDa, which is the region where activity was predicted. Samples that contained activity in the high molecular weight fractions corresponded to the aggregates.

The activity of the other samples, after subsequent gel filtration testing, was found exclusively in the molecular weight range corresponding to 44 kDa. These samples were LDL precipitates dissolved in 10 ml * 1 CHAPS in the presence of 0.5 L * 1 NaCl and further samples from DEAE pool, which were treated after column elution with 10 ml * 1 CHAPS. The results obtained confirm that at least 10 ml * 1 CHAPS should be used to prevent aggregation of PAF-AH samples. Increasing the CHAPS concentration from 1 ml * l to 10 ml * l after chromatography on a DEAE cellulose column, but prior to the next steps of the purification process, caused dramatic changes in the purification process. For example, the degree of purification of PAF-AH is determined in the column

S-Sepharose FAST Flou was increased from two to ten times. PAF-AH activity binds in 1 m CHAPS to a Blue Sepharose Fast Flou column irreversible, but when used with 10 ml * 1 CHAPS, a very high level of activity is eluted from the column. However, the chromatography on the DEAE column did not improve with the addition of 10 mM CHAPS over previous chromatographies.

Cu column chromatography followed chromatography to increase PAF-AH activity by 15-fold. It has also been found that PAF-AH activity can be restored after electrophoresis on a reducing SDS-polyacrylamide gel. however, the sample must not be cooked before electrophoresis. Activity of material obtained by elution from Cu column

Chelating Sepharosy. which Blue Sepharose Fast Flou,

Chelating Sepharose, after electrophoresis on an SDS polyacrylamide gel and subsequent silver staining, occurred in the main band of the protein.

B. Protocol for Purification of PAF-AH

The new protocol for purifying PAF-AH for amino acid sequencing targets comprises the following steps, which are carried out at 4 ° C. Human plasma was dispensed in 900 ml aliquots into 1-liter Nalgene bottles and the pH was adjusted to 8, G. LDL The particles were then precipitated by the addition of 90 inl 3, 85% sodium phosphovate, followed by 21 * 1 MgCl 2 ... The plasma was then centrifuged for 15 minutes at 3800 g. The pellet was resuspended in 800 ml of 0.2% sodium citrate. LOL The particles were reprecipitated by the addition of 10 g NaCl and 24 ml 2N PlgCl ₂ . LDL Particles obtained from 5 L of human plasma were dissolved in 5 L of buffer A (25 ml * 1 Tris-HCl, 10 ml * 1 CHAPS, pH 7.5) and allowed to stir overnight. The dissolved LOL particles were centrifuged at 3800 g for 1.5 hours. The supernatant was collected, then filtered through Uhatman 113 filter paper to remove any remaining solids. Solubilized LOL particles were loaded onto a DEAE Sepharose Fast Flou column (11 x 10 cm; resin volume 1 L; 80 mL / min) that was equilibrated with C25mPI Tris-HCl buffer, 1 mL * 1 CHAPS. pH 7.5). The column was washed with 8 L of a 0-0.5 µl NaCl gradient and 480 mL fractions were collected. This step was necessary to bind the compounds to the Blue Sepharose Fast Flou column as described below. Fractions were assayed for acetyl hydrolase activity by the method described in Example 4.

The fractions showing activity were pooled and CHAPS was added to give a concentration of 10ml * l over the entire fraction. All fractions obtained after chromatography on DEAE were loaded overnight at 4 ml / min. to a Blue Sepharose Fast Flou column (5 cm x 10 cm, 200 ml dead volume) equilibrated with 0.5 L * 1 NaCl buffer. The column was washed with equilibration buffer at a rate of 18 ml / ml until the absorbance was returned to the absorbance measured before sample application. PAF-AH activity was eluted from the column with step elution buffer A containing 0.5 M KSCN (Caotropic salt) and tn at a rate of 16 ml / ml. 50 ml fractions were collected. This step resulted in more than a thousand fold purification of the enzyme preparation. Active fractions were collected, then adjusted to pH 8.0 and tn with 11 * 1 Tris-HCl, pH 8.0 throughout their collected volume. The active fractions obtained after Blue Sepharose Fast Flou column chromatography were loaded onto a Cu Chelatin Sepharose C2 column, 5 x 2 cm, dead volume 10 ml; flow rate 4 ml / min), which hol. equilibrated with C buffer C25ml * 1 Tris-HCl, 10ml * 1 CHAPS. 0.5L * NaCl, pH R, 0. A buffer of pH 7.5) may also be used. The column was eluted with a volume of 50 ml of buffer C. PAF-AH activity was eluted from the 100 m column. 50 µl of imidazole in buffer C and 10-ml fractions were collected. The eluent with active PAF-AH was collected and allowed to analyze against buffer A. In addition, the activity of the enzyme preparation PAF-AH was increased 15-fold by this step. Chromatography on a Cu Chelating Sepharose column gave very little purification. The active eluent from the Cu Chelating Sepharose column was reduced in 50mL of DTT for 15 minutes at 37 ° C and then loaded onto a 0.75mm 7.5X acrylic amide gel. The gel plates were cut into strips with a width of 0.5cm and placed in single-use centrifuge micro-cells containing 20 µl. 25ml * 1 Tris-HCl, 10ml * 1 CHAPS, 150ml * 1 NaCl. The rod strips were then ground and left to incubate in the cuvette overnight at 4 ° C. The supernatant produced by centrifugation of these crushed gel bands was then tested for PAF-AH activity to determine which band on the SDS-PAGE gel contained PAF-AH. PAF-AH activity was found in a band that corresponds to a molecular weight of approximately 44 kDa. Duplicate gel proteins were electrically transferred to the CTmmnbilon-P PVDF membrane. Millipore) and stained with Comassie Blue. A photograph of the PVDF membrane is shown in Figure 1.

As shown in Table no. 1 below. 5 1 of human plasma was obtained about 200 .mu.g PAF-AH impure 2 x 10 ^Å. For comparison. Stafforini. and others (C1987). see above. described in his procedure the purification of 3 x 10 * activity.

Table 3

Vzn- volume Akt.1 - On the whole, Horses. spe- Tí · ná- fold saying, CML) WELCOMES Additive pro- C vr a t- naCistenia Ccpmx The activity teí- fic- the company 3.0 *) WELCOMES new Moon ka The activity Ccpmx C mg / The activity Vity 3 0 * ml) vľi ta Ccpmx C cpmx 10 10 *) step tot. step tot

plasma 5000 23 115 62 0. 37 100 3:00 1 1 L.DI. 4500 22 97 1, 76 12 84 84 33 33 DEAF 4200 49 207 3 .. 08 46 '212 3.78 3.7 124 Blue 3.65 891 14 0. 02 54200 70 1.26 11.90 1, 5x 3.0® Cu 3.2 12700 3:52 0. 3.5 82200 104 131 1. 5 2. 2x 3.0® SDS-PAGE - - - - - - - 3.0 ~ 2, 2x

10 = ⁵

In summary, the following steps are unique and critical for successful purification of PAF-AH 7 plasma for microsquencing: * 1) dissolution and chromatography on 10 ml * 1 CHAPS, (2) affinity chromatography on a blue 11gandom column, such as Blue Sepharose Fast Flou, C3) chromatography on Cu ligand affinity carrier, such as Cu Chelating Sepharose and C4) eluting PAF-AH from gel after SDS-PAGE.

Example 2

A PVDF membrane size band containing the PAF-AH sequence of proteins present in the Applied protein sequencer of about 44 kDa was excised from the Cp described in Example 1).

has been determined by

Biosystems 473A. Sequence analysis. The N-terminal region of proteins migrating in the region of about 44 kDa, which exhibits PAF-AH activity, indicated the presence of two major and two minor sequences. The ratio of the two major sequences was 1: 1, so it was difficult to interpret the sequence data.

In order to differentiate, the sequences of the two major proteins separated on an SDS gel, a duplicate of a PVDF membrane with a band of about 44 kDa was split and the upper and lower portions were sequenced in sequence.

The N-knn sequence obtained from the lower half of the membrane is as follows:

SEQ ID NO: 1

FKDLGEEAFKALVLIAF

After searching the protein databases, this sequence was identified by the n fragment of human serum albumin. The N-knock-down sectors determined for the material of the upper half of the same PVDF membrane are as follows:

SEQ ID NO: 2

IQVLMAAASFGQTKIP

This sequence had no counterpart in any protein from the databases examined and differed from the N-terminal amino acid sequence:

SEQ ID NO: 3 MKPLVVFVLGG described for erythrocyte cytoplasmic PAF-AH in Stafforini et al. (1993), supra. The new sequence (SEO Tf) NO: 2) was used in cloning r.DNA of human plasma.

PAF-AH as described in Example 3 below.

Example 3

A clone encoding full human plasma PAF-AH was isolated from a macrophage cDNA library.

A. Construction of a macrophage cDNA library

7. Poly A + RNA was isolated from peripheral blood macrophages derived from monocytes. Double stranded, blunt-ended cDNA was obtained using Invitrogen Copy Kit (Diego Diego, CA), ligated with BstXI adapters, and then inserted into a mammalian expression vector (pRcCCMV (Invitrogen)). The resulting plasmid was introduced by electroporation into E. coli XL-1 Blue. Transformed bacteria were seeded on 978 plates at a density of about 3000 colonies per plate. Plasmid DNA prepared separately from each plate was stored in individual pools, and was also pooled into larger pools, reprezeritizing 300,000 clans.

B. Library Testing by PCR

The macrophage library was tested by a polymerase chain reaction using degenerate antisense primers designed based on the new N-terminal amino acid sequence described in Example 2. The primer sequence is given in accordance with the IUPAC nomenclature, I 'means inosine.

SEQ ID NO: 4

5 'ACATGAATTCGGIATCYTTIGTYTGICCRAA 3'

Nuc. Acids Res., 19S: Nucleotides on Third For Testing Files

Codon selection table (for example, et al.,

1981-1986 (1991) was used to determine the position of each codon of this primer.

This primer was used in combination with a primer specific for either the SP6 or T7 promoter sequence located on both sides of the cloning site of the pRc / CI * IV vector. Each PCR reaction mixture was composed of 3.00 ng template cDNA. 1 µl of each of the two primers, 0, 125 ml * 1 of each of the four dNTPs, 1Π ml * 1 of Tris-HCl pH 8.4, 50 ml * 1 of MgCl 2 and 2.5 units of Taq polymerase. The initial step of at denaturačriý 4 minutes at 94 ° C it], followed by 30 cycles amplifl cation composed of 1 min at 94, 1 min at 6Π ° C and 2 minutes at 72 ^e C. The resulting PCR product, the alcohol nakloriovaný the vector pBluescript (Ctratagene, La Jolla, CA) and its nucleation sequence determined by the dinidene method. This PCR product contained a sequence deductible nd to the new peptide sequence corresponding to nucleotides 3 to 331. (CSFC1 ID Nfls 7).

The following are PCR primers specific for the above-described clonovirus PCR fragment and designed to find a full-length clan.

Sense primer CSFO 10 RΠ »R)

5 'TATTTCTAGAAGTGTGGTGGAACTCGCTGG 3'

Antisense primer CSEC3 TO ΝΠ «6)

5 'CGATGAATTCAGCTTGCAGCAGCCATCAGTAC 3'

PCR reactions using these primers were performed as described above and served for the first testing of cDNA sets with 300 0ΟΠ spans, and then for testing the corresponding smaller sets with 3000 clonml. Three sets of 3000 ions that gave the PCR product with the expected size rot used to transform the bacteria.

C. Testing the library by hybridization

The DNA from these bacteria was tested by hybridization using the original cloned PCR fragment as a probe. Colony holi. transferred to nitrocellulose, prehybridized and hybridized in 502 formam1.de, 0.75M sodium chloride, 0, Π75ΓΙ sodium citrate, 0, Π5Μ sodium phosphate, pH 6.5. 12 polyvinylpyrrolidine, 12 ficolls, 12 bovine serumaltium and 50 ng / ml sonicated salmon ONA. The hybridization probe was labeled with a raridoin priming procedure with hexamers. Hybridization was performed at. 42 ° C overnight. Filters were washed by 0.031 1 * with sodium chloride, sodium citrate 3niľl, 0.12 SOS at 42 ^o C. The nucleotide sequence was determined for the 10 hybridizing clones signal. One of these clones. sAH 406-3, contained a sequence deducible based on the original peptide sequencing of the protein with PAF-AH activity purified from human plasma. The DNA sequences and amino acid sequence derived therefrom are shown in SEO ID NO: 7 (DNA) and SEO ID NO: 8 (amino acids).

The sAH 406-3 clone contains an 1.52 kb insert with an open reading frame that encodes a putative protein of 441 amino acids. At the N-terminus, there is a relatively hydrophobic region of 41 amino acids in front of the N-amino acid determined by the sequence of the Clzoleucine protein at position 42 in SEQ ID NO: 8). The encoded protein may have either a long signal sequence or a signal sequence plus an additional peptide that is cleaved to yield a mature functional enzyme. The presence of a signal sequence is one of the traits of secreted proteins. In addition, in the protein encoded by the clone sAH 406-3 there is a consensus motif GxSxG (amino acids 271-275 in SEO ID NO: 8), which is believed to contain a series of active site for all known mammalian and microbial lipases and seroproteases, see Chapus et al., Bloahimie. 701 1223-1224 (1988) and Brenner, Nature, 334: 528-530 (1988).

Table 2, below, compares the amino acid composition of human plasma PAF-AH that is the subject of this patent, based on the Sequence ID NO: 8 with the amino acid composition of the purified material described by Stafforini et al. (1987), supra.

Table 2

Kloň sAH 406-3 Stafforini et al. Ala 26 24 Asp & Asn 48 37 Cys 5 14 Glu & Gin 36 42 Phe 22 12 Gly 29 58 His 13 24 Ile 31 17 Lys 26 50 Leu 40 26 Met 10 7 for 15 11 Arg 18 16 Ser 27 36 Thr 20 15 wall 13 14 Trp 7 undetermined ' Tyr 14 13

The amirio-acid composition of the mature form of human plasma PAF-AH which is the subject of this patent, and the amirio-acid composition of the previously purified material, which has been designated human plasma PAF-AH, is distinctly different.

Comparison of the nucleotide and deduced amino acid sequence of bovine brain cytoplasmic PAF-AH (Hattori et al., Supra) with the human plasma PAF-AH nucleotide sequence of the present invention revealed no significant structural similarity to these sequences.

Example 4

PCR performed with cDNA from macrophages and stimulated PBMC using primers hybridizing to the 5 'untranslated region (nucleotides 31 to 52 in SEQ ID NO <7) and regions containing translational termination coders at the 3' end of the PAF-AH cDNA (nucleotides 1465 to 1487 in SEQ ID NO: 7) found the putative splice variant of the human PAF-AH gene. This PCR reaction resulted in two bands on the gel. one corresponding to the expected size of the PAF-AH cDNA of Example 3 and the other shorter by about 100 bp. The larger band was identified by sequencing with the PAF-AH cDNA of Example 3. while the shorter band was absent from the exune 2 (see Example 5 below) of the PAF-AH sequence, which encodes the putative signal and propeptide sequence of plasma PAF-AH.

In the shorter clone, the putative catalyst residue and all cystelin residues are present. therefore, the biochemical activity of the protein encoded by this clorium is likely to be identical to that of the plasma enzyme.

To determine the biological significance of this splicing variant of PAF-AH, which is believed to encode a cytoplasmic active enzyme, the relative amount of both forms in inocyte-derived blood inacrophages was examined by the RNase protection method. Neither of the two mRNAs was present in the freshly isolated monocytes, but both mRNAs were found on day 2 during in vitro differentiation of moriocytes into macrophages and persisted for 6 days of culture. The number of mRNA admissions was approximately the same throughout the differentiation period. In contrast, a similar analysis of nerve tissue detected only full-length mRNA expression encoding the extracellular full-length form of PAF-AH.

Example 5

Human plasma PAF-AH genomic sequences were also isolated. The structure of the PAF-AH gene was determined by isolating lambda α 1 phage clones containing human genomic DNA. using stringency. Fragments and sequenced by

DNA hybridizations under high phage clone conditions were further cloned with primers associated at regular intervals with the cDNA clone of sAH 406-3. In addition, new sequencing primers were prepared homologous to the exon-bordering intron regions and used for upstream sequencing across the exon-intron boundaries to confirm the sequence. The exon / intron boundaries were defined as points where the genomic sequence and cDNA begin to differ. This analysis revealed the composition of the human

PAF-AH gene from 12 exons.

Exons 1, 2, 3, 4, 5, 6 and part of exon 7 were isolated from a male fetal placental library constructed in lambda FIX (Stratagene). The phage plaques were transferred to nitrocellulose, prehybridized and hybridized in 50% formamide, 0.75 µl sodium chloride, 75 ml * 1 sodium citrate, 50 ml * 1 sodium phosphate (pH 6.5). 1% polyvinylpyrrolidone. 1% ficola, 1% bovine serumalbumine and 50 ng / ml sonicated salmon milk DNA. The complete cDNA clone of sAH 406-3 was used as a hybridization probe to identify phage clones containing exons 2-6 and part of exon 7. The clone containing exor 1 was identified by a fragment derived from the 5 'end of this cDNA clone (nucleotides 1 to 312 in SED ID). NO: 7). Both probes were labeled with ^3S p by random priming with hexamers. Hybridization was performed at 42 ° C overnight, membranes were extensively washed with 30 ml * 1 sodium chloride, 3 ml * 1 sodium citrate, 0.1% SDS at 42 ^e C. DNA sequences of exons 1,2,

3, 4, 5 and 6 together with partial sequences of adjacent Introns are given in SEO ID NO: 9, 10, 11, 12, 13 and 14.

The remainder of exon 7 and exons 8, 9, 10, 11 and 12 were cloned from a PI clone isolated from a human PI genomic library. PI phage plaques were transferred to nitrocellulose, prehybridized and hybridized in 0. 751 * 1 sodium chloride, 50ml * 1 sodium phosphate (pH 7.4), 5mL EDTA, 1% polyvinylpyrrolidone, 1% ficola, 1% bovine serum albumin. 0.5% SDS and 0.1 mg / ml total human DNA. 2? 6 kb

An EcoRI fragment of genomic DNA derived from the 3 'end of the lambda clone isolated previously was used as a probe and labeled with ³² P by random priming with hexamer. This fragment contained exon 6 and part of exon 7 present in the phage clone. After overnight hybridization at 65 ° C, the membranes were washed as described above. The DNA sequences of exons 7, 8, 9, 10, 11 and 12, together with partial sequences of adjacent introns, are set forth in SEQ ID NOs: 15, 16, 17, 18, 19 and 20.

Example 6

Complete plasma PAF-AH cDNA clones were isolated from mouse, canine, bovine and chicken spleen cDNA libraries, the incomplete rodent clone was isolated from rat thymus cDNA library. These clones were identified by low-stringency hybridization with human cDNA (the hybridization conditions were as described above for exons 1 to 6 in Example 5. only formainide 20X1 was used instead of 50x1 formamide). A 1 kb Hind III fragment of the human PAF-AH cDNA clone sAH 406-3 (nucleotides 309-1322 in SEQ ID NO: 7) was used as a probe. In addition, an incomplete monkey clone was isolated from the brain cDNA otherwise if and by PCR and primers designed according to nucleotides 285 to 303 and 851 to 867 of SEQ ID NO: 7. Nucleotide and derived amino acid sequences of the mouse, cows, cows, hens, rats and cynomolgus monkeys are shown in SEQ ID NOs: 21, 22, 23, 24, 25 and 26, respectively.

Comparison of the deduced amino acid sequences of the cDNA clones with the human cDNA clorium gave data on the percentage of identical amino acids as shown in Table 1. 3 below.

Table 3

Man The dog mouse cow hen The dog 80 100 64 82 50 mouse 66 64 100 64 47 monkey 82 82 69 80 52 rat 74 69 82 69 55 cow 82 82 64 100 50 hen 50 50 47 50 100

About 38Z amino acid! residues are completely conserved in all sequences. The most variable regions are located on the N-konc1 (containing the signal sequence) and the C-konc1 and are not significant for the enzyme activity as shown in Example 10. The Gly-Xaa-Ser-Xaa-Gly motif (SEQ ID NOs 27) found in neutral lipases and other esterases is preserved in beef. canine, murine, rat and chicken PAF-AH. Serine. located in the middle of this motif serves as an active nucleophilic site by these enzymes. The putative aspartame and histldirium components of the active site (Example 10A) are also conserved. Human Plasma PAF-AH. which is the subject of this patent. thus, it is likely to utilize a catalytic triad and may occupy a hydrol □ ... hydrolase conformation typical of neutral lipases, although it otherwise does not exhibit sequence homology with lipases.

Human plasma PAF-AH should contain a region that specifically interacts with plasma lipoprotein particles (lou density and high density). The N-terminal portion of the molecule containing long stretches of highly interspecific conserved amino acids but not catalytic triads of the enzyme could mediate these interactions.

Example 7

The pRC / CMV expression construct was briefly expressed in COS 7 cells to determine the PAF-AH activity of the protein encoded by the sAH 406-3 clonoin cONA of human plasma PAF-AH (Example 3). PAF-AH activity in medium from COS cells was detected three days after transfection of these cells with DEAE dextran.

Cells were seeded at a density of 300,000 cells per 60 mm diameter culture dish. The following day, the cells were incubated for 2 hours in DMEM containing 0.5 mg / ml OEAE extran, 0, linM chloroquine and 5-10 µg of plasma DNA. The cells were then treated with 10 6 DMSO in PBS for 1 minute. washed with medium and incubated in DMEM containing 10X fetal calf serum in which endogenous PAF-AH activity was previously inactivated with diisopropyl fluorophosphate (DFP). After three days of incubation, PAF-AH activity was detected in transformed cell media. Measurements of this activity were performed in the presence and absence of either 10 mM EDTA or 1 mM DFP to determine whether the recombinant enzyme required calcium and whether. is inhibited by DFP, which is an inhibitor of seriresterases as described for PAF-AH Stafforirioni et al., (1987), supra. Negative controls included pRc / CMV transformed cells with no insert or with a sAH 406-3 insert in the opposite orientation.

PAF-AH activity in transformed cell superriatants was determined by the method of Stafforini et al., (1990), supra, with the following modifications. Briefly, PAF-AH activity was determined by measuring the hydrolysis of ³ H acetate from Eacetyl- ³ H 1 PAF (New England Nuclear, Boston, MA). The free ³ H acetate in the aqueous phase was separated from the labeled substrate by reverse-phase chromatography on octadecyl gel (Baker Research Products, Illinois, PA). Assays were performed with 10 µl of supernatant from transformed 7.2. in a reaction volume of 50 µl.

cells in 0.IM Hepes buffer, pH A total of 50 pmol substrate was used in one reaction with a ratio of labeled> unlabeled PAF of 1: 5. The reaction mixtures were incubated for 30 minutes at 37 ° C and stopped by adding 40 µL of 10 M acetic acid. The solution was then washed on a column of octadecyl silicagel, then rinsed with 0.1M sodium acetate. The eluate of each sample was collected and measured on a scintillation counter for one minute.

As shown in FIG. 2, media from cells transformed with sAH 406-3 contained PAF-AH activity with values four times higher than background. This activity was not affected by the presence of EDTA, but was inhibited by 1 ml * 1 DFP. These observations demonstrate that the sAH 406-3 clone drives an enzyme which activity is consistent with the human plasma enzyme PAF-AH.

Example 8

DNA encoding full-length human plasma PAF-AH, its various truncated forms, and chimeric mouse-human PAF-AH were, by recombinant methods, expression in E. coli and yeast, and permanent expression in mammalian cells.

A. Expression in E. coli

By PCR, a protein encoding a cDNA fragment of the sAH 406-3 clone for human plasma PAF-AH was generated, suitable for immediate further cloning in an expression vector in E. coli. This segment begins at the 5 'of the human gene with the codon for Ile I s (SEQ ID NO: 8), an N-amino acid amino acid residue of an enzyme purified from human plasma. The remainder of the gene was included in the construct, including the original terinination codor. The following 5 'sense primer was used:

SEQ ID NO: 28

5 'TATTCTAGAATTATGATACAAGTATTAATGGCTGCTGCAAG 3' that contained the XbaT cloning site translation initiation codon (underlined). The following 3 'antiserise primer was used:

SEQ ID NO: 29

5'ATTGATATCCTAATTGTATTTCTCTAT1CCTG 3

This primer included a termination codon of sAH 406-3 and contained an EcoRV cleavage site. PCR reactions were performed as described in Example 3. The resulting PCR product was digested with XbaI and EcoRV and further cloned into the pBR322 vector containing the Trp promoter (deBoer et al., PfMS, 80121-25 (1983)) just prior to cloning. place. The E. coli XL-1 Blue strain was transformed with an expression construct and cultured in L soil containing carbenicillin at a concentration of 100 µg / ml. Transformarities were grown overnight, centrifuged and resuspended in lysis buffer composed of 50mL Tris-HCl pH7.5, 50mM NaCl, 10mM CHAPS, 1mL * 1 EDTA, 100µg / ml lysosome and 0.05 trypsin inhibiting units (TIU) / ml of Aprotinin. After one hour incubation on ice and two minutes sonication, PAF-AH activity was determined in the lysates by the method described in Example 4. E. coli transformed with an expression construct (termed trp AH) produced a protein with PAF-AH activity. See tab. 6 in Example 9.

Constructs containing other promoters were also used to control expression of human PAF-AH in the E. coli tacII promoter (deBoer, supra), the arabiriose (ara B) promoter from Salmonella typhimurium L. Horitz et al., Gene, 14-309-319. (1981) 3 and the bacteriophage T7 promoter. Constructs containing the Trp promoter (pUC trp AH), the tacII promoter (pUC tac AH), and the araB promoter (pUC ara AID) were constructed based on plasmid pUC19 (neu Engalrid Blolabs. 1 * IA), whereas the T7 promoter construct was constructed from plasmid pET15B. (Novagen. Madison, WI) a hybrid promoter (pHAB / PH), consisting of araB (pET AH), was a construct with

The T7 promoter region was PET15B. All constructs of the ribosome binding site z also generated from the plasmid produced proteins in E. coli with PAF-AH activity in the range of 20-50 U / ml / 0D _o0 o. This activity corresponds to a total recombinant protein> 1% of all cellular proteins.

Several expression constructs were also evaluated. which produce PAF-AH in E. coli with extended N-termini. The N-terminus of natural human PAF-AH was determined to be β ₄₂ based on amino acid sequencing (Example 2). However, the sequence is right in front of Tle-is ·

3R does not match and no acid also makes us ard m

Destination point.O for cleaving the nehn signal pp.pf.1dn Γ tn means rule -A-1 is not met, since in position -1 not there is ski n present, (eg in vnn Heljne, Nim. Arrfrte Rfí.tt ,, 14 with 4RR3-4RRI.1 (1ARR11, .Tr may be that the cell secretion system is recognized by clasic and signal sequence fl * lj-A ₁₇ or l * l-P _{= 1} ) and then followed by another end The entire PAF-AH coding sequence starting with methionine (nuclides 1R? to 14R7 n of sequence RFC1? P NO: 7) is digested and engineered for expression in F, helper helper. ~ p prnmrttnra. Tentn knnštrnkt, the active PAF-AH. however, the acridinium t n is referred to in the Table. 4, lamb expression was around fifty both, the level of the original expresi.e knnštri also here, _the starting Tlc, Tietn results indicate that the extension of the prnteínii N-knot is not critical or necessary for the activity of recombinant PAF-AH in F, cr> 11.

Table 4

PAF-AH activity (Ι.Ι / Π1_Ί / 0η _ώΟΟ )

Knnštri.ik t 1 .yzát Médi um pl IC trp AH (Tle.-ja at the N-terminus i 177, 7 II, Π30 ρΙΙΠ trp AH l * years. 3, 1. Π, ΠΠ3 pl 1 (1 trp AH Val x o Fi4, R n, Π33

Also, shortened recombinant forms of TiHuman PAF-AH foamy plasmid with a low copy number on clay and a promoter that is induced by the addition of arabinose in culture medium can also be prepared in such forms of PAF-AH. truncated on the N-knot is the product of recombinant DNA expression encoding the amino acid residues Met? x to Asn _{? 4 of the} polypeptide encoded by the complete PAF-AH cDNA (SFI ID NO: A). For the production of rPH, 2 in bacterial cells, the plasmid d pRAR2 / PH, 2 nd nR pRR322, which contains (I) nucleotides 2A7 to 1487 of SEQ ID NO: 7, which encodes human PAF-AH starting with a codon, is now used. for the methionine at position 46, (2) the araB-C promoters and the araC gene from the arabnose operon Salmonella typhimurium, (3) the transcription termination sequence from bacteriophage T7, and (4) the phage fl1 origin of replication.

Specifically, pBAR / PH.2 comprises the following DNA regions: (1) a region from the deleted AAT1 site at position 1994 to the EcoRI site at position 6274, that is, a vector sequence containing a replication origin and genes responsible for resistance to apmicillin or tetracycline derived from a bacterial plasmid pBR322: (2) a region from the EcoRI site at position 6274 to the XbaI site at position. 131, i.e. Salmonella typhimurium arabnose operon DNA (Genbank Accession nuinber: 1 * 111045, M11046, 1 * 111047, J01797); (3) a region from an XbaI site at position 131 to an NcoI site at position 170, that is, a DNA containing a rlbosome binding site from pET-21b (Novagen, Madison, UI); (4) a region from the NcoI site at position 17D to the Xliol site at position 1363, that is, the sequence of the human PAF-AH cDNA, and (5) a region from the XhoI site at position 1363 to the deleted AATI site at position 1993. -21b (Novagen), containing the transcription termination sequence of bacteriophage T7 and the bacteriophage fl1 origin of replication.

Another form of PAF-AH, designated as rPF.9, is the product of recombinant expression of DNA encoding the amino acid residues of Met? _A to Ilen ₄ and? A polypeptide encoded by the complete PAF-AH cDNA (SE13 ID NO: 8). The DNA encoding rPH.9 was inserted into the same vector that was used to produce rPH.2 in bacterial cells. This plasmid was named PBAR2 / PH.9, consisting of the following DNA regions: (1) a region from the deleted AAT1 site at position 1958 to the EcoRI site at position 6239, i.e., a vector sequence containing an origin of replication and genes responsible for ampicillin resistance; tetracycline derived from the bacterial plasmid pBR322; (2) a region from the EcoRI site at position 6239 to the XbaI site at position 131, that is, the arabnose operon DNA from Salmonella typhimurium (Genbank accession number. M11045, M11046, M11047, J01797); (3) Area from

XbaI sites at position 131 to the NcoI site at position 170, i.e. DNA containing the ribosome binding site of pET-21b (CNagen, Madison, UI); (4) a region from the NcoI site at position 170 to the XhoI site at position 1363, that is, the sequence of human PAF-AH cONA, and C) a region from the XhoI site at position 1328 to the deleted AatII site at position 1958, i.e. the DNA fragment from pET -21b CNovagen), containing the transcriptional thermirectional sequence of bacteriophage T7 and the replication origin of bacteriophage fl.

The expression of PAF-AH proteins in pBAR / PH.2 and pBAR / PH.9 is under the control of the araB promoter, which is perfectly repressed in the presence of glucose and in the absence of arabinose, but functions as a depleted medium by addition

(M9 medium casein hydrolyzate).

strong promoter after addition of L-arabinose with glucose. Selection of cells with the plasmid ampicillin (or related antibiotic) or tetracycline into the medium. Many E. coli strains including C but not exclusively) strains prototrophic in arabinose metabolism such as e.g. U3110. DH5‘D, BL21. C600, JM101 and derivatives thereof, strains harboring proteolysis-reducing mutations such as e.g. CAG829, KY1429 and strains unable to degrade arabinose such as e.g. SB7219 and MC1051. The advantage of using a strain that cannot decompose arabinose. consists in that the inducer (Carabinosis) of PAF-AH expression is not pumped out of the medium during the induction period, resulting in higher levels of PAF-AH compared to those achieved with strains capable of metabolizing arabinose. The active PAF-AH forms are expressed in various strains of E. coli in any suitable medium and under any suitable conditions. Rich LB media, E0M295 supplemented with yeast extract and acidic as well as defined CA675 media, i.e. at least medium A adjusted to pH 5.75, with glycerol as a carbon source, supplemented with trace elements and vitamins) allow considerable production of rPAF-AH forms. Tetracycline is added to the media to ensure the presence of plasmid in the culture due to selection pressure.

E. coli strain MC1061 (CATCC 53338). carrying arabinose operon deletion and thus unable to metabolize arabinose was transformed with plasmid pBAR2 / PH.2. Strain 1C1061 is an auxotrophic ria leucine, was cultured by constant replenishment of media containing casein extract and complemerating the leucine mutation. PBAR / PH.2 transformed E. coli cells were grown at 30 ° C in growth media containing 2g / L glucose. Glucose has a double meaning - it serves as a carbon source for cell growth and as a repressor for the arabinose promoter. When the glucose level dropped to <50 mg / L, nutrient replenishment with glucose concentrate (300 g / L) started. Glucose replenishment increased linearly over 16 hours at a rate that limited the formation of acidic by-products. At this point, glucose replenishment was replaced by the addition of glycerol. L-arabirosis (500g / L) was added simultaneously to a final concentration of 5g / L. Glycerol replenishment was maintained at a constant height for 22 hours. Cells were harvested by hollow fiber filtration, whereby the cell suspension was concentrated approximately 1-fold. Cell sediment bo.l stored at -70 ° C. Total cell mass reached about 80g / L (OD 600 = 50-60) with a PAF-AH activity of 65-70 units / OD / ml, which represents about 10X of all cellular proteins. A bacterial culture with a final volume of about 75 liters contained 50-60g PAF-AH.

High levels of production of rPAF-AH forms can be achieved by expression of PBAR2 / PH.2 or PH.9 in strains SB7219 and MC1061. Other strains unable to degrade arabinose are also suitable for achieving a high density of bacterial culture. The following bacterial culture conditions are recommended. Fermentation tanks containing 2g / L glucose culture medium are inoculated with exponentially growing strains SB7219; pBAR2 / PH * 2 and SB7219spBAR2 / PH.9. After glucose has been consumed, the fermentation tanks are supplied with a glycerol solution containing trace elements, vitamins, magnesium and ammonium salts so. to maintain healthy exponential growth. The fermentor temperature is maintained at 30 ° C, the tanks are aerated and shaken. the dissolved oxygen level is above 15% saturation. When the density of the bacterial culture is higher than 11Dg / L (wet weight), the nutrients are replenished at a constant rate, L-arabiriose C (final concentration of about 0.5%) is added to the culture). Product formation is followed for 16-22 hours. The yield is usually 40-50 g / L (dry cell weight). Cells are harvested by ceritrifugation, stored at -70 ° C and rPAF-AH is purified for further analysis. Specific yields higher than 150 units / µl / OD 40 are commonly achieved.

B. Yeast expression

Human recombinant PAF-AH was also expressed in Saccharomyces cerevisiae. The yeast ADH2 promoter was used to control the expression of rPAF-AH, production reached 7 units / ml (OD.i.oo CTab.5).

Table 5

Korištrukt promoter tribe Cll / ml, Enzyme activity / OD) pUC tac AH tac E. coli U3110 30 PUC trp AH trp E. coli W3110 40 pUC ara AH Araq E. coli W3110 20 pET AH T7 E. coli BL21CDE3) C Novagen) 50 pHAB / PB Arabi / T7 E. coli XL-1 34 PBAR2 / PH.2 araB 1 * 101061 90 pYep ADH2 AH ADH2 Yeast BJ2.28 7

C. Expression of PAF-AH in Mammalian Cells

1. Expression of a human PAF-AH cDNA construct

Plasmids engineered to express PAF-AH utilize, with the exception of pSNF / PAFAH.1, a strong viral promoter derived from cytomagalovirus. a polyadenylation site from the cow's growth hormone gene and the SV40 virus replication origin to provide a high copy number plasmid in COS cells. The plasmids were. introduced into the cells by electroporation. First, a set of plasmids was prepared. in which there were either 5 'border sequences (pDCl / PAFAH.1). or both the 5 'and 3' flanking sequences of human PAF-AH cDNA (PDC1 / PAFAH.2), replaced by flanking regions of other genes that exhibit high levels of expression in mammalian cells. Trisection of these plasmids into COS, CHO, or 293 cells resulted in the production of PAF-AH at the same level (0.01 units / ml, thus 2-4 times higher than the background level). as achieved with the sAH-406 clan after transient expression of COS cells (Example 7). Another plasmid containing the Friend's spleen virus promoter instead of the cytomegalovirus promoter was constructed. The human PAF-AH cDNA was inserted into the plasmid ρπιΗ-neo (Hahn et al., Gene 127s 267 (1993)) under the control of the Friend's spleen virus promoter. After transfection of NSSO myeloma line cells with plasmid pSFN / PAFAH.1 and testing several hundred clones, two were found: transfectants (4B11 and 1C11), which gave 0.15-0.5 units / ml PAF-AH activity. The productivity of the two NSO transfectants corresponds to about 0.1 mg / liter. assuming a specific activity of about 5000 units / mg.

2. Expression of PAF-AH chimeric constructs produced by the mouse and human gene

PRC / 1S9 construct containing. The mouse PAF-AH cDNA in the mammalian oRc / CMV expression vector resulted in the production of a secreted form of PAF-AH at a rate of 5-10 units / ml (1000-fold above background level) after transfection into COS cells. Assuming that the specific activity of murine PAF-AH is approximately the same as that of the human enzyme, murine cDNA is expressed about 500 to 1000-fold more than human cDNA for PAF-AH.

To investigate the difference between the level of expression of human and mouse PAF-AH in COS cells, two chimeric human-mouse genes were constructed and screened for expression in COS hunks. The first structure, pRc / PH. HI * IC1, contains the coding sequence for 97 N-terminal polypeptides (SEO ID NO x 21) of the human PAF-AH amino acids of murine PAF-AH linked to the 343 C-terminal in the pRc / CI * IV expression vector (Invitrogen, San Diego, CA). Second chimeric pRc / PH gene. I * IHC2 contains the coding sequence for the 40 N-terminal amino acids of the murine PAF-AH polypeptide linked to the 400 C-terminal residues of human PAF-AH in pRc / CI * IV. After transfection of COS cells with plasmidoin pRc / PH. 1 HCl accumulated 1-2 units / ml PAF-AH activity in the medium. Plasmidido pRc / PH is used in conditioned media from trarisforum cell culture. 1 CH 2 was found only 0.01 Units / ml PAF-AH activity. These experiments show that the difference in expression level of the murine and human PAF-AH gene is at least in part due to the properties of the polypeptide segment delimited by the amino acid residues 40-97, or the corresponding region of RNA and DNA encoding this region of PA-AH protein.

3. Recoding the first 290 bp sequence of PAF-AH

One of the hypotheses explaining the low level of synthesis of human PAF-AH in tris-treated mammalian cells suggests that the use of codors in the natural gene is not optimal for efficient expression. However, it does not appear likely that the type of coders used could be responsible for the 500-1000 fold difference in expression levels between the human and mouse genes, since codon optimization only results in a 10 fold increase in expression. The second hypothesis explains the different levels of expression of the mouse and human PAF-AH gene in the presence of a secondary structure in the 5 'coding region of human PAF-AH mRNA. which leads to relatively rapid degradation of mRNA, or causes ineffective initiation or elongation of trarislation.

To verify these hypotheses, a synthetic fragment encoding the original human PAF-AH protein was constructed from the N-terminus to the amino acid residue 96 in which most codons were replaced (recoded) with the same amino acid codon but having a different sequence. A change from GTA in the second codon resulted in the creation of an Asp718 site. which is at one end of the synthetic fragment and which is also present in the mouse cDNA. The other end of the fragment contained a BamHI site found in codon 97 of the human gene. The Asp718 / BamHI fragment of about 290 bp was derived from a PCR fragment prepared by the dual asymmetric PCRs procedure for the construction of the synthetic genes described in Saridh et al., BioTec / miques, 12 s 14-16 (1992). The synthetic Asp718 / BamHI fragment was ligated with DNA fragments encoding the remainder of the human PAF-AH molecule, starting at nucleotide 463 of SECID ID NO. Then, the sequence encoding the native human PAF-AH enzyme was inserted into a mammalian expression vector pRc / CPIV (Invitrogen, Sari Diego) to create the plasmid pRc / HPH.4. The complete sequence of the recoded gene is shown in SEC) ID NO * 30. The region in pRc / HPH.4 adjacent to the 5 'end of the sequence encoding human PAF-AH originates from the murine PAF-AH cDNA at pRo / 1 * IC9 (nucleotides 1 to 116 of SEC) ID NO. 21).

transformation of COS pRc / HPH.4 will contain the gene

COS cells were transiently transformed with plasmid ml pRc / HPH. 4 (recoded human gene). pRc / 1 * IS9 (murine PAF-AH) and pRc / PH.1 / IHC1 (mouse-human hybrid sequence # 1) to determine expression of human PAF-AH from pRc / HPH.4. In conditioned media from transformed cell cultures, PAF-AH activity was determined to have values of 5.7 units / ml (mouse gene), 0.9 units / ml (mouse-human hybrid sequence # 1) and 2.6 units / ml (recoded human gene). Recoding the first 290 bp coding sequence for human PAF-AH proved to be a successful strategy and raised the level of expression of human PAF-AH from several nanograms / ml to about 0.5 micrograms / ml during transient cells. The recoded PAF-AH gene from the plasmid was inserted into a mammalian dihydrofolate reductase (DHFR) mammalian expression vector and transformed with the DHFR negative Chinese hamster ovary cell line. Transformed cells will be subjected to methotrexate selection. thus, gems will be obtained which make up a large amount of human PAF-AH through gene amplification.

Example 9

Human recombinant plasma PAF-AH (beginning with Ile ₄₃ ) expressed in E. coli was purified to the Coomassie level of blue stained ria SDS-PAGE by various methods. The activities exhibited by the native enzyme PAF-AH were also determined.

A. Purification of recoinbinant PAF-AH

The first purification procedure used is similar to that described in Example 1 for native PAF-AH. The following steps were performed at 4 ° C. Pellet from 50 ml E. coli culture. which produce PAF-AH (transformed with the trp AH expression construct) was lysed as described in Example 8. Solid particles were removed by centrifugation at 10,000g for 20 minutes. The supernatant was loaded at 0.8 ml / min onto a Blue sepharose Fast Flou column (2.5 cm x 4 cm; 20 ml fill volume)) which was equilibrated with buffer D (25 µl * 1 Tris-HCl, 10 ml * 1 CHAPS, D, 51 * 1 NaCl, pH 7.5). The column was washed with 10D ml of buffer D and eluted at a rate of 3.2 ml / min with 100 ml of buffer A containing 0.51 * 1 KSCN. 15 ml of the active fraction was loaded on a 1 ml Cu chelating Sepharose column equilibrated with buffer D. This column was washed with 5 ml of buffer D, followed by elution of 5 ml of buffer D containing 100 ml * 1 imidazole by gravity flow. Fractions with PAF-AH activity were analyzed by SDS-PAGE.

The purification results are shown in Tab. 6, in which the unit equals hydrolysis umol PAF per hour. The purification product obtained at 4 ° C appeared on SDS-PAGE as the only intensely stained band below the 43 kDa marker position, suggesting diffuse staining just below and above each other. The recombinant material is significantly purer and exhibits a higher specific activity compared to the PAF-AH preparation from plasma described in Example 1.

Table 6

Vzo- volume If you- On the whole, Koric. spe- % on the- fold saying, (Ml) WELCOMES Additive pro- C boiler return načistenla C jed- The activity teí- fic- nose 11 no t- welcomes new Moon ka The activity ka / ml (jed- (Mg / The activity Vity not- ml) WELCOMES ka / (jed- 10 = not-

ka / mg

step tot. step tot lysate 4.5 989 4451 15. 6 63 100 100 1 1 Blue 15 64 960 0. 07 914 22 22 14. 4 14, 4 Cu 1 2128 2128 0. 55 3869 220 48 4.2 61

When the same purification procedure was performed at room temperature, in addition to the 43 kDa marker band, a group of strips migrating below the 29 kDa marker position appeared. which coincided with PAF-AH activity measured in gel slices. These lower molecular weight bands may be proteolytic fragments of PAF-AH. that retained enzymatic activity.

A falsified purification procedure was also used at room temperature. A pellet (100 g) of E. coli producing PAF-AH C transformed with the expression construct pUC trp AH) was resuspended in 200 ml lysis buffer (25 ml * 1 Tris, 20 mm CHAPS, 50 ml * 1 NaCl, 1 ml * 1 EDTA, 50 ml). µg / ml benzamide (pH 7.5) and lysed three times by passing through a microfluidizer at 15,000 psi. The solids were removed by centrifugation at 14300 x g for one hour. The supernatant was diluted 10-fold in dilution buffer (25 ml 1 * IES (2-CN-morpholinolethanesulfonic acid), 10 ml * 1 CHAPS, 1 ml * 1 EDTA, pH 4.9) and loaded at 25 ml per minute onto a Sepharose Fast Flou column (200 ml). (cation-ion exchange column), which is equilibrated in buffer E (25ml * 11 * IES, 10ml * 1 CHAPS, 1m EDTA, 50ml * 1 NaCl, pH 5.5). The column was washed with 1 liter of buffer E and eluted with 11 * 1 NaCl. The eluate was collected into 50 ml fractions and the pH was adjusted to 7.5 with 0.5 ml of 21 * 1 Tris C base). Fractions containing PAF-AH activity were pooled and conditioned. to contain 0.51 * 1 NaCl. The fractions so combined were loaded at a rate of 1 ml / min onto a Blue Sepharase Fast Flou column <2.5 cm x 4 cm; 20 ml) equilibrated in buffer F <25 ml * 1 Tris, 10 ml * 1 CHAPS, 0.51 * 1 NaCl, 1 ml * 1 EDTA, pH 7.5). The column was washed with 100 ml of buffer r and F and eluted with buffer F containing 31 * 1 NaCl at a rate of 4 ml / min. This Blue Sepharose Fast Flou chromatography step was repeated to reduce the level of endotoxins in the sample. Fractions containing PAF-AH activity were pooled and dialyzed against buffer G <25ml * 1 Tris. pH 7.5. 0.51 * 1 NaCl. 0.1 * Tueen 80. lml * l EDTA).

The results of the purification are shown in Table 7, in which the unity is equal to µmol of PAF hydrolysis per hour.

Table 7

Vzo- volume The activity On the whole, Horses. spe- * on the- fold saying, C ml) WELCOMES Additive pro- C boiler return riačistenia <jed- The activity teí- fic- the company not- WELCOMES new Moon ka The activity ka / ml C jed- C mg / The activity Vity not- ml) WELCOMES ka / <jed- 10 riot- ka / mg step tot. step tot lysate 200 5640 1128 57. 46 98 100 100 1 1 WITH 111 5742 637 3. 69 1557 57 56 16 16 Blue 100 3944 394 0. 84 4676 35 62 3 48

The obtained purification product appeared on SDS-PAGE as a single intense band under the 43 kDa label with some diffuse staining directly below and above the band. The recombinant material is substantially cleaner and exhibits greater specific activity compared to plasma PAF-AH treatment as described in Example 1.

Another purification protocol is contemplated in the present invention. This includes cell lysis, purification and the first column step. Cells were diluted 1 * 1 in lysis buffer (25m Tris pH 7.5. 150mL * 1 NaCl, 1X Tueen 80. 2ml * 1 EDTA). Lysis was performed in a cooled microfluidizer at 15,000-20000 psi. A triple extrusion of the material through a microfluidizer was used to achieve> 992 cell breakage. The lysate was diluted 1 · 20 in dilution buffer (25ml * 1 Tris, pH 8.5, 1ml * 1 EDTA) and applied to a column packed with Q-Sepharosa Big Bead chromatography medium (Pharmacia). The column was equilibrated in 25mm Tris pH

8.5, lml * l EDTA, 0.0152 Tueeri SO. The eluate was diluted with ls10 in 25 ml * 1 µES pH 5.5, 1.21 * 1 ammonium sulfate, 1 ml * 1 EDTA and loaded onto Butyl Sepharose (Pharmacia) chromatography medium equilibrated with the same buffer. PAF-AH activity was eluted with 25ml * 11 * IES pH5.5, 0. 12 Tueen BO, 1ml * 1 EDTA.

In the present invention, a method for purifying the purification of an enzymatically active PAF-AH is further contemplated. This method comprises the following steps: a) preparing an E. coli extract which, after lysis in a buffer containing CHAPS, results in a supernatant containing solubllizarial PAF-AH; b) diluting the above supernatant and applying it to an anion-exchange column by equilibrating to a pH of about 8.0; c) eluting the enzyme PAF-AH from the above anion-exchange column; d) loading said adjusted eluate from said anion exchange resin column onto a blue dye ligand containing affinity column; e) eluting the above-mentioned blue dye ligand column with a buffer containing 3.01 * 1 salt; f) diluting the eluate from the column containing the blue dye ligand to a buffer suitable for performing hydroxylapatite chromatography; g) performing hydroxylapatite chromatography, wherein the washing and elution is carried out with buffers (with or without CHAPS); h) brightening the above hydroxylapatite eluate to a salt concentration suitable for a cation exchange column; i) applying the above-mentioned hydroxylapatite eluate to a cation-exchange column at a pH ranging from about 6.0 to 7.0; j) eluting PAF-AH from the above cation-exchange column with a suitable buffer of a suitable composition; k) performing cation-exchange chromatography in the cold; and 1) the composition of a liquid or frozen form of PAF-AH in the absence of CHAPS.

It is best to use totos in the step above

a) the lysis buffer has the following composition: 25ml * 1 Tris, 100ml * 1 EDTA, 20ml * 1 CHAPS, pH 8.0; in step b) the dilution of the anion-exchange chromatography supernatant is 3-4 times from 25ml * 1 Tris, 1ml * 1 EDTA, 10ml * 1 CHAPS, pH 8.0 and the column is (3-Sepharose column equilibrated with 25ml * 1 Tris, 1 ml of EDTA, 50 ml of NaCl, 10 ml * 1 of CHAPS, pH 8.0, in step c) the anionic-ionic column is eluted using 25 ml * 1 of Tris, 1 ml of EDTA, 350 ml of 1 NaCl, 10 ml of CHAPS. pH 8.0; in step d), the eluate of step c) is applied directly to the affinity column with a blue dye ligand; in step e) the column is eluted with a buffer containing 31 * 1 NaCl, 10 ml CHAPS, 25 ml * 1 Tris, pH 8.0; in step f) the Blue dye eluate is diluted for hydroxylapatite chromatography. The eluate is diluted with 10µl sodium phosphate, 100µl NaCl, 10µl * 1 CHAPS, pH 6.2; in step g), hydroxylapatite chromatography is performed using a hydroxylapatite j column equilibrated with 10ml * 1 sodium phosphate, 100ml NaCl, 10ml * 1 CHAPS. Elution is performed using 50mL sodium phosphate, 100mL NaCl (with or without 10mL * 1 CHAPS), pH 7.5; in step h) the dilution of the above-mentioned hydroxylapatite eluate for cation-exchange chromatography is performed with a buffer having a pH in the range of about 6.0 to 7.0 and containing sodium phosphate (with or without CHAPS); in step i) the column is s

S-Sepharose equilibrated with 50 mM sodium phosphate (with or without 10 ml * 1 CHAPS), pH 6.8; in step j), the elution is carried out with a buffer of a suitable composition, such as 50 ml * 1 potassium phosphate, 12.5 ml * 1 acid. aspartic, 125ml * 1 NaCl, pH 7.5 and containing 0.01% Tueen 80; in step k), cation-exchange chromatography is performed at 2-8 ° C. An example for a suitable buffer composition useful in step 1). which stabilizes PAF-AH is 50ml * l potassium phosphate, 12.5ml * l acid. aspartic, 125ml * i NaCl, pH about 7.4 (with or without Tueen 80 and / or Plurinic F68) or 25ml * l phosphate CK *) buffer containing (at least) 125ml * l NaCl, 25ml arginine and 0.01% Tween

C with Ci without Plurinick F68 at a concentration of about 0.1 to 0. 530.

Recombinant PAF-AH activity

The most prominent feature of PAF acetyl hydrolase is its distinct specificity for substrates with short residues at the sn-2 position of the substrate. This strict specificity distinguishes PAF acetyl hydrolase from other forms of PLA _and . Thus, in order to determine that the C1 recombinant PAF-AH degrades long chain fatty acid phospholipids at the sn-2 position, hydrolysis of 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (arachidoriylPC) was tested. This substance is the preferred substrate for a well characterized form of PLA _and . As predicted by previous studies with native PAF-AH, this phospholipid was not hydrolyzed when incubated with recoinbinant PAF-AH. In other experiments, arachidoriylPC was added to the standard hydrolysis assay solution at a concentration of 0 to 125 µL *. The aim of this was to find out that this addition inhibits the hydrolysis of PAF by recombinant PAF-AH. Even at the highest concentration of PAF-AH (CCo), the concentration was 5-fold higher than that of PAF), and no inhibition of PAF hydrolysis was observed. Thus, recombinant PAF-AH exhibits the same substrate specificity as the native enzyme - long chain substrates are not recognized. In addition, the recombinant enzyme PAF-AH rapidly degrades the oxidized phospholipid (CgluraroylPC), which has undergone oxidative cleavage of sn-2 fatty acids. Native plasma PAF-AH has several other properties that distinguish it from other phospholipases. including calcium ion-independent phospholipases and phospholipases resistant to substances that modify SH groups or cleave disulfides.

Native and recombinant plasma PAF-AH enzymes are sensitive to DFP, suggesting that serine forms part of their active serum. An unusual property of native plasma PAF acetyl hydrolase is its close association with lipoproteins in the bloodstream and the fact that its catalytic efficiency is influenced by the lipoprotein environment. When recombinant PAF-AH, as described herein, is incubated with human plasma Ca previously treated with DFP to remove endogenous enzymatic activity), it is associated with low- and high-density 11β-propellants in the same manner as native activity. This result is important because there is solid evidence that modification of low-density lipoproteins is necessary for the cholesterol deposition observed at atheroma and that lipid oxidation is an initiating factor in this process. PAF-AH protects low density II proteins from modification under oxidative conditions in vitro and may play this role in vivo. Administration of PAF-AH is therefore recommended to reduce lipoprotein oxidation in atherosclerotic plaques as well as in the treatment of inflammation. All these results confirm that the sAH 406-3 cDNA clone encodes a protein with human plasma PAF acetyl hydrolase activities.

Example 10

Other recombinant PAF-AH products were also expressed in E. coli. These products included arialogs of PAF-AH having a single amino acid mutation and fragments of PAF-AH.

A. PAF-AH products resulting from amino acid substitution

PAF-AH is a lipase. because it hydrolyzes the phospholipid PAF. Although there is no general similarity between PAF-AH and the other lipase-specific characteristics, conserved residues were found compared to the structurally characterized lipases. A serie has been identified in the active center. Serine, together with the remainder of aspartame and glidant, forms a catalytic triad that represents the active site of the lipases. These three residues are not contiguous in the primary amino acid sequence, but we know from the structural studies. that these three neighbors in three-dimensional space. Comparing the structures of mammalian IIpases, we know that the aspartame residue is usually a 24 amino acid residue from the C terminus, towards the active site with serine.

Individual codons of human PAF-AH that encoded sequences for an alanine residue were modified by site-directed mutagenesis and PCR. These mutants were expressed in E. coli. As shown in Table 8, where, for example, the abbreviation S108A means that the sparing new residue at position 108 has been replaced by alaniri,

Hiss _3S i completely destroyed between the individual centers are for PAF-AH and point mutations Ser ₂₇ 3, Asp _{S9 &} or PAF-AH activity. The amino acid distances are similar to those in the other 11pase active (from Ser to Asp is 23 amino acids; from Ser to His is 78 amino acids). These experiments have also shown that Ser _ffi 73. Asp _{29 (i} , or His _3S i are residues critical for activity and therefore also candidates for the triad of catalytic residues. Cysteir residues, by their ability to form disulphide bridges, are often critical for maintaining functional integrity Plasma PAF-AH enzyme contains five cysteine residues In order to determine which of the cysteine residues is critical for maintaining enzyme activity, cysteines were individually replaced by serine mutation and the resulting mutants were expressed in E. coli. preparations of these recombinant mutant forms of the enzyme are shown in the second column of Table 8, while the results obtained with multiple purified enzyme preparations are summarized in the third column of Table 8. The data show that all mutant forms showed almost the same activity, therefore We conclude that no cysteine residue is required for PAH-AH activity, and other mutants also had little or no effect on the catalytic activity of PAF-AH. In Table 8, ++++ represent the activity of the naturally occurring type

PAF-AH equal to 40 to 60 U / ml / CX) _and oo. ⁺⁺⁺ represent an activity of about 20 to 40 U / ml / OD 600, and 10 to 20 U / ml / OD 600. ⁺ 1 to 10 U / ml / QDaoo and the - sign indicate activity less than 1 U / ml / ODtf.oo.

Table 8

mutation PAF-AH activity Specific act. purifications PAF-AH Wild Cpriro- 6.9 mmol / mg / h dzene sa vysky- type) S108A ++++ S273 - 0286 - 0286 ++ D296 - D304 ++++ D338 ++++ H351 - H395. H399A ++++ C67S +++ 5.7 mmol / mg / h C229S + 6.5 mmol / mg / h C291S + 5.9 mmol / mg / h C334S ++++ 6.8 mmol / mg / h C407S +++ 6.4 mmol / mg / h C67S, C334S, C407S 6.8 mmol / mg / h

B. Fragmental PAF-AH products

Deletions at the C terminus were prepared by digestion with exonuclease III at the 3 'end of the PAF-AH coding sequence for various long periods of time. These truncated coding sequences were then ligated into plasmid DNA encoding stop codons in all three parts of the frame. Ten DNA deletion constructs were characterized using DNA sequence analysis, protein expression, and measurement of PAF-AH activity. Removal of the 21-30 amino acid residues from the C terminus strongly reduced catalytic activity, and removal of the 52 amino acid residues resulted in complete destruction of activity. This is shown in Figure 3.

Similar deletions were performed on the amino terminus of PAF-AH. PAF-AH fusion proteins with thioredoxin at the N-terminus were prepared in E. coli. This resulted in a consistently high level of expression of PAF-AH activity (LaVallie et al., Oiotechnolay, 1993, 111: 187-193). Removal of the nineteen amino acid residues from the naturally occurring N-terminus of the protein (Ile ₄₂ ) reduced activity by up to 99%. While removal of the 26 amino acid residues completely destroyed the enzymatic activity of the fusion protein (see Figure 3). The deletion of the 12 amino acid residues increased enzyme activity four times.

In the following purification processes for PAF-AH starting from fresh human plasma using methods similar to those described in Example 1 (two concentrations of the N-terminal N-terminal were identified to concentrate the Blue Sepharose eluate instead of the Cu column of Amicon llicrocon 30). amino acids alongside Ile _4a namely Ser _3s and Lys _5S . Heterogeneity may have a natural origin in the state of the enzyme in the plasma, whether it may result from a purification procedure.

The purified material described above was also subjected to glycosylation analysis. Purified native PAF-AH was incubated in the presence or absence of N-glycanase, a carbohydrate-removing enzyme bound to the N-terminus of glycoproteins. The PAF-AH treated samples were then subjected to electrophoresis in a 12X SDS polyarkylamide gel and then labeled with rabbit polyclonal antler in a Western blot. The protein that was not incubated with N-glycanase traveled in the gel as a diffusion band with a molecular weight of 45-50 kDa, while the glycanase-treated protein traveled as a sharply defined band corresponding to a molecular weight of 44 kDa. This demonstrates that native PAF-AH is glycosylated.

N-terminal heterogeneity was also observed in purified preparations of recombinant PAF-AH (Ile _42).

N-terminus). These enzyme preparations were a mixture of polypeptides with N-koncaml beginning with Ala ^ _x, Ile ₄ · s or an artificial initiating radical ľlet_i adjacent to Ile ".

Preliminary comparison of PAF-AH fragments with PAF-AH

Because of the observed heterogeneity of recombinantly prepared PAF-AH, other recombinant products were prepared, which were also tested for observed heterogeneity after expression and subsequent purification. The composition of recombinant expressed PBAR2 / PH.2 and pBAR2 / PH.2 products in E. coli strain MC1061 was analyzed at various time intervals during the production phase of the cell cycle. Partially purified samples of recombinant PH.2 and PH.9 taken at different time intervals between 5 and 22 hours after induction of protein expression were analyzed by laser desorption ionization mass spectrometry (1 * IALDI-1 * IS).

When a PH.2 expression vector was used, two peaks were observed in the spectrum of the partially purified protein, which corresponded to the mass value predicted for the rPAF-AH protein. these two peaks were observed at all time intervals, greater heterogeneity was observed only at the time interval corresponding to the stress action caused by acetate accumulation or depletion of oxygen in the medium during fermentation of the cells. The measurement accuracy of the MALDI-MS technique lies within approximately ± 0.3% by weight of one amino acid. The higher-weighted product observed, by its weight composition, corresponds to the expected total translation product of the PH.2 vector minus the weight of methionine. as the initiator of translation. which is believed to have been post-translationally removed. The lower mass peak had approximately 1200 atomic mass units less.

When the expression vector PH.9 was used, preferably a protein that corresponded to its expected mass of rPAF-AH protein was obtained in the partially purified protein spectrum. This single peak was observed at all times, nor did the protein heterogeneity increase over time. The observed mass of this protein was consistent with the presence of the expected translation product for the full-length PH.9 vector, again minus the initiating methionine.

2. Purification of PAF-AH fragments

Recombinantly expressed rPH.2 (the expression product of DNA encoding LLET _{A and 44X} to Asn) and rPH.9 (the expression product of DNA encoding LLET _{4A-Ile 439)} was purified active for further comparative studies with the product of PAF-AH (expression product of DNA encoding Ile ₄₃ to Asn ₄₄₁ ). RPH.9 was produced using an E. coli strain SB7219 and purified mostly by a chelated purification process using Zri. so. as described above. rPH.2 was produced in the ClClOS1 strain in E. coli, as described above, and was purified as described below. The transformed cells were lysed by diluting the cells with lysis buffer (ID01ml succinate, 10mm NaCl. 20ml CHAPS, pH 6. The resulting mass was mixed and lysed by cracking the cells under high pressure. The lysed cells were centrifuged and the supernatant containing rPH.2 was left for further processing. The clarified supernatant was diluted 5-fold with 25 ml of phosphate buffer containing 1 ml * 1 EDTA, 10 ml of CHAPS, pH 7.0, and the diluted supernatant was then applied to a Sepharose Q column. 1µl EDTA, 50mM NaCl, 1µml CHAPS, pH 7.0 (Wash 1), followed by a 10-fold column volume wash of 25ml * 1 Tris buffer containing 1mLl EDTA, 10mL CHAPS, pH 8.0 (Wash 2) followed by ten column volumes of 25mL. Tris buffer containing 1mL EDTA, IDOmL NaCl, 10mL CHAPS, pH 8.0 (Wash 3) Elution was terminated with 25mL Tris buffer containing 1mL EDTA, 350 The pH of the eluate from the Q Sepharose column was then diluted three times with its volume of 25 ml Tris. containing 1ml * 1 EDTA. 10 ml of CHAPS, pH 8.0, and was stacked on a Blue Sepharose column. The column was washed first with ten column volumes of 25 µl Trls. containing 10 ml EDTA, 10 ml CHAPS, pH 8.0. This was followed by washing with three column volumes of 25 ml of Tis, containing 0.5 µl of NaCl, 10 ml of CHAPS, pH 8.0. The elution was terminated by washing with 25 ml * 1 Tris containing 3.01 * 1 NaCl, 10 ml * 1 CHAPS, pH 8.0. The Blue Sepharose column eluate was diluted five times with 10 ml Na-phosphate buffer containing 10 ml * 1 CHAPS, pH 6.2, and then applied to the chromatography column. The column was washed with ten times the column volume with 10mL * 1 Na-phosphate buffer containing NaCl, 0.1X Pluronic F68, pH 6, 2. rPH. 2 was eluted from the column with 120 ml * 1 Na-phosphate buffer containing 100 µl * 1 NaCl and 0.1 X Pluronic F-68m, pH 7.5. In the case of the undiluted hydroxylapatite eluate, the pH was adjusted to pH 6.8 with 0.5 N-succinyl acid. This was followed by Sepharose SP column chromatography. The SP Sepharose column was washed ten times its volume with 50 ml * 1 sodium phosphate containing 125 ml * 1 NaCl, 0.1 X Pluronic F68, pH 6.8. Elution was performed with 50ml * 1 sodium phosphate with 125ml * 1 NaCl and 0.1X Pluronic F68, pH7.5. The elution of rPH.2 was finally adjusted by dilution to a final concentration of 4mg / ml with 50ml * 1 sodium phosphate containing 1251 * 1 NaCl, 0.15X Pluronic F68, pH 7.5. Next, Tueen 80 was added to a final concentration of 0.02X. The product formed was filtered through a 0.2 mn membrane and stored for further use.

3. Comparison of PAF-AH fragments with PAF-AH by sequencing

Purified preparations rPH.2 and rPH, 9 were compared to purified rPAF-AH compounds by N. terminal sequencing using an Applied Biosystems Model 473 Protein Sequencer sequencer (Applied Biosystems, Foster City, CA) and using a Protein Sequencer. with rPAF-AH less

C-terminal sequencing performed by Heulett-Packard Model G1009A C-terminal The prepared rPH.2 showed N-terminal heterogeneity in comparison. The N-terminal analysis of the prepared rPH.9 was also very similar to that of rPH.2, but compared to rPH.2, lower C-terminal heterogeneity was observed for rPH.9.

The purified rPH.2 protein showed most of the sequence. containing an N-terminal Ala ₄₇ (approximately 86-89%) and a smaller fraction of the sequences. having N-terminal Ala _4B (approximately 11-14)

*). This ratio was quite stable even under various cell fermentation conditions. Purified rPH.9 protein also contained in most sequences the N-terminal Ala ₄₇ - (between 83 to 90%) and a minor proportion with the N-terminal amino acid Ala <* _{with a} C of about 10 to 17%). In contrast. efforts to prepare polypeptides in bacteria starting with lle ₄₂ (rPAF-AH) resulted in a mixture of different polypeptides with N-terminal amino acids starting with Ala ₄₇ (20-53%), Ile ₄₂ (8-103) 3, and an artificially delivered initiator by metlonin fly -1 <37 to 72 30 in the vicinity of lle.q. ₂ . In the case of rPH.2 and rPH.9, the initiating inethionin was effectively removed by amnotherminal peptidase after bacterial peptide synthesis, leaving Ala at position 47 (or alanine at position 48) as the terminal residue.

C-terminal sequencing was performed very frequently on rPH.2, where C-terminal was detected as the main occurring sequence of HOOC-Asn-tyr (approximately 80 30, which is in good agreement with the predicted C-terminal sequence of the translation product, which the sequence of HOOC-Leu appeared only in 20% After further fractionation of rPH.2 on SDS-PAGE, further sequencing of the primary and secondary bands was performed, sequencing was determined as the C-terminal sequence. HOOC-Leu-fly from the lower secondary band (AHL described below to section B.5), which corresponds to a translation product truncated by 10 amino acid residues, which also shows a low HOOC-His level. Direct C-terminal sequencing was primarily determined as HOOC-Ile-His (approximately 78-91%, depending on batch) by r-sequencing of rPH.9. This result is in good agreement with the predicted C end of the translation product HOOC-lle ₄₂₉ -Hls _42e. However, this technique appears to have some background (noise), thus avoiding low levels of concentrations of other sequences.

4. Comparison of PAF-AH fragments with PAF-AH by the ľALDI-1 * IS method

The MALDI-1S method was applied to purified rPH.2 and rPH.9 products. The spectrum measured for rPH.2 showed two peaks.

which by their weight corresponded to the value expected for rPH.AH products (see Figure 4). This spectrum is similar to that obtained in the partially purified protein described in section B..1 above. The second peak, corresponding to the lower molecular weight, was present in about 20 to 30% of the majority of measurements. The spectrum of rPH.9 showed that the dominant peak corresponds to the predicted mass for the complete translation product of the vector PH.9, from which the mass of the initiating methionine is subtracted (see Figure 5). For rPH.9, a lower molecular weight minor peak was also observed. It was represented in about 5% of the total number of samples.

5. Comparison of PAF-AH fragments with PAF-AH by SDS-PAGE

Polyalkrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was applied to purified batches of rPAF-AH. rPH.2 and rPH.9. In the case of rPH.2, a very complex image of strips was obtained on the electropherogram, distributed around a site corresponding to the migration range expected for the rPAF-AH product. This site was determined using the appropriate molecular standards. Two or more dominant strips were observed on one or more of the gels, each having its two secondary strips in close proximity which were placed above and below the primary stripe. These upper secondary bands, middle primary bands and lower secondary bands were designated as AHU, ΑΗΙΊ, and AHL. All of these strips reacted with a monoclonal antibody prepared against rPAF-AH in a Western blot and were thus identified as rPAF-AH-related products. The upper secondary band of AHU increased its intensity as a function of protein storage time and probably represented a modified form of the rPAF-AH product. The results obtained from SDS-PAGE for rPAF-AH products are similar to those obtained by the same method for rPH.2. There are two larger stripes. which migrate near the predicted molecular weight for rPAF-AH, two minor bands above and the shadow below the major bands. In contrast, rPH.9 provides one dominant band on SDS-PAGE without apparent cleavage. Furthermore, poorly recognizable bands corresponding to a lower molecular weight and another band at a position corresponding to the weight of the dimer were also observed. However, no band corresponding to the AHU position was apparent.

The composition of the purified preparations of rPH.2 and rPH.9 was further analyzed by 2D gels and isoelectric focusing (IEF) in urea following SDS-PAGE in the second dimension. For rPH.9, five major spots distributed in the IEF direction are on two-dimensional electrophoresis. Heterogeneity in the number of charges appeared consistent across batches of rPH.9. In contrast to the above results, the 2D gel for rPH.2 appeared far more complicated as it contained about 15 spots distributed in IEF and SDS-PAGE.

6. Comparison of PAF-AH activity with that of PAF-AH fragments

Purified rPH.2 and rPH.9 products showed both enzymatic activity that was the same as for serum purified endogenous PAF-AH, both products also bind lipoprotein in the same manner as purified endogenous PAF-AH.

Example 11

Preliminary analysis of human plasma mRNA expression for PAF-AH was determined by Northern blot hybridization in human tissue samples.

one to three electrophoresis in

RNAs were prepared from human cortex, heart, kidney, placenta, thymus and tonsils using RNA Stat 60 Ctel-Test B, Friendswood, TX. RNA was also prepared from cells similar to the THP-1 human hematopoietic cell precursor (ATCC) TIB 202), in which differentiation into a macrophage-like phenotype was induced using phorbol ester of phorbolinyristyl acetate (PI * 1A). Tissue-derived RNA and RNA prepared from the THF-1 premylocyte cell line before and days after induction. were separated using a 1.2X agarous forinaldehyde gel and then transferred to a nitrocellulose membrane. A full-length PAF-AH cDNA clone, SAH406-3, prepared from human plasma, was labeled by random priming and hybridized to a membrane under conditions identical to those described in Example 3 for library browsing. The results obtained indicated that the PAF-AH probe hybridized to a band corresponding to 1.8 kb in thymus, tonsil, and, to a small extent, placental RNA samples.

PAF is synthesized in the brain under both normal and pathophysiological conditions. In view of the known inflammatory-inducing and potential neurotoxic properties of this molecule, it is believed that the mechanism of localization of PAF synthesis or its rapid catabolism is critical to maintaining the health of nerve tissue. The presence of PAF acetyl hydrolase in neural tissues indicates its protective role in these tissues. Is interesting. that both PAF-AH, i.e. bovine (bovine) heterotrimeric intracellular PAF-AH (its cloning is described in Hattori et al., J. Biol. Chem., 2G9C37) and pages 23150 to 23155 (1994) and PAF-AH according to of the present invention have been found in the brain.

In order to determine whether both enzymes expressed in the same or other parts of the brain, a human bovine cDNA homologue for intracellular PAF-AH was raclonated and samples of this brain expressing in the brain mRNA expressing PAF-AH mRNA according to the invention were compared using the same method, as described in the previous paragraph och. They were in the brain. Northern blot examined the following areas: small brain, medulla, spinal cord, basal nucleus, amygdala, pituitary, thalamus and tulle pole, frontal lobe and temporal lobe of the cerebral cortex. Traces of both enzymes were detected in each tissue. However, a heterotrimeric intracellular form of the enzyme was detected in a higher amount than the secreted form. Northern blot analysis also confirmed for other tissues and cells that the heterotrimeric form of the intracellular enzyme was very abundant in these tissues. The tissues where the enzyme was detected are as follows: thymus, prostate, testes, ovaries. small intestine, colon, peripheral blood leukocytes, macrophages, brain, liver, skeletal muscles, kidneys, pancreas and adrenal glands. This ubiquitous expression of this form of enzyme suggests that heterotrimeric intracellular PAF-AH has a general maintenance function in cells.

Furthermore, expression of RNA for PAF-AH in monocytes isolated from human blood and during their spontaneous differentiation of macrophages was investigated in culture. Little or no RNA was detected in fresh monocytes, but expression was induced and amplified during differentiation into macrophages. An accompanying phenomenon was also an increase in PAF-AH activity in the culture medium of differentiating cells. Expression of the human plasma RNA transcript for PAF-AH was also observed in THP-1 cells on the first day. but no longer on the third day following the induction. Baseline stage THP-1 cells did not express PAF-AH mRNA.

Example 12

PAF-AH expression in human and mouse tissues was examined by in situ hybridization.

Human tissues were obtained by National Disease Research Interchange and Cooperative Humari Tissue Netuork. Normal mouse brain and spinal cord, EAE spinal cord were obtained from S / JLJ mice. More normal mouse embryos were obtained from eleven to eighteen days after fertilization.

Parts of the tissues were placed in a Tissue Tek II cryoform (ils Laboratories, Inc., Naperville, IL) with a small amount of OCT compound (ils, Inc. Elkhart, IN). The samples were centered in the mold. the form filled with the OCT compound, then placed in a 2-methylbutane (C ₂ H ₅ CH (CH ₃ ) ₂ , ALdrich Chemical Company, Inc.) mixture and placed in liquid nitrogen. Once the tissue and the OCT compound are frozen in the cryoform, they are stored at -80 ° C until the block is cut. The tissue blocks were divided into sections of 6 µm thickness and attached to Vectabonds (CVector Laboratories, Inc., Burlingame, CA) coated slides and stored at -70 ° C and then placed at 50 ° C for approximately 5 minutes to remove condensation and then the samples are fixed in 4% paraformaldehyde for 20 minutes at 4 ° C, dehydrated with 70% C. 95%, 100% ethanol) for one minute for each step, and finally allowed to air dry for 30 minutes at room temperature. The filaments are hybridized in situ with radiolabeled single stranded mRNA generated from DNA derived from the internal 1 Kb HindIII fragment of the PAF-AH gene (308-1332 of SEQ ID NO: 7) by in vitro RNA transcription by incorporation of ^3S S-UTP (CAmersham) or from DNA derived from heterotrimeric intracellular PAF-AH cDNA identified by Hattori et al. Probes were used in different lengths from 250 to 500bp. Hybridization was performed overnight (C12 to 16 hours) at. 50 [deg.] C .; ^3S S-labeled C6x10 probe ^with cpm / inc), tRNA CO, 5 µg / inc), diethylpyrocarbonate-treated water (dep) was added to the hybridization buffer to adjust the concentrations of its components to 50% forinamide. 0.31 * 1 NaCl, 20ml * l Tris ph 7. 5. 10% dextran siren. 1x Denhardt's solution, 100mM ditintreitol CDTT) and 5ml * 1 EDTA. After hybridization, sections were washed for 1 hour at room temperature in 4x SSC / 10ml * 1 DTT, then 40 minutes at 60 ° C in 50% formamide / 1xSSC / 10mM DTT, 30 minutes at room temperature in 2 x SSC, 30 minutes at room temperature. temperature at 0.1 x SSC. The sections were then dehydrated, air-dried for 2 hours and then induced (after storage at 4 ° C in complete darkness) and stained with the hematoxyline / eosin contrast dye.

A. Brain floss

In both human and mouse brain, a strong signal was observed in the small brain Purkinje cell layer. then in the basket cells and in the central parts of individual neurons in nucleus dentate C one of the four nuclei in the cerebellum). Furthermore, a heterotrimeric form was also observed in these cells

PAF-AH. Furthermore, a signal was also found for plasma PAF-AH in granular and molecular layers of gray brain matter.

Hippocapmus

A strong signal was recorded in the hippocampus. especially in individual cells, which appeared to be neuronal cells. These cells were identified as polymorphic portions of the middle portion of neurons and granular cells. Also in the hippocampus a signal was found for the heterotrimeric iritracellular form of PAF-AH.

/ 7ozgroi / ý strain

As in human, taj and mouse brainstem, a strong signal was recorded in individual cells of gray matter.

crust

In the parts of the human cerebral cortex that were obtained from the main cerebral, tulle temporal cortex as well as in whole parts of the mouse brain, a strong signal was observed for individual cells. These cells were identified as pyramidal, star, and polymorphic cells. No differences were observed in the results obtained from the different layers of bark. The results obtained by the in situ hybridization method were different from those obtained by examining the cerebral cortex by Northern blotting. Probability differences result from higher sensitivity of the in situ hybridization method compared to the Northern blot method. In both the cerebellum and hippocampus, similar results were obtained confirming the expression of the heterotrimeric form of intracellular PAF-AH.

hypophysis

A slightly weaker signal was recorded on individual scattered cells of the distal portion of these human tissues.

B. Human colon

Both in normal and in the colon of patients with Crohn's disease, a signal appeared in the lymphatic aggregates present in the mucosal parts, the signal was slightly higher in patients with Crohn's disease. The samples from the colon of patients with Crohn's disease also showed a strong signal in the lamina propria. Similarly, a higher signal level was observed in diseased caecum samples, although a healthy intestine showed a low but still detectable signal. Samples from patients suffering from ulcerative colitis showed no signal, either in lymphatic aggregates or in the intestinal mucosa.

C. Human almonds and thymus

A signal signal was recorded for scattered cells in the almond and thymus germinal centers.

D. Human lymph nodes

For normal donor lymph node specimens, a strong signal was observed, whereas in the sample taken from the patient's lymph node in septic shock, the signal was very weak.

E. Human small intestine

Both the small intestine specimens from a healthy donor and the intestinal specimens from Crohn's disease patients gave only a weak signal in Peyer's plaques and intestinal mucosa. The signal was somewhat higher in the samples of the sick donors.

F. Human spleen and lungs

No signal was found in both spleen and lung spleen specimens and spleen specimens of patients suffering from spleen abscess and lung patients.

G. Mouse spinal cord

For both types of spinal cord, i.e. normal and AEE stage 3 spinal cord, a strong signal was recorded in the gray spinal cord mass. Expression was slightly higher in the EAE Stage 3 spinal cord. In the EAE Stage 3 spinal cord, cells in the white matter of the brain and perivascular connective tissue were likely to be infiltrated by macrophages or leukocytes and showed a signal that was not apparent in the normal spinal cord.

H. Mouse embryos

In the eleven-day embryo, the signal is evident in the central neural system in the fourth ventricle, which remains constant during the development of the cerebellum and embryonic brainstem. During embryo maturation, the signal becomes apparent in the central nervous system (spinal cord (day 12), primary cortex and Gasseri lymph node (day 14) and in the pituitary (day 16). In the peripheral nervous system, the signal becomes clear (beginning on days 14 and 15) in the nerves branching out of the spinal cord, and on day 17 a strong signal appears around the bases of the embryonic olfactory organs. Expression is also evident from day 14 and liver and lung, in the intestine from day 15 and in the posterior part of the mouth / throat from day 16. From day 18, the signal can be determined differentially as the signal in the cortex. hind brain (small brain and brainstem), in nerves branching from the lumbar region of the spinal cord, as a signal at the back of the mouth / throat, liver, kidney, as well as a possible weak signal in the lungs and intestine.

sum

PAF-AH mRNA expression in almonds, thymus, lymph nodes, Peyer's lungs, appendix and colon lymphatic aggregates is in good agreement with the conclusions. that the likely predominant source of PAF-AH in vivo is in the macrophage, since in all these tissues macrophages are strongly represented, serving as phagocytosis and antigen-presenting cells

The expression of PAF-AH in inflammatory tissues can be explained by the hypothesis that macrophage-derived monocytes play a major role in inflammatory processes. PAF-AH is believed to inactivate PAF and inflammatory-promoting phospholipids and thereby down-regulate the cascade of inflammatory processes initiated by these mediators.

PAF is detected in rats in all tissues and is secreted in the culture of small brain granular cells. In vitro and in vivo experiments have shown that PAF binds to specific receptors in nerve tissues and induces functional and phenotopic changes such as calcium mobilization. regulation of transcription that activates genes and differentiation of neural precursor cell lines, PC12. These observations clarify the role of PAF in the brain, and accordingly, experiments have been utilized using tissue culture cultures of the hyppocampus and analogs of PAF and its antagonists, which utilize PAF as an important backbone for long-term hypocampal enhancement. Therefore, in addition to the pathological role of PAF in inflammatory processes, PAF also appears to be involved in the routine neural signaling process. Thus, expression of extracellular PAF-AH in the brain can serve to regulate the duration and magnitude of PAF-mediated signaling.

Example 13

Using PAF-AH as the antigen produced in E. coli, monoclonal antibodies specific for human specific plasma PAF-AH were prepared.

The 1342 mouse was immunized on days 0, 19, and 40 with recombinant PAF-AH. As a dose just prior to the fusion, a dose of immunotherapy in PBS was used on immunized mice. Four days later, the mouse was sacrificed and its spleen sterile removed. This was then placed in a 10 ml serum-free RPMI 1640 medium dish. The cell suspension was prepared by rubbing the spleens between the two ends of frozen microscope slides immersed.

1640 with delivered 2 mM L-glutamine, units / ml penicillin and 100 µg / ml streptomycin (RPMI) (GIBCO. Canada). The cell suspension was filtered through a sterile 70-mesh Nltex cell filter (Becton Dicklnson, Parsippany, Neu Jersey), and then the cells were washed twice by centrifugation at 200 g for 20 min in serum free RPMI 1 mM sodium pyruvate medium, and pellets were resuspended again. in 20 ml of serum-free RPIII medium. Spleen cells from three native Balb / c mice were prepared in a similar manner. NS-1 myeloma cells were maintained in logarithmic phase in RP µl with 11 2 fetal bovine serum (FBS) (Hyclorie Laboratories, Inc., Logan, Utah) for three days prior to fusion, centrifuged at 200 g for 5 minutes and pellet twice washed in the same manner as described above.

Once 10 ^θ spleen cells were combined with 2.0 x 10 ⁷ NS-1 cells. centrifuged and the supernatant was aspirated. The sediment in the cuvette was split by tapping two cuvettes and then 1 ml of 37 ° C warm PEG 1500 (502 in 75 ml Hepes. PH 8.0) (Boehringer Llanheim) was added over one minute with stirring, followed by 7 ml of serum-free medium. for 7 minutes. This was followed by the addition of 8 ml RPIII and the cells were centrifuged at 200 g for 10 minutes. After discarding the supernatant, the pellet was resuspended in 200 µl RPµl containing 15 2 FBS, 100 µl sodium hypoxanthria, 0.4 µl aminopterin, 16 µl thymidine (HAT) (Gitico). 25 units / ml IL-6 (Boehringer ľlarinheim) and 1 x 10 5 thymocytes ^R / inl and the cells were plated on ten plates Corning flat-bottom 96-well plates (Corning, Corning New York).

On the second, fourth and sixth day following the fusion, 100 µl per well was exchanged for fresh. On day 8, the fusion was tested by ELISA. it was tested for the presence of mouse IgG fluttering with recombinant PAF-AH. Immulon 4 aliquot plates (Dynatech, Cambridge, IL) were coated with recombinant PAF-AH at a concentration of 100ng / plate diluted in 25 ml of TRIS, pH 7.5.

Incubation was performed at 37 ° C for two hours. Then, the coating solution was aspirated and 200 µL of blocking solution (0.52 fish skin gelatin (Sigma) diluted with C 11 F-PBS) was added dropwise and the blocking solution was incubated for 30 minutes at 37 ° C. Plates were then washed three times with PBS containing 0.052 Tween 20 (PBST) and 50 µl of culture supernatant was added to each well. After a further 30 minute incubation at 37 ^e C and subsequently washing three times was added to wells of a goat anti-mouse conjugate added IgG <fc) horseradish peroxidase CJackson ImmunoResearch. West Grove, Pennsylvania) diluted in PBS 1 3500. The plates were incubated as described above, washed four times with PBST followed by incubation with peroxidase substrate (100 µl / well) dissolved in substrate buffer. The solution had this composition (1 mg / ml o-feriylenedlamine (Sigma) and 0.1 µl / ml 30% H _and 0 _and in 100 ml citrate pH 4.5). After five minutes, the color reaction was stopped by adding 50 µl of 15% HeSCU. On a photometer reading the absorbance values in a CDynatech reader solution, the absorbbaricity was read at 490 nm - A ^ ₉₀ .

Selected wells from the fusion were double-purified by limiting dilution to a 96-well plate. After five days of incubation, the number of colonies per well was visually assessed. Hybridomas. which were cloned were the following: 9001E, 90E3A, 90E6C, 90G11D (ATCC HB 11724) and 90F2D CATCC HB 11725).

Isotypes of inonoclonal antibodies. produced by hybridomas were determined using the Isostrip CBoehringerannhelm system. Iridianapolis, IN). The results showed that the morioclonal antibodies produced by fusion 90 hybridomas were of the IgG _t type.

All monoclorial antibodies produced by hybridomas from fusion 90 could be used in ELISA assays but did not bind to PAF-AH in Western blots. To prepare antibodies that could recognize PAF-AH in a Western blot, mice were immunized with a recombinant enzyme. Hybridomas were prepared by the method described for fusion 90. but testing was by Western blot and not by ELISA. Thus, clones were selected that could be used for detection by this method.

For Western analysis, recombinant PAF-AH was mixed with an equal volume of sample buffer containing 125 ml * 1 Tris, pH 6, 8.4% SDS. 100 ml * 1 dithiothreitol and 0.05% brofenol blue. The sample was boiled for 5 minutes prior to electrophoresis. Bo), using a 12% SDS polyacrylamide gel (CNovex). Electrophoresis was performed at 40inA, followed by electrotransfer of the protein onto a polyvinylidene fluoride membrane (Pierce). The transfer took 1 hour at 124 V in a solution of 192mL glycine, 25mM Tris base, 20% methanol and 0.01% SDS. The membrane was allowed to incubate overnight in 20 mM Tris. 100mM NaCl (TBS) containing 5% bovine serum albumin (BSA, Sigma). The blot was further incubated for 1 hour at room temperature with rabbit polyclonal antiserum undiluted 1: 8000 with TBS buffer containing 5% BSA, followed by washing in TBS and incubation with alkaline phosphatase conjugated goat anti-mouse IgG conjugated in TBS for 1 hour at room temperature. temperature. The blot was followed by incubation with a substrate for alkaline phosphatase, i.e. 5-bromo-4-chloro-3-indolyl phosphate at concentrations of 0.02% and 0.03% tetrazolium blue in Tris-HCl, pH

9.5, 5 mM NaCl and lOOmM _and MgCl. The reaction was stopped by repeated washing with water.

containing 5% BSA washed again with TBS

Selected fusion wells whose supernatants were positive in Western analysis were processed as described above. Hybridone 143A reactive with PAF-AH in Western blots was cloned (ATCC HB 11900).

Polyclonal anti-serum specific for human plasma PAF-AH were prepared during 3-month immunization of rabbits with 1011 µg of purified recombinant enzyme in Freund's adjuvant.

Example 14

To assess the in vivo therapeutic effect of recombinant PAF-AH prepared according to the invention, experimental studies were conducted in an acute inflammatory model in the case of rat swelling (Henriues et al. Br. J. Pharmacol. 106: 579-589 (1992). Results of these Studies have shown that rPAF-AH blocks PAF-induced edema, and parallel studies have been conducted to compare the efficacy of PAF-AH with two commercially available PAF antagonists.

A. Preparation of PAF-AH

E. coli transformed with the PAF-AH expression vector puc trp AH were lysed in a microfluidizer. the solids were centrifuged and the cell supernatants were loaded onto a S-Sepharose column (Pharmacia). The column was washed extensively with a buffer containing 50 ml * 1 NaCl. 10ml * 1 CHAPS, 25ml * 1 * 1ES and 1ml * 1 EDTA. pH 5.5. PAF-AH was eluted with increasing NaCl, up to 11 * 1. Affinity chromatography on a Blue Sepharose column (Pharmacia) was used as the next step of the purification process. Before application of PAF-AH to the Blue Sepharose column, the sample was diluted 1 - 2, thereby reducing the salt concentration to 0.51 * 1 and adjusting the pH to 7.5. After extensive washing of the Blue Sepharose column with a buffer containing 0. 51 * 1 NaCl. 25ml * l Tris. 10ml CHAPS and 1ml * 1 EDTA. pH 7. 5. PAF-AH was eluted from the column with increasing NaCl concentration up to 3.01 * 1.

range 5000 performed with eridotoxin and

The purity of PAF-AH isolated by this method was usually 95%. so. as determined by SDS-PAGE with activity at up to 1000 U / ml. Additional quantitative controls of PAF-AH preparations included determination of hemolytic activity levels with freshly prepared rat erythrocytes. A buffer containing 25 ml * 1 Tris, 10 ml * 1 CHAPS was used as both the preservation buffer and the carrier for administration of the preparation. 0.51 * 1 NaCl, pH 7.5. The dosages used in the experiments were based on the determination of enzyme activity in assays directly prior to the experiments.

B. Edema induction (edema)

Long Evans rats (Charles River, Uilmlngtori, 1 * IA) between 6 and 8 weeks of age, weighing between 1 and 200 g, were used in all experiments. Prior to the experiment, the animals were anesthetized with a mixture of Ketaset anesthetic preparations (Fort Dodge Laboratories, Fort Dodge, IA), Rompun (11 * Shlesunee Mlsslon, KS) and AcePromazine (Aveco, Fort Dodge, IA). All anesthetics were administered subcutaneously at doses of approximately 2.5 mg

Ktasetu. 1.6 mg Rompun. 0.2 mg of AcePromasin per dose given to one animal. Swelling was induced in the feet by administration of either PAF or zymosan as described below. PAF for the realization of each stored in the mixture

The required volumes were fresh for each experiment and the animals (Sigma -1402) were freshly prepared from the 19, 1 ml * 1 chloroform / methanol (9 1 1) stock solution at -20 C. C.

dried under nitrogen, undiluted with 1 * 1000 in buffer containing 150 ml * 1 NaCl, 10 mM Tris. pH 7.5 and 0.25% BSA and sordated for five minutes. The animals were injected subcutaneously between the abdominal thigh with 50 µl of PAF (final dose 0. 95 nmol). Swelling occurred in an hour, in some cases after two hours. Zymozan A (Sigma A-8800) was prepared as a 10 mg / ml suspension in PS. subcutaneously between the tummy on the hind foot of 50 µl zymosan (final dose 500 µg) and swelling appeared after two hours.

Swelling was quantified by measuring the volume of the foot immediately before administration of PAF or zymosan and then at that time after administration of PAF and zymosan. Swelling was quantified by increasing the foot volume in milliliters. Volume measurement was performed on a plethysmometer (UGO Basile, model 7150), which can be measured. the volume of water displaced by the immersed foot. In order to be comparable in size to the submerged parts of the foot at admiration of time intervals, the back of the foot was marked with irreversible ink where the heeling ends. Repeated measurements of the same foot using this technique indicated measurement accuracy with an error of up to 5%.

C. Methods of Administration and Dosage of PAF-AH

PAF-AH was injected locally between the tummy on the foot or systemically by intravenous injection into the tail vein. When administered locally, the rats received 100 µl of PAF-AH (4000-6000 U / ml) subcutaneously between the right hind abdomen. The left leg served as a control, injected with a 100 µl dose of vehicle (buffered saline). For systemic administration of PAF-AH, rats were given the indicated amount of PAF-AH units in 300 µl of carrier by tail vein injection. Control animals received the same volume of vehicle intravenously into the tail vein.

D. Topical Administration of PAF-AH

Rats (N = ^s 4) were injected subcutaneously between the tummy of the right foot with a 100 µl injection of PAF-AH (4000-6000 U / ml). Only the vehicle (100 µl of buffered saline) was injected into the left foot. Four other rats received only the dose of vehicle. All rats were immediately subjected to PAF by subcutaneous injection and the volume was measured one hour after administration. Figure 6, where swelling size is expressed by the increase in foot volume (ml) ± SEM for each treatment group, shows that PAF-induced swelling is blocked by local administration of PAF-AH. In the group of animals treated with PAF-AH prior to PAF administration, there was a decrease in swelling compared to the control group of animals. In the PAF-AH treated mice, an increase in foot volume of only 0.08 ml ± 0 was observed. 008 (SEM). The increase in foot volume was due to injection of PAF into the foot. This increase in volume was not observed in soles injected solely with the vehicle.

E. Intravenous administration of PAF-AH

Rats (N = 4 per group) were treated by intravenous injection of PAF-AH (2000 U in 300 µl vehicle) 15 minutes before PAF administration. Swelling occurred 1-2 hours after PAF administration. Figure 7, in which the swelling size is expressed in volume increase in (ml) ± SEM for each study group, shows that intravenous application of PAF-AH blocks the development of swelling due to PAF administration one to two hours after administration. The group that was treated with 20QUU PAF-AH by the intravenous route showed a decrease in inflammation within two hours. The mean increase in the volume of liquid treated with PAF-AH was 0.10 ± 0.08 (SEM), compared to an increase of 0.56 ml ± 0.1 in the vehicle-treated control group.

F. Comparison of PAF-AH Effect in Preventing PAF or Zymozan-induced Swelling

Rats (N = 4 per group) were treated by intravenous injection of PAF-AH (2QQ0U in 300 µl vehicle) or vehicle alone. 15 minutes prior to this injection, the animals received PAF or zymozan A and the foot volume was measured after one or two hours. Figure 8 shows the swelling size as the mean volume increase in (ml) ± SEM for all treatment groups. It can be seen from the figure that the administration of PAF-AH (2000 U) was effective in reducing PAF-induced foot edema, but is ineffective in blocking edema formation with zymosan administration. The mean volume increase for the PAF-AH group was 0.08 ± 0. 02 (SEI * I), versus an increase of 0.49 ± 0.03 measured for the control group.

G. Administration of an effective dose determined by titration of PAF-AH. that gives protection

In two separate experiments, rats (N = 3 to 4 per group) were administered intravenously with PAF-AH in a series of dilutions or vehicle only in 300 µl volumes 15 minutes prior to dosing with PAF. Both feet of the animals were treated with PAF in the same manner as described above, the swelling was evaluated after one hour. Figure 9 shows the swelling size as the mean volume increase in (ml) ± SEM for all treatment groups. It can be seen from the figure that administration of increasing doses of PAF-AH also prevents an increasing tendency to produce swelling induced by PAF injection in the rat. The experiment was found I0 _of PAF-AH given by injection between 40 and 80 U per rat.

H. Efficacy of PAF-AH in vivo as a function of time after administration

In two separate experiments, rats (N = 3 to 4 per group) were administered intravenously with PAF-AH (200 U in 300 µl vehicle) or vehicle only. After administration of PAF-AH, rats received a dose of PAF at time intervals beginning 15 minutes after PAF-AH administration up to 47 hours after PAF-AH administration. The swelling was then assessed one hour after PAF administration. Figure 10 shows swelling size as the mean volume increase in (ml) ± SEI * 1 for all treatment groups. From the figure, it is evident that administration of 200U PAF-Ah will protect rats from induced PAF swelling for at least 24 hours.

I. Pharmacokinetics of PAF-AH

Four rats received a dose of 2000 PAF-AH by intravenous injection in 300 µl volume. Plasma was collected at various time points and stored at 4 ° C. Plasma concentration of PAF-AH was determined by ELISA, using a type of double-binding mAb assay. Briefly, the 90G11D monoclonal antibody (Example 13) was diluted in 50 ml of carbonate buffer pH 9.0 to a concentration of 100 ng / ml and allowed to bind to the Immulon 4 microtiter plate overnight at +4 ° C. After extensive washing with PBS containing 0.05% Tueen 20, the plates were blocked for 1 hour at room temperature with 0.5% fish skin gelatin (Sigma) diluted in PBS. Serum samples diluted in PBS containing 15mL of CHAPS were plated in duplicate on the washed plate and incubated for 1 hour at room temperature. After washing, the 90F2D monoclonal antibody conjugate with biotirioin (Example 13) was added to each well at a concentration of 5 µg / ml. Conjugate was allowed to incubate for one hour. After washing, 50 µl was added to each well.

Extra avldine (Sigma) at 1: 1000 dilution. Avidin was incubated for one hour at room temperature. After washing, the OPD substrate was added and allowed to develop staining, which was measured on a photometer. The enzyme activity was then read from the calibration curve according to the measured values. From Figure 11, where the dots represent mean ± SEI * 1, it is evident that in plasma collected an hour prior to administration of PAF-AH levels, they predict concentrations in plasma samples of 5-6 ml in rats weighing 180-200 grams, average 374 U / ml ± 58.2. After one hour, the concentration levels steadily decrease until they reach an average value of 19.3 U / ml ± 3.4 after 24 hours, which is still appreciably diluted in PBS. room temperature.

higher than the concentration of endogenous PAF-AH, which reached approximately 4 U / ml in enzymatic assays.

J. Efficacy of PAF-AH compared to PAF antagonists

Rats (N = 4 per group) were treated with one of three potential anti-inflammatory agents: PAF antagonist CV3988 (Biomol L-103) administered IP (2mg in 200µl EtOH). PAF antagonist Alpazol (Sigma A-8800) administered IP (2 mg in 200 µl EtOH) or PAF-AH (2000 U) administered IV. A control group of rats received 300 µl of carrier IV. PAF antagonists have been administered IP because they are soluble in ethanol. Rats treated with either CV3988 or Alprazole were given PAF 30 minutes after the administration of these antagonists, to allow PAF antagonists to circulate, while PAF-AH and rat treated carriers were administered 15 minutes after administration of the enzyme. PAF-AH-treated rats showed a reduction in PAF-induced swelling until additional CV3988 and Aprazolain antagonists were placed behind them. This is shown in Figure 12, where swelling is shown as the mean volume increase in (inl) ± SEI * 1 for all groups studied.

Summarizing the results obtained, it is clear that rPAF-AH effectively blocks in vivo PAF-induced edema. Administration of PAF-AH products can be either local or systemic by IV injection. In dosing studies, doses of IV injections ranged from 160-2000 U / rat. These benefits dramatically reduce PAF mediated inflammation, while a dose of 01 _S was between 40 and 80 ϋ / rat. Calculations based on plasma volume for a rat weighing 180-200 grams provide a good assumption that plasma concentrations of 25-40 U / ml effectively prevented PAF-induced edema. These assumptions were supported by preliminary pharmacokinetic studies. A dose of 2000U of PAF-AH was found to be effective in blocking PAF-induced edema for at least 24 hours. Plasma PAF-AH concentrations of approximately 25 U / ml were detected 24 hours after administration. PAF-AH blocked PAF-induced swelling much more efficiently than both testing of said other PAF antagonists. Taken together, these results confirm that PAF-AH effectively blocks PAF-induced inflammation and could be of therapeutic importance in the treatment of diseases where PAF is the primary mediator.

Example 15

The recombinant PAF-AH of the invention was tested in vivo in a second model, pleural inflammation (pleuritis) induced by PAF. As previously shown, when applied to the pleural space, it induces vascular leakage. (Hebriques et al., Supra). Female rats (Charles River, 180-200 g) were injected into the tail vein with 200 µl of 1% Evans Blue dissolved in 300 µl of recombinant PAF-AH (1500 µl / ml / hour, prepared as described in Example 14), or with just the same volume of control buffer. After 15 minutes, 100µl rats were injected with PAF (2nmol) into the pleural space. After one hour of PAF treatment they were. rats are killed and aspirated by washing the pleural cavity with 3 ml of heparinized PBS. Vessel leakage was measured by the amount of Evaris blue in the pleural space by measuring absorbance at 620 nm. In the rats initially given PAF-AH, the leakage of the vessels was much less than that of the control group of animals (representing more than 80% reduction in inflammation).

Previous results support the possibility of using recombinant PAF-AH of the invention as a medicament for pleural inflammation.

Example 16

The efficacy of the recombinant PAF-AH enzyme of the invention was also tested in a model of antigen-induced eosinophil formation. The accumulation of eosinophils in the airways is a characteristic feature of the late phase of the immune response that occurs in asthma. rnititis and eczema, unlike BALB / c (Charles River), were sensitized by two intraperitoneal injections containing 1 µg ovalbumin (OVA) in 4 mg aluminum hydroxide (Imject alum, Pierce Laboratories, Rockford, IL) given at two week intervals. On the fourteenth day following the second immunoglobulin, the sensitized mice were subjected to an immunoassay with aerosolized OVA or saline as a control.

Prior to the test, mice were randomly divided into four groups of four mice. Plushes in Groups 1 and 3 were treated with 140 µl of control buffer containing 25ml * 1 Tris, 0.51 * 1 NaCl, 1ml * 1 EDTA and 0.1Z Tueen 80 administered by intravenous injection. Plushes in Group 2a 4 were treated with 750 units of PAF-AH (5500U / ml activity administered in 140 µl of PAF-AH buffer). Thirty minutes after PAF-AH or buffer administration, mice in Groups 1 and 2 were exposed to aerosolized PBS. as described below, while mice in groups 3 and 4 were exposed to aerosolized OVA. 24 hours later, the mice were. re-treated a second time with either 140 µl of buffer (groups 1 and 3) or 750 units of PAF-AH in 140 µl of buffer (groups 2 and 4). The compounds were injected intravenously.

Trachea eosifile infiltration was induced in sensitized mice by exposure of animals to aerolized OVA. Sensitized mice were placed in 50 µl conical centrifuge tubes (Cormimg) and forced to breathe aerolized OVA (50 mg / ml) dissolved in 0.9% saline for 20 minutes using a nebulizer (Model 646, DeVilblss Corp., Somerset, PA). Control mice were treated in the same manner except that only 0.9% saline was used in the nebulizer. 48 hours after exposure to aerolized OVA or saline, mice were sacrificed and trachea removed. Tracheas from each group were firmly captured in OCT and stored at -70 ° C until the slides were cut.

To evaluate eosinophil trachea infiltration, tissue sections from four groups of mice were stained with either Luna solution and haematoxylin-eosin solution or peroxidase. Twelve six-micron sections were cut from each group and numbered accordingly. Sections numbered odd were stained with Luna according to the following procedure. The sections were fixed for 5 minutes at room temperature, rinsed with running water three times for 2 minutes at room temperature and then rinsed in two changes of distilled water (dH ₂ O) for one minute at room temperature. Tissue sections were stained with Luna for 5 minutes at room temperature (Luna is composed of 90 ml Ueigert's iron hematoxylin (Ueigert's Irori heinatoxylin) and 10 ml 12 Bierbrich Scarlet). The stained sections were immersed in 12 acidic alcohol 6 times, rinsed in running water for 1 minute at room temperature, immersed in 0.52 of lithium carbonate solution, rinsed five times with running water for 2 min at room temperature. Sections were dehydrated with ethanol at concentrations of 70 - 95 - 2 - 100 2, left for 1 minute at room temperature, further cleaned twice with xylene for 1 minute at room temperature and attached to Cytoseal 60.

For peroxidase staining, segments were fixed with acetone at 4 ° C for 10 minutes and air dried. 200 µl of DAB solution was added to each portion and left at room temperature for 5 min. Sections were rinsed with tap water at room temperature for 5 min and 2 drops of 12 osmium acid were added to each segment for 3-5 s. Sections were rinsed with tap water for 5 min at room temperature and counterstained with Mayer's hematoxylin at 25 ° C at room temperature. Then, the sections were rinsed with running water for 5 min and dehydrated with an ethanol series 70 of 2 - 95 2 - 100 for 1 minute at each concentration, at room temperature. Sections were further cleaned twice with xylene for 1 minute at room temperature and mounted in Cyboseal 60.

The number of eosinophils in submucosal tracheal tissue was evaluated. The trachea of the mouse first and second groups contained very few eosinophils alone in submucosal tissue. Supposedly, the tracheas of the third group of mice pre-treated with buffer and exposed to OVA dust were a large number of eosinophils in subniucosal tissue. In contrast, trachea mice. The fourth group, which had been pre-treated with PAF-AH and exposed to OVA dusting, contained only a few eosinophils in submucosal tissue compared to the control group and groups 1 and 2.

Therefore, therapeutic treatment of PAF-AH may be indicated in cases of a late immune response in which eosinophils accumulate, as in the case of asthma and rhinitis.

Example 17

The patented PAF-AH product has been tested for two different rat models in the treatment of necrotic enterocolitis (NEC), acute haemorrhagic intestinal necrosis that occurs in fetuses of low birth weight and causes significant morbidity and mortality. Previous experiments have shown that glucocorticoid treatment reduced the incidence of NEC in animals and premature infants and glucocorticoid activity is believed to occur through elevated plasma PAF-AH temperature.

A. Activity in PAF-induced NIJEC rats

1. Prevention

Recombine-tan PAF-AH product, rPH.2 (25,000 units in 0, 3 ml, groups 2 and 4) or buffer alone (25ml * 1 Tris, 0.51 * 1 NaCl, 1ml * 1 EDTA and 0, IX Tueen 80) (groups 1 and 3) were administered to the tail vein of female Uistar rats (n = 3) weighing 180-200 g. 15 minutes after administration of rPH.2 or buffer alone, the animals were injected as described by Furukau, et al. (J. Pediatr. Res. 34 * 237-241 (1993)), into the abdominal aorta at the level of the superorous mesenteric artery, either BSA alone (0.25 X) - saline (Groups 1 and 2) or PAF (0.2 µg) (100 mg) suspended in saline containing BSA (groups 3 and 4). The small intestine was removed from the caecum after two hours by severing the Trietz ligament, thoroughly rinsed with cold saline, and evaluated. For microscopic observation, upper, middle and lower small intestine samples were obtained. The tissues were fixed in buffered formalin and samples prepared for microscopic evaluation by hematoxiline and eosin staining. The experiment was repeated three times.

A cursory examination of the intestines revealed normal findings in BSA-treated saline groups. Similarly, injected rPH.2 in the absence of PAF had no effect on the findings in the intestines (Cgross findings). In contrast, injection of PAF into the descending aorta resulted in rapid and severe discolouration and haemorrhage of the serous intestinal surface. A similar haemorrhage was observed when the small intestine segments were observed on the mucosal side and the small intestine was completely necrotic. When rPH.2 was injected into the tail vein 15 min before PAF was administered to the aorta, the findings were normal.

The microscopic observation of the intestines in groups 1, 2 and 4 revealed a normal villus architecture and a normal population of cells in the lamina proprla. In contrast, in the group treated with PAF alone, strong mucosa rheumatism and haemorrhage were found throughout the mucosa. Plasma PAF-AH activities were determined in the same rats as in the previous experiment. PAF-AH activity was determined as described below. Blood samples were taken before injection into the tail vein. In addition, samples were taken from the vena cava immediately prior to PAF injection and at the time of sacrifice. Blood was collected at about 50 µl into heparinized capillary tubes. Plasma was obtained by centrifugation (C980xg for 5 minutes). The enzyme was determined by Yasuda and Johnston. Endocrinology, 1301 708-716 (C1992).

Plasma PAF-AH activity in all rats before injection was 75 ± 2.5 units (unit 1 equals 1 nmol x min ^-1 x ml ^-1 plasma). PAF-AH activities measured in plasma were minutes after injection of buffer alone 75, 2 ± 2.6 units in Group 1 and 76.7 ± 3.5 units in Group 3. After 15 minutes, PAF-AH activity was detected in animal plasma . injected 25,000 rPH.2 units, 2249 ± 341 units in Group 2, and 2494 ± 623 units in Group 4. Activity in Groups 2 and 4 remained elevated (1855 ± 257 units) until sacrifice (2 1/4 hours). after injection (rPH.2) (group 2 = 1771 ± 308; group 4 = 1939 ± 478). These results indicate that PAF-Ah activity in the plasma of injected rats (Groups Ia and 3a). did not change during the experiment. For animals injected with PAF. developed NEC, while all rats given rPH.2 and then injected with PAF. were completely protected.

2. Dosage-dependent NEC prevention

In order to determine the dose-dependency of NEC protective activity in rats. animals were treated with increasing doses of rPH.2 15 min before PAF administration. The rPH.2 doses ranged from 25, 5 to 25,500 units that were. administered into the tail vein of rats. PAF (0.1 µg in 0.2 saline with BSA) was then injected 15 min after rPH.2 administration into the abdominal aorta. 2 hours after PAF administration, the small intestine was removed and NEC was evaluated. Plasma PAF-AH activity was determined before exogenous enzyme administration and 15 minutes and 2 1/4 hours after rPH.2 administration. Results are the mean of 2-5 animals in each group. All rats that received less than 2,000 enzyme units developed an NEC. PAF-AH activity in the plasma of animals receiving the lowest protective doses of enzyme (2040 units) reached 363 units per ml of plasma after 15 minutes, a 5-fold increase from baseline. When rPH.2 was administered at a dose of less than 1020 total units, the mean plasma enzyme activity was about 160 and lower, and NECs were found in all animals.

3. Duration of protection against NEC

In order to determine the length of the exogenous action of the PAF-AH product required to protect against NEC, rats were injected once with a fixed amount of enzyme and subsequently administered PAF at various time intervals. rPH.2 (8,500 units in 0.3 ml) or drug-free solution alone was administered to the rat from the tail vein and PAF (0.36 ng in 0.2 ml BSA in saline) injected into the abdominal aorta at various time intervals after administration of the enzyme. Small intestines were removed 2 hours after PAF injection and examined and histologically observed for NEC. Plasma PAF-AH activity was determined at various intervals after enzyme administration and two hours after PAF administration. Mean values of enzyme activity ± standard deviation were determined in each group.

The results showed that no rat developed NEC within the first 8 hours after rPH.2 injection, whereas 100 X animals that were subsequently treated with PAF 24 and 48 hours after enzyme injection developed NEC.

4. Reversal of NEC

To determine whether PAF-AH administration could reverse the development of NEC induced by PAF injection, 25,000 units of enzyme were injected into the vena cava two minutes after successful PAF (0.4 µg). NEC was not formed in any animal. However, when rPH.2 was administered in this manner 15 minutes after PAF injection, NEC was formed in all animals. which corresponds to the rapid course of development of NEC induced by PAF administration, as described previously by Furukav et al. (see above). Taken together, these results indicate that a relatively small increase (5-fold) in plasma PAF-AH activity is able to prevent NEC. These observations, together with previous reports that PAF-AH activity in rabbit fetal plasma Clakki et al., Proc. Natl. Acad. Sci. (USA) 85: 728-732 (1988)] and untreated children

CCaplan et al., J. Pediatr. 116: 908-964 (1990)] was relatively low, suggesting that the prophylactic administration of formulations containing the human recombinant PAF-AH product to children of low birth weight can be used in the treatment of NEC.

B. Activity in neonatal NEC model

Efficacy of PAF-AH. rPH.2. was evaluated in the NEC model for newly born stressed rats by the following procedure by administration of compound feed and asphyxia. which are two risk factors for human disease. In this model system, 70-80% of the animals had evident and microscopic bowel damage by day 3 of life, similar to neonatal NEC. Rat neonates were obtained from breeding Sprague-Dauley breeding CHarlari Sprague-Dauley rats. Indianapolis. IN), under CO ₂ anesthesia and born with abdominal infections. Newborn animals were collected. dried and placed in the newborn infant incubator throughout the experiment.

First, the dosage and absorption characteristics of rPH.2 were determined in each group. Normal neonatal rats were administered enteral or intraperitoneally at one time with three doses of rPH.2 (31 µgda, 1Slambda, or 75 µgda) and blood was then collected for determination of PAF-AH activity in plasma after 1 h. 6h and 24h. PAF-AH activity was measured by incubation with substrate CCray et al. Nature, 374 (549 «1995)] and ELISA using anti-human rPAF-AH monoclonal antibodies for each sample (C90F2D and 90G11D, as described in Example 13). . Immunohistological assays were performed using standard techniques using antibodies 1-100 and incubated overnight.

In normal-born rat pups, no increase in plasma PAF-AH activity was observed after either rPH.2 intestinal administration with either substrate incubation method or ELISA. For intraperitoneal administration of rPH.2, circulating PAF-AH activity was measurable within one hour after dosing by both methods and peaked 6h after dosing. Higher doses of rPH.2 (Cod 3 to 75 µm, 10 to 250 U) elicited higher PAF-AH activity in plasma. Following enteral administration, immunohistochemical analysis revealed the presence of rPAF-AH product in intestinal mucosal epithelial cells. Activity was concentrated mainly in intestinal villas with minimal dye presence in cryptic cells. The ileum was stained more intensively than the jejunum, and some rPAF-AH product was detected immunochemically in the colon sections. No observable staining was found for the control samples and the samples obtained from animals treated with intraperitoneally. Thus, enteral administration of rPAF-AH resulted in local accumulation in the mucosal epithelium. meanwhile, administration of rPAF-AH increased levels but not local accumulation in the mucosa.

what intraperitoneally circulating enzyme,

In the NEC model, NEC according to Caplan et al. Was induced in neonatal rats. Pathol. 14: 1017-1028 (1994). In this procedure, the animals were fed every three hours with a tube of a soluble infant feed mix (Esbiliac, Borden Inc.).

the feed dose of 0.1 ml / feed was later increased to 0.4 ml / feed until day four. In animals, asphyxia% nitrogen was induced twice a day for 50 seconds at sealed

Initial tolerance of the case all by inhalation of the 100 plastic chamber and the animals were then exposed to cold (4 ° C) for 10 min. Intestinal and bladder function was stimulated by gentle manipulation after each feeding. The animals were fed for 96 hours or until they showed signs of anxiety. Diseased animals inall abdominal dlstenzlu. bloody stools, breathing difficulties, cyanosis, letharia and were killed by decapitation. After sacrifice, the intestines of all rats were evaluated for the presence of necrosis and fixed in formalin for later histological analysis. The samples were embedded in paraffin, cut with a microtome. stained with hematoxylin and eosin and evaluated by two independent observers. Intestinal damage was designated as 1+ for detached or detached epithelial cells, 2+ for peeling epithelial cells into the middle villus layer, 3+ for whole villain necrosis and 4+ for transmural necrosis.

To determine the efficacy of rPH.2, three groups of rats were administered enteral, intraperitoneally, or both. RPH.2 contained 0.8 mg / ml protein and about 4000 units / mg PAF-AH with an endotoxin / protein ratio <0.5 EU / mg. For enteral use of rPH.2, animals were given 251 µg (C80U) of the compound diluted in the ration by the orogastric tube (every three hours). For intraperitoneal use, animals were injected 75 µg by intraperitoneal injection twice daily. Control animals (20 mM NaPCU, pH 7.4) were administered without rPH.2 and each experimental group. Mortality and symptoms of NEC in each treatment group and differences were analyzed by Fischer's exact test. Values at p <0.05 were considered significant. The results are shown in Table 9.

the corresponding buffer volume was monitored simultaneously with

Table 9

NEC dead control (i.p. administration) 7/10 8/10 rPH.2 (240 U i.p., twice daily) 6/11 8/11 control (enteral administration) 19/26 21/26 rPH.2 (80 U enteral, after 3h) 6/26 7/26 control (i.p. + enteral administration) 10/17 12/17 rPH.2 (240 U i.p. twice daily a 3/14 7/14

U enteral after 3h)

The data represent the cumulative results of four independent experiments at i. p, administration, four enteral administration experiments and three i.p. + enteral administration.

Enteral administration of rPH.2 significantly reduced both NEC and animal mortality compared to controls. The results of four different experiments showed that pretreatment of rPH.2 animals reduced NEC from 19/26 (control) to 6/26 (p <0.001).

Iritestinal damage to treated and control animals was varied, but in most cases it was midvillous necrosis in several segments, complete villus necrosis in other parts, or the same sites with transmutral necrosis, the remaining parts had normal histological findings. The highest NEC grade for both treated and control animals with intestinal impairment was similar (median 2, 8 for controls and 2, 4 for pre-treated rPH.2. P> 0.05).

Intraperitoneal administration of rPH.2 had no significant effect on NEC production and animal mortality in this model system. The onset of symptoms in this group and the control was similar (40 ± 5 hours for the control. 36 ± 7 hours in the rPH.2-treated group) and the degree of NEC was also similar in both groups (mean 2, 6 in the control and 2 groups). 5 in the group of rats treated with rPH.2).

Additional experiments were performed in which rats were administered rPH.2 both enterally and intraperitoneally at the same dosages as when administered by either route (251 µgda rPH.2 at each 3 hour feed and 75 µg intraperitoneal injection twice daily). The results are shown in Table 9. Although there was no significant difference in the number of animals killed between treatment and control, treatment with rPH.2 significantly reduced the incidence of NEC (10/17 for control and 3/14 for rPH.2-treated rats, p = 0.04). Six out of seven dead animals from the rPH.2-treated group were found to have a positive E. coli blood count just prior to death.

These results confirm the role of PAF-AH products in the neonatal model of NEC that is not induced by PAF. Enteral treatment of the rPAF-AH product prevented NEC, while intraperitoneal treatment at these doses had no apparent effect. These results suggest. that a PAF-AH product added to an infant formula for newborns at risk of developing NEC could reduce the incidence of this disease.

Example 18

The efficacy of PAF-AH product in acute respiratory distress syndrome (ARDS) was studied in guinea pigs.

Intravenously irrigated PAF induced severe lung inflammation in guinea pigs resembling initial ARDS in humans. Within minutes after intravenous administration of PAF, the lung parenchyma was filled with constricted bronchi and bronchioles (Lellouch-Tubiana et al., Supra). Platelets and polymorphonuclear neutrophils began to marginalize and cell aggregates were readily detectable along the pulmonary arterioles of Lellouch-Tubiana Br. J. Exp. Path., 66: 345-355 (1985) 3. PAF infusions also damaged bronchial epithelial cells, which separated from the walls and accumulated in the airways. Such damage to the epithelial cells in the airway is consistent with the formation of hyaline membranes that occur in humans during ARDS. The ilginalization of neutrophils and platelets is rapidly followed by diapedesis of these cells into the alveolar septum and alveolar lung space. PAF-induced cellular infiltrates are accompanied by significant vascular leakage and the development of EKirsch airway edema. Exp. Lung Res., 18: 447-459 (1992). The edema was also confirmed by in vitro studies where PAF induced extravasation fibrinogériu ^IAS labeled I in a dose-dependent (10-1000 ng / ml) for the washed guinea pig lung EBasran, Br. J. Pharmacol. 77: 437 (1982) 3.

Based on the above observations, an ARDS model was developed in guinea pigs. The cannula is placed under anesthesia in the cervical vein of male Hartly guinea pigs (approximately 300-400 g) and PAF diluted in a 500 µg PBS with 0.25% bovine serum albumin (PBS-BSA) infused over 15 minutes in a total dose 100-400 ng / kg. At various intervals after PAF infusion, the animals were sacrificed and lung tissue was removed. In the case of guinea pigs infused with PAF, time-dependent lung damage and inflammation, which lasted up to 60 minutes, were clearly evident within 15 minutes. Neutrophils and red blood cells are present in the guinea pig alveolar compartments given PAF, whereas their presence was not detected in control animals. Epithelial cell damage resembling hyaline membrane formation in human ARDS patients has also been demonstrated. In bronchoalveolar lavage (BAL) samples taken from guinea pigs that were infused with PAF, a strong accumulation of proteins in the inflamed lungs and vascular leakage was detected.

In this guinea pig model of ARDS, rPH.2 was found to completely prevent PAF-induced lung damage. Groups of guinea pigs were pretreated with either rPH.2 (2000 units in 500 µl) or only PAF-AH buffer alone (500 µl). After 15 minutes, guinea pigs were infused with PAF (400 ng / kg in 500 µl) and administered for 15 minutes. The guinea pig control group was infused with 500 µl PBS-BSA. After completion of the PAF infusion, animals were sacrificed and BAL fluid was collected by lavage lung X with 10 ml saline containing 2 µ / ml heparin to prevent clotting. In order to determine the protein content of the BAL, samples were diluted 1 - 10 with saline and OD 280 was determined. A protein concentration of 2.10 ± 1.3 mg / ml was found in the BAL of guinea pigs treated with buffer alone. In contrast, BAL fluid from animals infused with PAF contained

12.55 ± 1.65 mg / ml proteins. In guinea pigs pre-treated with rPH.2, a protein content of 1.13 ± 0.25 mg / ml was found in the BAL fluid, which is comparable to the control and demonstrates that the PAF-AH product completely prevents PAF-induced pulmonary edema.

Example 19

The efficacy of PAF-AH, rPH.2, was evaluated for two model systems of acute pancreatitis.

A. Activity in the rat pancreatitis model

Male Uistar rats (200-250 g) were purchased from Charles River Laboratories (Ullmlngton, 11A). The rats were housed in a controlled climate room of 23 ± 2 ° C, a 12 hour light / dark cycle, and fed standard laboratory feed and water ad libitum. Animals were randomized into control and experimental groups. The rats were anesthetized with 50 mg / kg sodium pentobarbltal administered intraperitoneally and then a polyvinyl catheter (V3 size, Biolab Products. Lake Havasu, AZ) was inserted into the jugular vein. The catheter was pulled subcutaneously and opened in the dorsal cervical region. The animals were allowed to wake up with anesthesia. The rats had free access to water but were left without food overnight. The experiments were carried out the following day on conscious animals. Until the beginning of the experiment, the catheter was filled with a constant infusion of saline (0.2 ml / h). That day. when the experiment was performed, the animals were injected intravenously with rPH.2 or the portable control alone and subsequently infused either (1) 5 µg / kg caerulein for an hour over 3.5 hours, or (2) 10 µg / kg caerulein for an hour over 5 hours , (Research Plus, Bayonne, N.J.). Immediately after the end of the infusion, the animals were anesthetized with sodium pentobarbltal. the abdominal cavities were opened, and 5 ml of blood was aspirated from the inner vein of the cava for further determination. The animals were then sacrificed by bleeding. Serum amylase serum lipase and serum bilirubin and pancreas collected were measured. Portions of the pancreas were either fixed in 42 phosphate formaldehyde buffer solution for histological activities or immediately frozen at -80 ° C to measure myeloperoxidase activity. Other portions of the pancreas were used to determine the water content of the pancreas, pancreas amylase and trypsin, as described below. Myeloperoxidase activity, which shows the degree of neutrophil staining, was determined in the pancreas and lungs as described below. Data were statistically evaluated using an unpaired Student's t-test. mean + S.E. 1 * 1. least

The values shown represent three different experiments. Results at p <0.05 are considered significant.

1. Water content in the pancreas

The pancreas segments were dried and weighed (fresh weight) and then dried for 34 hours at 120 ° C and reweighed (dry weight). Pancreas water was calculated as the difference between Fresh and Dry weight and expressed as a percentage of Fresh Weight. Increased water content in the pancreas indicated edema.

2. Serum and pancreatic amylase

Serum amylase activity was measured by substrate

4,6-ethylidene (G ₇₎ -p-nitrophenyl (G) -oŕiD-maltoplastid (ET-G ^ PNP) (Sigma Chemical Co., St. Louis, LLO) by Pierre et al .. Clin. Chem., 22, 1219 (1976). Amylase activity in pancreatic tissues was measured by the same method after tissue homogenization in 10 ml of phosphate buffer, pH 7.4.

3. Pancreatic trypsin

Trypsin activity was measured fluorimetrically using the substrate Boc-Gln-Ala-Arg-11CA. In a cuvette, 200 µl of sample and 2.7 ml of 50 ml Tris Purge (pH 8.0) containing 150 ml of NaCl, 1 ml of CaCl ₂ and 0.1 * bovine serum albumin were mixed. 100 µl of substrate was added to the sample and after 20 seconds preincubation the reaction was initiated. Fluorescence was measured at 380 nm excitation and 440 nm emission and expressed by the slope of the line. In order to evaluate the various experiments together, trypsin activity in fractions was expressed as a percentage of total trypsin activity.

4. Histology and morphometry

For light microscopy, sections from the anterior, middle and posterior pancreas were randomly selected and fixed in 10% neutral phosphate formalin buffer. Paraffin-attached segments of 5 µm were stained with hematoxylin-eosin (H&E) and evaluated by the morphologist tested. Damage or necrosis of acellular cells was determined by either (a) the presence of no acres in the case of acellular cells or (b) the vacuolization and swelling of the acinar cells and destruction of the whole or only part of the histo-architecture, both events must be related to the inflammatory response. The number of wounded or rhecotic cells and the total area formed by the acinar cells was quantified inorfometrically using video and computerized planimetric image analysis (CCD-72 model. Dage-MT1, Michigan city, IN) equipped with NIH-1200 image analysis software. Ten randomly selected microscope fields were evaluated for each sample. The extent of damaged or necrotic cells was determined as a percentage of the total area of acinar cells with areas meeting the criteria for damage or necrosis.

5. Measurement of myeloperoxidase activity (MPO) in pancreas and lungs

Neutrophil staining in the pancreas and lung was determined based on tissue myeloperoxidase activity. Samples were taken at the time of sacrifice stored at -70 ° C until analysis. Samples (50 mg) were thawed and homogenized in 1 ml of 20 µM phosphate buffer a (pH 7.4) and ceritrifuged (10,000 x g, 10 µL, 4 ° C). The obtained pellet was resuspended in 50 mM phosphate buffer (pH 6.0) containing 0.52 hexadecyltrimethylammonium bromide (Sigma, St. Louis, MO) and frozen and thawed four times in succession. The suspensions were then disrupted by sonication for 40 seconds and centrifuged (101,000 x g. 5 min, 4 ° C). The reaction mixture containing the extracted enzyme, 1.6 mM tetramethylbenzidine (Sigma Chemical Co., St. Louis, MO), 80 mM phosphate buffer (pH 5.4) and 0.3 mM hydrogen peroxide, was incubated at 37 ° C for 110 seconds and the absorbance measured at 655 nm with CobasBlo automatic analyzer. This absorbance was then recalculated according to the dry matter content of the tissue sample.

6. Measurement of pulmonary vascular permeability

Upchanle of the common blliopancreatic canal usually results in severe pancreatltis associated with lung damage, which can be quantified by vascular permeability of the lungs and histological evaluation.

hours before fluorescein

Animals received two 5 mg / kg CFITC-albumin. Sigma Chemical Co. microvascular permeability was leakage of FITC-albumin from bronchoalveolar spaces.

by sacrificing intravenous isothiocyanate albumin Louis, 1 * 10). Pulmonary Assessment Based on Vascular Compartments by Brief Description of the Method * Right after sacrifice, right bronchus was clamped and the trachea opened. Then the right lungs selected by cannula were inserted into the trachea. Three saline lavages (60 ml per lavage) were pooled and serum FITC fluorescence and lavage was measured at 494 nm excitation and 520 nm emission. The calculated ratio of lavage to blood fluorescence then expressed a measure of the lung microvascular permeability. The lungs were also stained with H&E and histologically evaluated.

7. Effect of Caerulein falling and rPH.2

Infusion of caerulein alone at 5 µg / kg / h for 3.5 hours induced typical mild induced pancreatitis. characterized by hyperamylase, pancreatic edema measured by pancreatic water content, and histological changes including marked vacuolization and pancreatic edema. Saline infusion did not induce any of these biochemical or histological changes in control animals. Intravenous administration of rPH.2 at 5, 10, or 20 mg / kg 30 minutes prior to the start of caerulein infusion did not significantly affect the severity of changes in pancreatic edema (water content) and the histological finding induced by infusion of caerulein alone. Also, administration of rPH.2 did not affect caerulinoin-induced pancreatic trypsinogen activity or amylase content.

Infusion of higher doses of caerulein. 10 µg / kg / h for 5 hours, induced severe pancreatitis in rats and characterized by a marked increase in serum amylase activity, pancreatic edema development, a strong increase in pancreatic activity ΙΊΡΟ, and a significant increase in trypsinogen activation and pancreatic amylase activity. Histology of the pancreas revealed not only pancreatic edema and vacuolization of the alcanic cells, but also several necrotic spots and several infiltrated cells.

Treatment with rPH.2 at dose 5 resulted in a reduction in the incidence of caerulein measured according to

Administration of rPH.2 (5 or 10 mg / kg intravenously) 30 min before the start of caerulein infusion (10 µg / kg / h) improved most of the pancreatic changes induced by caerulein infusion alone. The results are presented in Table 10. see below. Treatment with rPH.2 at 5 mg / kg resulted in a decrease in serum amylase activity (from 1098 ± 1412 to 6763 + 1256). Higher doses of 10 mg / kg rPH.2 no longer induced an improvement in hyperamylasia.

or 10 mg / kg also had a water content induced pancreatic edema (90.61 ± 0.27 for caerulein alone and 88.21 ± 0.61 for caerulein with 5 mg / kg rPH.2). rPH.2 at 5 mg / kg caused a strong improvement in pancreatic NPO activity (2.92 ± 0.32-fold for caerulein alone and 1.19 ± 0.21 for caerulein together with rPH.2, p <0.05) . Higher doses of rPH.2 did not elicit any further improvement in I * IPO activity. Also, the dose of rPH.2 did not significantly alter the extent of trypsinogen activation or amylase content in the pancreas. After rPH.2 pretreatment, histological analysis revealed an improvement in microscopic pancreatic necrosis and infiltration.

Lung damage associated with pancreatitis has been observed both clinically and in several pancraetitis models. Infusion of caerulein at a dose of 5 µg / kg / h for 3.5 h, which induces a mild form of pancreatitis, did not result in significant lung involvement. However, infusion of caerulein at a dose of 10 µg / kg / h for 5 h, which induced more severe pancreatitis, caused lung damage quantitatively by increased vascular lung permeability (0.31 ± 0.04 to 0.79 ± 0.09), IL-10 activity in the lungs (corresponding to neutrophil staining) ) and neutrophil infiltration.

Administration of rPH.2 at 5 mg / kg 30 min before caerulein infusion significantly improved lung MPO activity induced by infusion of caerulein alone (3.55 ± 0.93 for caerulein alone and 1.51 ± 0.26 for caerulein with rPH.2 ). Treatment with rPH.2 significantly reduced the severity of microscopic changes observed in lung tissue after caerulein infusion. The increased vascular permeability caused by caerulein infusion was reduced by rPH.2, but the difference was not statistically significant. A higher dose of rPH.2 (10 mg / kg) did not reduce caerulein-induced lung damage more than the lower doses.

Table 10

inspection (no CER) caeruleininduced (CER) 10 µg / kg / h CER + 5 mg / kg rPH.2 CER + 10 mg / kg rPH.2 serum amylase (U / L) 961 + 174 10984 ± 1412 6763 ± 1256 8576 ± 1024 water content in pancreas (X wet weight) 72. 71 ± 0.64 90. 61 ± 0. 27 88. 21 ± 0.61 89.00 ± 0. 94 pancreatic ILPO (multiples control) 1.0 2, 92 ± 0.32 1, 19 ± 0. 21 1. 42 ± 0.19 activity pancreatic trypsin (LOOOxsklon / ug ONA) 0.12 ± 0. 06 9. 70 ± 2. 50 8, 33 ± 1. 75 9. 15 ± 1. 28 Contents pancreatic amylase (U / ug DNA) 0.28 ± 0.66 0. 42 ± 0. 07 0. 45 ± 0. 04 0, 46 ± 0. 044

Table 10 - continued

inspection (no CER) caeruleininduced (CER) 10 µg / kg / h CER + 5 mg / kg rPH.2 CER η- 10 mg / kg rPH.2 vascular permeability Lung (lavage / serum 2) 0, 31 ± 0, 04 0. 79 ± 0. 09 0.70 ± 0.09 0.70 ± 0.77 ΙΊΡΟ lungs (multiples control) 1. 0 3. 55 ± 0. 93 1. 51 ± 0.26 1.64 ± 0.22

B. Opossum pancreatitis model activity

Healthy, randomly captured American Opossums (.Didelphis i / irginiana) of both sexes (2.0 kg to 4.0 kg) were obtained from Scott-Haas and kept in controlled atmosphere rooms of 23 ± 2 ° C, 12 hour light / dark and fed standard laboratory food and water ad libitum. Following overnight fasting, the animals were anesthetized with 50 mg / kg sodium phenobarbital i.p. (Veterinary Laboratories Inc., Leriexa, KS). Cellotomy was performed by means of a central incision under sterile conditions and a channel common to the bile and pancreas was ligated to induce necrotic pancreatitis. In addition, it was cystic so that the gallbladder could not function as a reservoir, randomly divided into control and groups. Beginning on the second day after ligation of the pancreatic canal, the experimental group was administered rPH.2 at a dose of 5 mg / kg body weight per day (administered with a 4 mg / ml solution) intravenously into the tail vein. whereas the control group received intravenous placebo in the same volume. After 1 and 2 days of treatment (days 3 and 4 after ligation of pancreatic bile-ligated bile. Experimental channel animals), the animals were sacrificed by an overdose of sodium phenobarbital. Blood samples were collected from the heart for the determination of serum amylase, serum llpase and serum billrubin and pancreas were collected. Portions of the pancreas were either fixed in 4% phosphate forinaldehyde buffer for histological observation or immediately frozen at -80 ° C to determine myeloperoxidase activity. Other parts of the pancreas served to determine the water content and pancreatic amylase content, respectively. as described above in section A of this example, llyeloperoxidase activity, a measure of neutrophil staining, was determined in the pancreas so. as described above. Pulmonary vascular permeability was also determined by the above method.

The results presented represent the mean ± mean error of the mean (CSE1) obtained from three or more experiments. The significance of the changes was evaluated by the Student's t-test. for data organized in two groups and variance analysis (ANOVA) when three groups or more were compared. If ANOVA indicated a significant difference, the data were analyzed by Turkey as a post hoc test for group differences. The difference was considered significant if p <0.05.

The results are shown in Table 11. Congestion of the common biliopancreatic duct resulted in severe necrotic pancreatitis, characterized by hyperamylase, hyperlipaseemia, and extensive pancreatic necrosis. In addition, clogging of the common biolopancratic duct resulted in a strong increase in serum billrubin values. Intravenous administration of rPH.2 (5 mg / kg / day) beginning the second day after pancreatic duct ligation improved most of the pancreatic duct-induced pancreatic changes and compared to placebo-treated animals. One day treatment with rPH.2 reduced serum amylase levels compared to control animals, however. that the difference was not statistically significant and the two-day application of rPH.2 (day 4 after pancreatic duct ligation) significantly reduced amylase levels compared to placebo. One-day and two-day rPH.2 treatment reduced serum lipase levels to control levels.

however, the difference was not statistically significant. A two-day application of rPH.2 reduced pancreatic amylase content to control levels, but a one-day administration increased pancreatic amylase. Treatment with rPH.2 did not affect serum bilirubin levels, pancreatic myeloperoxidase activity or pancreatic water content.

The main characteristic changes induced by obstruction of the bile-pancreatic duct included necrosis, inflamed cell infiltration, acinar cell vacuum, and a pronounced expansion of the acinar lumens. Morphometric observation of damaged pancreatic acinar cells showed a strong protective effect of rPH.2 on a day or two advance treatment. At rPH.2 day treatment, acinic cell damage was reduced to 23 2 of total acinar tissue, compared to 48 2 of placebo-injured animal tissue. This decrease in the injured cells was even more rPH.2. when there was a reduction in apparent after two days of injured cell application to 35 2 compared to 60 2 in placebo-treated animals.

Vascular permeability of the lungs, quantified by injection of FITC, in the rPH.2 treated group, was significantly different from the placebo group at one and two days. Histological observations showed severe lung injury in all placebo-treated groups. Lung damage was characterized by a widespread inflammatory response with interstitial and intraalveolar infiltration mainly by macrophages, lymphocytes and neutrophils and irregular but marked interstitial edema and thickening of alveolar membranes. Administration of rPH.2 significantly reduced inflammatory cell infiltration and reduced interstitial edema at both application times.

The summary of these results clearly shows that intravenous administration of rPH.2 at 5 mg / kg / day starting 48 hours after pancreatic duct IgG1 resulted in a significant reduction in elevated levels of amylase and llpase and acinar cell damage quantified by morphometric analysis of H & E stained sections there was a significant decrease in the severity of pancreatltid-induced lung injury. In this clinically appropriate model of pancreatitis, rPAF-AH product showed a beneficial effect in reducing the severity of pancreatitis.

Table 11

After one day treatment After two days treatment (killing day 3) (Day 4) placebo rPH.2 5 mg / kg placebo rPH.2 5 mg / kg bilirubin serum (Mg / dl) 5, 49 + 0. 96 7, 10 ± 0. 60 6, 54 + 0.55 9110, 79 serum amylase (U / L) 5618 + 899 4288 + 675 6538 + 1355 3106 + 467 ** serum lipase (U / L) 22261554 1241 + 263 14241257 10231295 water content in pancrease (X) 81, 10 ± 0.56 81, 52 ± 0.79 80, 0511, 07 79 pancreatic Γ1ΡΟ C OD / fraction solids) 1345 + 286 1142 + 83 11491232 1033 + 130 panreaktlcká amylase (U / ug DNA) 706 + 92 11011105 950185 712 + 131 vascular permeability Lung (FITC) lavage / serum X) 7610, 09 21 + 0. **** 04 57 + 0.13 0. 23 + 0, 04 **

Table 11 - continued

After one day treatment After two days treatment (day 3 sacrifice) (day 4 sacrifice) placebo rPH.2 mg / kg placebo rPH.2 mg / kg damaged 48 23 acinar cells (% of total acinar tissue)

35 * p = 0.02 versus placebo ^MW p <0.001 versus placebo

Example 20

A study was conducted to evaluate the effect of PAF-AH product, rPH.2, on neurotoxicity associated with HIV infection. Infection of the central nervous system HIV-1 (human immunodeficiency irus, type 1) causes neuronal loss by apoptosis. When monocytes infected with HIV-1 are activated by various antigenic stimuli, including contact with nerve cells, they secrete high levels of neurotoxic cytokines, including PAF, inducing inflammation.

The effect of rPH.2 on the neurotoxicity of conditioned media from HIV infected monocytes was studied.

Monocytes were infected with HIV and activated as described below. Monocytes were obtained from peripheral bone marrou cells (PBMCs) of HIV and hepatitis B seronegative donors after leukophoresis and purified by 098%) by centrifugation (Countercurrent centrifugal eluriatiori) so. as described by Genis et al., J. Exp. Med., 176: 1703-1718 (1992). Cells were cultured (1x 10 * cells / ml in T-75 flasks) in DMEM / Sigma, St. Petersburg. Louls, MO) with human recombinant macrophage colony stimulating factor

100

CMSCF (Recombinant Human Macrophage Colony Stimulators Factor) CGenetic Institute, Inc. Cambridge, MA). Under these conditions, monocytes differentiated into macrophages. After 7-10 days of culture, macrophages were exposed to HIV-1 _ftDft C under number 1 * 160472) in excess of infectious agent (1 * 1014) of 0.01 infectious virions / target cell. Under these conditions, 20-50% of the monocytes were infected 7 days after inoculation with HIV-1, as confirmed by immunofluorescence methods and 1n situ hybridization (Kalter et al., J. Immunol., 146 »298-306 C1991)]. The cultures were supplemented with fresh nutrient medium every 2-3 days. Five to seven days after infection with HIV, the culmination time of the reverse transcriptase activity CIO ⁷ cpm / ml) determined in Kalter et al. _r see above, monocyte cultures, both HIV-1 infected and uninfected monocyte parallel cultures, stimulated with LPS (10 ng / ml) or transfer agent alone for 30 miri, at 37 ° C and then rapidly frozen to -80 ° C and maintained until neurotoxicity testing.

Cultures of human cerebral cortical neuronal cells were derived according to the following procedure. Human fetal brain tissue was obtained from telencephalon in the second trimester. C13-16 weeks of pregnancy) by the modified procedure of Banker and Couan, Braln Res., 1361 397-425 (C1977). Brief description of the method »brain tissue was collected, washed with 30 ml of cold Hank's BSS C medium containing Ca ³ ' ¹ ' and Mg ^and * + 25ml * 1 HEPES and 5X gentainicin), separated from adherent meninges and blood and cut into 2 pieces. mm ³ . The tissue was passed through a 230 µL Nitex bag and gently passed through a flame-burnt Pasteur pipette 10-15 times. Next, the tissue was centrifuged at 550 rpm. 5 min. 4 ° C and the pellet was resuspended in 5-10 ml of 1 * IEI * 1-hipp CD-glucose, 5 g / L; L-glutamiri, 2ml * 1 with HEPES. 10 ml * l; AN pyruvate, 1mM; KCl, 20 min) containing N1 components C insulin. 5 mg / ml; transferrin, 5 mg / ml; selenlt. 5 µg / L. progesterone, 20 µl * 1; putrescine, 100 µM), as well as 10% fetal serum (CFCS), PSN antibiotic C penicillin, 50 mg / L; streptomycin 50 mg / L; neomycin 100 mg / l) and fungizone <2.5 g / l). Cell number and cell viability were determined by dilution with Hanks BSS containing 0.4% tryptophan

101

X blue (1 → 1 v / v) and cells were counted using a hemocytometer. The cells were washed five times with a 10 ml flask and seeded at a dilution equivalent to a density of 105 cells / 12 mm coverslip that had been pre-coated with poly-L-lysine (70K-150K lW, Sigma, St. Louis, MO) and placed in the cells. 24-well culture plates. 1 ml of medium was then added to each culture well. Cells were incubated and cultured for 10-28 days at 370 ° C in a humidified incubator composed of 5% CO 2 _and / 95% air. Medium was changed every three days. Under these conditions, the cultures were> 60-70% homogeneous for neurons, further the culture contained 20-30% star cells, <1% microglia and approximately 10% macrophages and stained microglia. After 14 to 28 days of culture, the neuronal culture expressed a sufficient amount of N-methyl-D-aspartate (NMDA) or nori-NMDA receptors were killed after administration of excitotoxic doses of NMDA or alphaamino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( AMPA).

The rheotoxicity test was carried out as follows. Test samples, including conditioned media from LPS-stimulating HIV-1 infected moriocytes. (b) control media, (c) conditioned media with added rPH.2 at a concentration of 51 µg / ml, or (d) conditioned media with added carrier for rPH.2, were applied to neuronal culture cells at a concentration of ls10 v / v for a period of time 24 hours. Neurotoxicity was measured by identifying apoptotic nuclei in situ on neurons growing on coverslips that were fixed with 4% formaldehyde. A commercial kit (Apop Tag; ONCOR, Gaithersburg, MD) was used for this assay, using terminal deoxynucleotidyltransferase (TdT) to bind digoxigenin-dUPT to the 3'-OH end of the newly digested DNA (TUNEL staining). Digital processing of TUNEL stained neuron images in more than 15 randomly selected microscopic fields was analyzed for the number of TUNEL stained nuclei / total number of neurons detected in 50 microscopic fields by computer morphometry (MCID, Imaglng Research, St. Catherine, Ontario, Canada).

102

Data were expressed by TUNEL + SEI * I staining and statistical evidence to reduce neurons.

The% between the nuclei of the positive neurons are shown in Figure 13. Tests between control and experimental treatment were performed by ANOVA or paired t-tests, with a p <0.05 probability. Quantification of these cultures confirmed that conditioned medium from HIV-infected and activated monocytes induced neuronal cell death in the range of 25% of the total cerebral cortex neuron population. rPH.2 was capable of this toxicity to less than 5% of the total number of rPH.2 itself was not neurotoxic, since at 50 µg / ml rPH.2 had no effect on neuronal cell death compared to culture treated with control medium. These results clearly demonstrated that PAF must be the major neurotoxicity-inducing component in the administration of conditioned media from HIV-1 cells infected with activated monocytes, since neurotoxicity can be almost completely suppressed by co-incubation with PAF-AH, the enzyme responsible for PAF metabolism in central nervous system. These findings outline possible therapeutic uses in the treatment of CNS neurological diseases associated with HIV-1 infection.

Example 21

Almost 4 percent of the Japanese population has low or even undetectable levels of PAF-AH activity in plasma. This distinction is correlated with severe respiratory symptoms in children suffering from asthma. Fliua et al. (J. Clin. Invest., 82s 1983-1991 (1988)) have pointed out that these conditions may be caused by a deficient member inherited in an autosomal recessive manner.

To determine whether this deficlencla comes from inactivated. but the enzyme present, whether it is due to the impossibility of PAF-AH synthesis, plasma was collected from many patients deficient in PAF-AH activity (using the method described in Example 10 for transfectants) and further tested for PAF-AH using monoclonal antibodies 90G11D (Example 13). ) using the EL1SA method described below

103

Flat-bottom Immunolon 4 (Dynatech, Chantilly, VA) was coated with monoclonal antibody 90G11D at 100 ng / well and allowed to incubate until morning. The wells were then blocked for 1 hour at room temperature with 0.5% fish skin gelatin (Sigma) diluted in CMF-PBS. Next, the wells were washed four times. Patient plasma was diluted in PBS containing 15ml * 1 CHAPS and added to each well of the plate (50 µl / well). Plates were incubated for 1 hour at room temperature and then washed four times. This was followed by the addition of 5 µl of monoclonal antibody 90F2D at a concentration of 5 µg / ml, which was biotinylated by a standard method and diluted in PBST to each well. The plate was incubated for 1 hour at room temperature and then washed three times. This was followed by the addition of 50 µl of Extra Avidin (Sigma) undiluted 1 with 1000 in C 1 H-PBST to each well and the plate was again incubated for 1 hour at room temperature. This was followed by induction of activity.

Direct correlations were found between PAF-AH activity and enzyme concentration. If no activity was measured in the patient's serum, this was always accompanied by the inability to detect the presence of the enzyme. Similarly, plasma samples showing half the normal activity value contained half of the normal level of PAF-AH. These findings confirm. That the deficiency of PAF-AH activity is due to the inability to synthesize the enzyme or is due to the fact that the monoclonal antibodies do not recognize an inactive form of the enzyme.

Further attempts have been made to determine whether the deficiency is due to a genetic disorder in the human plasma PAF-AH gene. Genomic DNA from PAF-AH deficient individuals was isolated and used as a template for PCR reactions with primers specific for the PAF-AH gene. Each of the exon coding sequences was first amplified and sequenced from one individual. Exon 9 revealed a single nucleotide replacement (G to T at position 996 of SEQ ID NO: 7). Nucleotide replacement resulted in an amino acid substitution of phenylalania for valine at position 279 of the sequence PAF-AH (V279F). Exon 9 was amplified from genomic DNA for a further 11

104

PAF-AH deficient patients in whom the same point mutation was found.

To determine whether this mutation altered the enzyme, a construct with this mutation showing no PAF-AH activity was expressed in E. coli, whereas a control construct that did not contain the mutation. was fully active. Replacement of this amino acid is likely to result in a structural modification that causes an observed deficiency in activity as well as an inability to immunoreactivity with PAF-AH antibodies generated according to the invention.

Thus, the PAF-AH specific antibodies of the invention can be used in diagnostic methods to detect abnormal serum levels of PAF-AH (normal concentrations are between 1 and 5 U / ml) and to monitor the pathological conditions associated with PAF-AH. What more. the determination of genetic damage in the PAF-AH gene allows genetic research into PAF-AH deficiency in Japanese patients. The mutation causes a restriction endonuclease site (MAEII) to be obtained, thus allowing the application of a simple method, Restrition Fragment Lenght Polymorphlsm (RFLP), which is able to distinguish between normal and mutant allelines. (information on this application can be obtained with Leuln, pages 136-141 in Geries

V. Oxford International Press, New York (1994)).

Using the DNA II erase enzyme digestion method, genomic DNA was investigated from 12 patients deficient in PAF-AH, followed by Southern blotting and hybridization with a probe complementary to exon 9 (nucleotides 1 to 396, according to SEQ ID NO: 17). All patients were found to have RFLPs consistent with mutant alleles.

While the invention is described by terms specific to an embodiment, it will be understood that variations and modifications may be apparent to those skilled in the art. Therefore, only the limitations that appear in the appended claims can be considered to be within the scope of the invention.

105

Sequence Listing (1) General Information;

(Cl) Applicant: ICOS CORPORATION

C11) Title of the invention: Platelet-Actlvating Factor Acetylhyrolase

C111) Number of Sequences: 30 (iv) Mailing Address:

(A) Address: Marshall, O'Toole. Gerstein. Murray &

Borun (B) Street: 6300 Sears Tower. 233 South Uacker Drive (C) Location: Chicago (D) Country: Illinois (E) Country: United States of America (F) Postcode CZIP): 60606-6402 (v) Form Compatible:

(A) Media Type: Floppy Disk (B) Computer: IBM PC compatible (C) Operating System: PC-DOS / MS-DOS (D) Software: PatentIn Release # 1.0. Version # 1.25 (vl) Current application data:

(A) Application number:

(B) Registration date:

(C) Classification:

(viii) Attorney / Agent Information:

(A) Name: Rin-Laures. Li-Hsien (B) Registration number: 33,547 (C) Reference / number of court cases: 27866/34026 (ix) Telecommunication information:

(A) Telephone: (312) 474-6300 (B) Fax: (312) 474-0448 (C) Telefax: 25-3658 (2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids (B) TYPE: amino acid CD) TOPOLOGY: linear (s) MOLECULA TYPE: peptld (xi) SEQUENCE DESCRIPTION. SEO ID NO: 2:

Phe Lys Asp Leu Gly Glu Glu Asn

Phe

106

C2) INFORMATION ON SEO ID NO: 2:

(Cl) SEQUENCE CHARACTERISTICS:

CA) LENGTH: 16 amino acids

CB) TYPE: amino acid CD) TOPOLOGY linear

Cii) MOLECULA TYPE: peptide

Cxi) SEQUENCE DESCRIPTION: SEQ ID NO. 2:

Gin Val Leu Met Ala Ala Ala Phe Gly Gin Thr Lys Pro 15 10 15

C2) SEO ID NO. Third

Ci) SEQUENCE CHARACTERISTICS.

C A) LENGTH: 11 amino acids

CB) TYPE. amino acid CD) TOPOLOGY linear

Cii) MOLECULA TYPE, peptide

Cxi) SEQUENCE DESCRIPTION. SEO ID NO. Third

Met Lys Pro Leu Gly Gly 15 10

C2) SEQ ID NO. 4th

C (i) SEQUENCE CHARACTERISTICS.

CA) LENGTH. 32 base pairs

CB) TYPE. nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY: linear

(Cii) MOLECULA TYPE. DNA

Cix) FEATURES:

C A) NAME / KEY: Modified-place

CB) LOCATION, Group C13, 21. 27)

CC) ADDITIONAL INFORMATION: / note = The nucleotide at each of these positions is inosine

Cxi) SEQUENCE DESCRIPTION. SEO ID NO. 4th

ACATGAATTC GGNATCYTTG NGTYTGNCCR AA 32

C2) SEQ ID NO. 5:

Ci) SEQUENCE CHARACTERISTICS.

CA) LENGTH. 32 base pairs

CB) TYPE. nucleic acid (C) NUMBER OF STRINGS, one

CD) TOPOLOGY. straight

107 (ii) MOLECULA TYPE * DNA (xi) SEQUENCE DESCRIPTION * SEO ID NO * 5 *

TATTTCTAGA AGTGTGGTGG AACTCGCTGG 30 (2) SEO ID NO * 6 * (i) SEQUENCE CHARACTERISTICS * (A) LENGTH * 32 base pairs (B) TYPE * nucleic acid (C) NUMBER OF STRINGS * one (D) TOPOLOGY. linear (ii) MOLECULA TYPE * DNA (xi) SEQUENCE DESCRIPTION * SEO ID NO * 6.

CGATGAATTC AGCTTGCAGC AGCCATCAGT AC 32 (2) SEO ID NO * 7 * (i) SEQUENCE CHARACTERISTICS.

(A) LENGTH * 1520 base pairs (B) TYPE * nucleic acid (C) NUMBER OF STRINGS, one (D) TOPOLOGY. linear (ii) MOLECULA TYPE * cDNA (ix) CHARACTERISTICS.

(A) NAME / KEY. CDS (B) LOCATION. 162..1484 (xi) DESCRIPTION OF THE SEQUENCE. SEO ID NO. 7th

GCTGGTCGGA GGCTCGCAGT GCTGTCGGCG AGAAGCAGTC GGGTTTGGAG CGCTTGGGTC 60 GCGTTGGTGC GCGGTGGAAC GCGCCCAGGG ACCCCAGTTC CCGCGAGCAG CTCCGCGCCG 120 CGCCTGAGAG ACTAAGCTGA AACTGCTGCT CAGCTCCCAA ATG GTG CCA CCC 173

Met Val Pro Pro 2

AAA TTG Lys Leu 5 CAT GTG CTT TTC TGC CTC TGC GGC 221 His Val Leu Phe Cys Leu Cys Gly Cys Leu Val Val Tyr 15 20 CCT TTT GAC TGG CAA TAC ATA AAT CCT GTT GCC CAT ATG AAA TCA TCA 269 for Phe Asp Trp gin Tyr Ile same time for wall Ala His Met Lys Ser Ser 25 30 35 GCA TGG GTC AAC AAA ATA CAA GTA CTG ATG GCT GCT GCA AGC TTT GGC 317 Ala Trp wall same time Lys Ile gin wall Leu Met Ala Ala Ala Ser Phe Gly 40 45 50 CAA ACT AAA ATC CCC CGG GGA AAT GGG CCT TAT TCC GTT GGT TGT ACA 365 gin Thr Lys Ile for Arg Gly same time Gly for Tyr Ser wall Gly Cys Thr 55 60 65

108 GAC TTA ATG TTT GAT CAC ACT AAT AAG GGC ACC TTC TTG. CGT TTA TAT 413 Asp Leu Met Phe Asp His Thr same time Lys Gly Thr Phe Leu Arg Leu Tyr 70 75 80 TAT CCA TCC CAA GAT AAT GAT CGC CTT GAC ACC CTT TGG ATC CCA AAT 461 Tyr for Ser gin Asp same time Asp Arg Leu Asp Thr Leu Trp Ile for same time 85 90 95 100 AAA GAA TAT TTT TGG GGT CTT AGC AAA TTT CTT GGA ACA CAC TGG CTT 509 Lys Glu Tyr Phe Trp Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu 105 110 115 ATG GGC AAC ATT TTG AGG TTA CTC TTT GGT TCA ATG ACA ACT CCT GCA 557 Met Gly same time Ile Leu Arg Leu Leu Phe Gly Ser Met Thr Thr for Ala 120 125 130 AAC TGG AAT TCC CCT CTG AGG CCT GGT GAA AAA TAT CCA CTT GTT GTT 605 same time Trp same time Ser for Leu Arg for Gly Glu Lys Tyr for Leu wall wall 135 140 145 TTT TCT CAT GGT CTT GGG GCA TTC AGG ACA CTT TAT TCT GCT ATT GGC 653 Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr Ser Ala Ile Gly 150 155 160 ATT GAC CTG GCA TCT CAT GGG TTT ATA GTT GCT GCT GTA GAA CAC AGA 701 Ile Asp Leu Ala Ser His Gly Phe Ile wall Ala Ala wall Glu His Arg 165 170 175 180 GAT AGA TCT GCA TCT GCA ACT TAC TAT TTC AAG GAC CAA TCT GCT GCA 749 Asp Arg Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp gin Ser Ala Ala 185 190 195 GAA ATA GGG GAC AAG TCT TGG CTC TAC CTT AGA ACC CTG AAA CAA GAG 797 Glu Ile Gly Asp Lys Ser Trp Leu Tyr Leu Arg Thr Leu Lys gin Glu 200 205 210 GAG GAG ACA CAT ATA CGA AAT GAG CAG GTA CGG CAA AGA GCA AAA GAA 845 Glu Glu Thr His Ile Arg same time Glu gin wall Arg gin Arg Ala Lys Glu 215 220 225 TGT TCC CAA GCT CTC AGT CTG ATT CTT GAC ATT GAT CAT GGA AAG CCA 893 Cys Ser gin Ala Leu Ser Leu Ile Leu Asp Ile Asp His Gly Lys for 230 235 240 GTG AAG AAT GCA TTA GAT TTA AAG TTT GAT ATG GAA CAA CTG AAG GAC 941 wall Lys same time Ala Leu Asp Leu Lys Phe Asp Met Glu gin Leu Lys Asp 245 250 255 260 TCT ATT GAT AGG GAA AAA ATA GCA GTA ATT GGA CAT TCT TTT GGT GGA 989 Ser Ile Asp Arg Glu Lys Ile Ala wall Ile Gly His Ser Phe Gly Gly 265 270 275 GCA ACG GTT ATT CAG ACT CTT AGT GAA GAT CAG AGA TTC AGA TGT GGT 1037 Ala Thr wall Ile gin Thr Leu Ser Glu Asp gin Arg Phe Arg Cys Gly 280 285 290 ATT GCC CTG GAT GCA TGG ATG TTT CCA CTG GGT GAT GAA GTA TAT TCC 1085 Ile Ala Leu Asp Ala Trp Met Phe for Leu Gly Asp Glu wall Tyr Ser 295 300 305 AGA ATT CCT CAG CCC CTC TTT TTT ATC AAC TCT GAA TAT TTC CAA TAT 1133

109

Arg ile for gin for Leu Phe Phe White Asn Ser Glu Tyr Phe Gin Tyr 310 315 320 CCT GCT AAT ATC ATA ATA AAA AAA TGC TAC CCT GAT AAA GAA 1181 for Ala same time Ile Ile Lys Met Lys Lys Cys Tyr Ser Glu 325 330 335 340 AGA AAG ATG ATT ACA ATC AGG GGT TCA GTC CAC CAG AAT TTT GCT GAC 1229 Arg Lys Met Ile Thr Arg Gly Ser Val Hls Gin Asn Phe Ala * Asp 345 350 355 TTC ACT TTT GCA ACT GGC AAA ATA ATT GGA CAC AAG 1277 Phe Thr Phe Ala Thr Gly Lys White Gly His Met Leu Lys Leu Lys 360 365 370 GGA GAC ATA GAT TCA AAT GTA GCT ATT GAT CTT AGC AAC AAA GCT TCA 1325 Gly Asp Ile Asp Ser Asn Val Ala Asp Leu Ser Asn Lys Ala Ser 375 380 385 TTA GCA TTC TTA CAA AAG CAT TTA GGA CTAT CAT AAA GAT TTT GAT CAG 1373 Leu Ala Phe Leu gin Lys Hls Leu Gly Leu Lys Asp Phe Asp gin 390 395 400 TGG GAC TGC TTG ATT GAA GGA GAT GAT GAG ACC 1421 Trp Asp Cys Leu Ile Glu Gly Asp Asp Glu Asn Leu White For Gly Thr 405 410 415 420 AAC ATT AAC ACA ACC AAT CAA CAC ATC ATG TTA CAG AAC TCT TCA GGA 1469 same time Ile same time Thr Thr Asn Gin Kis Met Leu Gin Asn Ser Ser Gly 425 430 435 ATA GAG AAA TAC AAT TAGGATTAAA ATAGGTTTTT ΤΑΆΑΆΑΑΑΑΑ AAAAAA 1520 Ile Glu Lys Tyr same time 440 (2) INFORMATION SEQ. B. (1) SEQUENCE CHARACTERISTICS: (AD i LONG: 441 amino acids (B: »TYPE« amino acid (D: i TOPOLOGY. straight (Ii) TYPE MOLECULES, protein (Xi) DESCRIPTION OF THE SEQUENCE. SEQ ID NO. B. Met wall for for Lys Leu His Val Leu Phe Cys Leu 1 5 10 15 Ala wall wall Tyr for Phe Asp Trp Gin Tyr White Asn Pro Val Ala His 20 25 30 Met Lys Ser Ser Ala Val Asn Lys White Gin Val Leu Met Ala Ala 35 40 45 Ala Ser Phe Gly gin Thr Lys White Pro Gly Asn Gly Pro Tyr Ser 50 55 60 wall Gly Cys Thr Asp Phu Asp His Thr - Asn Lys Gly Thr Phe 65 70 75 80

110

Leu Arg Leu Tyr Tyr 85 for Ser Gin Asp Asp Asp Leu Asp Thr Leu 90 95 Patience For Asn Lys Glu Tyr Phe Trp Lys Phe Leu Gly 100 105 110 Thr His Trp Leu Met Gly Asn Leu Arg Leu Leu Phe. Gly Ser Met 115 120 125 Thr Thr For Ala same time Trp Asn Ser Pro Leu Arg Pro Gly Glu Lys Tyr 130 135 140 For Leu Val Val Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr 145 150 155 160 Ser Ala White Gly Ile Asp Leu Ala Ser His Gly Phe Val Ala Ala 165 170 175 Val Glu His Arg Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp 180 185 190 Gin Ser Ala Ala Glu Ile Gly Asp Lys - Ser Trp Leu Tyr Leu Arg Thr 195 200 205 Leu Lys Gin Glu Glu Thr His ile Arg Asn Glu Gin Val Arg 210 215 220 Arg Ala Lys Glu Cys Ser Gin Ala Leu Ser Leu White Leu Asp and Asp 225 230 235 240 His Gly Lys Pro wall Lys Asn Ala Leu Asp Leu Lys Phe Asp Met Glu 245 250 255 Gin Leu Lys Asp Ser Ile Asp Arg Glu Lys White Ala Val ile Gly His 260 265 270 Ser Phe Gly Ala Thr Val ile Gin Thr Leu Ser Glu Asp Gin Arg 275 280 285 Phe Arg Ile Ala Leu Asp Ala Trp Met Phe For Leu Gly Asp 290 295 300 Glu Val Tyr Arg Ile Pro Gin Pro Leu Phe Phe Asn Ser Glu 305 310 315 320 Tyr Phe Gin Tyr for Ala Lys Met Lys Lys Cys Tyr Ser 325 330 335 For Asp Lys Glu Arg Lys Met ile Thr ile Arg Gly Ser Val His Gin 340 345 350 Asn Phe Ala Asp Phe Thr Phe Ala Thr Gly Lys White Gly His Met 355 360 365 Leu Lys Gly Asp Asp Ser Asn Val Ala Asp Leu Ser 370 375 380 Asn Lys Ala Ser Leu Ala Phe Leu Gly Leu His Lys 385 390 395 400

111

Asp Phe Asp Gin Tzp Asp Cys Leu Clay Glu Gly Asp Asp Glu Asn Leu 405 410 415 Clay Pro Gly Thr Asn Clay Asn Thr Thr Asn Gin His Clay Met Leu Gin 420 425 430

Asn Ser Ser Gly White Glu Lys Tyr Asn 435 440

C 2) INFORMATION ON SEO ID NO.

(1) SEQUENCE CHARACTERISTICS · (A) LENGTH · 1123 base pairs (B) TYPE · nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY · Linear

C11) MOLECULA TYPE: DNA (genomic)

Clx) FEATURES:

CA) NAME / KEY: exun

CB) LOCATION »Not determined

Cxl) SEQUENCE DESCRIPTION: SEO ID NO. 9 '

ΑΆΑΤΑΤΑΑΑΤ TTTAATAACA CCACACATAA

TGAAGTTTTA AATGAGTGTG TTTTTAATTT

GAAGATGAAG GAAGGCGTTT CAGTTAAACC

AAACGGCTCC TTCTAGCTCC ATTTCTCCTC

ATTGTCATTT CCAGGGAGAA ATGACACCAG

GGTCCACACA ACTTCCGAAT TGGTGTTCAG

CAGGGCATTG CCAACTATAG ATGCTCGGAG

ACAAGGAAAA CCGGCCTGAC TGGGGGGTGA

AGGTCTGCGG AAAGGAGCTG GTGAGCACGA

GGCGGGCTAA GTTAACCTCG GGTCCAGGTG

GCGCTCGAGC AGGCTACGTC GGGAGCCGCC

CCTCCCCGGT ACCTCCTCCA GCATCACCAG

AGGCGGACCC AGACACAGCC GCGCGCAGCC

GCCCGCAGCC AGGGGGACAC CGGCTGGTCG

TCGGGTTTGG AGCGCTTGGG TCGCGTTGGT

CCCGCGAGCA GCTCCGCGCC GCGCCTGAGT

GGGAGGAAGG GCACGGTCGC CGCGCTGGAG

GTGGGCTCGC TGAGTCGCAC CCGCTCTGCT

ATTTCAAACT ACTTTCCCTA AGTTTCTAGC 60

ATTAGAAAGT GGATTGAAGA GAAAACATTG 120

CCAAATAACT CTGTGTTACA CTGAGCTATG 180

AGACCTAAGT GCTATTCCTG ATTGTCCTTC 240

CACAGTGGCA GGCCTTCCAA TCTGGAGCAC 300

TGTAAAGTGT ATCGGAGTGC GGAAAATGCG 360

TAATTCAGTG TATTCAGAGA ACACGGTGAA 420

ATTCAGCAGG GAGTAAATCT GATCGGCATC 480

CACCACCAGG CATTGCCTGG CTCTCTCCGC 540

CGGGCCATGG TCTTGGGGAG GGTGCTGGGT 600

GCTGCTAGTG AGAGCCGGGC CACACACGCT- 660

GGGAGGAGAG GGTCGGGCAC AAGGCGCGCT 720

CACCCGCCCG CCGCCTGCCA GAGCTGCTCG 780

GAGGCTCGCA GTGCTGTCGG CGAGAAGCAG 840

GCGCGGTGGA ACCCCCCAGG GACCCCAGTT 900

GAGGAGGGGC CCCGGGGGCG AGGCGGGAGT 960

GTCGGGACCC CGGAGCGGCG ACCGGCCGGG 1020

GGCCGGTCCT GGGCTCACAG TCCCTGCAGC 1080

112

CCTCGGAAAC AGCGCTAGGA TCCTTCGGGA GAGGAGAGAT GAC 1123

C2) INFORMATION 0 SEQ ID Nb. 10th

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 417 base pairs (B) TYPE. nucleic acid (C) NUMBER OF STRINGS, one (D) TOPOLOGY. linear (ii) MOLECULA TYPE. DNA (genomic) (ix) PROPERTIES.

(A) NAME / KEY. exon (B) LOCATION. 145..287 (xi) DESCRIPTION OF THE SEQUENCE. SEO ID NO. 10th

GTACCAATCT AAAACCCAGC ACAGAAAAAT ACATGTTTTA TTTTTTCCAA GTGTTACTAG 60

TACCTCAGCC TTTCTTGATT TGTCAGCTTA TTTAAGGCCT CTTCATTGCA TACTTCTTTT 120

TTCTTTTAAT CATCTGCTTC GAAGGAGACT AAGCTGAAAC TGCTGCTCAG CTCCCAAGAT 180

GGTGCCACCC AAATTGCATG TGCTTTTCTG CCTCTGCGGC TGCCTGGCTG TGGTTTATCC 240

TTTTGACTGG CAATACATAA ATCCTGTTGC CCATATGAAA TCATCAGGTA AGAGGTGTAT 300

TTGTTCAAGG TCTTGAGCAA CTGATCTGTC GCCATACTTC AAGTGGGCCC CAAGAAGTTG 360

CACATCTGCA CATCTAAACA AGTCCTATTT AAAGGCTTAT GGAGATCCTG TATTCTC 417 (2) INFORMATION ABOUT SEO ID NO. 11th

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 498 base pairs (B) TYPE. nucleic acid (C) NUMBER OF STRINGS, one (D) TOPOLOGY. linear (11) MOLECULA TYPE. DNA (genomic) (1x) FEATURES.

(A) NAME / KEY. exon (B) LOCATION. 251..372 (xi) SEQUENCE DESCRIPTION. SEO ID NO. 11th

CATTAGGAGG TAACAGTCCA AGGCAGCTGA GAGAAAGGCT ATGTCTACTT TCATCTCTTT 60

ACCCTCCAAA ACCCCTACAC AGTGTTTGAA ACAGAGAGAC CCTCAATAAT TGCATATCTT 120

ACTTGTTAGG TTGAGAAAGA AAGAAGGCCA GAAACTATGG GAAGTAACTT GATTCCGTTG 180

GAATTCTTTT GCATAATAAA ATCTGATATG TAATGGATGA CAAATGAGAT AATATTTACC 240

TGTTTTTCAG catgggtcaa CAAAATAGAA GTACTGATGG CTGCTGCAAC GTTTGGCCAA 300

113

ACTAAAATCC CCCGGGGAAA TGGGCCTTAT TCCGTTGGTT GTACAGACTT AATGTTTGAT 360

CACACTAATA AGGTAATGCT TTGATTTATA CAACTTATCC TGATACTCTA ATATTGTCTG 420

TCGCTATGGA CCACTAGAAG GTGTTCAAAT GTGACCTTGC CCTCACCTGA GAATGACTCA 480

TTTTCGAATT TGTATTGT 498

C 2) INFORMATION ON SEO ID NOs 12 »(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 433 base pairs (B) TYPE. nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY. straight

C11) MOLECULA TYPE. DNA (Genomic)

Clx) CHARACTERISTICS.

CA) NAME / KEY. exon (B) LOCATION. 130..274

Cxl) DESCRIPTION OF THE SEQUENCE. SEO ID NO. 12th

CAGCAGCCTA AAGTCTTAGA CTTTGTGAAC ACAGAGGTAT TGAGTCCCAC TAATTAATAT 60

CGAAAATAGC TGCTGGAATA TGTTTGAGAC ACAACTTCTC TAAAAGTGCA TTAATTTCTT 120

TCTTAACAGG GCACCTTCTT GCGTTTATAT TATCCATCCC AAGATAATGA TCACCTTGAC 180

ACCCTTTGGA TCCCAAATAA AGAATATTTT TGGGGTCTTA GCAAATTTCT TGGAACACAC 240

TGGCTTATGG GCAACATTTT GAGGTTACTC TTTGGTAAGA TTTCTGTTGA TCCTTCTTTG 300

TAGGCTCTTG CATGTATGAA AACCTTGAAA ACAACAAGAA CTTCAAGTAG TTAAGACCAA 360

AGTAGATTTT TCTTCAGTCC AAATAGCTCC TAAAATGATA AGGAAAGTAT TTCTTTAAAG 420

CCCAGGCAAC TAC 433

C2) SEO ID NO. 13 »

CHARACTERISTICS OF THE SEQUENCE.

CA) LENGTH. 486 base pairs

CB) TYPE. nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY. straight

C11) MOLECULA TYPE. DNA (Genomic)

CIX) CHARACTERISTICS.

CA) NAME / KEY. exon

CB) LOCATION. 164..257

Cxi) SEQUENCE DESCRIPTION. SEO ID NO. 13 «

TTGGTGGGTA TCTAGTAGCA GTCTTTTTAA TGAATCTACT ATTCATCCAT AAAAAAGTAG 60

ATATAAATCA GATGGGTCTG CATTTTATGC TAATGAGATA TGAATTAAAT TCACTAGCAA 120

114

CACTCAGAGA AAACCTTAAC TATAACCTTC CATTGTTGTC TAGGTTCAAT GACAACTCCT 180

GCAAACTGGA ATTCCCCTCT GAGGCCTGGT GAAAAAATATC CACTTGTTGT TTTTTCTCAT 240 ggtcttgggg CATTCAGGTA atgtttgaga 'GGTTGAACAA TTTTGGCTTC CAGGAATAAA 300

TGACAATTTT TTTATTCAAG AAAGAAATAG CAGAGTTTGG AATGTCATGC AGGCCCTTGT 360

CTGGAGGAGT TGGGGTTCCT CAATAATTGG CTGTGGGTCT ATTGATCAGT CCTAGACCTG 420

TCTGGTCAAG TAGTTTTTTC CCTACTATCA GCTCATTGGG ATTAGCCTCA CAGCAGAGAA 480

GAAAGG 486

C 2) INFORMATION ON SEQ ID NO. 14 »(1) SEQUENCE CHARACTERISTICS» (L) LENGTH »363 base pairs (B) TYPE» nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY. straight

(A) MOLECULA TYPE. DNA (Genomic)

Cix) FEATURES:

CA) NAME / KEY. exon

CB) PLACEMENT: 113..181

Cxi) SEQUENCE DESCRIPTION:.

CCCCAGGCTC TACTACAGGG TGTAATGGCC TCCATGTTCC CAGTTTTATT AGTGACTCAG 60

CCTTGTAATT CATGACTGGT AGTTGTAATT CTTCCCTCTT TTTGTTTTGA AGGACACTTT 120 Attctgctat TGGCATTGAC CTGGCATCTC ATGGGTTTAT AGTTGCTGCT GTAGAACACA 180

GGTATGTTAC CTGATATAAT TGGGCTCTTT GGCCAACTAC AGGGAATGTC AATGCTCATA 240

ACTATGTTTC TAATTTTCAT AAAAGTTTAT TTAAAATGTT GATGGAACTT TCAAGTATGG 300

TAACATCATG AGCAAAAAAG GAGATTGAGT TTTATCGACT TAAAAGACTT AAAAGCACCT 360

AAC 363

C2) INFORMATION ON SEO ID NO: 15.

(Cl) SEQUENCE CHARACTERISTICS.

CA) LENGTH. 441 base pairs

CB) TYPE. nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY. straight

(Cii) MOLECULA TYPE. DNA (Genomic)

Clx) CHARACTERISTICS.

CA) NAME / KEY. exon C B) LOCATION »68..191

Cxi) SEQUENCE DESCRIPTION. SEO ID NO. 15 «

115 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

GAACTGAGAA ACATGGTCAG ATGAGGAAGG GAAGGAGCAT GCATAAATAA TTTTGCTTGT 60

ATTATAGAGA TAGATCTGCA TCTGCAACTT ACTATTTCAA GGACCAATCT GCTGCAGAAA 120

TAGGGGACAA GTCTTGGCTC TACCTTAGAA CCCTGAAACA AGAGGAGGAG ACACATATAC 180

GAAATGAGCA GGTACATTGC AGTGAAAGGA GAGGTGGTTG GTGACCTAAA AGCATGTACA 240

AAAGGATGAC ATTTGTTAAT TTAATTTTAC ACCTGGCAAG TTATGCTCCT AGCTCTCCTA 300

TTTCCCATTC CCAAAAGATC TGTCAATAGA TTCCTGGAGC AGTAAAATTC CCTTAATGGA 360

ATATCTAGTT CATAGTAAAA ACAAAGGCAA ATACAAAAAT TTGGGAGATG ACAGTGAATA 420

TTCAGAATTC CTCGAGCCGG G 441

C 2) INFORMATION ON SEQ ID NO: 16:

(Cl) SEQUENCE CHARACTERISTICS.

CA) LENGTH: 577 base pairs

CB) TYPE. nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY. linear Cil) MOLECULA TYPE: DNA Genomic)

CIX) CHARACTERISTICS.

CA) NAME / KEY: exun

CB) LOCATION. 245..358

Cxl) DESCRIPTION OF THE SEQUENCE. SEO ID NO. 16th

GGTTAAGTAA ATCGTCTGAA GTCACATAGT AGGTAAGGCA AAACAGAGCC AGGATTTGGA 60

CTAAGGCTAT ACCTATGTGC AAAGCTGGGG CCTGTGTCAT TATGGTAGCA AGTAATAGTC 120

ACTAATCAGA TTTCCAGTTT ATAACTGACC AACGATTTTT CCCAAATACA GCTTCTACCT 180

AAACTTTAAA ATAAGTGTTA TAACTTTTTA CTTTGTCATT TCCTTCTTCT AATAATTATA 240

TTAGGTACGG CAAAGAGCAA AAGAATGTTC CCAAGCTCTC AGTCTGATTC TTGACATTGA 300

TCATGGAAAG CCAGTGAAGA ATGCATTAGA TTTAAAGTT GATATGGAAC AACTGAAGGT- 360

AAGCTATAAA AAGTAATTTT TCTCTTGTCC TACAGTTCTT TATTGTTTTT TGTCATTTAA 420

TTTTCTGCCT ATATTGCAAG GTACAATATG ATAAAGGGCT GCAACCAGCC CCTCCCCAAT 480

GCGCACACAC AGACACACAA AGCAGTACAG GTAAAGTATT GCAGCAATGA AGAATGCATT 540

ATCTTGGACT AGATATGAAT GCAAAGTTAG TCAGTTT 577

C2) INFORMATION ON SEQ ID NO: 17.

(Cl) SEQUENCE CHARACTERISTICS.

CA) LENGTH. 396 base pairs

116 (B) TYPE. nucleic acid (C) NUMBER OF STRINGS, one

CD) TOPOLOGY. straight

C11) MOLECULA TYPE. DNA (genomic) (ix) PROPERTIES.

(A) NAME / KEY. exon (B) LOCATION. 108..199 (xi) SEQUENCE DESCRIPTION. SEO ID NO. 17th

ATCAATGTAT TTACCATCCC CATGAAATGA ACAATTATAT GATTGACAAA TCATTTCTTC 60 TAACACCACG AAAATAGCTAT AAAATTTATAT CATGCTTTTT CAAATAGGAC TCTATTGATA 120 GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AACGGTTATT CAGACTCTTA 180 GTGAAGATCA GAGATTCAGG TAGAAAATA AGATAGTAAA GCAAGAGAAT AGTAAATTAT 240 TGGAAGAAAT TATATTGTGA GATATAATTT TTATTCAAAT TCTTAGTGAA GGAAGGGGAT 300 CTCTTGGAGT TTATAAGGCT ATTCTTTTGC CCCCATAAAA TACTCTATAT ACATTTTCCT 360 AGGCTAAAAC ATCTCCTCTC CTGCTATTAA AATCTC 396 (2) INFORMATION ON SEO ID NO. 18th

(i) SEQUENCE CHARACTERISTICS (A) LENGTH. 519 base pairs (B) TYPE. nucleic acid (C) NUMBER OF STRINGS, one (D) TOPOLOGY. linear (ii) MOLECULA TYPE. DNA (genomic) (ix) PROPERTIES.

(A) NAME / KEY. exon (B) LOCATION. 181..351 (xi) SEQUENCE DESCRIPTION. SEO ID NO. 18th

CTTACAAAGT TAATCATATC CCTTTCCCAC ATTGAAGTAT GATACCTCTT TATTCCAATC 60

AGATAACCCA TAATAAACTG GTATGGTGCG TGTCCACCAA TCCTAGCATT ATTAGGATGT 120

CCTCAATGTT GGCTAGTATG TAACCAGTTT AATTTCATCA TTGTCAACAA ATATCTACAG 180

ATGTGGTATT GCCCTGGATG CATGGATGTT tccactgggt GATGAAGTAT ATTCCAGAAT 240

TCCTCAGCCC CTCTTTTTTA TCAACTCTGA ATATTTCCAA TATCCTGCTA ATATCATAAA 300

AATGAAAAAA TGCTACTCAC CTGATAAAGA AAGAAAGATG ATTACAATCA GGTAAGTATT 360

AGTGACTTAT TTCATTATGT GAAACAAACT TGAAGCTTGG GTAAATATCA ATCGATATCA 420

TTTGGTAACT ATTAAAGAAT TGCTGAATTG GTTGTTTAGA CTTTGAATAA GGAGAGAATT 480

AGATAATCTC AGTTTCTAAG TACATTTAGT CTACTCTTT

519

117 (2) INFORMATION ON SEO ID NO. 19th

(i) SEQUENCE CHARACTERISTICS.

CA) LENGTH. 569 base pairs (B) TYPE. nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY. straight

(Cii) MOLECULA TYPE. DNA (Genomic)

CIX) CHARACTERISTICS.

C A) NAME / KEY. exon

CB) LOCATION. 156..304

Cxi) SEQUENCE DESCRIPTION. SEO ID NO. 19th

TGAAACACAT CTAAGTAGAT CAAATTACAA GTTTTATTTC TTCTTTGGTT TTCAGTAAAC 60 AGACCAACAA GACCAGTACC TTTCCTTACA CTCTAACTAA ΑΑΑΑΑΤΑΆΤΑ ATTTTATCAA 120 ACAATGTGAC TTTTAAATGT CTTGTTCTCT TTTAGGGGTT CAGTCCACCA GAATTTTGCT 180 GACTTCACTT TTGCAACTGG CAAAATAATT GGACACATGC TCAAATTAAA GGGAGACATA 240 GATTCAAATG TAGCTATTGA TCTTAGCAAC AAAGCTTCAT TAGCATTCTT ACAAAAGCAT 300 TTAGGTAAGA AACTATTTTT TTCATGACCT AAACCGAGAT GAATCTCGAG GACAAAGCTG 360 TCTATCTTAA TACAGCTTTA GTACTATTTA AACTATTTCC AGTTGGTTTA CAATGGAACA 420 AAGCAGTATA TCAATTTGAA AACAGAAATT TGAGAAAGTC AATTTTGCTG CTTTACATCT 480 CTATATCATA GAAAGCAAAT CAACTGTTAA AGGTAATATT CTTTGTATGA GCTAGAGTGA 540 CTCATGTGAG GATATCGAAC gacggtgct 569

C2) INFORMATION 0 SEO ID NO. 20th

Ci) SEQUENCE CHARACTERISTICS.

C A) LENGTH. 469 base pairs

CB) TYPE. nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY. straight

(Cii) MOLECULA TYPE. DNA (Genomic)

CIX) CHARACTERISTICS.

C A) NAME / KEY. exon CB) LOCATION. 137..253

Cxi) SEQUENCE DESCRIPTION. SEO ID NO. 20th

GATACAGAGG CACATCGTCT CTACCATCCT AACGGAACTT GTGTAATTTG TAAATCTTTA 60

TTGCCACCTA GGGGCATCCA AACTGTTTAA TGCTCTCAAA AGTTTAATAT GTTGATTAAC 120

ACTTTATATT TTATAGGACT TCATAAAGAT TTTGATCAGT GGGACTGCTT GATTGAAGGA 180

GATGATGAGA ATCTTATTCC AGGGACCAAC ATTAACACAA CCAATCAACA CATCATGTTA 240

118

CAGAACTCTT CAGGAATAGA GAAATACAAT TAGGATTAAA ATAGGTTTTT TAAAAGTCTT 300

GTTTCAAAAC TGTCTAAAAT TATGTGTGTG TGTGTGTGTG TGTGTGTGTG AGAGAGAGAG 360

AGAGAGAGAG AGAGAGAATT TTAATGTATT TTCCCAAAGG ACTCATATTT TAAAATGTAG 420

GCTATACTGT AATCGTGATT GAAGCTTGGA CTAAGAATTT TTTCCCTTT 469

C2) INFORMATION ON SEO ID NO: 21.

(Cl) SEQUENCE CHARACTERISTICS.

CA) LENGTH. 1494 base pairs

CB) TYPE. nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY. straight

Cii) MOLECULA TYPE: cDNA

CIX) CHARACTERISTICS.

CA) NAME / KEY. CDS

CB) LOCATION. 117..1436

Cxi) SEQUENCE DESCRIPTION. SEO ID NO. 21st

GGCACGAGCT AGGATCTGAC TCGCTCTGGT GGCATTGCTG CGCTCAGGGT TCTGGGTATC

CGGGAGTCAG TGCAGTGACC AGAACATCAA ACTGAAGCCA CTGCTCAGCT CCTAAG 116

ATG GTA CCA CTC AAA CTG Met 1 wall for Leu Lys 5 Leu CCA TGG GTC CAT CCT TTT for Trp wall HLS 20 for Phe AGG CCG TCA GTA ATG TTT Arg for Ser 35 wall Met Phe GGC TCT GGC CAT AGT AAA Gly Ser 50 Gly HLS Ser Lys GGT TGT ACA GAT CTG ATG Gly 65 Cys Thr Asp Leu Met 70 CGT TTG TAC TAC CCA GCT Arg Leu Tyr Tyr for 85 Ala ATC CCA AAC AAA GAA TAT Ile for same time Lys 100 Glu Tyr CCC AGT ATT GTA GGC AAT for Ser Ile 115 wall Gly same time

CAG GCG CTT TTC TGC CTC gin Ala Leu Phe 10 Cys Leu CAC TGG CAA GAC ACA TCT HLS Trp gin 25 Asp Thr Ser CAC AAG CTC CAA TCG GTG His Lys 40 Leu gin Ser wall ATC CCC AAA GGA AAT GGA Ile 55 for Lys Gly same time Gly 60 TTC GGT TAT GGG AAT GAG Phe Gly Tyr Gly same time 75 Glu CAA GAT CAA GGT CGC CTC gin Asp gin Gly Arg Leu TTT TTG GGT CTT AGT ATA Phe Leu Gly 105 Leu Ser Ile ATT TTA CAC CTC TTA TAT Ile Leu HLS Leu Leu Tyr

120

CTC TGC TGC CTC 164

Cys Leu

TCT TTT GAC TTC 212

Ser Phe Asp Phe

ATG TCT GCT GCC 260

Met Ser Ala Ala

TCG TAC CCC GTC 308

Ser Tyr Pro Val

AGC GTC TTC GTG 356

Ser Val

GAC ACT GTT 404

Asp Thr Val Trp

TTT CTT GGA ACA 452

Phe Leu Gly Thr

110

GGT TCT CTG ACA 500

Gly Ser Leu Thr

125

113

ACT CCT GCA AGC TGG AAT TCT CCT TTA AGG ACT GGA GAA AAA TAC CCG 548 Thr For Ala Ser 130 Trp same time Ser 135 for Leu Arg Thr Gly Glu Lys Tyr for CTC ATT GTC TTT TCT CAT GGT CTC GGA GCC TTC AGG ACG ATT TAT TCT 596 Leu 145 Val Phe Ser HLS 150 Gly Leu Gly Ala Phe Arg Thr Ile Tyr Ser 160 GCT ATT GGC ATT GGC TTG GCA TCT AAT GGG TTT ATA GTG GCC ACT GTC 644 Ala Gly White Gly 165 Leu Ala Ser same time Gly 170 Phe Ile Val Ala Thr 175 wall 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 185 Thr Phr Glu 190 Asp gin GTG GCT GCA AAA GTG GAA AAC AGG TCT TGG CTT TAC CTG AGA AAA GTA 740 wall Ala Ala Lys 195 wall Glu same time Arg 200 Ser Trp Leu Tyr Leu Arg Lys wall ΑΑΆ CAA GAG TCG GAA AGT GTC CGG AAA GAA CAG GTT CAG CAA AGA 788 Lys Gin Glu Glu 210 Ser Glu Ser 215 wall Arg Lys Glu Gin Val 220 gin gin Arg GCA ATA GAA TGT TCC CGG GCT CTC AGT GCG ATT CTT GAC ATT GAA CAT 836 Ala 225 Glu Cys Ser Arg 230 Ala Leu Ser Ala Leu Asp 235 Ile Glu His 240 GGA GAC CCA AAA GAG AAT GTA CTA GGT TCA GCT TTT GAC ATG AAA CAG 884 Gly Asp Lys Glu 245 same time wall Leu Gly Ser 250 Ala Phe Asp Met Lys 255 gin CTG AAG GAT GCT ATT GAT GAG ACT AAA ATA GCT TTG ATG GGA CAT TCT 932 Leu Lys Asp Ala 260 Ile Asp Glu Thr Lys 265 Ile Ala Leu Met Gly 270 His Ser TTT GGA GGA GCA ACA GTT CTT CAA GCC CTT AGT GAG GAC CAG AGA TTC 980 Phe Gly Gly Ala 275 Thr wall Leu gin 280 Ala Leu Ser Glu Asp 285 gin Arg Phe AGA TGT GGA GTT GCT CTT GAT CCA TGG ATG TAT CCG GTG AAC GAA GAG 1028 Arg Cys Gly Val Ala Leu Asp 295 for Trp Met Tyr Pro Val 300 same time Glu Glu CTG TAC TCC AGA ACC CTC CAG CCT CTC CTC TTT ATC AAC TCT GCC AAA 1076 Leu 305 Tyr Ser Arg Thr Leu 310 gin for Leu Leu Phe White Asn 315 Ser Ala Lys 320 TTC CAG ACT CCA AAG GAC ATC GCA AAA ATG AAA AAG TTC TAC CAG CCT 1124 Phe Gin Thr Pro Lys 325 Asp Ile Ala Lys Met 330 Lys Lys Phe Tyr gin 335 for GAC AAG GAA AGG AAA AAT GAT TAC AAT CAA GGG CTC AGG CAC CAG AAC 1172 Asp Lys Glu Arg 340 Lys same time Asp Tyr same time 345 gin Gly Leu Arg His 350 gin same time TTT GAC GAC TTT ACT TTT GTA ACT GGC AAA ATA ATT GGA AAC AAG CTG 1220 Phe Asp Asp Phe 355 Thr Phe wall Thr 360 Gly Lys Gly 365 same time Lys Leu

120

ACA CTG AAA GGA GAA ATC GAT TCC AGA GTA GCC ATC GAC 1268 Thr Leu 370 Lys Gly Glu Ile Asp 375 Ser Arg Val Ala Ile Asp 380 Leu Thr Asn AAA GCT TCG ATG GCT TTC TTA CAA AAG CAT TTA GGG CTT CAG AAA GAC 1316 Lys Ala Ser Met Ala Phe Leu gin Lys His Gly Leu Gin Lys Asp 385 390 395 400 TTT GAT CAG TGG GAC CCT CTG GTG GAA GGA GAT GAT GAG AAC CTG ATT 1364 Phe Asp Gin Trp Asp for Leu wall Glu Asp Asp Glu Asn Leu White 405 410 415 CCT GGG TCA CCC TTT GAC GCA GTC ACC CAG GCC CCG GCT CAG CAA CAC 1412 for Gly Ser Pro Phe Asp Ala wall Thr Gin Ala For Ala Gin Gin His 420 425 430 TCT CCA GGA TCA CAG ACC CAG AAT TAGAAGAACT TGCTTGTTAC ACAGTTGCCT 1466 Ser for Gly Ser gin Thr gin same time 435 440

TTTAAAAGTA GAGTGACATG AGAGAGAG 1494

C 2) INFORMATION ON SEQ ID NO. 22nd

C (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 2191 base pairs (B) TYPE. nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY. straight

(Cii) MOLECULA TYPE. cDNA

CIX) CHARACTERISTICS.

CA) NAME / KEY. CDS

CB) LOCATION. 92..1423

Cxi) SEQUENCE DESCRIPTION. SEO ID NO. 22nd

CCGCGCGCTC CGGCCGGGGG ACCCTGGTTC CGGCGAGCGG CTCAGCGCGG CGCCCGGAAG 60

TTTAAGCTGA AACCACTGCT CAGCTTCCAA G

Met Leu Pro Lys Leu His

5

GCG Ala CTT TTC TGC CTC TGC Leu Phe 10 AGC TGC CTC 160 Ser Cys 15 Leu Thr Leu Val His 20 for Asp TGG CAA GAC CTA AAT CCT GTT GCC CAT ATT AGA TCA TCA GCA TGG GCC 208 Trp gin Asp Leu same time for wall Ala His Ile Arg Ser Ser Ala Trp Ala 25 30 35 AAT AAA ATA CAA GCT CTG ATG GCT GCT GCA AGT ATT AGG CAA AGT AGA 256 same time Lys Ile gin Ala Leu Met Ala Ala Ala Ser Ile Arg gin Ser Arg 40 45 50 55 ATT CCC AAA GGA AAT GGA TCT TAT TCT GTC GGT TGT ACA GAT TTG ATG 304 Ile for Lys Gly same time Gly Ser Tyr Ser wall Gly Cys Thr Asp Leu Met

65 70

121

TTT GAT Phe Asp TAT ACT Tyr Thr 75 AAT AAG GGC Gly ACC TTT TTG TAT CCA TCG 352 same time Lys Thr Phe Leu Arg Leu Tyr Tyr 85 Pro Ser CAA GAG GAT GAC CAC TCT GAC ACG CTT TGG ATC CCA AAC AAA GAA TAT 400 gin Glu Asp Asp His Ser Asp Thr Leu Trp Ile for same time Lys Glu Tyr 90 95 100 TTT TTT GGT CTT AGT AAA TAT CTT GGA ACA CCC TGG CTT ATG GGC AAA 448 Phe Phe Gly Leu Ser Lys Tyr Leu Gly Thr for Trp Leu Met Gly Lys 105 110 115 ATA TTG AGC TTC TTT TTT GGT TCA GTG ACA ACT CCT GCG AAC TGG AAT 496 Ile Leu Ser Phe Phe Phe Gly Ser wall Thr Thr for Ala same time Trp same time 120 125 130 135 TCC CCT CTG AGG ACT GGT GAA AAA TAT CCA CTG ATT GTT TTT TCT CAT 544 Ser for Leu Arg Thr Gly Glu Lys Tyr for Leu Ile wall Phe Ser His 140 145 150 GGT CTT GGA GCA TTC CGG ACA ATT TAT TCT GCT ATT GGC ATT GAT CTA 592 Gly Leu Gly Ala Phe Arg Thr Ile Tyr Ser Ala Ile Gly Ile Asp Leu 155 160 165

GCA Ala TCA CAT GGG TTC ATC Ile GTT GCT GCT ATA GAA CAC 640 Ser His 170 Gly Phe wall Ala 175 Ala Ile Glu His Arg Asp Gly Ser GCC TCT GCG ACT TAC TAT TTC AAG GAC CAG TCT GCT GCA GAA ATA GGG 688 Ala Ser 185 Ala Thr Tyr Tyr Phe 190 Lys Asp gin Ser Ala Ala Glu Ile Gly AAC AAA TCT TGG TCT TAT CTT CAA GAA CTA AAA CCA GGG GAT GAG GAG 736 same time 200 Lys Ser Trp Ser Tyr 205 Leu gin Glu Leu Lys 210 For Gly Asp Glu Glu 215 ATA CAT GTT CGA AAT GAG CAG GTA CAG AAA AGG GCA AAG GAG TGC TCC 784 Ile His wall Arg Asn Glu gin wall gin Lys 225 Arg Ala Lys Glu Cys 230 Ser CAA GCT CTC AAC TTG ATT CTG GAC ATT GAT CAT GGA AGG CCA ATT AAG 832 gin Ala Leu Asn Leu Ile Leu Asp Ile 240 Asp His Gly Arg Pro 245 Ile Lys AAT GTA CTA GAC TTA GAG TTT GAT GTG GAA CAA CTG AAG GAC TCT ATT 880 same time wall Leu 250 Asp Leu Glu Phe Asp 255 wall Glu gin Leu Lys Asp 260 Ser Ile GAC AGG GAT AAA ATA GCA GTA ATT GGA CAT TCT TTT GGT GGA GCC ACA 928 Asp Arg Asp 265 Lys ile Ala wall 270 Ile Gly His Ser Phe Gly Gly Ala 275 Thr GTT CTT CAG GCT CTT AGT GAA GAC CAG AGA TTT AGG TGC GGG ATT GCC 976 wall 280 Leu gin Ala Leu Ser 285 Glu Asp gin Arg Phe 290 Arg Cys Gly Ile Ala 295 TTG GAT GCA TGG ATG CTT CCA CTG GAT GAT GCA ATA TAT TCC AGA ATC 1024 Leu Asp Ala Trp Met 300 Leu for Leu Asp Asp 305 Ala Clay Tyr Ser Arg ile 310

122

CCT CAG CCC CTC ATT AAC TCG GAA GTC TTC CAA TTT CCT GAG 1072 for Gin Pro Leu 315 Phe Phe Asn Ser 320 Glu Arg Phe gin Phe Pro 325 Glu AAT ATC AAA AAA ATG AAA AAA TGC TAC TCA CCT GAC AAA GAA AGA AAA 1120 same time Ile Lys Lys Met Lys Lys Cys Tyr Ser for Asp Lys Glu Arg Lys 330 335 340 ATG ATT ACA ATC AGG GGT TCA GTC CAT CAG AAC TTT GCT GAT TTC ACT 1168 Met Ile Thr Ile Arg Gly Ser wall His gin same time Phe Ala Asp Phe Thr 345 350 355 TTT ACA ACT GGC AAA ATA GTT GGA TAC ATA TTC ACA TTA AAA GGA GAT 1216 Phe Thr Thr Gly Lys Ile wall Gly Tyr Ile Phe Thr Leu Lys Gly Asp 360 365 370 375 ATA GAT TCA AAT GTA GCA ATT GAT CTT TGC AAC AAA GCT TCA TTG GCA 1264 Ile Asp Ser same time wall Ala Ile Asp Leu Cys same time Lys Ala Ser Leu Ala 380 385 390 TTT TTA CAA AAG CAT TTA GGA CTG CGG AAA GAT TTT GAT CAG TGG GAT 1312 Phe Leu gin Lys His Leu Gly Leu Arg Lys Asp Phe Asp gin Trp Asp 395 400 405 TCT TTG ATT GAA GGA AAA GAC GAA AAT CTT ATG CCA GGG ACC AAC ATT 1360 Ser Leu Ile Glu Gly Lys Asp Glu same time Leu Met for Gly Thr same time Ile 410 415 420 AAC ATC ACC AAC GAA CAT GAC ACT CTA CAG AAC TCT CCA GAA GCA GAG 1408 same time Ile Thr same time Glu His Asp Thr Leu gin same time Ser for Glu Ala Glu 425 430 435

AAA TCG ΑΑΤΤΑ GAT TAAAAGCACT TTTTTAAAGA TCTTGTTTAA AAACTGTCAA 1463

Lys Ser Asn Leu Asp

440

AAAATGTGTG TATGACTTTT AATATATTTT CTCAAATAAC TCATATTGGA AAATGTAGGC 1523

TATCCCATAA AAGTGATTGA AGCTTGGACT AGGAGGTTTT TTTCTTTAAA GAAAGATTGG 1583

TGTCTATCGA AATCATGCCA GCCTAAATTT TAATTTTACT AAAATGATGC TGTGTCAAAA 1643

TTAATAACTA CTTTTACATT CTTTAATGGA CAAGTATAAC AGGCACAAGG CTAATGAAAA 1703

CGTGTTGCAA TGACATAACA ATCCCTAAAA ATACAGATGT TCTTGCCTCT TTTTTCTATT 1763

ATAATTGAGT TTTAGCAACA TGTTATGCTA GGTAGAATTT GGAAGCACTT CCCTTTGACT 1823

TTTGGTCATG ATAAGAAAAA TTAGATCAAG CAAATGATAA AAGCAGTGTT TTACCAAGGA 1883

TTAGGGATAC TGAACAATTT CACTATGGTA ACTGAATGGG GAGTGACCAA GGGTAAAAAT 1943

ATTAAAGCCA AGGCAAAGGC AGCAGATTAG AATGGATTAA AGAGAGTTTA TAATTTGTTT 2003

GCATTTACTT GATGGTTTAT CTCATGGATT CATGAGTCAA GAAAGGTGCG TAGGACAGGC 2063

CAGGGATTCC AGTTATAACA CATTATTCAC CCAAAGGGTT CTTTAATTCT GTATGAGTAT 2123

TGGGAGTGGA TTAGCACAAT AGAGGCATAT GTTGCTTTAA AAAAAAAAAA AAAAAAAAAA 2183

123

ΑΑΑΑΆΑΑΑ 2191 (2) INFORMATION Ο SEO ID NO. · 23 «(1) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 1533 base pairs (B) TYPE. nucleic acid (C) NUMBER OF STRINGS, one

CD) TOPOLOGY. linear (ii) MOLECULA TYPE: protein (ix) CHARACTERISTICS.

CA) NAME / KEY. (B) LOCATION. 62..1394 (xi) SEQUENCE DESCRIPTION. SEO ID NO. 23 «

CCGCGAGCAG TTCACCGCGG CGTCCGGAAG GTTAAGCTGA AACGGCAGCT CAGCTTCGGA 60

G ATG TTA CCG TCC AAA TTG CAT

Met Leu Pro Lys Leu His Ala Leu Phe Cys Leu Cys Thr Cys

10 15

CTT Leu GCA CTG GTT TAT CCT TTT GAG TGG CAA GAC CTG AAT CCA GTT GCC 154 Ala Leu Val Tyr Pro 19 Phe Asp Trp gin 25 Asp Leu same time for wall 30 Ala TAT ATT GAA TCA TGG GTC AGT AAG ATA CAA GCT CTG ATG GCT 202 Tyr Badly Glu Ser Pro Ala 35 Trp Val Ser 40 Lys ile Gin Ala Leu 45 Met Ala GCT GCA AAC ATT TCT AAA ATC CCC AGA GGA AAT GGA TCT TAT 250 Ala Ala Asn Gly Gin 50 Ser Lys Ile for Arg Gly same time 60 Gly Ser Tyr. TCC GTC GGT TGT ACA GAC TTG ATG TTT GAT TAC ACT AAT AAG GGC ACC 298 Ser Val Gly Thys Asp 65 Leu Met Phe Asp Tyr Thr same time Lys Gly Thr TTC TTG CGT CCA TCT CAA GAT GAT GAT CAC TCC GAC ACC 346 Phe 80 Leu Arg Leu Tyr Tyr 84 Pro Ser gin Asp Asp Asp 90 His Ser Asp Thr 95 CTT TGG ATC CCA AAC AAA GAA TAT TTT TTG GGT CTT AGT AAA TTT CTT 394 Leu Patience For Asn Lys 100 Glu Tyr Phe Leu 105 Gly Leu Ser Lys Phe 110 Leu GGA ACA CAC TGG CTT GTG GGC AAA ATT ATG GGC TTA TTC TTC GGT TCA 442 Gly Thr His Trp Leu Val 116 Gly Lys Ile 120 Met Gly Leu Phe Phe 125 Gly Ser ATG ACA ACT CCT GCA GCC TGG AAT GCA CAT CTG AGG ACT GGG GAA AAA 490 Met Thr Thr Pro Ala Ala 130 Trp Asn Ala His Leu Arg Thr 140 Gly Glu Lys TAC CCA ATT ATT TTT TCT CAT GGT CTT GGA GCA TTC AGG ACG ATT 538 Tyr For Leu Ile Phe Ser His Gly Leu Gly Ala Phe Arg Thr Ile

124

145 150 155

ΤΑΤ Tyr 160 ATT GGC ATT GAT CTG GCA TCC CAC GGG TTT 586 Ser Ala Ile Gly Ile 165 Asp Leu Ala Ser His Gly Phe White Val Ala 170 175 GCT GTA GAA CAC AGG GAT GGC TCT GCA TCC TCG ACA TAC TAT TTC AAG 634 Ala wall Glu His Arg Asp Gly Ser Ala Ser Ser Thr Tyr Tyr Phe Lys 180 185 19U GAC CAG TCT GCT GTA GAA ATA GGC AAC AAG TCT TAT CTC AGA 682 Asp gin Ser Ala wall Glu Ile Gly Asn Lys Ser Trp Leu Tyr Leu Arg 195 200 205 ACC CTG AAG CGA GGA GAG GAG GAG TTT CCT TTA CGA AAT 730 Thr Leu Lys Arg Gly Glu Glu Glu Phe Pro Leu Arg Asn Glu Gin Leu 210 215 220 CGG CAA CGA GCA AAG GAA TGT TCT CAA GCT CTC AGT TTG ATT CTG GAC 778 Arg gin Arg Ala Lys Glu Cys Ser Gin Ala Leu Ser Leu White Leu Asp 225 230 235 ATT GAT CAC GGG AGG CCA GTG ACG AAT GTA CTA GAT TTA GAG 826 Ile Asp His Gly Arg for wall Thr Asn Val Leu Asp Leu Glu Phe Asp 240 245 250 255 GTG GAA CAG CTG AAG GAC TCT ATT GAT AGG GAT AAA ATA GCC ATT ATT 874 wall Glu gin Leu Lys Asp Ser Ile Asp Arg Asp Lys ile Ala ile ile 260 265 270 GGA CAT TCT TTT GGT GGA GCC ACA GTT ATT CAG ACT CTT AGT GAA GAC 922 Gly His Ser Phe Gly Gly Ala Thr Val ile gin Thr Leu Ser Glu Asp 275 280 285 CAG AGA TTC AGG TGT GGC ATT GCT CTG GAT GCA TGG ATG TTT CCC GTG 970 gin Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala Trp Met Phe Pro Val 290 295 300 GGT GAT GAA GTA TAT TCC AGA ATT CCT CAA CCC ATTC AAC 1018 Gly Asp Glu wall Tyr Ser Arg Ile Pro Gin for Leu Phe Phe White Asn 305 310 315 TCG GAA CGA TTC CAA TAC CCT TCT AAT ATC ATA AGA ATG AAA AAA TGC 1066 Ser Glu Arg Phe gin Tyr for Ser Asn Zle Ile Arg Met Lys Cys 320 325 330 335 TTC TTA CCT GAT AGA GAA CGA AAA ATG ATT ACA ATG AGG GGT TCG GTC 1114 Phe Leu for Asp Arg Glu Arg Lys Met íle Thr Il Arg Gly Ser Val 340 345 350 GAT CAG AAT TTT GTT GAC TTC ACT TTT GCC ACT AGC AAA ATA ATT GGC 1162 His gin same time Phe wall Asp Phe Thr Phe Ala Thr Ser Lys White Clay Gly 355 360 365 TAC CTA TTC ACA CTG AAA GGA GAC ATC GAT TCC AAT GTA GCC ATC AGC 1210 Tyr Leu Phe Thr Leu Lys Gly Asp Asp Ser Asn Val Ala Ile Ser 370 375 380 CTT AGC AAC ΑΆΑ GCT TCC TTA GCG TTC TTA CAA AAA CAT TTA GGA CTT 1258 Leu Ser same time Lys Ala Ser Leu Ala Phe Leu Gin Lys His Leu

385 390 395

125

CAG AAA GAT TTT GAT CAG TGG GAT TCT TTA GTT

Gin Lys Asp Phe Asp Gin Asp Ser Leu Val Glu Gly Glu Asp Hls

(+420) 400 405 410 415

AAT CTT ATT CCA GGG ACC AAC ATT AAC ACA ACC AAC CAC CAA GCC ATT 1354

Asn Leu White For Gly Thr Asn White Asn Thr White Asn His Gin Ala White

420 425 430

CAG AAC TCC ACA GGA ATA GAG AGA CCA AAT TTA GAT T AAAAGAGCTT 1404 Leu Gin Asn Ser Thr Gly White Glu Arg Pro Asn Leu Asp

435 440

TTTAAAAAGT TTTGTTTACG AACTTGTCTA AAAGTGTGTG TGTGTATGAT TTAAATGTAT 1464

TTTCTCAAAT AGCTCATATT AAAAAATGTA GGCTATAGCA CAAAAAAAAA ΑΑΑΑΑΑΑΑΑΑΑ 1524

ΑΑΑΆΆΑΑΆΑ 1533

C 2) INFORMATION ON SEO ID NOs 24.

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 1876 base pairs (B) TYPE. nucleic acid (C) NUMBER OF STRINGS, one

CD) TOPOLOGY. straight

C11) MOLECULA TYPE, protein (1x) PROPERTIES.

CA) NAME / KEY. CDS

CB) LOCATION. 468..1734

Cxl) DESCRIPTION OF THE SEQUENCE. SEO ID NO. 24th

CGGCGGGCTG CTGGCCCTTC CCGGCTGTTC GTAGAGCCGG ATCCTGCAGC GCCCCTGAGA 60

CGAACCGCCC CGATGCGGTG CTCCTCAGCG CCACGGGACG CAGCCGGGGC CGGCCGTGTT 120

GGCGCAGCTC CCACGACGTA CGCTTCCTTT CCAGGCTCGA GGAAAGCCTC TCCCACAAAC 180

ACCGTCCCAG CTGGGAAGTG AGGCGGAGTT TTGGTCCCTC CCCTCCGGCA GCGCCCGGCA 240

TTCCGTCCGT CCGTCCGTCC GTCCGTGCGG CGCACGGCGC CCTGCAGAGC CGGGACACCG- 300

CAGCAGGGTA GGAGGACCCG GAGGTGGTGT GCAGCCACAG GTTTCCATCC TGCCCCCACC 360

TCCCGGGGAG CAGCCCTGTG CTATACCCAA CCCCCCGCAC AGAGCACTGA GCCGGCTGCT 420

GCCTGCCTGC ACCCCGCCGT GGGACCTTCT GCTCTTCCCA ACAAGTG ATG GCA TCG 476

Met. Ala Ser

GTC AGG GTC AGG AGG AGG AGG AGG AGG AGG AGG AGG AGG AGG AGG AGG AGG AGG

Leu Trp Val Arg

10 15

TTC TCG GCG AAG GCG GCG ACG GAG ATG GGG AGC GGC GGC GCG GAG AAG 572

126

Phe 20 Ser Ala Lys Ala Ala 25 Thr Glu Met Gly Ser 30 Gly Gly Ala Glu Lys 35 GGC TAT CGG ATC CCC GCC GGG AAG GGC CCG CAC GCC GTG GGC TGC ACG 620 Gly Tyr Arg Ile for 40 Ala Gly Lys Gly for 45 His Ala wall Gly Cys 50 Thr GAT CTG ATG ACC GGC GAC GCG GCC GAG GGA AGC TTT TTG CGC CTG TAT 668 Asp Leu Met Thr 55 Gly Asp Ala Ala Glu 60 Gly Ser Phe Leu Arg 65 Leu Tyr TAC CTA TCG TGT GAC GAC ACA GAT ACT GAA GAG ACA CCC TGG ATT CCA 716 Tyr Leu Ser 70 Cys Asp Asp Thr Asp 75 Thr Glu Glu Thr for 80 Trp Ile for GAT AAA GAG TAC TAC CAG GGG CTG TCT GAC TTC CTC AAC GTG TAC CGG 764 Asp Lys 85 Glu Tyr Tyr gin Gly 90 Leu Ser Asp Phe Leu 95 same time wall Tyr Arg GCC CTG GGA GAA AGG CTT TTC CAG TAC TAC GTT GGC TCA GTG ACC TGT 812 Ala 100 Leu Gly Glu Arg Leu 105 Phe gin Tyr Tyr wall 110 Gly Ser wall Thr Cys 115 CCT GCA AAA TCA AAC GCT GCT TTT AAG CCA GGA GAG AAA TAC CCA CTG 860 for Ala Lys Ser same time 120 Ala Ala Phe Lys for 125 Gly Glu Lys Tyr for 130 Leu CTC GTT TTT TCC CAT GGA CTT GGA GCT TTT CGG ACC ATC TAT TCT GCT 908 Leu wall Phe Ser 135 His Gly Leu Gly Ala 140 Phe Arg Thr Ile Tyr 145 Ser Ala ATC TGC ATA GAG ATG GCT TCT CAA GGC TTT CTA GTG GCA GCT GTG GAG 956 Ile Cys Ile 150 Glu Met Ala Ser gin 155 Gly Phe Leu wall Ala 160 Ala wall Glu CAC AGA GAT GAA TCG GCT TCA GCA ACG TAT TTC TGT AAA AAG AAG GCT 1004 His Arg Asp Glu Ser Ala Ser 170 Ala Thr Tyr Phe Cys 175 Lys Lys Lys Ala GAT TCT GAG CCA GAG GAG GAT CAA ACA TCA GGC GTG GAG AAG GAG TGG 1052 Asp 180 Ser Glu for Glu Glu 185 Asp gin Thr Ser Gly 190 wall Glu Lys Glu Trp 195 ATC TAC TAC AGG AAG CTC AGA GCA GGA GAG GAG GAG CGC TGT CTG CGT 1100 Ile Tyr Tyr Arg Lys 200 Leu Arg Ala Gly Glu 205 Glu Glu Arg Cys Leu 210 Arg CAC AAG CAG GTA CAG CAG AGA GCA CAG GAG TGC ATC AAA GCG CTC AAC 1148 His Lys gin wall 215 gin gin Arg Ala gin 220 Glu Cys Ile Lys Ala 225 Leu same time CTC ATT CTT AAG ATC AGT TCA GGA GAG GAA GTG ATG AAT GTG CTG AAC 1196 Leu Ile Leu 230 Lys Ile Ser Ser Gly 235 Glu Glu wall Met same time 240 wall Leu same time TCA GAC TTT GAC TGG AAC CAC CTG AAG GAT TCT GTT GAT ACT AGC AGA 1244 Ser Asp 245 Phe Asp Trp same time His 250 Leu Lys Asp Ser wall 255 Asp Thr Ser Arg ATA GCT GTG ATG GGA CAC TCT TTT GGT GGT GCT ACA GTT ATT GAG AGC 1292 Ile Ala wall Met Gly His Ser Phe Gly Gly Ala Thr wall Ile Glu Ser

127

260 265 270 275

CTC Leu AGC AAA GAA ATT TGT GGC ATT GCC CTT GAT GCG Asp Ala 290 TGG Trp 1340 Ser Lys Glu White Arg 280 Phe Arg Cys Gly 285 Ile Ala Leu ATG CTC CCG GTA GGC GAT GAC ACT TAC CAA AGC AGT GTG CAG CAA CCA 1388 Met Leu Pro Val Asp Asp 295 Tyr 300 gin Ser Ser Val Gin Gin for CTG CTC TTT ATT AAT TCC GAA AAA TTC CAG TGG GCT GCC AAT ATC TTA 1436 Leu Leu Phe White Asn Ser 310 Glu Lys 315 Phe gin Trp Ala Ala Asn ile Leu AAG ATG AAG AAG CTT AGC TCC AAT GAT ACC AAC AAG AAA ATG ATC ACC 1484 Lys Met Lys Lys Leu Ser 325 Ser Asn 330 Asp Thr same time Lys Lys Met íle Thr ATC AAA GGA TCG GTA CAT CAG AGC TTT CCT GAT TTT ACT TTT GTG AGT 1532 Ile 340 Lys Gly Ser Val His 345 Gin Ser Phe for Asp 350 Phe Thr Phe Val Ser 355 GGA GAA ATC ATT GGA AAG TTT TTC AAG TTA AAA GGA GAA ATA GAC CCA 1580 Gly Gly White Gly Lys 360 Phe Phe Lys Leu 365 Lys Gly Glu Asp 370 for AAT GAA GCT ATT TGC AAC CAC GCT TCA TTG GCC TTC CTG CAG 1628 same time Glu Ala Ia Asp Ia 375 Cys Asn His 380 Ala Ser Leu Ala Phe Leu gin AAA CAT CTG AGT CTT AAG AGA GAT TTT GAT AAG TGG GAT TCA CTC GTG 1676 Lys Hls Leu Ser Leu Lys 390 Arg Asp Phe Asp Lys Asp 400 Ser Leu wall GAT GGC ATA GGA CCC AAT GTT ATT TCT GGT ACC AAT ATC GAC TTA TCT 1724 Asp Gly White Gly Pro Asn Val ile Ser Gly Thr Asn ile Asp Leu Ser

405 410 415

CCA ACT GAG T AAGGAGTACA AGAAGTACTG CAAAGGCCAC CAGCAGCAGG 1774

For Thr Glu

420

ACACCAACGT TGGCCACACA TTGCTTGGAG CTGAGATAGC ACTGGCCTCC CACACAGCTT 1834

TTGGAGTGTG AAACAACAAA AAAAAAAATC ACAGGGGAGC CG 1876

C 2) INFORMATION ON SEO ID NO: 25.

(1) SEQUENCE CHARACTERISTICS. (A) LENGTH: 517 base pairs

CB) TYPE. nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY: linear

C11) MOLECULA TYPE. cDNA

Clx) CHARACTERISTICS.

C A) NAME / KC.ÚČ. CDS CB) LOCATION: 2..514

128

Cxl) DESCRIPTION OF THE SEQUENCE. SEO ID NO. 25th

GGG CAT TCT TTT GGA GGA GCA ACA GTT TTT CAA

Gly Hls Ser Phe

10 15

GAC Asp CAG AGA TTC AGA TGT GGG ATT Gly Wrong GCC Ala CTT GAT CCG TGG ATG TTT CCC 94 gin Arg Phe Arg 20 Cys Leu 25 Asp-Pro Trp Met Phe 30 for GTG AGT GAG GAG CTG TAC TCC AGA GTT CCT CAG CCT CTC TTC TTT ATC 142 wall Ser Glu Glu Leu Tyr Ser Arg wall for gin for Leu Phe Phe ill 35 40 45 AAC TCT GCC GAA TTC CAG ACT CCA AAG GAC ATT GCA AAA ATG AAA AAC 190 same time Ser Ala Glu Phe gin Thr for Lys Asp ill Ala Lys Met Lys same time 50 55 60 TTC TAC CAG CCT GAC AAG GAA AGG AAA ATG ATT ACG ATC AAG GGC TCA 238 Phe Tyr gin for Asp Lys Glu Arg Lys Met ill Thr ill Lys Gly Ser 65 70 75 GTG CAC CAG AAT TTT GCT GAC GGG ACT TTT GTA ACT GGC AAA ATA ATT 286 wall His gin same time Phe Ala Asp Gly Thr Phe wall Thr Gly Lys ill ill 80 85 90 95 GGA AAC AAG CTG TCA CTG AAA GGA GAC ATA GAC TCC AGA GTT GCC ATA 334 Gly same time Lys Leu Ser Leu Lys Gly Asp ill Asp Ser Arg wall Ala ill 100 105 110 GAC CTC ACC AAC AAG GCT TCC TTG GCT TTC TTA CAA AAA CAT TTA GGA 382 Asp Leu Thr same time Lys Ala Ser Leu Ala Phe Leu gin Lys His Leu Gly 115 120 125 CTT CAT AAA GAC TTT GAT CAG TGG GAC TGT CTG GTG GAG GGA GAG AAC 430 Leu HLS Lys Asp Phe Asp gin Trp Asp Cys Leu wall Glu Gly Glu same time 130 135 140 GAG AAC CTC ATC CCG GGG TCA CCC TTT GAT GTA GTC ACC CAG TCC CCG 478 Glu same time Leu ill for Gly Ser for Phe Asp wall wall Thr gin Ser for 145 150 155 GCT CTG CAG AGT TCT CCC GGA TCA CAC AAC CAG AAT TAG 517 Ala Leu gin Ser Ser for Gly Ser HLS same time gin same time

160

C2) SEO ID NO. 26 »

C (i) SEQUENCE CHARACTERISTICS.

CA) 580 base pairs

CB) TYPE »nucleic acid

CC) NUMBER OF CHAINS, one

CD) TOPOLOGY. straight

C11) MOLECULA TYPE. cDNA

Clx) CHARACTERISTICS.

C A) NAME / KEY. CDS

129 (B) LOCATION. 1..580 (xi) SEQUENCE DESCRIPTION. SEO ID NO. 26th

CAA GTA CTG ATG GCT GCT GCA AGC TTT GGC GAA CGT AAA ATC CCT AAG 48 gin wall Leu Met Ala Ala Ala Ser Phe Gly Glu Arg Lys Ile for Lys 1 5 10 15

GGA AAT GGG CCT TAT TCC GTT GGT TGT ACA 96 Gly same time Gly for 20 Tyr Ser wall Gly Cys Thr Asp Leu Met Phe 30 Asp Tyr ACT AAA AAG GGC ACC TTC TTG CGT TTA TAT TAT CCA TCC CAA GAT GAT 144 Thr Lys Lys 35 Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser 45 gin Asp Asp GAT CGC CTT GAC ACC CTT TGG ATC CCA AAT AAG GAG TAT TTT TGG GGT 192 Asp Arg Leu Asp Thr Leu Trp 55 For Pro Asn Lys Glu Tyr Phe Trp Gly CTT AGC AAG TAT CTT GGA AAA CAC TGG CTT ATG GGC AAC ATT TTG AGT 240 Leu 65 Ser Lys Tyr Leu Gly Lys His Trp Leu Met Gly same time Ile Leu Ser 80 TTA CTC TTT GGT TCA GTG ACA ACT CCT GCA AAC TGG AAT TCC CCT CTG 288 Leu Leu Phe Gly Ser Val Thr Thr Pro Ala Asn Trp same time Ser Pro Leu 95 AGG CCT GGT GAA AAA TAC CCA CTT GTT GTT TTT TCT CAT GGT CTT GGA 336 Arg for Gly Glu 100 Lys Tyr for Leu Val Val Phe Ser His Gly 110 Leu Gly GCA TTC AGG ACA ATT TAT TCT GCT ATT GGC ATT GAC CTG GCA TCT CAT 384 Ala Phe Arg 115 Thr ile Tyr Ser Ala il Gly 120 Asp Leu 125 Ala Ser His GGG TTT ATA GTT GCT GCT GTA GAA CAC AGA GAT AGA TCT GCA TCT GCA 432 Gly Phe 130 Ile wall Ala Ala wall 135 Glu His Arg Asp Arg Ser Ala Ser Ala ACT TAC TAT TTC AAG AAC CAA TCT GCT GCA GAA ATA GGG AAA AAG TCT 480 Thr 145 Tyr Tyr Phe Lys Asn gin Ser Ala Ala Glues 155 Gly Lys Lys Ser 160 TGG CTC TAC CTT AGA ACC CTG AAA GAA GAG GAG GAG ATA CAT ATA CGA · 528 Trp Leu Tyr Leu Arg Thr Leu Lys Glu Glu Glu Glu Ile His Arg 175 AAT AAG CAG GTA CGA CAA AGA GCA AAA GAA TGT TCC CAA GCT CTC AGT 576 same time Lys gin wall Arg Gin Arg Ala Lys Glu Cys Ser gin Ala Leu Ser

180 185 190

CTG A 580

Leu (2) INFORMATION ABOUT SEO ID NO. 27th

Ci) SEQUENCE CHARACTERISTICS.

130 (A) LENGTH: 5 amino acids CB) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

Gly Xaa Gly Xaa Ser

5 (2) INFORMATION ON SEO ID NO. 28:

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 41 base pairs (B) TYPE «nucleic acid (C) NUMBER OF STRINGS, one (D) TOPOLOGY. linear (ii) MOLECULA TYPE. DNA (xi) SEQUENCE DESCRIPTION: SEO ID NO: 28.

TATTCTAGAA TTATGATACA AGTATTAATG GCTGCTGCAA G ⁴¹ (2) INFORMATION ON SEO ID NO. 29th

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 32 base pairs (B) TYPE «nucleic acid (C) NUMBER OF STRINGS, one (D) TOPOLOGY. linear (ii) MOLECULA TYPE. DNA (xi) SEQUENCE DESCRIPTION: SEO ID NO: 29.

ATTGATATCC TAATTGTATT TCTCTATTCC TG 32 (2) SEO ID NO: 30.

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 1335 base pairs (B) TYPE. nucleic acid (C) NUMBER OF STRINGS, one (D) TOPOLOGY: linear (ii) MOLECULAR TYPE. cDNA (xi) DESCRIPTION OF THE SEQUENCE. SEQ ID NO. 30:

ATGGTACCCC CAAAGCTGCA CGTCCTGTTT TGTCTGTGTG GATGTCTCGC CGTCGTGTAC 60

CCCTTCGATT GGCAGTATAT CAACCCCGTG GCTCACATGA AGAGCAGCGC CTGGGTGAAT 120

AAGATCCAGG TGCTCATGGC CGCACCAAGC TTCGGTCAGA CCAAGATTCC TAGAGGCAAC 180

GGCCCCTACA GCGTGGGCTG CACCGATCTG ATGTTCGACC ATACCAACAA AGGAACTTTT 240

131

CTGAGACTGT ACTACCCCAG CCAGGACAAC GACAGACTGG ATACTCTGTG GATCCCAAAT 300 AAAGAATATT TTTGGGGTCT TAGCAAATTT CTTGGAACAC ACTGGCTTAT GGGCAACATT 360 TTGAGGTTAC TCTTTGGTTC AATGACAACT CCTGCAAACT GGAATTCCCC TCTGAGGCCT 420 GGTGAAAAAT ATCCACTTGT TGTTTTTTCT CATGGTCTTG GGGCATTCAG GACACTTTAT 480 TCTGCTATTG GCATTGACCT GGCATCTCAT GGGTTTATAG TTGCTGCTGT AGAACACAGA 540 GATAGATCTG CATCTGCAAC TTACTATTTC AAGGACCAAT CTGCTGCAGA AATAGGGGAC 600 AAGTCTTGGC TCTACCTTAG AACCCTGAAA CAAGAGGAGG AGACACATAT ACGAAATGAG 660 CAGGTACGGC AAAGAGCAAA AGAATGTTCC CAAGCTCTCA GTCTGATTCT TGACATTGAT 720 CATGGAAAGC CAGTGAAGAA TGCATTAGAT TTAAAGTTTG ATATGGAACA ACTGAAGGAC 780 TCTATTGATA GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AACGGTTATT 840 CAGACTCTTA GTGAAGATCA GAGATTCAGA TGTGGTATTG CCCTGGATGC ATGGATGTTT 900 CCACTGGGTG ATGAAGTATA TTCCAGAATT CCTCAGCCCC TCTTTTTTAT CAACTCTGAA 960 TATTTCCÄAT ATCCTGCTAA TATCATAAAA ATGAAAAAAT GCTACTCACC TGATAAAGAA 1020 AGAAAGATGA TTACAATCAG GGGTTCAGTC CACCAGAATT TTGCTGACTT CACTTTTGCA 1080 ACTGGCAAAA TAATTGGACA CATGCTCAAA TTAAAGGGAG ACATAGATTC AAATGTAGCT 1140 ATTGATCTTA GCAACAAAGC TTCATTAGCA TTCTTACAAA AGCATTTAGG ACTTCATAAA 1200 GATTTTGATC AGTGGGACTG CTTGATTGAA GGAGATGATG AGAATCTTAT TCCAGGGACC 1260 AACATTAACA CAACCAATCA ACACATCATG TTACAGAACT CTTCAGGAAT AGAGAAATAC 1320

AATTAGGATT CTAGA

Claims (13)

PATENT CLAIMS Purified and isolated human plasma platelet-acetyl hydrolase polypeptide fragment of platelet-activating factor acetylhydrolase (PAF-AH) lacking the first twelve N-terminal amino acids of the human PAF-AH amino acid sequence described in SEQ ID NO: 8 . The PAF-AH polypeptide fragment of claim 1 selected from the group consisting of a) a polypeptide. having LLET _4d, SEQ ID NO: 8 as the initiating N-terminal amino acid b) a polypeptide having Ala _4A N-terminal amino acid c) a polypeptide having Ala ^ ₇ N-terminal amino acid. SEO ID NO> 8 as an initiator SEO ID NO: 8 as an initiator The PAF-AH polypeptide fragment according to any one of claims 1 or 2, which lacks up to 30 C-terminal amino acids of the SEO ID NO: 8 amino acid sequence. A PAF-AH polypeptide fragment according to any one of claims 1 or 2, having as its C-terminal residue a residue selected from the group consisting of: (a) Ile429 (b) Leu ₄₃₁ ; c) Asn _4. * i A variant of the PAF-AH polypeptide fragment according to claim 1 having an amino acid substitution in SEQ ID NO: 8 and selected from the group consisting of. 4¾ a) WITH 108 A b) WITH 273 A c) D 286 A d) D 286 N e) D 296 A f) D 304 A g) D 338 A h) H 351 A i) H 395 A j) H 399 A k) C 67 S 1) C 229 WITH m) C 291 WITH n) C 334 S a about) C 407 WITH. 6th Human PAF-AH polypeptide variation SEQ ID NO: 8 replaced the amino acid groups composed of: a) D 286 A b) D 286 N c) D 304 A. 7th An isolated polynucleotide encoding a fragment having in the sequence which is selected from a polypeptide fragment PAF-AH, modified or modified according to claims 1 or 6. according to any of the 8. An isolated polynucleotide encoding a human or altered fragment having the fly ₄ &apos; SEQ ID NQ &gt; residue and Ile ₄₂ ? or Asn ₄₄ as the C-terminal residue of the PAF-AH 8 fragment as the N-terminal The polynucleotide of any one of claims 7 or 8. A DNA vector comprising the DNA of claim 9. The host cell stably transformed or transfected with the DNA of claim 9, which allows the expression of the PAF-AH polypeptide fragment, variant, or altered fragment in the above host cell. A method of producing a PAF-AH polypeptide fragment, variant or modified fragment of plasma PAF-AH, which comprises growing host cells according to claim 11 in a suitable nutrient medium and isolating the aforementioned PAF-AH fragment, variant or modified fragment from the aforementioned cells. or their growth media. A PAF-AH polypeptide fragment, variant or modified fragment produced by the method of claim 12. A pharmaceutical composition comprising a PAF-AH fragment, variant or modified fragment according to any one of claims 1, 6 or 13 and a pharmaceutically acceptable diluent, adjuvant or carrier. 15. A method of treating a mammal easily susceptible to or suffering from pathological conditions caused by PAF. The treatment comprises administering the pharmaceutical compositions of claim 14 in amounts sufficient to complement the activity of PAF-AH and inactivating the pathological effects of PAF on the above mammal. The method of claim 15 wherein the above pathological condition is pleuritis. asthma, rhinitis, necrosis eriterocolltis. acute respiratory distress syndrome, acute pancreatitis or neurological diseases associated with HIV infection.