Patent Application: US-21828105-A

Abstract:
the present invention is directed to methods for the identification and uses of receptors that interact with anti - inflammatory compounds derived from eicosapentaenoic acid . the receptors are of the g - protein coupled receptor family , and are useful to screen candidate substances for anti - inflammatory activity , especially substances that are analogs of epa . such analogs are termed “ resolvins ”; and are typically di - and tri - hydroxy epa analogs . one analog herein denoted resolvin e1 was identified in humans and prepared by total synthesis . in nanomolar range resolvin e1 reduces dermal inflammation , peritonitis , dendritic cells migration and il - 12 production . also described herein is a receptor denoted reso er1 that interacts with resolvin e1 to attenuate cytokine induced activation of inflammatory pathways mediated by transcription factor - kb . treatment of dcs with small - interfering rna specific for resoe1 eliminated the ligand &# 39 ; s ability to regulate il - 12 . assays of anti - inflammatory activity based on these discoveries are also described .

Description:
in the detailed description that follows , citation is made to various references that may aid one of skill in the art to understand or practice the invention in its fullest scope . each such reference is incorporated herein by reference , to the extent the teaching of those references do not conflict with the teachings provided herein . clinical assessment of dietary supplementation with omega - 3 polyunsaturated fatty acids ( w - 3 pufa ) indicate their beneficial impact in certain human diseases particularly those in which inflammation is suspected as a key component in pathogenesis ( 1 - 3 ). their molecular bases of action in reducing disease and local inflammation is important and of interest given the heightened awareness that inflammation and resolution is a major mechanisms in many diseases including cardiovascular disease , arthritis , alzheimer &# 39 ; s disease , asthma and periodontitis ( 4 , 5 ). w - 3 pufas are widely held to act via several possible mechanisms , such as preventing conversion of arachidonate to proinflammatory eicosanoids , or serving as an alternative substrate producing less potent products ( 1 ). of interest , fish leukocytes rich in w - 3 generate mediators from eicosapentaenoic acid ( epa ) that play signaling roles ( 6 ). however , the pathophysiological role of leukotriene and prostanoid - like compounds from epa remains uncertain in humans as many of these molecules &# 39 ; role ( s ) are unknown . recently , the present inventors discovered a novel family of aspirin - triggered bioactive lipids biosynthesized during the spontaneous resolution phase of acute inflammation in vivo . this family of bioactive lipids have been termed the resolvins ( resolution - phase interaction products ), are described in more detail in u . s . patent application ser . no . 10 / 639 , 714 , filed aug . 12 , 2003 , entitled “ resolvins : biotemplates for novel therapeutic interventions ” and in pct application no . pct / us03 / 25336 , filed on aug . 12 , 2003 and entitled the same , which are incorporated herein by reference in their entirety . the resolvins are potent autacoids , which now can provide molecular means that underlie w - 3 pufa &# 39 ; s protective actions ( 7 , 8 ). at local sites , aspirin treatment enables epa conversion to the novel 18r series of oxygenated products that carry potent counterregulatory signals . one of the main compounds of this 18r series , namely 5 , 12 , 18r - trihydroxyeicosapentaenoic acid ( termed resolvin e1 ) can arise via cell - cell interactions in murine inflammatory exudates , also exemplified with human vascular endothelium carrying aspirin - acetylated cyclooxygenase ( cox )- 2 and leukocytes possessing 5 - lipoxygenase ( lo ) ( 7 ). here , resolvin e1 was generated in healthy human volunteers given epa and aspirin , plasma values ranging 0 . 1 to 0 . 4 ng / ml for 6 donors using liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) ( fig1 ). formation is consistent with the scheme that endothelial cells expressing cox - 2 treated with aspirin transform vascular epa and release 18r - hepe . when leukocyte and endothelial cell interact within the vasculature , 18r - hepe is rapidly converted to resolvin e1 via transcellular biosynthesis ( fig2 a ). to assign the complete stereochemistry of the main 18r series resolvin e1 and establish its biological activities , biogenic resolvin e1 was prepared ( 7 ), and matched with synthetic resolvin e1 ( 5s , 12r , 18r - trihydroxy - 6z , 8e , 10e , 14z , 16e - eicosapentaenoic acid ) having complete stereochemistry that was prepared by total organic synthesis from isomerically pure precursors ( fig2 b ). a geometric isomer carrying all - trans conjugation at both carbon 6 and 14 positions in native resolvin e1 was also prepared by organic synthesis to establish chromatographic properties as described in the supplementary examples of this description . since resolvin e1 is produced in subnanogram amounts in vivo , both synthetic and biogenic materials were prepared for matching their physical properties using uv spectroscopy , lc - ms / ms , gc - ms , and importantly to compare biological activities . the matching synthetic compound eluted beneath a single peak in hplc with uv absorbance maximum 271 nm and 234 nm , indicative of conjugated triene and diene in the molecule ( fig2 c ). ms / ms fragmentation ions were essentially identical with the biogenic material namely a parent ion at m / z 349 =[ m - h ]- and diagnostic product ions at m / z = 291 and 195 ( fig2 d ). results of physical matching studies are summarized in the supplementary examples . administration of as little as 100 ng / mouse of synthetic resolvin e1 stopped leukocyte infiltration into inflammatory loci by 50 - 70 % in tnf - a induced dorsal air pouch , which proved to be as potent as the biogenic material ( fig2 e ). for comparison in this model , local administration of dexamethasone ( 10 mg / mouse ) gives 60 % inhibition ( fig2 e ) and aspirin ( 1 . 0 mg / mouse ) gives 70 % inhibition of leukocyte recruitment ( 9 ), indicating that resolvin e1 at 100 ng / mouse is orders of magnitude more potent than dexamethasone or aspirin in stopping leukocyte infiltration . also indomethacin ( 100 ng / mouse ) gave 25 % inhibition and resolvin e1 ( 100 ng / mouse ) gave 50 - 60 % inhibition of leukocyte recruitment in zymosan - induced peritonitis as described in the supplementary examples . the18s isomer gave essentially equivalent activity as native resolvin e1 containing 18r , whereas the 6 - trans , 14 - trans isomer showed reduced potency (˜ 70 %) for reducing leukocyte infiltration in zymosan - induced peritonitis . based on matching of physical and biological properties , the 18r series resolvin e1 , a potent anti - inflammatory lipid mediator , was assigned the complete structure 5s , 12r , 18r - trihydroxy - 6z , 8e , 10e , 14z , 16e - eicosapentaenoic acid . the murine airpouch is widely used to assess dermal inflammation and arthritis ( fig2 e ). the murine airpouch is characterized by a cavity and a lining composed of both fibroblast - like and macrophage - like cells ( 10 ). intrapouch application of tnf - a evokes leukocyte infiltration by stimulating local release of chemokines and chemoattractants that are often produced by fibroblasts and phagocytes via regulation of nuclear factor ( nf )- kb transcription factors ( 11 ). systemic administration of resolvin e1 dramatically attenuated leukocyte recruitment ( fig2 e ), meaning that receptor target for resolvin e1 was expressed in those cells which counterregulates tnf - a induced nf - kb activation . resolvin e1 and lipoxin ( lx ) a4 have different structures , are formed via different biosynthetic pathways and precursors ( epa vs arachidonate ), yet they appear to share redundant beneficial properties that dampen excessive leukocyte recruitment ( 12 ), hence the present invention is based , at least in part , on recognizing that resolvin e1 receptors share similar structural features to lo - derived eicosanoid receptors such as lxa4 receptor ( alx ) and leukotriene b4 receptor ( blt ) ( 13 ). fig3 a shows a branch of the phylogenetic tree of human alx with closely related g - protein coupled receptors ( gpcrs ). expression plasmids of each gpcr were introduced into hek293 cells and the ability of resolvin e1 to inhibit tnf - a stimulated nf - kb activation was monitored by co - transfection with nf - kb response element - luciferase reporter plasmid . this permitted analysis of the activation of the relevant post ligand - receptor “ stop ” signaling for downregulation of nf - kb activation as for example demonstrated with alx - transfected cells and its ligands ( 14 ). among those screened ( fig3 b ), a putative orphan receptor denoted earlier as dez / chemr23 ( 15 ) was specifically activated by resolvin e1 and at 10 nm inhibited nf - kb activation ( fig3 b ). in view of these results , the dez receptor is herein termed “ reso er1 .” reso er1 shares 36 . 4 % identity with alx in deduced amino acid sequences and of note contains a highly conserved domain within its second intracellular loop ( 75 %) and seventh transmembrane region ( 69 . 5 %) ( fig3 c ). resolvin e1 gave concentration dependent inhibition of tnf - a induced nf - kb activation with an ec50 of ˜ 1 . 0 nm in reso er1 transfected cells but not in mock transfected cells ( fig3 d ). in this system , 1 mm aspirin , a known inhibitor of nf - kb at high concentrations namely millimolar range ( 16 ), gave non - receptor dependent inhibition of 26 . 2 + 4 . 9 % for reso er1 transfected cells . neither epa nor 18r - hepe at 100 nm , both metabolic precursors of resolvin e1 , inhibited nf - kb in reso er1 transfected cells ( fig3 e ). the isomer 6 - trans , 14 - trans at 100 nm showed reduced potency for nf - kb inhibition that was essentially the same magnitude reduction in vivo . the functional interactions between reso er1 and g proteins using ligand - dependent binding of [ 35s ]- gtpgs , a hydrolysis resistant gtp analog were also examined . specific [ 35s ]- gtpgs binding in isolated membranes obtained from cells expressing reso er1 increased selectively with resolvin e1 in a concentration - dependent manner ( fig3 f ). these results indicate that resolvin e1 transmits signal as a selective agonist via reso er1 and counterregulates tnf - a stimulated nf - kb activation . tissue distribution of human reso er1 was determined with dot blots containing mrnas from human tissues that showed expression of reso er1 in several tissues such as cardiovascular system , brain , kidney , gastrointestinal tissues and myeloid tissues as is illustrated in fig8 . also , a murine receptor counterpart was found in developing bone using in situ hybridization ( 17 ). among the human peripheral blood leukocytes , reso er1 was abundantly expressed in monocytes , with lower amounts in neutrophils and t lymphocytes ( fig4 a ), findings consistent with the observation that this receptor is expressed in antigen - presenting cells ( apc ) such as macrophage and dendritic cells ( 15 ). both monocyte reso er1 and cox - 2 transcripts were highly upregulated by treatment with inflammatory cytokines such as tnf - a and ifn - g , and reso er1 showed delayed induction to that of cox - 2 ( fig4 b ). resolvin e1 increased phosphoryation of extracellular signal - regulated kinase ( erk ) mitogen - activated protein ( map ) kinase both in peripheral blood monocytes and hek293 - reso er1 cells , but not in mock - transfected hek293 cells ( fig4 c ). in addition , treatment of hek293 - reso er1 with pertussis toxin ( ptx ) abolished resolvin e1 dependent erk activation and nf - kb inhibition , indicating coupling to gai / o - protein for the signal transduction ( fig4 d ). as shown in fig9 , resolvin e1 did not evoke a calcium mobilization with either human peripheral blood monocytes or hek - reso er1 stable transformants , and at 100 nm did not inhibit calcium mobilization by 100 nm ltb4 ( data not shown ). these results demonstrate that resolvin e1 activates reso er1 , evokes erk phosphorylation and regulates gene expression , through gi / o - protein . given expression of human reso er1 in apcs , and since apc function is influenced by dietary w - 3 pufa supplementation ( 18 ), the activity of resolvin e1 on apc function was examined using a microbial pathogen model . injection of pathogen extract derived from toxoplasma gondii ( stag ) causes activation of splenic dendritic cells ( dcs ) to mobilize to t cell enriched areas where they produce high amounts of il - 12 ( 19 ). addition of increasing concentrations of resolvin e1 to isolated mouse splenic cd11c + dcs markedly inhibited il - 12p40 production by stag within the nanomolar range ( fig5 a ). sirna experiments were carried out to reduce reso er1 in splenic dcs . the mouse resoer receptor ( 17 ), which shares 80 . 3 % identity with human reso er1 , was also present in splenic dcs . resolvin el &# 39 ; s action in regulating il - 12 production from dcs was eliminated by treatment with a sirna specific for the mouse reso er1 ( fig5 b ). it was confirmed that this sirna treatment dramatically reduced reso er1 mrna expression in dcs ( fig5 b , inset ) and cell - surface expression of recombinant reso er1 in hek293 cells as described in the supplementary example . these results confirm that resolvin e1 &# 39 ; s anti - inflammatory action is mediated via reso er1 . in vivo treatment with resolvin e1 also blocked il - 12 production ( fig5 c ) as well as dc migration into t cell areas of the spleen ( fig5 d - g ). acute inflammation is a protective host response to foreign challenge or tissue injury that could lead to , if unopposed , loss of tissue structure as well as function . in many chronic disorders , prolonged and unresolved inflammation is believed to contribute to pathogenesis ( 4 ). resolution of inflammation is an active process controlled by endogenous mediators that can counterregulate pro - inflammatory gene expression and cell trafficking , as well as stimulate inflammatory cell clearance ( 11 , 20 ). the observation that cytokines upregulated reso er1 as well as cox - 2 in monocytes indicates that in scenarios where cox - 2 is induced during inflammation , monocytes as well as endothelial cells treated with aspirin can also potentially convert w - 3 epa into resolvin e1 in concert with pmn ( 7 ), that may serve an autocrine and / or paracrine message to terminate further nf - kb activation and cytokine production in a temporal and spatially regulated fashion . resolvin e1 is generated in healthy volunteers taking epa and aspirin ( fig1 ). these results are consistent with the notion that cox - 2 is also constitutively expressed in healthy vasculature in vivo ( 21 , 22 ). also , the results presented here support the notion that aspirin , in addition to its well - appreciated action to inhibit prostanoid formation , can exert its beneficial actions , in part , via epa catabolic synthesis of 18r series resolvin e1 that in turn interacts with receptors such as reso er1 to dampen further proinflammatory processes . it is likely that in vivo , resolvin e1 can also interact with additional receptors , in addition to reso er1 . indeed , resolvin e1 can , at higher concentrations (˜ 0 . 5 mm ), interact with recombinant ltb4 receptor blt1 ( 7 ) and could potentially antagonize blt1 and blt2 receptors ( 23 ) in vivo . endogenous chemically redundant anti - inflammatory lipid autacoids act with high affinities ( nm range ) and stereoselectivity on structurally related receptors as does aspirin triggered lipoxin a4 generated from arachidonic acid ( 24 ) to enhance resolution by “ stopping ” pmn recruitment and il - 12 production from apc . together , the present findings provide an endogenous agonist driven and hst - protective molecular mechanism that can underlie some of the beneficial actions of co - 3 epa observed in many clinical situations ( 1 - 3 ) as well as identify novel components in endogenous anti - inflammation / resolution , exemplified by resolvin e1 and one of its receptors reso er1 that are of interest as new checkpoint regulators ( 20 ) in the pathogenesis of a wide range of human diseases . studies reported here were performed using protocols approved by harvard medical area standing committee on animals and human subjects in accordance with the brigham and women &# 39 ; s human research committee . human plasma samples were collected at 4 hours after oral administration of fish oil supplement ( fish oil concentrate , walgreens ) containing epa ( 1 g ) and dha ( 0 . 7 g ) followed by aspirin ( 160 mg ) at 3 h in six healthy volunteers . plasma samples were extracted by c18 solid phase extraction with d4 - ltb4 ( cascade ) as internal standard for lc - ms / ms analysis ( 7 ) using a finnigan lcq liquid chlomatography ion trap tandem mass spectrometer equipped with a luna c18 - 2 ( 100 × 2 mm × 5 mm ) column and uv diode array detector using mobile phase ( methanol : water : acetate at 65 : 35 : 0 . 01 ) from 0 to 8 min , ramped to methanol 8 to 30 min , with a 0 . 2 ml / min flow rate . dorsal air pouches were raised on male fvb mice ( 6 - 8 wk ) by injecting 3 ml of sterile air subcutaneously on days 0 and 3 . on day 6 , 100 ng / mouse of compounds were injected into tail vein . inflammation in the air - pouch was induced by intrapouch injection of mouse recombinant tnf - a ( 100 ng / pouch ), and pouch lavages were collected at 4 h and cells were enumerated . for peritonitis , 100 ng / mouse of resolvin e1 or related structures was injected into tail vein and followed by 1 ml zymosan a ( 1 mg / ml ) into - the peritoneum . peritoneal lavages were collected at 2 h and cells were enumerated . gpcr cdnas were cloned by rt - pcr using specific primers designed according to the genbank ™ database ; human fpr ( p21462 ), alx ( p25090 ), fprl2 ( p25089 ), gpr1 ( a55733 ), gpr32 ( 075388 ), dez ( q99788 ), crth2 ( q9y5y4 ), c3ar ( q16581 ), c5ar ( p21730 ), blt1 ( q15722 ). mouse reso er1 ( u79525 ). the phylogenetic tree was constructed using the “ all program ” at the computational biochemistry server at ethz ( http :// cbrg . inf . ethz . ch / server / allall . html ). hek293 cells ( 1 . 0 ′ 105 cells ) were transiently transfected with 50 ng pnf - kb - luciferase ( stratagene ), 500 ng of either pcdna3 or pcdna3 - gpcrs and the internal standard prl - tk ( promega ) using superfect transfection reagent ( qiagen ). after 24 h , cells were exposed to the test compounds for 30 min , stimulated with recombinant human tnf - a ( 1 . 0 ng / ml , bd pharmingen ) for 5 h . luciferase activity was measured by the dual - luciferase reporter assay system ( promega ). basal induction of luciferase activity by tnf - a was & gt ; 150 - fold in this system . efficient expression of gpcrs to the cell surface was observed by immunostaining using ha - tagged gpcr constructs . for ptx treatment , hek293 cells were treated with ptx ( 200 ng / ml ) for 24 h before stimulation . hek293 cells stably expressing human reso er1 were homogenized in ice - cold ted buffer ( 20 mm tris - hcl ph7 . 5 / 1 mm edta / 5 mm mgcl2 / 1 mm dtt ). membrane fraction ( 10 mg ) was incubated in 400 ml of gtp - binding buffer ( 50 mm hepes , ph7 . 5 / 100 mm nacl / 1 mm edta / 5 mm mgcl2 / 1 mm dtt ) containing 0 . 1 nm [ 35s ]- gtpgs (& gt ; 1000 ci / mmol , amersham ) and 10 mm gdp for 30 min at 30 ° c . the bound and unbound [ 35s ]- gtpgs was separated by rapid filtration through gf / c filters , and counted by liquid scintillation . nonspecific binding was determined in the presence of 50 mm unlabeled gtpgs . basal [ 35s ]- gtp - gs binding was 81 . 6 + 1 . 5 cpm / mg protein . hybridization to mte array ( clontech ) was carried out using 1 . 1 kb . p . fragment encoding open reading frame of reso er1 following the manufacturer &# 39 ; s protocol . primers used in amplifications are 5 ′- atgagaatggaggatgaaga - 3 ′ and 5 ′- tcaaagcatgccggtctcc - 3 ′ for human reso er1 , 5 ′- atggagtacgacgctta caa - 3 ′ and 5 ′- tcagagggtactggtctccttct - 3 ′ for mouse reso er1 , 5 ′- gctgactatggctacaaaagctgg - 3 ′ and 5 ′- atgctcagggacttgaggagg gta - 3 ′ for cox - 2 , 5 ′- gaccacagtccatgacatcact - 3 ′ and 5 ′- tccaccaccc tgttgctgtag - 3 ′ for glyceraldehyde - 3 - phosphate dehydrogenase ( gapdh ). amplified products were confirmed by direct sequencing . map kinase activation in monocytes and hek293 cells after treatment with 100 nm of each compound was determined . after incubations , cells were lysed in cold lysis buffer ( 50 mm tris - hcl , ph 8 . 0 , 150 mm nacl , 0 . 5 mm edta , 1 . 0 % np - 40 , 0 . 5 % sodium deoxycolate , 10 mm naf , 10 mm sodium pyrophosphate ) containing protease inhibitor cocktail ( sigma ). 40 mg of protein was separated on sds - page and immunoblot was performed using anti - phospho - p44 / 42 map kinase ( cell signaling ) and anti - erk ( santa cruz ) antibodies . for ptx treatment , hek - reso er1 cells were incubated with or without ptx ( 200 ng / ml ) for 24 h at 37 ° c . and erk activation was monitored by addition of resolvin e1 ( 100 nm ) for 5 min . experiments were performed essentially as in ( 19 ). stag was prepared from sonicated t . gondii ( rh strain ) tachzoytes . for isolated dc experiments , 70 - 85 % cd11c positive dcs were isolated from spleen . cd11c + dc suspensions ( 1 . 0 × 106 cells / ml ) were spread into 96 - well plates and incubated for 24 h with resolvin e1 before the addition of stag ( 5 mg / ml ). after overnight culture , supernatants were collected and el - 12p40 was measured with a sandwich elisa . for in vivo treatments , c57bl / 6 mice ( n = 3 per group ) were injected intravenously with 100 ng resolvin e1 . after 18 h the animals were challenged intraperitoneally with pbs ( 0 . 2 ml / mouse ), stag ( 5 mg / ml ) and sacrificed after an additional 6 h . cd11c + dcs were isolated from spleen and il12 - p40 secretion was measured at 24 h . for dc migration , splenic frozen section from mice treated as above but given 10 mg of resolvin e1 or vehicle were stained for cd11c and counterstained with hematoxylin . chemically synthesized sirna for mouse reso er1 ( 5 ′- aacacugugugguuugucaacdtdt - 3 ′) and non - specific control ix sirna ( 5 ′- auuguaugcgaucgcagacuu - 3 ′) were from dharmacon research . spleen cells ( 1 . 0 × 106 cells / ml ) were transfected using chariot ( active motif ) following manufacturers &# 39 ; instructions . briefly , sirna was mixed with chariot transfection reagent and incubated at room temperature for 30 minutes . spleen cells were plated in serum - free rpmi medium , 200 ng sirna / chariot solution was added and incubated for 2 h at 37 ° c ., followed by adding 10 % fcs rpmi to the cultures . to assure effective inhibition of gene expression , cells were further incubated for 30 h at 37 ° c . before stag stimulation . fig6 illustrates results obtained from chromatographic analysis of synthetic and biogenic resolving e1 . for note ( a ) lc - ms / ms was performed with finnigan lcq liquid chromatography ion trap tandem mass spectrometer equipped with a luna c18 - 2 ( 100 × 2 mm × 5 mm ) column and a uv diode array detector using isocratic mobile phase ( meoh : h2o : acoh at 65 : 35 : 0 . 01 ( vol : vol : vol ), with a 0 . 2 ml / min flow rate ). for note ( b ) gc - ms was performed with a hewlett - packard 6890 equipped with a hp 5973 mass detector . a hp5ms cross - linked 5 % me siloxane column ( 30 m × 0 . 25 mm × 0 . 25 mm ) was employed with a temperature program . the helium flow rate was 1 . 0 ml / min and the initial temperature was 150 ° c ., followed by 230 ° c . ( 2 min ), and 280 ° c . ( 10 min ). trimethylsilyl derivatives were prepared with each compound following treatment with diazomethane . for note ( c ) spectra were recorded in methanol . 18r - hepe ( 100 ng ), resolvin e1 ( 100 ng ), or indomethacin ( 100 ng ) was injected intravenously into mouse tails followed by zymosan a into the peritoneum . mice were sacrificed , and peritoneal lavages were collected ( 2 h ) and cells enumerated ( n = 3 ). effect of pertussis toxin ( ptx ) on resolvin e1 - induced erk activation ( a ) and nf - kb inhibition ( b ). fig4 d illustrate effects of ptx on resolvin e1 induced activation and nf - kb inhibition . in -( a ) hek - hreso er1 cells were incubated with or without ptx ( 200 ng / ml ) for 24 h at 37 c and erk activation was monitored by addition of resolvin e1 ( 100 nm ) for 5 min . in ( b ) hek293 cells were transiently transfected with pcdna - hreso er1 , pnf - kb - luciferase and prl - tk . after 24 h with or without ptx ( 200 ng / ml ), cells were exposed to resolvin e1 ( 50 nm ) for 30 min , stimulated with tnf - a ( 1 . 0 ng / ml ) for 5 h , and luciferase activity was measured . hek293 cells ( 5 . 0 × 105 cells ) were transiently co - transfected with haemagglutinin ( ha )- tagged mouse reso er1 expression plasmid ( phm6 - mreso er1 , 0 . 5 mg ) and sirna ( 1 . 5 mg ) using superfect ( qiagen ). after 48 h , cells were harvested and stained with anti - ha monoclonal antibody 3f10 and fitc - anti - rat igg ( roche ) and analyzed for cell - surface expression of ha - mreso er1 by flow cytometry . 1 . de caterina , r ., endres , s ., kristensen , s . d ., schmidt , e . b . eds . n - 3 fatty acids and vascular disease . bi & amp ; 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