Patent Application: US-201013513465-A

Abstract:
the present disclosure provides in vitro methods of diagnosing a plasmodium infection in a subject . the methods include measuring the serum concentration of at least one secreted phospholipase a 2 selected from the group consisting of giif , gv and gx spla 2 s , in a blood sample from the subject . the disclosure also relates to a kit for carrying out the disclosed methods and pharmaceutical compositions comprising a recombinant mammalian giif , gv or gx spla 2 , or a combination thereof .

Description:
evaluation of anti - plasmodium activities of the human spla 2 s purified recombinant human spla 2 s were prepared as described in singer et al . ( 2002 ). the accession number in the genbank database of the mrna encoding the different spla 2 s are given below : apis mellifera ( bee ) venom spla 2 , naja mossambica mossambica venom spla 2 and high quality grade biochemical reagents used throughout this work were purchased from sigma ( st quentin fallavier , france ). plasmion was from fresenius kabi france ( sèvres , france ). albumax ii ® was from invitrogen ( cergy pontoise , france ). nefa - c kit and phospholipids b kit used for quantitative determination of non - esterified fatty acids ( nefas ) and phospholipids ( pls ) respectively were from wako chemicals gmbh ( neuss , germany ). 3h - hypoxanthine ( 37 mbq / ml ) was from ge healthcare ( france ). diff - quick ® stain was purchased from medion diagnostics gmbh ( düdingen , switzerland ). ly329722 ( sodium [ 3 - aminooxalyl - 1 - benzyl - 2 - ethyl - 6 - methyl - 1h - indol - 4 - yloxy ]- acetic acid ) ( smart et al ., 2006 ). the chloroquine - resistant p . falciparum fcb1 / colombian strain was used . under the following culture conditions , the in vitro life cycle of the fcb1 strain was 48 hours . cultures were grown in complete medium consisting of rpmi 1640 ( life technologies , inc .) supplemented with 11 mm glucose , 27 . 5 mm nahco3 , 100 ui / ml penicillin , 100 μg / ml streptomycin , adjusted to ph 7 . 4 before addition of heat - inactivated human serum ( a + blood group , 8 % final ) or albumax ii ® ( 0 . 5 % final ), according to the procedure of trager and jensen ( 1976 ). parasites were routinely grown at 37 ° c . in human a + rbcs ( red blood cells ) at a 2 % haematocrit and 2 - 6 % parasitemia , in a 3 % co2 , 6 % o2 and 91 % n2 atmosphere . semi - synchronized cultures were established by sorbitol ( lambros ) treatment . highly synchronized cultures ( 4 hour - window synchronization ) were obtained by successive plamion ( pasvol ) and sorbitol treatments . parasitaemia and stage distribution were determined by optical examination of diff - quick stained culture smears . parasitaemia ( expressed in percent ) is hundred times the number of parasitized erythrocytes divided by the total number of erythrocytes . recombinant spla 2 s were routinely checked for enzymatic activity through hydrolysis of autoclaved 3 h - oleic acid - labelled e . coli ( franson et al ., 1974 ). dried preparations of the purified recombinant human spla 2 s were re - suspended at high concentration ( usually 50 μm ) in rpmi 0 . 05 % bsa , and then tested individually for their capacity to inhibit the in vitro intra - erythrocytic development of p . falciparum . dose - response assays based on 3 h - hypoxanthine incorporation by growing parasites were performed as in guillaume et al . ( 2004 ). radioactivity was measured with a 1450 microbeta counter ( wallac , perkin elmer ). percentage of growth inhibition was calculated from the parasite - associated radioactivity compared with control without spla 2 . values for the ic 50 were determined from dose - response curves . purification , oxidation and enzymatic hydrolysis procedures were performed under sterile conditions . non - fasted human plasma was aliquoted and frozen at − 20 ° c . just after blood drawing . three weeks before enzymatic hydrolysis experiment , one aliquot of plasma was thawed for purification of cm ( chylomicrons )/ vldl ( very low - density lipoprotein ), ldl ( low - density lipoprotein ) and hdl ( high - density lipoprotein ) fractions by differential centrifugation , according to the procedure described in havel et al . ( 1955 ). the lipoprotein fractions were extensively dialyzed at 4 ° c . against phosphate - buffered saline ( pbs : 0 . 15 m nacl , 10 mm sodium phosphate buffer , ph 7 . 2 ). to prepare minimally oxidized lipoproteins , air / light oxidation was induced by storage of the fractions in a transparent flask at ambient temperature and under sterile air exchange for 18 - 20 days . native lipoproteins were prepared from another aliquot of the plasma just prior to the hydrolysis assay . when necessary , the lipoprotein fractions were stored at 4 ° c . under n 2 atmosphere and in the dark after addition of 50 μg / ml gentamycin . phosphatidylcholine ( pc ) content of each lipoprotein fraction was measured by the use of the phospholipid b dosage kit ( wako chemicals ), following manufacturer &# 39 ; s instructions . decreasing concentrations of each native and oxidized lipoprotein fraction were tested following procedure of the dose - response assay as described above . positive control for the parasite growth was without lipoprotein . kinetic analysis of human plasma hydrolysis by spla 2 s was performed by mixing on ice 50 μl of crude plasma with recombinant spla 2 s at various final concentrations ( respectively 2 ×, 5 × and 10 × the ic 50 value ). at time zero , the mixture was transferred to 37 ° c . samples ( 7 μl ) were taken after 5 , 15 , 30 and 60 minutes of incubation and quickly frozen at − 20 ° c . an aliquot of plasma without enzyme was similarly processed . concentration of non - esterified fatty acids ( nefas ) in each sample was measured by a discontinuous enzymatic method using the nefa - c test kit from wako , following manufacturer &# 39 ; s instructions . the amount of nefas in each enzyme - containing plasma sample was subtracted for nefas in enzyme - free plasma sample . specific activity for each of the spla 2 s was determined from the linear part of the curve [ nefa ]= f ( t ). experiment was repeated three times with plasmas from different donors and different batches of spla 2 s . crude human plasma was incubated with 3 nm of recombinant human gx spla 2 , 15 nm of the full length or the truncated form ( iifδc ) of recombinant human giif spla 2 , and 15 pm of the bee venom spla 2 , for 48 h at 37 ° c . aliquots ( 7 μl ) were taken at times 0 h , 6 h , 22 h , 30 h and 48 h of incubation . nefas were measured in each aliquot by using the nefa - c kit ( wako ). experiment was repeated twice with different plasmas . plasma and purified ldl and hdl were adjusted to 1 . 67 mg phospholipid ( pl )/ ml final concentration in pbs . because purification yielded low amount of cm / vldl , the cm / vldl fraction was adjusted to 1 . 00 mg pl / ml . cacl 2 ( 1 mm final ) was added to the lipoprotein fractions . 200 μl of each fraction were deposited in a 96 - well plate and incubated for 15 hours at 37 ° c ., alone or in the presence of recombinant human gx spla 2 ( hgx pla 2 , 25 . 0 nm ), recombinant human giif spla 2 ( hgiif pla 2 , 125 . 0 nm ), bee venom spla 2 ( bvpla 2 , 0 . 18 nm ) and naja mossambica mossambica venom spla 2 ( naja pla 2 , 0 . 05 nm ). nefas were measured in triplicate using the nefa - c kit ( wako ). value from each fraction was normalized by subtracting the nefa amount in the corresponding fraction without enzyme . two independent experiments with lipoproteins from different plasmas were performed , which gave similar qualitative results . according to the experiment , maximum hydrolysis reached 50 % or 65 % of total pls in the fraction . 1 . 2 . 7 ) involvement of enzymatic activity in gx spla2 - induced inhibition of parasite growth a schizont - enriched culture of p . falciparum was grown for 24 h in the presence of 50 nm recombinant human gx spla 2 , or 50 nm gx spla 2 plus 37 . 5 μm ly329722 , a potent inhibitor of the gx spla 2 enzymatic activity ( 75 nm ic 50 , smart et al ., 2006 ); controls in normal culture conditions and in the presence of ly329722 alone ( ly ) were processed in parallel . parasitaemia (%) before incubation , and after 24 h of incubation , was determined from diff quick - stained smears . parasite growth was expressed as the percent delta of parasitaemia , using the following formula : δp =[( p x − p 0 )/( p 100 − p 0 )]× 100 ; where δp is in percent , p 0 is the parasitaemia (%) in the initial culture , p 100 is the parasitaemia (%) after 24 h in normal culture conditions , and p x is the parasitaemia (%) after 24 h in experimental conditions . 1 . 2 . 8 ) sensitivity of the p . falciparum blood stages to gx spla 2 a parasite culture was synchronised on a 4 h - window . parasites aged 0 - 4 h post - invasion ( rings ), 18 - 22 h post - invasion ( trophozoites ), and 34 - 38 h post - invasion ( schizonts ) were adjusted to 1 % parasitaemia and 2 % haematocrit in complete medium , and were then incubated in a 96 - well plate in a candle jar at 37 ° c ., with or without 100 nm of recombinant gx spla2 . after 15 hours , cells were centrifuged at 900 × g for 2 min , washed in rpmi and re - suspended at a 2 % haematocrit in fresh complete medium for further growth . when re - invasion had occurred ( p . falciparum invades new rbcs when its 48 hour - development cycle is completed ), parasitaemia (%) in each well was determined from diff quick - stained smears . gx spla 2 inhibitory activity on the complete parasite cycle was assessed by incubating rings with the recombinant enzyme for 48 hours . 1 . 2 . 9 ) gx spla 2 lipolysis of infected red blood cells 100 μl it of packed non - infected rbcs ( 1 . 1 × 10 9 rbcs ) were washed in pbs at room temperature , then lysed in 10 volumes of iced 5p8 buffer ( 5 mm sodium phosphate , ph 8 . 0 ) and centrifuged at × 14 . 000 g for 15 minutes at 4 ° c . pelleted ghosts were washed several times in iced 5p8 to fully remove haemoglobin , and re - suspended in 5p8 to the initial rbc volume ( 100 μl ). phospholipids ( pc ) in ghosts were measured by using the phospholipids b kit from wako , following manufacturer &# 39 ; s instructions . pc content was also measured in crude plasma . from at least four independent measurements ( erythrocytes and plasma from different donors ), pc content was estimated at 1 . 45 g / l ( volume of packed erythrocytes ) in rbcs and 2 . 20 g / l in plasma . a parasite culture was enriched in young parasite stages ( rings + early trophozoites ) by sorbitol treatment . parasitaemia ( 2 . 5 - 3 %) and stage distribution were determined from diff - quick staining of culture smears . one half of the culture was processed immediately as described below . the other half was maintained in normal culture conditions for a further 24 hour - period for the parasite to reach the schizont stage , and then the culture was processed similarly . erythrocytes were centrifuged for 2 minutes at × 900 g . one volume ( 100 μl ) of packed erythrocytes was washed in rpmi and re - suspended in 1 vol . of rpmi bsa 0 . 05 %. 50 μl of the suspension were distributed into 3 wells of a 96 - well microplate . non - infected erythrocytes which had been maintained in culture conditions for 24 h prior to the experiment were processed similarly . 25 μl volumes of plasma diluted × 4 . 4 in rpmi were also distributed into wells . a maximum 2 μl volume of recombinant gx spla 2 and bee venom spla 2 in pbs bsa 0 . 02 % were added to respectively 30 nm and 0 . 2 nm final concentrations . control wells for endogenous generation of nefas were without enzyme . the microplate was incubated in a candle jar for 5 hours at 37 ° c . plasma samples and cell supernatants were taken and frozen at − 20 ° c . ghosts were prepared from pelleted erythrocytes and frozen at − 20 ° c . samples were thawed on ice just prior to triplicate measurement of nefas using the nefa - c kit from wako . nefas in erythrocyte samples are expressed as the sum of nefas from ghosts and corresponding supernatant . parasites were grown for several cycles in culture medium supplemented either with 8 % human serum or with 0 . 5 % albumax ii ®. cultures were semi - synchronized by sorbitol treatment 2 - 3 days prior to the experiment . when they contained mainly mature parasites and had reached 2 . 5 - 3 % parasitaemia , cells from both cultures were washed in rpmi , re - suspended in rpmi 0 . 05 % bsa at a 50 % haematocrit , and distributed in wells of a 96 - well microplate in the presence or absence of 50 nm of recombinant gx spla2 . the microplate was incubated at 37 ° c . for 6 h in a candle jar . incubations of non - infected rbcs and serum were performed in parallel as respectively negative and positive controls of the gx spla 2 activity . supernatants were taken and ghosts were prepared from cells . nefas were measured using the nefa - c kit ( wako ). values for healthy rbcs ( rbc ) and infected cultures ( irbc ) are the sum of nefas from paired ghosts and supernatants . 1 . 2 . 10 ) evaluation of the in vitro chemosensitizing activity of platelet - activating factor ( paf ) quantitative analysis of the increased activity of recombinant hgx spla 2 when combined with paf was done by comparing concentration - response curves for hgx spla 2 alone and in the presence of several fixed , sub - inhibitory concentrations of paf . effects of each fixed concentration of paf on the response of the parasites ( ic 50 ) to hgx spla 2 were expressed as the response modification index ( rmi ) ( oduola et al ., 1998 ). the rmi was calculated by the following formula : rmi = ic 50 ( a , b )/ ic 50 ( a ), where drug a is hgx spla 2 and b is paf . an rmi of 1 . 0 represents no change in the ic 50 for the recombinant hgx spla 2 combined with paf . the rmi values & lt ; 1 . 0 represent chemosensitization ( including possible synergy ). sub - inhibitory concentrations of paf in the assay were : 25 μm , 37 . 5 μm and 50 μm . recombinant human spla 2 s from groups ib , iia , iid , iie , iif , iii , v , x , xiia and xiib were assayed for inhibition of the p . falciparum development in vitro . results are shown in table i below . anti - p . falciparum activities of human spla 2 s . ic 50 values are the mean of independent dose - response assays using human red blood cells and serum from different donors for the p . falciparum culture . at least two assays were performed with the gib , giia , giid , giie , giii , gxiia and gxiib non - toxic spla 2 s . ic 50 values for gx and giif spla 2 s are the mean of four independent determinations . group - x and - iif enzymes were found inhibitory , with ic 50 values of 2 . 9 ± 2 . 4 nm and 14 . 3 ± 10 . 4 nm , respectively . group - v spla 2 exhibited a low inhibitory activity , with ic 50 values of 162 . 5 ± 90 . 9 . remarkably , the inflammatory spla 2 , i . e . giia spla 2 , whose level has been shown to increase in the plasma of malaria patients , was clearly inactive against the parasite . group - iif spla 2 is structurally unique among spla 2 s , in that it has an unusually long , proline - rich c - terminal extension ( valentin et al ., 2000b ). this extension mediates the giif spla 2 binding to mammalian cells ( wijewickrama et al ., 2006 ). by contrast to the full length form , the c - terminus truncated form of giif spla 2 ( giifδc ), although catalytically active ( singer et al ., 2002 ), did not inhibit plasmodium , suggesting that the attachment of truncated giif spla 2 to the erythrocyte membrane and / or lipoprotein surface might be impaired , leading to a drop in toxicity . 2 . 2 . 1 ) anti - plasmodium activities of human spla 2 s are lowered in the absence of serum the toxicity of venom spla 2 s toward p . falciparum has been attributed mainly to enzymatic hydrolysis of serum phospholipids because those spla 2 s are largely inefficient at killing p . falciparum in the presence of albumax ii ®, a phospholipid - poor serum substitute ( guillaume et al ., 2004 ). anti - plasmodium activities of gx and giif spla 2 s were thus assayed in albumax ii ® to assess for their requirement in exogenous pls . ic 50 values of both enzymes were found to increase in albumax ii ®. group - x spla 2 ic 50 value exhibited variable increase according to the experiment , ranging from 25 - fold to 150 - fold its value in serum . giif spla 2 ic 50 value was increased by approximately 17 . 5 - fold . therefore , toxicities of both gx and giif spla 2 s appeared to be lowered in the presence of albumax ii ®, suggesting that their mechanisms of action rely to some extent on the presence of extra - cellular pls . hydrolysis of serum by human spla 2 s has been reported for gv and giia spla 2 s : the gv spla 2 was found active , the giia spla 2 was found inactive ( rosengren et al ., 2006 ). the specific activities of gx , giif and gv spla 2 s on whole human plasma was determined by measuring nefas released upon incubation with different enzyme concentrations and incubation times . activities of the bee ( apis mellifera ) and naja mossambica mossambica venom spla 2 s ( respectively bvpla 2 and naja pla 2 ) were measured for comparison . results are shown in table ii . specific activities of spla 2 s on human plasma . hydrolysis of plasma phospholipids by human and venom spla 2 s was assayed using different incubation times and enzymes concentrations . hydrolysis was assessed by measuring the amount of nefas in samples using the colorimetric assay developed by wako ( nefa - c kit ). enzymatic production of nefas was corrected for endogenous production of nefas measured in the absence of spla2 . specific activity for each of the spla 2 s was determined from the linear part of the kinetic curve . values are the mean of three independent experiments . sd : standard deviation . anti - p . falciparum activities ( ic 50 ) of the respective spla 2 s are given for comparison . human enzymes exhibited lower activities on plasma than venom enzymes . among the human spla 2 s , gx spla 2 was 9 - times and 30 - times more active than giif and gv spla 2 s , respectively . in line with what has been reported in serum , high concentration ( 1 μm ) of giia spla 2 was inefficient at hydrolysing plasma ( not shown ). of note , spla 2 s &# 39 ; activities in plasma are in line with their toxic properties against p . falciparum , indicated by their ic 50 values . nefas released from plasma by ic 50 concentrations of gx , giif and bee venom spla 2 s were measured on a time scale relevant to the dose - response assay , i . e . 48 h . three independent experiments with plasmas from different donors were performed that gave similar qualitative results . results from one experiment are presented in fig1 . each spla 2 at its ic 50 concentration hydrolysed plasma at constant rate for at least 20 h , with gx spla 2 being the most active . this indicated that in the presence of the human spla 2 s , the parasite is potentially submitted to continuous release of nefas and lysophospholipids , most probably at the origin of toxicity , as proven in the case of the bee venom enzyme . the non toxic , truncated form of giif spla 2 was largely inefficient at hydrolysing plasma , reinforcing the idea that hydrolysis of pls is involved in the anti - plasmodium activity of native giif spla2 . giia , giif , gv and gx spla 2 s are known to readily hydrolyse purified ldl and hdl in their native state ( ishimoto , et al , 2003 ; pruzanski et al ., 2005 , sato et al ., 2008 ), although giia spla 2 was found less efficient than the other spla 2 s ( gesquiere et al ., 2002 ). malaria patients exhibit elevated oxidation of lipoproteins as compared to normal subjects ( sibmooh et al ., 2004 ), and it is known that such modifications can modulate the susceptibility of lipoproteins to spla 2 s ( eckey et al ., 1997 ; pruzanski et al ., 1998 ). the ability of recombinant gx and giif spla 2 s to hydrolyse oxidized lipoproteins in vitro was then analysed . prior to the experiment , because it was also previously reported ( guillaume et al ., 2006 ) that cm / vldl oxidized by prolonged exposure to air and light ( minimally oxidized lipoproteins ) are inhibitory to the development of p . falciparum in vitro , it was analysed whether similarly oxidized ldl and hdl would be toxic as well . results are shown in fig2 . it can be seen that all three classes of lipoproteins are inhibitory when oxidized , with respective ic 50 values of 85 μg pl / ml ( cm / vldl ), 200 μg pl / ml ( ldl ), and 300 μg / pl / ml ( hdl ). noticeably , parasite growth was enhanced in the presence of native hdl , confirming the nutrient properties of this particular lipoprotein ( grellier et al ., 1990 ). assuming that pc is the major phospholipid in either class of lipoproteins , enzymatic hydrolysis of lipoproteins was performed at a given enzyme - to - pc ratio after pc measurement in lipoproteins . the bee and naja venom spla 2 s were assayed in parallel to the human enzymes . each spla 2 was used at approximately 10 × ic 50 . experiment was performed twice with lipoproteins from different donors and gave similar qualitative results . results from one experiment in triplicate are shown in fig3 : group - iif spla 2 hydrolysed lipoproteins in the same preferential order than gx spla 2 , but , unlike gx spla 2 , it hydrolysed poorly the triglyceride - rich lipoproteins ( cm / vldl ); contrasting with the human spla 2 s , the bee and naja enzymes hydrolysed lipoproteins in the following preferential order : ldl & gt ; hdl & gt ; cm / vldl . except in the case of the triglyceride ( tg )- rich lipoproteins , substantial amount of nefas was generated by either enzyme from the oxidized lipoproteins . however , it was noticed that the amount was smaller than that generated from the lipoproteins in their native state . 2 . 3 . 1 ) the catalytic activity of gx spla 2 is involved in the anti - plasmodium mechanism inhibition of parasite growth was removed in the presence of ly329722 , a potent inhibitor of the recombinant gx spla 2 ( smart et al ., 2006 ) ( fig4 a ), establishing the prevalence of enzymatic activity in the anti - plasmodium activity of gx spla2 . in accordance with this , high concentrations of the catalytically inactive h48q mutant of gx spla 2 inhibited poorly the p . falciparum development ( 30 % inhibition at 1 . 25 μm ). insofar as the h48q mutant exhibits unaffected interfacial binding capacities , this confirmed that phospholipid hydrolysis is essential to the gx spla 2 toxic mechanism . 2 . 3 . 2 ) all blood stages of p . falciparum are sensitive to gx spla 2 . as it can be seen in fig4 b , a 15 h - treatment with recombinant gx spla 2 is inhibitory to either of the parasite stages inhibition was largely irreversible . among the gx spla2 - treated stages , only rings succeeded into a 25 % re - invasion , whereas less than 10 % trophozoites and schizonts completed the cycle . 2 . 3 . 3 ) gx spla 2 exhibits membranolytic activity against infected erythrocytes . it is well known that alteration of the host erythrocyte membrane occurs during parasite intracellular maturation ( maguire et al ., 1991 ). those plasmodium - induced changes can modulate the membranolytic activity of spla 2 s ( moll et al ., 1990 ). the membranolytic activity of the recombinant gx spla 2 on rbcs infected by either young ( ring / young trophozoite ) or mature ( late trophozoite / schizont ) parasites was analysed . because it was previously shown that bee venom spla 2 does not interact with the infected erythrocytes ( guillaume et al ., 2006 ), this enzyme was used as a negative control of erythrocyte pl hydrolysis . parasite cultures were incubated with gx spla 2 in the absence of serum for 5 h and nefa production was measured thereafter . values were normalized according to non - specific release of nefas in the absence of enzyme . group - x spla 2 induced the release of nefas from the parasite culture with predominance for the schizont stages ( fig5 , a and b ). release increased with the parasite maturation . healthy erythrocytes were left intact . as expected , and contrasting with the human enzyme , the bee venom spla 2 hydrolysed poorly the erythrocytes , either infected or not . 2 . 3 . 4 . the anti - plasmodium effect of gx spla 2 is mediated mainly by hydrolysis of exogenous pls . it was found that gx spla 2 does not efficiently inhibit plasmodium growth in albumax ii ®, regarding its capacity to hydrolyse infected erythrocytes in culture ( see example 2 . 2 . 1 ). to check for possible inhibition of the enzyme by albumax ii ®, hydrolysis of e . coli membranes was carried out in the presence of albumax ii ®. hydrolysis occurred at the same rate in albumax ii ® and in control without albumax ® ( not shown ), indicating that albumax ii ® does not prevent the gx spla 2 enzymatic activity . it was further examined whether culture in the absence of serum might induce modifications of the erythrocyte membrane that would in turn prevent its hydrolysis by gx spla 2 . the membranolytic activity of gx spla 2 on a schizont - enriched parasite culture maintained in albumax ii ® was analysed . as illustrated in fig6 , nefas were released at the same rate from the albumax ii ®- derived culture and the serum - derived parasite culture , demonstrating that erythrocytes maintained in albumax ii ® do not undergo gx spla2 - inhibiting membrane modifications . taken together , these results indicate that neither enzyme inhibition nor erythrocyte membrane alteration can explain the albumax ii ®- induced drop in toxicity . 2 . 4 ) evaluating the in vitro chemosensitizing properties of paf on the anti - plasmodium activity of gx spla2 . the acetylated phospholipid platelet - activating factor ( paf ) is a potent pro - inflammatory mediator exhibiting multiple physiological and pathological actions . it is produced by various inflammatory cells and by endothelial cells . the actions of paf are abolished by hydrolysis of the acetyl residue , a reaction catalysed by paf acetylhydrolase ( paf - ah ), an atypical spla 2 associated to plasma lipoproteins . it was shown that recombinant gx spla 2 efficiently hydrolyses paf ( gora et al ; 2006 ), and it has been proposed that the enzyme be involved in the neutralization of the newly synthesized paf during ldl oxidation , thereby eliminating its biological activity . the effects of paf on the gx spla 2 anti - plasmodium activity were evaluated . it was first examined whether paf and / or its degradation products ( lyso - paf and acetate ) might affect the plasmodium intra - erythrocytic development . paf was found to exhibit high ic 50 value ( 82 . 0 ± 1 . 2 μm ) in standard culture conditions . in conditions where the serum paf - ah was inhibited by pefabloc , the paf ic 50 value did not vary ( 95 . 0 ± 2 . 8 μm ), making improbable that paf or products of paf generated in normal plasmatic conditions might have any effect on the parasite development . the effects of paf on the anti - plasmodium activity of gx spla 2 were assessed by gx spla 2 and paf interaction studies , in which fixed sub - inhibitory concentrations of paf were combined with pre - serially diluted gx spla 2 . concentration - response curves for gx spla 2 alone and in the presence of paf were compared . the data were expressed as the response modification index ( rmi ) ( as in oduola et al ., 1998 ). rmi values were found & lt ; 1 ( fig7 ), indicating that paf potentiates the activity of gx spla 2 against plasmodium . diagnosis of a plasmodium infection in a patient using a time - resolved fluoroimmunoassay ( tr - fia ) a blood sample from a patient having a non - specific febrile illness is analysed for determining the serum concentration of giif , gv and gx spla 2 s by tr - fia . the tr - fia for human giif , gv and gx spla 2 s is used as described in nevalainen et al ., 2005 . anti - giif , gv and gx spla 2 igg isolated from a rabbit are obtained and then labelled . recombinant human giif , gv and gx spla 2 s are used as standards in the tr - fia . a serum concentration of the giif , gv and / or gx spla 2 s respectively superior to 4 , 11 and 2 μg / l is indicative that said patient is infected with plasmodium . identification and quantification of individual spla 2 isoforms including hgiia , hgiif , hgv , and hgx in the plasma of malaria patients , spla 2 isoform profiling and relationship with parasitaemia and disease activity spla 2 enzymatic activity was measured using radiolabeled e . coli membranes as substrate , as described in rouault et al ., 2007 . briefly , 2 to 3 μl of plasma were incubated for 30 min in 60 μl of spla 2 activity buffer ( 0 . 1 m tris ph 8 . 0 , 10 mm cacl2 and 0 . 1 % bsa ) containing 100 , 000 dpm of [ 3h ]- oleate - radiolabeled e . coli membranes . reactions were stopped by addition of 80 μl of stop buffer ( 0 . 1 m edta ph 8 . 0 and 0 . 5 % fatty acid - free bsa ). mixtures were centrifuged at 10 , 000 g for 5 min , and supernatants containing released [ 3h ] oleate were counted in a wallac perkin - elmer microbeta counter . tr - fia was performed as described in nevalainen et al ., 2005 . briefly , 3 to 5 μl of plasma were diluted in 100 μl of delfia assay buffer ( perkin elmer wallac ) and added to giia , giif , gv and gx spla 2 s igg - coated microtiter wells previously washed with tr - fia washing solution ( tbs ph 7 . 8 , 0 . 04 % nan3 , 0 . 02 % tween 20 ). after incubation at room temperature with constant shaking for 30 minutes , wells were washed 4 times with tr - fia washing solution , incubated with 100 μl of eu - labeled human spla 2 igg tracer ( 0 . 5 μg / ml diluted in delfia assay buffer ) and washed again 4 times as above . then 100 μl of delfia enhancement solution was added to wells , incubated at room temperature for 5 min with shaking and thereafter for 10 min without shaking . time - resolved fluorescence was measured using a wallac envision perkin elmer plate reader and optimized optical modules for delfia assays . plasma from patients infected with plasmodium falciparum ( n = 26 , parasitaemia ranging from 0 . 01 % to 4 . 0 %) were analyzed for total spla 2 activity on e . coli membranes and for the presence of individual spla 2 isoforms including hgiia , hgiif , hgv and hgx by tr - fia . analysis of 15 plasmas from non infected subjects ( control group ) was performed in parallel . enzymatic activity on e . coli membranes was higher in plasma from infected patients than the control group ( p = 0 . 0284 , using mann - whitney test , two - tail p value ). however , the level of enzymatic activity was not predictive of parasitaemia or disease activity . tr - fia analysis of hgiia spla 2 showed a significant elevation of the enzyme mass level in plasma of patients with either low (& lt ; 0 . 5 %) or high parasitaemia (& gt ; 0 . 5 %) ( p = 0 . 0148 and p = 0 . 0170 , respectively , using mann - whitney test , two - tail p value ). the level of hgiia spla 2 mass is therefore not predictive of parasitaemia or disease activity . the level of hgiia spla 2 mass measured by tr - fia was clearly associated with the level of total enzymatic activity ( spearman r = 0 . 6327 , p & lt ; 0 . 0001 ). independent of these measurements , significant increases of hgv and hgiif spla 2 s were found in patients with low parasitaemia ( p = 0 . 0285 and p = 0 . 0113 , respectively , using mann - whitney test , two - tail p value ), but not in patients with higher parasite levels , as compared to the control group . no increase in hgx was seen in either group . this is probably because of the small cohort of patients in the present study . interestingly , correlation studies between plasma concentrations of the different spla 2 s showed a very strong correlation between the respective levels of hgv and hgiif spla 2 s ( spearman r = 0 . 5412 , p = 0 . 0003 ) and a slight but significant correlation between levels of hgiia and hgiif spla 2 s ( spearman r = 0 . 3889 , p = 0 . 0120 ). no correlation was found between the levels of hgiia and hgv spla 2 s . the above results indicate that the specific quantification of individual spla 2 isoforms in the plasma of malaria patients can provide an independent set of data and / or a combined set of data ( spla 2 isoform profiling ) which is associated with the level of parasitaemia . defining a spla 2 isoform profile may thus be useful for diagnosis of disease activity , therapeutic follow - up or prediction of patient &# 39 ; s outcome . three spla 2 isoforms , namely hgiia , hgiif and hgv were found to be significantly increased in patients infected by p . falciparum . no spla 2 s other than hgiia have been reported to be present in the plasma from normal subjects or septic shock patients ( nevalainen et al ., 2005 ). although preliminary , the above results indicate that the levels of hgiif and hgv spla 2 s are specifically increased after infection with the malaria parasite , and thus that these individual spla 2 isoforms may constitute novel biomarkers of disease activity , especially in patients with low parasitaemia . the correlation observed between the plasma levels of hgiif and hgv spla 2 s as well as that observed between hgiia and hgiif spla 2 s suggest that the combined measurement of spla 2 isoforms ( spla 2 isoform profiling ) represents a highly specific biomarker of malaria disease activity . for instance , it can be speculated that a specific combined increase of hgiif and hgv spla 2 s enzymes in plasma of patients with low parasitaemia may contribute to maintain parasitaemia under a deleterious threshold , and may be predictive of patient &# 39 ; 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