Bhu'himh, ~ 1998) 80. 379-390 0 Socidt6 i'ran~aise de biochimie et biologic mo16cukm'c / EIsc~ IcL Paris

B omed cai @p| cat ons of maghenfi e ferroflu d

A Halbre icDL J R o g e r ~', JN Pens b, D G e l d w e r t D , M F Da S i l v a a, M R o u d i e r ~, JC Bacr i a

~aLaboratoire des Milieux D~sordonnt;s et H6tdre:gbnes, case 78: b i,~#~oratom' des Liquhh's hmiques el b~le~.'/~-r'es Cha~,~ds. case 63, univer~'il~; Pierre-et-MaHe-Curie, 4. place Jussieu. 75252 Paris c~-'dex 05:

Chtstitut de Bioh~gie Phvsico-Chimique {IBPCL 13. rue Pier,e-el-Marie-Curie. 7,5005 Paris. France: dDepartameltlo ~h, Fisi('a, I, htiverskk~& ~h, Brasilk~, Com/,,tl.~; Universilario, Asa Norle, Caixa Postal (M455. ('EP-70910-9~Z Brasi/ia DE Brazil.

eService ~h, Gdronloiogie Clinique. H~)pilal CharLes Richel. 95540 Villier~-h,-B,,I. f"mm,'e

i Received 8 December 1997, accepted 27 April 1998)

Summary - - The use of cell-largeted I'errofhlid in the characterization of modil'ic~ltions of ceil membranes is reviewed. Maghemite ferrofluk! was synthesized by the Massart nlelhod, complcxcd with dimercaplosuccmic ackl (FF). Cell targeting by Ft- ~,~ls deve!ol~d by coupling FF to v~u'ious biological effectors such as antibodies, iectins, etc, which enabled magnetic cell sorting. Modificalk~ns m erythrocyle membranes were sludied using FF l~und to recombinant human annexin V (AnxFF) which i~, ~ery sensitive, compared to other Anx-based Ivagenls. in the early ddecuon of phosphatidylserine (PS) exl~)sition on lhe outer leallet of the plasma membnme. Thus PS exD~nition on I1)t}use RBC was detected already after a 24-h storage at 4°C and, transiently, 24 il after tlleir infection by Pktsmodiunt pm'asites, at which time the parasites are still confined to tlae tivet; thus leading to the i~cruimlent of young RBC and tile accumulation of a species, intermediate I~tween rcticul~ytes and eo'thr~:ytes, and the actual RBC tat'get of pike+me, dial invasion. AnxFF ~vealed PS exposition on RBC li'om sickle cell anemia patterns, ioilowing v;u'ious it~flammations and already atier 20 days of human bltxxl stonlgc under bkx~ ballk conditions. Such a sensitive detection slmuld ix: simil~u" to that of rnacml~hages which recognize extx~sed PS on ceils and bring al:~)ut the latter's eliminatkm lixm~ the circtdation. AnxFF binding detemaination was combined with that of cell eieclxophoretic mobility, glycerol resistance and filterability to characterize RBC membrane m(~.tificalions in Al~heimer's disease patienls which .suggested a continuous damage and regeneration in RBC of these patients. A logistic analysis suggested that several tiwee-parameler combinations could permit diagnosis of AlzheJmcr's dis,.:a~ with up IO 95~'~ accuracy. THPI cells and macrophagcs, derived themselves by incubation with retinoic acid, were l~mnd to ~ and placed in a ntdit) fi'equency alterllating nlagnelic Iiekl. Magnelt~.'ylolysis was asso~..'iated will) FF attachnlenl to the cells without damage to non-t'~mnd cells and without healing of the sttiTotmdmg solution (() S(~-idt6 li'an~'aise tie bit~'himie et biologie moldculaire / Eiseviel; Paris ~.

ferroflnid / au|ileXill-|'~rroflllid / llnllttrxi|| V / hhmd stonlge / blood sedimt'nt¢ltion rllle / AIzheimer's dist'~t~t' / malarial / imignetic ~.ell snr i ing I nu!gnetocytolysis

I n t r o d n ¢ ! i o n

Ferrofhtids Ill or magnetic fluids 12I are colloidal soluo lions of monodomain magnetic nanoparticles (usually 5.~ 15 nm in diameter) in a suitable liquid. The magnetic particles, most frequently used, are ferrites, having the general composition MFe204 (with M being a bivalent metal cation such as Ni, Co, Mg or Zn and including mag- netite Fe304) and nmghemite Fe203. Initially produced by grinding large particles ir! suitable organic solvents and sieving 131, ionic ferrofluids are now prepared chemically, mostly by Ibllowing the Massart method 141 which does not include an obligatory requirement el" organic solvents or surfactants as in other methods, in the absence of sur- factants, the stability of a fcrrol'luid depends on the small size of the particles and on an equilibrium between their charge repulsion and their Brownian motion. For the ob- taining e r a stable ferrofluid (FF) in physiological media, ie at neutral pH and appropriate ionic strength, it is necess- ary to condition the particle surface. To this end the par- ticles are most freqttently coated with dextran 15-91,

albunl in II()-121ol ° symhclic Imlymcr.~ ~uch a~ niclllacry- l~Ilc.s ~md orgmlo.,,ihm¢.,, I 13~ 15 I. The eff¢~;It~r fittest fre- quently an ~mtihody) is ~lttached It) the pm°lic!e through a covalent bond to the coating polymer, ~md the st~bility of this coating determines the stability of the effectoropm~tiele complex. Indeed, as pointed out by Groman and Josephson 1161. a partial desorption of these coatings has been obo served in rive, particularly where the coating was not very well tolerated, and the result was a shedding of the coat° ing-bound effector followed by an accelerated elimination of the particles. Also, the copolymerization with the coat° ing results in larger sized particles favoring their upt~ke by hepatic Kupfer cells. To overcome these problems, tt difo ferent type of particle was developed in our i~lb. These consist of bare maghemite particles in complex with dimer° caplosuccinic acid (DMSAI which stabilizes the fcrrofluid at physiological pH and ionic conditions and permits bind- ing of effectors directly to the particle core through the DMSA, using hetero-bifunctional reagents, such as N-suco cinin|idyl 3-(2-pyridyldithio) propionate (SPDP) as dee scribed I 17. i 81.

380

Two biomedical applications were studied in this work: the characterization oferythrocyte membrane modifications in several physiological and pathological situations and magnett~ytolysis which signifies a destruction of specific

aetic field. "tides with ing a rela-

tively stable union, is a prerequisite for these applications, and was developed using magnetic cell sorting h~ vitro.

80

10

~' I I I . . . . ! " ' ' - I . . . . . . . . . . . . . . . . 1

~ l l l . l l l le1,,ll l s I I I I l i ~ l

| I l l l l l l l l l l l l l ~

lllIllIIIlll|l|!

II llllIlllllllll ~

5i 0 ~

i

! I

,lIllIllIllIllIlllllIlllIllllIllllIllllllllll~llIIIIllllllllIlllIllllllIlllIll

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I I I I I l t I l t l l t l t o t l I I I i l l l l l I IO I I I I I I I I t l liOlllllllIlll lllIl lllIl llIllOllll

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ill~|llllllllllll I llll~lll II| IIIIIIIII III IIIIIIIIIIIIIIII.IIIIIII IIIIIIIIIII

' ............... i .................. I ................ . . . . . . . . . . . . . . .

' i i III~| Ill IIIIIIIIIIII~II IIIIIIIl It till

10 15 ;9 25 30 Diameter in nm

@

0,5 1 1,5 2

Time (ms)

Ftlt I, Slt~ distributitm o1" DMSA-coalcd maghemitc particles as detennil~ed by elcctl"~m micruscopy (tl} aud by magnclobirefriu- gcnce { h},

Synthesis and characterization of ferrofluid

The magnetic nanoparticles were synthesized as described pre- viously 14, 17 I. Briefly, this involves coprecipitating a mixture of ferrous and ferric chlorides in an anamoniacal solution, oxi- dation of the resulting magnetite with ferric nitrate at 90°C to yield maghemite (Fe203). A cationic ferrofluid with an iso- electric point at pH 7.5 was obtained by dispersing the pm'ticles in nitric acid; their complexation with meso 2,3-dimercapto- succinic acid (DMSA) in an acidic medium followed by pep- tization of the flocculate in an alkaline medium and neutralization yielded a l~rrofluid which is stable at pH values between 3 and 9 and in physiological saline (0.15 M NaCI at pH 7.4). Binding of the effector vhs heterobifunctional tea= gents is tollowed by masking or neutralization of excess l~ac- tire groups and by surlhce inodifications (lor use in i,il,o), which suppress non-specific interactions and diminish consid- erably the non-specific trapping of the particles in hepatic Kupfer cells. Particles, currently synthesized in our laboratory, have a diameter Dm = 7.5 + 3.15 nm as measured by electron microscopy (fig l a). Magnetic birefringence, which measures a hydrodynamic diameter, yielded a value Dn = 72 nm (fig ! b) in good agreement with a Dll of 74 nm observed by nanosizing (not shown). In these latter methods, the size polydispersity of the preparation (t~ = 0.4) is mote heavily weighted towards the large particles. This and the solvation layer are responsible Ibr the large difference between Dm and DH.

Some physical properties of magnetic fluid

S!alic' l ' [ l ' rc fs '

I:ronl a I~ficros¢opic poin! of view, maguclic par!icl¢,~ nf a FF alV lUagll~lically n11modonlai11 with a diameter hetwccll d ~ 3 nm and d ~ 12 rim, These panicles l~a,r u lllilBnl~li¢ l||OlHl#!it m~ of the ordor of 1(~ I. Bohr l UUglletOllS for d ~ I0 nm.

Depending on the ratio o1' the magnetic anisotropic en- ergy E~ to the thermal energy k13T, the particles are either i'crmmagnetic or superparamagnetic, This anisotropic en- ergy represents the height of the barrier which opposes a flip-flop t~f the magnelic moment,

if E~ > kaT, the particles are ferromagnetic, and their magnetic moments lbllow the easy magnetic axis. The par- ticles can rotate in a field until thc i~ magnetic tnoment is aligned with the magnetic field H,

If E~ < klff, the particles are superparamagnetic and their magnetic moments can rotate freely inside the grains.

E,~ can be proportional either to the volume of the particle E~, = K,V or to its surlhce Ea= K,S where V, S ate respec- tively the volume and the surface of the particle, K,., K, the anisotmpic constants for the volume or surface.

The magnetization curve is the first data which charac- terizes a magnetic fluid. The magnetization Ibllows a classi- cal Langevin law if the volume fraction ¢ ot" tile particles is not large: ¢ < 8%. M = ¢ M, L (~) where ~ = Bo in, H / kaT

is thc__~ange~l parameter tL t~ i = cofl~ ~ - t i t~!l. AI t,~x field M = Xo IT wi lh Xo = ~ Md l l , / 3 kilT.

Tile inieraclion belween a magnetk" dipole n~, and a homo- gelleOtlS magnetic field given a lOltlUe 4c'ling on Ihe parlicle (for I'ciTonlagnclic |~t~.ii<lit'ie)or Oli lhe nl4~2neiil." nlonlelll (for superparanlagnelic pariicle); Ibis lo~ttuo varies whh !.~0 m~ H and has, lor example, a vahie of 10 kn~T for H - 10 4 Aim.

A force is exerled on a magnetic particle h~ a gradienl of nia~nel:ic field Ib l lowln, , an Ol'ienl¢ilion of its nla~rlellc mo- Illellt aIOI! o ~ ,

Fp - t.i0 ( ino?)oH with lu = uru,, L (~)

This force is very weak, on Ihc order of 3' l0 is N, in a realistic experiment: It. =4~t'10~: m,= M,V (M, = 3"1~1~ A/nIL But ;I i]lrce ~,|Clillg 1111 ~<1 nlagnelic particle ;il |he inter° face IlelWeell a niagnelic Ill.lid and a noli-niagnelic one (a llleilibrane for example) can allain abotll IO is N. "Pile rea- son is thai lhe gradient of field is nol lh¢ exlernal ont~, btil a local one which is equal !o MIH) tb tvhe|'e M i t t ) i~ ihc nlagne~i;'.alion of the il lagnclic f luid and b a t:haraclcrislic length representing the nlean distance belween lwo p:u'iicles (b ,.- d (ll-I/.l; (~) iS tile volulne fraetioll Of lhe FF).

CelLs .s'orlilig with magneli~" parlil'h,s

Induction of a nlagnetoi)hor¢lic effect ou the particles in ;ill Oxlcrllal gradient o f il lagnelic field is nol praclical with a sial)It !:1( il can, however, be shown thai this effccl is indeed po~sib!c with Ilit!logic'al cells covered with niaglielic par- Iiclc~, In the case whore N parlicle~ arc bound on Ihe cell Ih¢ inagnelic force is given I~y:

i : , , : N lill I lk I . I ~ ) V I !

h! l!lc CXl)crii!!cilt, the co!In arc iiti¢cicd in a tube el Icnglh 1 and diameter a with a wdum¢ flow rate Q. The tube is immersed in il constant gradient of magnetic field VH, perpendicular Io file flow in the tube. The nlcan Iransit lime is given by it, = t / v where {7 is the inean velocity of the llow (Q = 7 it a : /41 .

t . = I / ~; = t it p a -~ 1 4 Q

We have to conlpare this iransil t ime ~tt to the time of trapping 17p which represents lhe mean tinle reqoired I'or a cell io cover hal f a diameter wi ihoul l'low.

'~p = ii / 2 Vp, where Vp repl'eSelllS the nlagnetophorelic velocity. Vp is determined by the balance between the mag- netic force F,, and the viscous one F, = 3 ix 11 D vp where ! 1 and D are respectively Ihe viscosity of the carrier fltiid and the diameler of tile cell.

t i, = (312) (it 11 a D/He N VH

For trapping the cells on lhe tube. we ueed to have l:p < ttr which corresponds Io:

Q < 14~ N V tt l a m, L(~) t 6 q D

In practice, a pood Irappia~ of lhc cell,, is oblaiaod for q = I0 ~ Pa..,,, a = ] ram, D = ll) um. I = 5 ~ } c m . # , , = 4 ~ I0-7, N ~ I06, m~ = M~ V = 3 I0 -Is Alto 2 V H ~ 107.Aim -~

with Q = 15 mmVs (~7 = 2 cmis.

Dynamic el.]'eels q[./~wro/hdd

Two eli:eels conlrol ihe magnetic dynamics of the parallel susceptibility Xz/((0). depending on the magnetic anisotropic energy of ~he particle. If the pariicle is ferromagnetic (E;, > kltT), file Brownian lime 1;B is dominant: lh~ = 11 V / ki(P, ii' Ihc parl icle is superparanlagneilc, die NCel l ime 1;x is donlinanl: 1;N = 1;o exp KV/KBT ( lypical ly, 1;ct ~ l() `j st.

A sinlplc Debye lay,,, can take l!11o accotilll l:hesc relaxa- tion times for lhe magnetic susceptibil i ty X((o):

)OA(o) = X d (1+i¢o1;) ~ i l h 1; = ei~. 1;,,

The transverse susceplibility X r~__( ~ ) has a resonance corresponding to Ihe t~rron/agnelic resonance IFMR)of the luacrospin which is the equivalent of the spin resonance of 111o protons in nuclear magnetic resonance.

X! = lLlo '7 M, (os/(( (oZ o-(o2) - 2icz0h co)

where y is the ~yroinagnelic factor of the electron. (~ is a faclor faking into accounl Ihe dissipalion of Ihe magnetic momoill inside Ihe crysial. (el is Ihc L;irm~r frcqucilcy:

(,it = 7 II,

B, can be induced by the ;inis~triq~ic field v¢iih ;ill illl¢rll;ii field It;i -- i{ .L/ IlK Ill i,, c;illcd lhc il l lr innic I :MR) t t r by ;it1 exicrnii l f ield (ihc cxli°ii!isic • PMRI. And forn!al!y the iolat nlagnclic stiseerttibility is given by:

Z ( (o ) = Z~J((oh 2Z~(co) t 3

Figure 2 presents the real tlild i inaginary paris of X(Oii, showing the N6el relaxation and the intrinsic FMR.

f e l l t a r g e t i n g wi th efl~etok- b o u n d f ern) f lu id

Cell targeting by fcr|'ofittid (FF) i , vivo requires reagent~ that are, on one hand, suMciently free of nonospccific inter° actions with non-targeted tissues, nt)iably the hepatic KLipo for cells and, on the other hand, can cros,~ ihc endothelial walls, recognize the desired target and f ix lhcnis¢lves Io it. Particles wi thout el'feeler (bill otherwise lreaied to ¢ounlero act non-specific interactions) were administered to mice at doses of up to 100 Hmol Fe/kg and, in preliminary experio menls, were observed 1o circulate for al least 4 h wi!holil excessive hepatic uptake (unpublished resulis). They seem

382

to pass through endothelial walls and possibly through the blood-brain barrier as well. Intravenous administration of up to 400 ~mol Fe/kg to mice did not reveal any overall toxicity over extended periods.

. . . .

~10Ws a convenient separation of ~,binding cells from non- binding cells inn magnetic field gradient, and the collection of the two cell populations[ 17]. As shown previously, binding of an enzyme, h o ~ i s h ~roxidase, to the p'~icles, either directly using SPDP or indirectly through an avidin-biotin bridge, did not alter its enzymatic activity 118]. Likewise, binding appropriate !¢ctins to FF yielded reagents that recog- nized correctly and very specifically the corresponding blood groups (results not shown). One of these lectin-FFs, Ulex eu- ropeaus I-IF, was also used for the purification of endothelial cells [ i 8] in a reaction that could be competed out completely by ob L-fuco~. The purified cells could be cultured after remo- val of the p~icles by EDTA treatment. This demonstrated the efficacy of efl~ctor bound ferrofluid in targeting and sorting specific cells and paved the way to the use of annexin V-fer- rofluid in the detection and characterization of modifications in erythrocyte membranes.

Annexin V bound fi, rtY~tluid in the characterization qf cell membranes

Recombinant human annexin V was bound to FF particles and the ensuing AnxFF complex was used to characterize phospholipid distribution in cellular membranes, Annexin V belongs to a family of homologous proteins of 27-~55 kDa whidt are ubiquitous intracellular proteins that participate in nlany devdopm~nial pl'O¢osses illohiding pl'Olociioil of pla~ntue l'ronl thrombolytic activity, initiation and in!or. ruption of lactation, ere (for review see t~tynal and Pollard [191L The common feature of the annexins is their ability to bind to membranous acidic phospholipids (PL) in the presence of C#*, Annexin V binds preferentially to phos- phatMylsefine (PS)[191 and, in view, to cardiolipin as well [201, It is possible that their binding to PS in the cell cyto- plasm is part of their activity in signal transduction (eg tall), as it is frequently found to ~ concentrated along the cytoplasmic side of the plasma membrane 1221. Normally, phospholipids are organized asymmetrically in cell mere. branes so that PS is found on the cytoplasm-facing side, and phosphatidylcholine and sphingomyelin on the outer leaflet of the plasma membrane, This asymmetric distribution is an active dyt amtc process, accomplished by the continuous action of an enzymatic system 1231 and a considerable in° vestment of metabolic energy which counteract the natural

d . . . . b " tendency towards an equilibrated istri utica of membrane constituents in all its compartments, Inhibition of this enzyme(s) by incubating the cells with N-ethylmaleimide 1241 results in a gradual exposure of PS on their surface, it is reasonable to assume that interruption of cellular ATP

production also results in PS exposure, but this has not been so far demonstrated. Physiologically, PS exposure on the cell surface is associated with blood coagulation, erythro- cyte aging [25] and apoptosis [26l (it is possible that the disruption of the inner mitochondrial transmembrane poten- tial [27l and the randomization of PS distribution in the mitochondrial membranes are also associated with a de- regulation of ATP metabolism). In rive, PS, on the outer leaflet, is recognized by specialized macrophage receptors [28, 291, thus leading to the elimination of the erythrocytes or apoptotic cells by the reticuloendothelial system. PS ex- posure on aged or sickled erythrocytes was revealed, in early work. through its activation of prothrombinase activ- ity or by accessibility to hydrolysis by phospholip~ses II 91, and. in later work, by the use of t2~l- or FlTC-labeled an- nexin V 130, 311.

06

0,5 . . . . . . . . . .

04 ...................................

X '° O a . . . . . .

O! ....... 1 0

|f,031 1 [~ ttl,} |ftOfi | g *L)fi I~*0] 1 (:~ * L'IL~ t~+O~ IE+IO

%

Y" 05

0

,05

, 1 5

!E*03 I E:.Oa tE*OS IE®06 IE,O? IE~08 IE*09 IE+IO

Fig 2, Real X" and imaginary X;" paris of the initial ,~usceptibility very'us the frequency t" = o~/2~:, )( and X" represent respcctivdy the real and imaginary pans of the initial susceptibility vep:vus fre- quency (f = co/2~), X °" and Z" show successively the rdaxation and the ferromagnetic resonance (l-MR).

383

Our interest in erythrocyte membrane modifications stemmed from our studies of cerebral malaria in mice which revealed modifications in the characteristics of red Nood cells (RBC) already during tile hepatic stage of plasmodiam infection, well before any RBC, were parasitized [321. as- sociated with an accumulation of a subpopulation of RBC intermediate between reticulocytes and erythrocytes, and prone to plasmodial infestation [33]. Recombinant human annexin V was thus bound to DMSA-complexed FF, and the ensuing AnxFF reagent was used to probe for PS exposure on RBC i!7, 34]. In this assay, PS exposure ts revealed by magnetic cell sorting and estimated from the observed frac- tion of cells, which are retained in a magnetic field gradient following their incubation with AnxFE As seen in figure 3, freshly collected mouse blood does not contain any detect- able AnxFF-binding RBC whereas 10% of the cells bound AnxFF already after storage for 24 h. Similarly, AnxFF binding to RBC increased steadily during storage of human blood under blood bank conditions (fig 4).

AnxFF binding to RBC is also enhanced in various pa- thological situations 11341. As can be seen in table 1, blood from patients with sickle cell anemia or those displaying a high blood sedimentation rate contained up to 50% AnxFF binding cells compared to up to 15% in normal controls.

For stored blood and sickled erythrocytes, the values in figure 4 and table I, using AnxFF, for both the control and the experimental cases, are 10-20 times higher compared with values reported by Kuypers et al [351 and by Wood et a11361, both using Anx-F!TC, while confirming the general sense of their results. Likewise, Taverne et al 1371 did not observe any enhanced binding of FITC-Anx V to RBC at any time ibllowing the infectio,i of mice with plasmodial parasites. On the othe," hand, we had observed 1331 modifi. cations of tile size, cell electrophoretic mobility and resist° ance to glycerol-induced lysis of eryth,'ocytes from mice during the early hepatic stage of their hdection with plas- modia, at a time when Ihe RBC themselves were not yet

subpopulattt n, inter- parasitized, and described an RBC • , ' 3 mediate between reticulocytes and erythrocytes, which is the actual target for plasmodial invasion. We thus inquired [381 whether the appearance of this sub-population was not preceded by an enhanced PS exposure on RBC, the removal of which could trigger a recruitment of new cells in the bone marrow. Indeed, as seen in figure 5, a transient enhancement of AnxFF binding by RBC was observed 24 h after the infection of Balb/C or C57/bl mice by P Berghei Anka, and this was followed by a peak of reticulocytes by day 3 and of the RBC sub-population by day 4 ! 181, just in time to be invaded by parasites emerging from the liver. This scenario is similar to the one operating in the evolution oi' normal RBC 125, 281 whereby aging erythrocytes, exposing PS, are recognized by macrophages and eliminated in the reticu- Ioendothelial system. They are replaced by young cells emerging from the hematoporetic system as reticulocytes, and maturing into erythrocytes, thus maintaining a low level of PS exposing cells. The higher sensitivity of AnxFF, corn-

100- % R B C

8 0 -

~d 24 H

Baib

6 0 -

4 0 -

2 0 - Control Balbtc = 0%

Non-retained Retained

Fig 3. Annexinqerrofluid binding to mouse erylhmcytes as a func- tion of their storage in vilro, "Pail blood was coilecled fl'om Balb/C mice into citrate buffer and RBC were separaled by centrifugafion and removal of the buffy coal. AnxFF binding |o RBC was tested, immediately or following an overnight storage at 4"C, by a 30omm incubation at 37'~C of 30 IJL of a 3% RBC suspension with 30 ~L AnxFF in 0.35 mL of Tris buffered saline containing 2.5 mM CaCI2 and O. I% detipidated BSA. The reaction mixture was diluted with buffer and passed through an electromagnetic field gradient to sep- arate the AnxFFobound cells from the non-bound cell as described previously [17. 341. Omission of Ca 2+ ions in any of the steps abolished AnxFF binding. Reprinted from [34j with permission.

pared to FITC-Anx, leads to a different description of the pm tlmgenic process. This is also trite in the case of thymocyte apoptosis where PS exposure is detected by FITC~An× later

F

6 0 - % Bound

............. y ~ 2.3728 + 19 987 log(x) R ,, 0.99436

4 5 - ,J

1 5 - ~°.e .°'

1 10 100 Storage (days, logarithmic scale)

Fig 4. Effect o1" tile duration of human RBC storage in vitro on the extent of Anx-FF binding, Normal blood samples, collected by the 'Centre de la Transfusion Sanguine', were tested fl~llowing storage in viov for varying limes under the blood bank conditions as de- scribed in the legend to figure 3. Reprinted I't'onl 134] with per* mission.

384

Table I. Percentage of AnxFF binding to flesh RBC fronl human subjects with various pathologies compared to healthy persons. Anonymous b l ~ sample, of persons requiring blotxl analysis were obtained from a local laboratory and used within 48 h of collection. Other conditions were as described in legend to figure 3. SR. sedimentation rate; other pathology, normal SR blood flora patients with either a deforming rheumatic disease era cancer necessitating chemotherapy. Reprinted from 1341 with permission.

Control Normal SR Sickle cell anemia High SR Other l,tlthoh,,~,'y

19,60 0.(30

14.80 12.(~ 11.90 18.00 0.0O

12.60 14,00 5.90

! 2110 8,70

Mean ~ SD 10.7 :t: 5.9 . . . . . . . . . . . .

21.73 50.00 36.94 16.75 49.00 36.37 ! 9.56 (~.00 51.26 27.59 35.00 54.49 32.1X) 49.00 49.85

53.20 46.80 65.70 71.00

49.76 63.00 63,00

23,5 ± 6, I 5(1.(} ± 9.3 51,5 ± 12.2 58.5 ± 7.6

than by a pmthmmbinase assay 1391 (cfM Sorice, lx~rsonal communication). The apparent lower sensitivity of FITC-Anx is also compatible with the fact that results obtained with this reagent correlated with those obtained by a prothrombinase assay only when ionophore-treated RBC were utilized 1351. This interpretation was confirmed in a comparison of 1he dif- ferent m e t h ~ l s of detecting PS exposure 1401. AnxFF binding to RBC, stored Ibr various lengths ol' time. correlated well with

10 ......

0 24 45 70

Time after infection (hours)

FIR $, AnllC~illof~rr~ffluid binding to mouse erytlm~cytcs immcdi° ately at~er hlt~ction with malaria l~U,asit~s, Balbtc mice wcrc in- I't.'cted with Ph~smmlhu, voelii tclosed circles) or injected with Ballqc mouse erylhmcyles infested with P Berghei ANKA ~open ci~les), Controls (o~n ~uares~ were el!her exl~,~sed to non.inl'cctcd ,losquit~s or injected ip with uniu~k,;~:d ~:(y~h~. ~-~,~:s, Tail bh~d was colh,'ck, d and its AnxFF binding was measulxxl as descfil'~'d in the legend to figure 3, Reprinted with permission fix,, 1381,

the estimation o1" PS exposure by either anti PS-IgG or a prothrombinase assay, whereas significant ~-'-Sl-Anx or FITC-Anx binding to the same samples was detected only after 80~ I IX) days of storage when extensive hemolysis was already evident. The same Kd of I-~sI-Anx binding to RBC was measured throtlghout the storage of blood (Geldwerth et al, in preparation), indicating that the same low aMnity sites arc detected at all time points with this reagent, alld these sites be¢ollle preponderant only after prolonged storage.

While being caused by many dil't~rent, and thu~ non-spe- cil'ic causes, measuring PS cxposulv o|1 RBC by AnxFl:: bind° tug i~lay ncvcllhclcss olTcr an assL,'alWC as to the viability in rive of the Iransl'used RBC. Also, viral (itlcluding tt!V and hepatitis C} alld bacterial illl'~clions nlanifcsl thelu~elves very rapidly by strong inflamlnatory reactions. Determining the fi'action of AnxFF-binding RBC in blot~ donations can thus be uselid during the time lapse (currently a minimum of 15 days for HIV) between inl~clion and seroconvcrsion.

Eryth. . 'yte membrane mod(lh'alhms in Alzheimer k disease

We had already developed previously a baUcry of tests to characterize cell membrane modifications in malarial infec- tions 133 I, and considered the possibility that this battery of tests could provide a diagnostic tool Ibra specific disease ~uch as Alzheimer's disease,

Alzheimer's disease (AD} is a neumdegenerative disease with an as yc! unknown etiology which usually appears rela- tively late in Ill}, AD is routinely diagnosed by a ncuro-psy- chological evaluation of patients according to the criteria proposed in 'DSM IV' for dementia 14 i I, by Mac Kahnn et a11421 and by Panisset et a11431 including a 'Mini Mental Stale Score' (MMS} 1441, Definitive diagnosis must, how- ever, await histological analysis of cerebral tissue (usually

385

postmortem). This reveals in AD, but no| in other demen- tias, an extensive degeneration of brain tissue with neural plaques composed of insoluble ~-amyloid (ilself resuhing of an abnormal processing of the lransmembrane amyloid precursor pro|ein lAPP) and neurofibrillary tangles contain- ing phosphorylated tau peptide (for review see I45]). Incu- bation of cells in v#n~ with ~-amyloid results in cell death [461, but the mechanism initiating the abnormal APP pro- cessing is not known, Several genetic variants of the APP and presenilin are associated with an early onset AD, but these concern only a small fraction of patients while age is the major risk factor for this disease. Polymorphic variation at the apoE locus is associated as a risk or protective factor for late onset AD [47,481. Other genetic risk factors for AD include presenilin intronic polymorphic variants 149, 501, and serotonin transporter polymorphisnl [51 I. Gene|ic mar- kers hold considerable promise to improve diagnosis [52, 53], However, considering apoE phenotype as a screen or diagnostic marker tbr AD would miss many Irtle cases and could misclassil~, many normais as AD 1541. Therapeutic interventions are limited to symptomatic treatment and, re- cently, to a transient augmentation of cerebral acetylcholine or a direct activation o1' nicotinic receptors !551. Objective surrogate markers would be very ht22"d in diagnosis (par- ticularly in early stages) and follow up of patients, and could thus contribute to a better understanding of the under- lying pathogenic processes. It might eventually enable a rationalization of therapeutic interventions.

Numerous works have described extra neuronal ano- malies in AD including abnormal aging of erythrocyte membranes 156-591, lipid peroxidation I(~01 and con fl icling reports exist on the internal fluidity of cell membranes 161-

651. O|" parLicuiar imeresl was the ~eporl of Walter m~d Widen of an ahered electrophorefic mobility of RBC from AD pafiems in certain polymer solutions I66, 671.

Modifications in RBC membranes from probable AD pa- tients were studied 168] using cell electrophoretic mobility measurements in polymer (CEMp), AnxFF binding, celt fil- terability (mean transit time, Mtt), which estimates cell membrane fluidity [69, 701, and resistance of the RBC to lysis in a glycerol medium (glycerol resistant cells, GRC). Both AD and non-AD patients were hospitalized in a geria- tric hospital department in a northern suburb of Paris. All 24 AD patients met the criteria of dementia [41 ]. Diagnosis of a probable AD was established according to the gui- delines proposed by Mc Khann et al 1421. The patients had an MMS score below 21/30, they all had amnesia, visuos- pathd disorientation and aphasia 143], and their educational level corresponded to that of a primary school. All ! 8 age- and sex-matched control patients were hospitalized lbr o f thopedic problems but were free of any neurological symp- toms and had an MMS score above 22/30. A duly signed inlbrmed consent was obtained from the patients and re- sponsible parties in accordance with the regulations of French hospitals. A group of 18 healthy, 30-52-year-old volunteers served a~ a healthy young control.

Many of the patients, hospitalized in a geriatric laci!ity, received various medications, mostly 'comfort ' drugs. Medication included antidepressive, antisecretory and anti° Iwpertensive drugs. The effect of these drugs was tested in vitro for the dill)rent l)atures of cell membranes studied (table il). it was assumed, for simplicity, that the prescribed dose was distributed homogeneously in vivo in the whole body. Only the effect on CEM 1711 and AnxFF binding !341

Tahle !1. El'lecl of various prescril~ed drugs, when added in vm'¢~o on the elcclrophoreli¢ mobility (('I:.M)and ;mnexin-|crrolh.d (Anxl:[:) binding of norlnlll RBC. Control RBC were incubated for 2 i~ at room temperature with diti'c!¢nl ¢oac¢~tr~tion.~ u[ drug. bcl~v¢~:n 11, i ;lilt[ l0 times of the estimated 'correcl' concenlralion, washed, and the vat'iotls l~altlrt~,s were measured in comparison with t:l)lreated RBC~

Drugs ¢'EM / tnxF l : himting

Generic name Nature ¢ ~tms¢' t V t c m I Celt ('~ t

None !.031 2.t~5 Fluoxetine Anti depressive 1.064 Theophiline Anti asmatic i. !()(1 Thioridazine Neuroleptic 1.057 Enoxaparine Anti clotting 1.085 Tiapride Neuroleptic 1.083 Nitroglycerine Vasodilator Lysis Oazepam Tramluilizer I. 108 Amiodamne Vasodilator 1.099 Omeprazole Enzyme inhibitor I.O37 Hydmxyzine anti depressive 1.071 Enalapryl ACE inhibitor 1.04 I FeSO4 B6 Anti anemic 1.002 Heptaminol Anti hypertensive 1.007

1.025 Paracetamol Analgesic Paracetamol - codeine Analgesic 1.028

7.10 46.(10

I00.(10 8,fit) 7.t~5

ND 2,70

3.8O 6.1(I 1.73 0.84

ND 7.111

ND

3 ~

is shown in table 11. Patients or controls who required the drugs producing an effect on the studied parameters were excluded from analysis.

The mean values of the RBC membrane features in the - and

r i c e s

rand in the extent of AnxFF binding, cell electrophoretic mobility in polymer(CEMp), and the fraction of glycerol resistant cells (GRC), while the difference in filterability (Mtt) was only of limited significance. Statistical significance is presented only for the differences between AD patients and their hospitalized age,. and sex-matched controls (ND). The changes in AD ery- throcyte membrane features were seen repeatedly over time in ~veral patients who were tested over an 18-month period, thus

epls ~dtc modifications which distinguishing this picture from '~, ' " are associated with acute inflammatory events (as discussed earlier). The magnitude of changes in RBC features was, in this limited cohort, not different in '- s patient, of relatively e,'u'ly (MMS of !5-20) and late stages (MMS < !0) indicating that the changes may be produced early and persist throughout disea~ progression. Judging from this ~mple of probable AD patients, the~ results indicate that the modifications in RBC membranes are indeed associated with Alzheimer's disease. The changes in CEM, filterability and glycerol resistance might be inherent to the alterations in membrane structure in this disease, However, in other physiological and pathological situations these modified features at,, characteristic of young, newly recruited RBC 133, 68, 71 !, As discussed earlier in this pal~l; PS, exposed on aging or damaged cells, is recognized by specialized receptors on macrophages 128,201 and the cells are eliminated in the reticuloendothelial system 1251, Results presented in I'i~urcs 4 and 5 are compatible with tiffs picture We, therefore, separated RBC fractions enriched for young, glycerol resistant ~ells by centrifugation in suitable density gradients, Unex~ctedly, howevel; the separated young cells

exhibited the same extent of AnxFF binding as the whole population. This is compatible with an assumption that PS exposure in AD erythrocytes is brought about by oxidative dins- age that is expected to act with the same efficiency on young and old cells, especially in view of the apparently high preponde- rance of young ~ C in AD pauents.

The overlap in test values between AD patients and their age- and sex-matched controls, seen in table III, would not permit the use of any one indiwdual feature as a diagnostic tool. However, a multiparametric ~ogistic analysis 1741 sug- gested a rather specific predictive value for several three- feature combinations in this limited cohort [6811. The probability of the patients belonging to the AD group may be estimated by the formula:

Prob I A D ! = I/( 1,4-e ~,~,)

where x can be calculated by one el lhe following combi- nations (with 75, 80 and 95% correct classification respec- tively):

Xt = 30.4 + 0.2 x GRC + 0.06 x AnxFF - 35.6 x CEMp;

X2 = 45.5 + O, 12 x AnxFF - 41.8 x CEMp - 4.7 x MTI;

X~ = -97. I + 0.21 x AnxFF + 50.65 x M~'~ + 0.5 x GRC.

The logistic regression using subsets of features per- mitred quite a good classification assuming t!lat the histo- logic examinat ion will confirm the AD diagnosis . Characterization of RBC from a large, multiccntric sample of patients including cases of other dementias and ncuro~ logical problems will be required in order to de~ermine the diagnoslic value of this test battery and estimate it~ positive and negative predictive values.

The observed modificatiou~ oI' erylhrocyte membrane prol~rties present a seemingly contradictory picture; on one

Table III, Biochemical and biophysical characterization of red bk~d cells from Alzheimer's disease patients. 5 mL blood s',unpies were ~:ollected in citrate vacutaincrs t'rom AD patients, age° and sex-matched non-demented (ND) patients, and from young controls, RBC were separated by centrifugation and aspiration of the bully coat, and used in determining An×FF binding [3:q, CEM [71 !, Mtt [701 and GRC 172l, L~vels of significance are shown tbr the t:omparisons of values fi'om AD and ND patients,

A,~e Atolt'A'iPI- V I' l¢clroldtort,t it ' I~lobililit, s Mt'ZO~ transit Glyt '¢ro! t yem's ~t bimfing f t:~,, J f t~oli ,vet' t V t cot) time ¢ MFr~ resistant cells

h, .valh~e in polym(,rs fins) (%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Al~heimer's patients (AD) 84 45.5 1,039 ! .028 1.45 30.8 n = 24 ± 7 ± 18,0 ± 0 . 0 1 8 ± 0.t)22 ± O, 12 ± I I , I

Age-matched non-demented 84 27, I i ,(M3 1,046 i .55 I 9,6 patients (ND) :t 7 ± 14,7 ± 0.020 ± 0.022 ± O. I I ± 6.4

Healthy young contrails 35 2,7 1,0f~5 1.053 1.54 10.2 ~I = 18 ±6 ± 1,9 :E 0,014 _+0,021 ±0.11 ± 3.1

P = AD vs ND 0,36 0,(X)3 0,48 0,02 0, I 0.026 ........................................................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

hand, that of an extensive damage 1o RBC membranes is seen in the enhanced AnxFF binding, and, on the olher hand, a high content of young, l~cently recruited RBC can be gleaned from the higher resistance to glycerol lysis, higher fluidity mid lower electrophomtic mobility 133, 70, 731. We assume that these changes reflect similm" changes in neuro- nal cells or result from them. Disruption of membrane asymmetD, and the consequent exposure of PS on the sur- face of any ceil, includh, g neural cells, could result from oxidative damage [ 19| or, at least initially, from an interrup- tion of energy metabolism. A diminution of mitochondrial energy metabolism has been associated with aging 1751. It has also been observed in cerebral tissue l~om AD patients 1761 along with a decrease in the expression of the milo- chondrially encoded cylochrome oxidase genes 1771. Mam- malian cells contain a large excess capacity of energy metabolism I78I and can function normally at 0.4-0.5 of the normal ATP concentration [79I provided that the metabolic rate is suMcient to prevent an accumulation of ADP and/or AMP 178-801. This excess capacity results from a large number of mitochondria per cell, each with an independent mitochondrial genome (up to 50/cell). Depletion of cellular ATP through mutational inactivation of mitochondria thus requires many independent mutational events and such an eventuality should have a rather low probability. This could explain both the relatively late onset of AD and its modula- tion by various genetic factors. A disruption of the mem- brane asymmetry of neural cells would lead to their interaction with the specil'ic receptor(s) on macrophages (nticmglia in the brain). Unlike activated systemic macro- phages, which atv evacuated in the reticuh)endothelial ceils, brain macmphages remain in place and produce NO or cyto- kines such as TNF or !!2 which, in turn, may be a source ol" a continuous insult to neighboring cells including the damage to RBC (which thus become a suitable surrogalc nlarkerl alld ;lls~, as relxmed, to T lymphocytes 1591.

Magnetocytolysis of ferrofluid-bearing cells

Physical basis

Magnetocytolysis is the destruction o f ferrofluid bearing cells following incubation in an alternating magnetic field. A wide spectrum of hyperthermia methods exists in the radio frequency range (10-100 MHz), microwave range (> 300 MHz), infi'a-red range using the imaginary part of the dielectric constant or ultrasonic waves using the acous- tic absorption in the tissues. Howeve,', the use of magnetic nanoparticles in a radio frequency magnetic field actually seems the best way (Ibr complementary information see the excellent review paper of Jordan et al 1811).

This phenomenon of hyperthermia in FF is based on the response of the imaginary part (fig 2) of the magnetic sus- ceptibility of the solution X" = Z0 t0x / (l+t021:z).

.~87

C

L

To /

Fig 6. A protot) pc of a magnemoc>tolysis apparatus. C and L are the capacitance and selI" hlductance of the coil for the l'e~onance circuit. Ti and To are the temperatures reside and outside the test tube.

When a particle is rigidly bound to a membrane, only the Ndei response is possible with the characteristic time !: = I:N = 1:o exp (E,,/klaT). The maximum of energy dissipa- tion occurs for ml:N ~ I. For maghemite particle td ~ 10 nm), ml:N ~ 1 is in the MHz range.

In order to test this phenomenon the prototype described in figure 6 was consmtcted: a resonant series circuit with a capa~ citance C and a sell" inductance L. The electronic power sup° ply permits us to obtain a magnetic field of 0.(11 T inside the solenoid.

For a given magnetic field (B 0.(11 T) and a ~ivcn frctitlcncy ( f :::: I MIIz)l'igui'c 7 picscnls the inct'¢a~c of the

50

40

30 I,,- C1=

W a. 2B Ig

IO

0

jm ~

/

'1

VOLUME FRACTION i

+ F .F i O 8 .805 O.Ol 0 .015 O.B2

Fig 7. Solution heating in an alternating magnetic fMd a.s a ftmc- lion ot' the volume fraction of t~m)fluid.

388

temperature AT as a function of ¢ the volume fraction of the FE It is possible to describe AT by:

a2 Ao @ A T -

8 ~ I + ¢ ( ~ : p - K . , . ) whe~ A ~ is the rate of heat generation; Ks and % are re- spectively the thermal conductivity of the solvent and of the panicle.

A,@ = toLI2x" (to) / 2 VF

where I, L V~ are respectively the current, the self induct- ance and the volume of the sample of the magnetic fluid.

it is clear (fig 7) that for a high volume traction 0 > !0~, a macroscopic heating is induced. This would not be inter- esting since it is non-specific. Interestingly, however, as seen in the legend to table IV, magnetocytolysis was pro- duced with FF concentrations smaller than I0 ~,~ (5 IJM Fe), with no macroscopic heating, and only when the particles were linked to the membrane,

Magnetocytolysis of THP I human monocytic cells and mac.,i, hages

In the initial experiments, described in table IV, a non-spe- cific binding of FF to ceils was achieved by exposing -SH on DMSA--coated FF by DTT treatment, Presumably this

Table IV, Magnelocylolysis of humml THPI monocytes aud macrophages, FF was reduced with dilhiolreiloi (DTT) mtd exccs~ DTT was removed by t'locculalion of !he parlicles at pH 2, The resultin8 FFDT particles underwent pepti~ation, after retention uu tl pel'mal+el, mtl~.eh by muspel+ding Ihem iu 1), 15 M NaCIo(), I M Tris--HCI, pH 9 and adjustmel, to pl=l 7.i,l. Particles were sterilized by a {),1 ~tlU filtration except where otherwise stated. Mouse nla~rophllges ~,ere induced by an intral~ritoneal injection of 0,~ mL thio~ly¢ol~te bl~th, harvested aseptically 48 h later and sus~nded in RPM11640 culture medium supplemented with ! 0% fetal calf serum, TPHI human monocytes were cultivated in the same cultu~ medium at 37°C in a 5% (702 atlnosphere, Retinoic acid t~almel'lt consisted of 2~3 day,' s of incubation of O,5~l × IO t, cellstmL with 0,2=2 × !0 ~ M retinoic acid, For magnett~ytulysis, 3~8 × 10 ~ ~ells Wel~ incubated for I h with or without 5 pM of the various ferrofluid ptepal'ations and subsequently introduced into the apparatus described in figure 5, and treated tbr I0 rain by an alternating magnetic field of I{~ Oe at a frequency el" - I Mhz. Cell survival was estimated 2, 4 and 24 h later by a differential cell count using the Trypan blue exclusion te~t and by a dye oxidation test using a WST° I kit (B~hringer), Result present the percentage of surviving cells f~dlowing magnetocytolysis Cul.pared to cells not treated in the luagnetic field.

?)v.tme.t THPI cells IYIPl celt,v+ Mouse ........................ ret#loic acid maCrOldmges

None 99,3 ~ I,t) 97,8 :k 2.8 I00 FF 91.8 ± I 1,5 8,9, I ~ 9,6 nd FFDT 7~,3 ~ 8.4 54.0 ~ 8,0 48.5 Non-filter~,d FF ~).4 46.5 42,4

enabled the particles to bind to membrane proteins through mixed S-S bridges. As seen in table IV, THPI monocytes and macrophages, obtained by incubation with retinoic acid or harvested from mice, were destroyed in an alternating magnetic field only when preincubated with DTT-treated FF. The FF in these experiments was routinely filtered through a O. 1 ~ filter to remove aggregates or larger par- ticles. Incubation of macrophages, but not undifferentiated THPI cells, with unfiltered FF resulted in their destruction by magnetocytolysis (table IV) presumably owing to their ingestion by the macrophages, Cell death, seen either by vital counting using Trypan blue or by the WSTI kit, was evident only 3-6 h after the lO-min incubation in the mag- netic field.

In later experilnents, still in progress, 4-hydroxytamox- il'en or estradiol were fixed onto FF and used to produce a specific magnetocytolysis of MCF7 human cancer ceils whereas THPI cells were unaffected under these conditions (results not shown).

Conclusion

The dilTerent lines of experimentation, described in this paper, were undertaken with the perspective of using speci- fically targeted ferrofluids both in the elaboration in vitro of cellular mechanisms such as apoptosis, membrane con- stitution, elc and in i,il,o lbl" diagnostic (eg MRI) and tllera- peutic (maglletocytolysis) applications which depend on the magnetic properties of tile particles. The targeting potential of this l'errtdhdd has beeu established in the magnetic cell ~ol'ting apl~!ica!i(ms and i~ !row heing incorporated iu the nlagtletocytolysis experiments. II is hoped lhal Ihis ap- proach will buth diminish non ,~pecil'ic e!'t~cts on 11eighbur° ing c~lls and also facil i tate an c labora l iou o{' the mechanisms involved in maglletocytolysis.

Acknowledgment

Financial support was prm, ided by Assodatiun pour la Recherche contre le Cancer ( R().

References

I R,t~,',cusxxcig RE ~ 1~85~ Fcn~hydmdymmm'v ('amhridge University Pm,ss, Cambridge

2 Shlmu.n Mi (1~)74) Sin' Phv,~-USP (Euglish translaliou) 17, 153 .t Kaiser R, R,asensweig RE ~ i969) Stu~& ojJi'rmmagneta'fluhL NASA

cr , 1.1,7 4 Massart R (1982) Magm'th'fluids mulpmcc.~',~'for obtaining them, US

Paten! 4 329 241 5 |lasegawa M, Hokkoku S t 1978) Magnetic iron oxhh,.&',~'tra, com.

ph,x mid Imr'esx flu" its pmducthm. US Patent 4 I 01 435 6 Dutton AH, "l~kuyasu KT, Singer J (1979) lron-dextran antib~ly con-

jugates: General ineth~.l for for simullaueous staining of two c~mlpo-

ili;nls ill i l i , 'h r t 's l ihi lhl l i i l l i~l l l l l lo¢leclroll i l l icros¢op\. Pr~,~i Nial] , lc~d Sci USA 76, 3392-339(~

7 Griffin T, Mosbach K, Mosbach R t it,~81~ Aftmit b absolbenls fi~ Magnetic biospccific iluumnog~obulin and e n ~ m c imkltion, ..~ppl Bioi'lwm Biotcctmo! fi, -t~ a ",o~ ~ . : ~ <~

8 Mokhlv RS, Macken,,ie D li ,',l_l |mmunospccific lmxomagnctic irtln-deMran reagents for the labeling and magnetic ..eparatio~l of ceils. J hmmlnol Mem,,d., 52. 353-387

9 Scrildcr U (1983) ,sl4agm, iic carbohydraw particles as carriers ./br i!t'#'inily separa#io~l pm?t}m~, eg cell sepm~i6<m. PCT lni Appl WO 83 03 426

Ill Widder K J, Scnyci AE I ik}S{)) M<qhod q(utog~li'lh' ,vep#u'olio~l o/t'e//x mid lll~' like, ~uld mh'rosphen's ,Ibr use therein, El_! Palenl () 016 552 AI

II Kandzia J, Haas W. Leyhausen G. bdi i l ler-Rudlholz W (1984) ( 'ell .~'(,INIril l ioll: Coul l )ar i .~o l l hi'/Wt, l'll IIIo,l~ll('li(' i i l l l l l l l l l {" illiClTL%l)hCtt'.% (MIMSJ a,d/~w.v hlleriuiihulal Meeling on Cell Ele¢lropht,'esis. Ros- lock, GDR

12 Renshaw PI::. O\~.en CS. Mcl~aughlin AC. F|'c).!R (li}8{~) i ci'romag ilelic COlilraM agl2111s: /% lie\\' approach. ~.l/lt.t411 Ri.~ Med 3. 217 _-~ ~ ....

13 Whilehead RA. (?haguon MS. Grtl l l iall I'~V..hlscphson L (! t)871 M.g . re'lie I;arlich',~Ibr use ill ,~¢porcllioti.~. tlS Palelll 4 695 392

14 Okada M, Ashihara Y, Yano A, Oishi M, ~'k~shioka K, Nakamural'l ] t)91}} hnmunocl.~ay using magli¢li( l)artwh'.~. I ' t ! Palent I)420 186 ..\2

i5 lIVallg Ct t . Shah I ) ( ) ( l i)911 Mat411clw(d#v rcv)o.~il '( ' /h. . /c~lc/ i t I)olvuler pclrlh'le.~ aml al)l)ih'olicJ;I lhr'rc~41{ In lernal Palenl k~i/() t) I/(1914 !

I() Gi'll1111111 EV, Josephson 1. (1993} Low t l lo l i ' i ' l l h l r li'Pi.t~hl ivt~//-bo]D.'- dilil('.~" a.~ (iddl/l'l'l..\ Io .tllii)ilie¢ ul!'ht/o.l*&h ° rolllpo.~i/huk ~/. [ IS Pa|eill 5 248 492

17 Halbreich A, Sabtllovic' D, Sesticr C, Gcld~verlh !), Pens JN, Rt)gcr J ( 191)5} NculOl~articilh's mogm;liqlte~ cou/)h;e.~ d de/'a,m'~-im' ~.1/rio" I#ili.~cllhui. I)enlllnde do brcvel FR 9507865 iPCT/Iq~96/lll1964, no de publieaihul 2 7M~ 197t INPi. Paris

114 t la!hrcMI A, I~llgt'i" ]. llluis iN. l)a Silva Mi;. tlasillt l l iay I:]. Rtuldicr M. Bllyuard I%'1. St'slier C. Anu'i A. Gchlwerih I). Ferlil B. Bacri J('. Sabohivic' I) I !gtJel Maguclic iua~heiii i le uauoparlicles: Ihei|" prcp- iirli!iou iuld apl~l!ic!ilitlii in cell sorlii lg and charlit:leri/ali~ui i! l cellular iI!ellllll'ill112s/ti l*iltt!, hl; I/c'h'H/i/h' and ('lUll~'id AI)plw, lic.l.t I#l ]l+/..tf- lll '/h' (.77'llrlii'r.~ tHai'eli U . ' / , I . ells} I)leiuilu Press. Nt'\v York. 399.-417

llJ Ray!ial I l, ! lt i lhird l IB i It)IM) Aliue+%inn: Ihe prtdllcul i l l iissc~siilg ihc hitlhlgi¢ii! i'olt ~ l i lr il gcile I i ini i ly ill* i iui l l i lui lc l i l lnal eal t ' iu i i i and illitlsll!iltlil!itl-Iliiltlilig llrllleinn. Bio~'hi , I lli,Jl)ll~,~ :l~'/. I Iq7, t~ t t)3

20 Rt~lll~lig~per~c'l' (7 t ! l i t) l) i(J,~i' Ot ittl t!tl!it i~iLtltthttll ti,~ 0 i#lii,l~!li!)llll ~tgeilL hll)rlltlliOl!l!l Piileill W()l)I/()9h28

21 Riiyniil It Ihl l l in E R:t~al~-Tholnlis JM. Iqivel J, (71till! I I I l!Jl)3! All ~ nexili 5 its ii pol0nlitil r0giihilor o1' iUili~)xinn I phosphllryhilii!n b), pl'll,, I¢iu khltlse C, hi iqlll~ inhibiliiui t'olnpal'¢d ~,iili qutuililiiliv¢ dala till lillnt~xiii di,~ll°iblliitlll in hiinliui t~iidoiheliill cells, llh!~qil,ut ,I 21)2.751) ....... 7(15

22 Kostcr J J, I!hluslelid CM, Midtllelt}n (?A, Walker ,11,1 (Itit)3)The sub- eelluhu' loci, dizalitln tff aitilcxin V in cultured chick-enlbrytl fibro- bhlsls. Biochem ,# 291,595-61111

23 I)evaux PF (1992) Prtllein invtl lvcnieni ill irallsUleull~rane lipid asyln- 1111211".%/. AIIl l Nl'l ' l i J o I g l y S I l i o u l o I S l r i R ' l l l l ' e 2 I, 4 1 7 - 4 3 t)

24 I,e DT, Rapaptlrl S!, Ratl INM (1995) Studies till Ihe inechanisnl for enhtinced cell surl'll¢c flicltlr \'ill lissue fil¢lOr ticlivalion of lilCllir x till fibrobhisls idler Ilit?il" exptlstli'e Ill N-elhyhualeinl ide. ]'lllluHl~ l i c i t 11103'/ 72, 8 4 8 - 8 5 5

25 Schrtlit A J, Madsen JW. Tanaka Y 11985} In t'/i.o recognilitm and clearance tff red hhlnd cells ctlnlaiuiug phtlsphalidylserille in Iheir phlsnla nleinbran¢..I Biol ('heul 260. 513 I.-5 I.t}{

26 Ktlol~iuan G. Reul¢lil igsperger CPM. Ktl i j len GAM. K12ehiicn RMJ. Pals S'I; van Oers MItJ f 1994) Anlie~in V Ior f low cyloni121ric t112iec ~ lion of pllosphalidylseril|¢ expr12ssit!n tin B cells und12rgtlillg liptlp- tests. Blood 84, 1415--1421}

27 Casledo M, Itirsch T. Susin SA. Zalnzanli N. Marchelii P, Macht) A. Kroen~er G 11996) Sequential aCtluisilion tit nlilochondrial and

bmmmM 157.5 ~ _--~. _ ~ "~ ~ 2S Mc | i vo ) L \ViBiam~on P, Schk'gci RA t ~ 6 ~ Mcmbiauc p h ~

pho~ipid as~mmuqr~ as a dck'rmimmt o~ cr\'lhmc~,~tc ~*c~ui~io~ b,,

29 Sambram~ GR. Steinberg D ~ g995} Rccognituon of mida tnch dam- aged and apoptotic cells b,, an oxidized h)~ dentil) tiD~pr~tcin ic- ceptor on mouse peritoneal macrophages: role of rnembr~:me phosphat[dylserine. Proc Nat /Acad Sci USA 92, 2179-2 IS4

3() kubin B, Chiu D, Bastack 3 J, Roelfsen B, Van Dcenen LLM (I981i Abnormalities in membrane phospholipid organization in sickled er)- throcyles. J C/in Invest 67, 1643-1649

31 Tail iF, Gibson D { 1994}. Measurement of membnme phospholipid asymrnetry in normal and sickle-cell erythrocytes b~ means of atl- nexin V binding. J Laib Med 123. 741-748

32 Moumaris M. Seslier C. Millgen F, Halbreidl A, Sabolox ic' D ( 101)51 I~l'f~cl Of l'aH 3 acids trealment in cerebral malaria susceptible and non-susceptible slrains of mice, .1 tMt'aqtol 8t, 997-9~}9

3?4 Sabtdm, ic D. Moumaris M. Millgen F. Scsticr (', Halbreich A i 191)5) ('haracterizatitnl of a sub-pol.~ulathm of mouse RBC as at prefere|itial tall'get for illi~l]{l|°iill ill\alSittlll. !#11 ('t~-iI~,,r{',~.s ~ O[ Ele~erol~l"m',si,L Paris. .t1}.8-2,9. lot}5

.14 Sestier C, Sabohl\,ic" D, ( ]e ld~er lh I), M<minaris M. R<lgcr j, pons .IN. Albreich A ( 1995} line of annexin V-lerr~ffluid to ¢tlt|lllC.l~iile cl*%- ihrocyie>, damaged in ~arious paiholugicn or during Ml~'l'agC t'tl rill~ t 'R Alt[~d .St 1 i~Jtl~l*i3 31 S, ] ] .41 "*" ] I '4(}

35 Kuypcrs FA, Lewis RA. Hua M, Scholl MA, Discher D. Ernst JD. lalbin BH ( Iq96) Detection of altered membrane phosphoqipid asym- racily ill subi}~pulalions ~tl lltlmall red blaod cells USillg flutnescei~lt) labeled annexin V. Blood 87. ! i 79-187

36 Wood BL, Gibson Di::. Tail JF (1996). Increased erythrocyle phos- phalidylserme cxpmure in sickle cell disease: Flow-cytomelric incax~ tlrelllelll alitl clinical asstwialitms. Bh,od 88. 1873-1880

3? Tavenle .I. van Schie R. i)]aylair J, Reutelinspergcr CPM t 1~95} Ma ~ laria: Phosphalidyiserine exprcssitm is I1OI incFoased till tile slit I+a~;¢ tit parasilized erylhrocyles, Para,~itol 7bdav II, 2914-,-2q9

38 Haibrcich A, Sabulovic' I), Scslicr, Amri A, Pore, iN, Roger J, GckF ~\cr lh i) (li')t){i) AIIIl t 'xhl V binding hi lilt*line cl<yilu~l~')lC., I'~,~llo~a inbi mle¢liou ~.~,illi l ' l . u m r l i m , palil~iten. I'.r.,~il,d l~.hl~ 12. 2~;12-20 ~,

~t{) i'athlk VA, iiir;l|lllll I)llqqt'~} tA+StiB ,Smmm'r ~ ,ml,'rc., c (m M , d r , . h." Bi,q#n'~" ~ ,!I( 'Hl . l . r Mi'mh,',m,'v Sa~,lo|v. Rhcr, Vcmuml 1 !%A _(I---,.5 ]lily. Itlq/~

4{I ( k !hh~ | l h I), j)It~ all ~, pl', sahtlht\ i~' I)~ t lc l lc) I), I{,tgcl J, l'~l|>, ,.IN I IC%4nill¢l .IM. I(ll)l~u'r~ I A. I.llhil| B I I S,,i<c M Ilalbl¢ich .%( I ~ t i t [;~pfis|lrtt ¢11' phl:!~i]li!llid}~l~ierme at It1¢ ~l.lrlal;t.' ~l[' htt[lli.tll erylhrot:yt¢~ during .~tol'ago ill vitro. I'ASEB Summer Re,searrh {'ot4[?retlC,, o~t Moh'ctllur Biophv,~ic,s Of ('ell,he, Mollbt~uw,~, Sa.~lon~ Rhier, V¢|*~, nit!st, USA, 2(1=25 Jllly, lqki(}

J ! Aulerican Psychialrit~ Assodalitm (li:lq4) lJia,g,m,~ti~ , m l Stuli~ti~,lt Mattttal 01' M('tltal l)i.u,der,~. 41h edI!, APA, Washinglori I](~

42 Mc Kahnn G. Drachniarl D. Folsh,,iu MI;. Rat/marl R, Price I), Sladhut EM (1984) Clinical tliagimsin el Alzh¢ini¢i'°~ di,~¢a,~c. In: Rcpott ~tf the NINCI)S-ADRDA Work Group under lh¢ auspices el ihe I)¢Du'l iil12111 t i t Ileallh and I-hllllali Services Task I'or¢c till Alzh¢inier°s lJi,~,, case. Ne,rology 34. 931)-~q44

,13 Panissel M. Roudier M, S;IXlI, II! ,I, l]oller F( 191)41 Severe hl!pairllt¢ltl I~aller). A IlellltlqllsychohIgii;al te~! 1or ~¢V¢l'ely deitl¢tlled pali¢|il~, Arch Neto'ol 51.4 I-M5

• .t4 IMIstein MF. l:olstcin SI!. Mcuph PR l ie75} Mini Menial State"; a practical nlellmd for grading lh¢ engnitiv¢ slate of palienls hlr lh¢ clinician..I Psv~'hhar Re s 12. 189- Iq14

-IS I.ti%eMtllte S. Reynii lds ( ' H I ! tjtje) The phosllhi ir)hi l i t in i l l lau; ii ¢lili.. cal slage in n¢tirl)d12vehlpulenlai aiid ileUllld¢~i.'ll¢l*iili~¢ pl't~*c~¢~. N¢IIIII.~'I'iPII('(' 7~. 3(19-324

46 ltardy J (1997} Amyhfid. Ihc prcsenilins alld AI/h¢ilner',, tli,.~¢a.~¢, Tremls Neurosci 20. 154-15q

-17 Sulkava It. Kainulainen K. Vel'kkttliiellii A. N i in i .q . l . . 511b¢l I{o [)~i. vauipur Z. Polk iktwski T. Ital l ia M. Kouluhi I~ (ll)lJfl) APOE lillelc~

3 ~

in Alehein~r's disease and vascular dementia in a population aged 85÷. Neumbiol Aging 17, 373-376

48 Halisalmi S. Linnaranta K. Lehtovirta M. Mannersmaa A. Heinonen O, Ryynanen M, Pielkkinen P. Soininen H (1996) Apolipoprotein E polymorphism in patients with different neurodegenerative disorders. Neurosc~tt 205.6 I+64

49 Kehoe P. Williams J, Holmans P. Liddel M~ Lovestone S. Homes C. ,.,r G; Owen+" ( 1 ~ ) Association between

a PS, I iht~,ic ~lymo~hiSm and late onset Aizheimer's di~ase. Neu~rcpon 7; 2155-2158

50 Keho¢ P. Williams J. Lovestone S. Wilcock MG (1996) UK AIz- heimer'~: ~ a s e Collaborative Group. Confimmtion of an associ- ation between a presenilin-! intronic polymorphism and late onset Alzhetmer's di.~ase, ~ncet 347, 1185

51 Li T. Holmes C. Sham ~ . Vallada H. Birkett J. Kirov G, Lesch KP. Powell J, Lovestone S. Collier D (1997) Allelic functional variation of .~rotonin transporter expression is a susceptibility factor lbr late onset Alzheimer's di,~a~, Neumreport 8. 683,686

52 Relkin NR. Tanzi R. Breitner J. Farter L. Gandy S. Haines J. Hyman B. Mullah M. Poirier J. Strittmatter W. Folstein M. Farlow M. Mayeux R. Petersen R, Roses A. Schenk D. Small G. Vangtx~! W, Cookdeegan R, Fleck L. Kapp M. Karlinsky H. Pericakvance M. Po~t S. Wolpert C. Berg L Blass J, Fletcher J. Hegele R. Khachaturian Z. Selkoe D. Thai L. Whitehouse P, Kwon YG (1996) Apolipoprotein E genotyping in Alzheimer's disease. Lancet 347. 109 I= 1095

53 Ro~s AD11995) Apolipoprotein E genotyping in the differential di- agnosis, not prediction, of Alzheimer's disease. Ann Neuroi 38, 6= 14

54 Facrcr LA, Brin MF, Bisas L, Goate A, Kennedy J. Mayeux R. Myers RH. Reilly P, Risch NJ (1995) Statement on use of apolipoprotein E testing in Alzheimer disea~ J Am Med Asset 274. 1627

55 Maeli~k¢ A. Albuquerque BX (1906) New approach to drug therapy in Al~heimer's dementia. Drug Discs.cO, '/bd~o' I, 53~59

~6 Bosman G J, Viss~r FE, De Man A J, Bat'tholomeus IG. De Grip WJ (1~931 ~Pythro~yte membrane chan~es of individuals with Down's syndrome in various stages o1' Alzh¢imerqype d~mentia. N¢,ttrohiol Agl~+g 14, '~3 ~'~m

++ Bt~+sman GJ, llartholomeu.~ lit, De Man A J, Van Kaimthlmut PJ. De Grip WJ (1991) |~rylhro+yte membrane ehm'acterist{¢s indicate ahnm'~ real cellular a~lng tn pattenls with Al~heimer's dtsea~e. Net,tJhiol Agltlg 12, 13.o!8

58 Soldall I., Alberonl M, Blun~:l~I (}. Cmml N, Franc'heM M 1 t~1~J611h~y thro~yil¢ calpain~calpaslattn system in Aleh,~tnler's disease. Agotg CI/n I~xp R¢.~' I~, 100=tt)8

~9 Shiilti I~ $1~.dnl B, Br{~dle C, Roll B, ltul~rman M (1995) T lympho: eyie subpopulttioi~s lind aettvatioi~ illark~rs ¢orrelale wilh s~verily of Alzhetmer's disease. Clin I,tttllOlOt I~tloliitlol~tlhol 7~, 246=2~0

6(1 van Ren~burl Si, Dimi~ls WM, vim zyl J> I~t~hnik FC, Van der W~li J, 'l~tljaa~.l II { I ~ ) Lipid pert~xidation and platel+t membrane fluidity o impli~aiion.~ tttr Al~heimcr's disease? Neltmre/tort 5. 2221: 2~24

61 Zubenko GS. Teply L Winwo~l 1~. Huff FJ. Moossy J. Sunderland 1". Martinet AJ (19%) I~ospectiv~ study of increased platdet membrane fluidity as a risk l~ctor for Alth~imer's disease: results at five years. Am J P<t~'ehitllPy 153. 4 2 ~ 2 3

62 Lejo~¢eu~ M, Adds M, Roudier M, Davelt~)se D, Viret J. Matin.re JC (1~3) No change in viscosity of lipid phase of RBC membrane in Al~heimer's disease. Psy('hitttO, Res 46, 20.++92I)5

63 Eagger S, Hajiohammadreza !, Fletcher K, Levy R. Brammer M (lgt~t) Platelel membrane fluidity, I'amily history, severity a,d age of on~+t in Altbeinler's disease, bit J Gt, r P,~'ychfil¢ 5, ?.95-41t0

64 Hajimohmmadreza I. Brammer MJ. Eagger S. Burns A. Levy R 11990) Platelet and erythrocyte membrane changes in Alzheimer's disease. Biochim Biophys Acta 1025.208-214

65 Hicks N. Brammer M J. Hymas N. Levy R (19871 Platelet membrane properties in Alzheimer and multi-infarct dementias. AIzheimer Dis Assoc Disord !. 90-97

66 Walter H. Widen KE. Read SL (1993) Red blood cells from Alzheimer patients and from normal subjects discerned by cell electrophoresis in an aqueous polymer solution. Biochem Biophys Res Common 194. 23-28

67 Walter H. Widen KE (1995) Differential electrophoretic behavior in aqueous polymer solutions of red blood cells from Alzheimer's pa- tients and from normal individuals. Biochim Biophys Acta 1234. ! 84- 190

68 Sabolovic' D. Roudier M. Boynard M. Pautou C. Sestier C. Fertii B. Geidwerth D. Roger J. Pens JN. Amri A. Halbreich A (1997) Mem- brane modifications of red blood cells in Alzheimer's disease..I Germ tel Bhd Sci 52A. B217-B220

69 Hardeman MR. Bauersachs RM. Mciselmau HJ (1~)88) RBC laser diffractometry and RBC aggregoluetry with a relational viscouleier: Comparison with rheoscope and myrenne aggregometel'. ('li, ih.mof heel 8. 581-593

7(1 Boynard M. Ribier E Guiilct R. Driss E Perrotin P. Gaudcy E Pautou C. Nalpas B (1994) Assessment of red blood ceil delbrmability in healthy adult subjects: influence of sex. age. cigarette smoking, men- strual cycle and oral contraceptives, f3i, Hemotheol 14. 225-262

71 Iwaguchi T. Shimizu M. Mort 1". Nakajima T ( ! 984) Analysis of dec- trophoretic mobility histogram of mouse thymocytes during tumor development, hmmmology 52. 359-365

72 Sauer A. Kurzion T. Meyerstein D. Meyerstein N (1991) Kinetics of hemolysis of normal and abnormal red blood cells in glycerol-con. raining media. Biochim Biophys Acta 11)63. 203-208

73 Hayashi H. Fujii M. Yoshikumi C. Kawai Y. Watanabe N. Watanabe K. Fnsama M. Shislaido H (1987) Changes in etectrophorctic mobility pattern of erythrocyles in patients with paroxymual nocturnal hemo~ glol',.dinuria. J OJ, l.ah bmuumd 23.77-81

74 Hosmer DW. Stanley L I It)891. Applied L,gi.~'tic Regressio,. Wiley and Sons Inc, New York

75 Shi[Ictu~ga MK. li~gen TM. Ame~ ItNt I~tl+tl O~idativc damage and nlil~wht,ldrial decay in aging. Pmr IV~#I A,'ad S,'i USA t}l. 111771-- 111778

7h Muiinya I~.M. IBti~'lil~g AC. Ileal MI: ~lqt}41. ('~,'lieal cyltWhl't|lnC oxidt|s¢ aclivily is I'¢dtl¢¢d ill A!eh¢iulcr's disca~c..1 Ncluochcmislrv

7"/ Chandrasekaran K. Utordmm T. Bl~nly DR, Stoll J. Martin IJ. Rais- in+at SI (1994) Impairment in mttocho,driul cytm:hrome oxidase gene expression in Alzheimer disease. Mol Bmi, R+,s 24, 336934I}

78 Weisent~rg if. ttalbreicla A. Mager J (198(1) Bitvchemical lesions in copper+deficient rats caused by secondary iron deficiency. Biochem Y 188. 633~6d I

79 Freudent~arg H. Halbrcich A. Mager J i 1970t On the nlechunism of inhibition of protein synthesis caused by intracellular ATP depletion and its rdation to tile fluorideoinduccd inhibition. Israel d ('hem 8. 131 p

80 Hamburger~Heyd J. ilulhreich A. Mager J ( 1%7 t Reduced rate of ATP catat~lism and the enhanced anaiuo acid incorl~wating activity of regenerating rat liver inicmsomes. I~iochem Biophvs Rcs (',mmtm 26. 471 +=476

81 Jordan A. Wust P. Scholz R. Faehling H. Krause 1. Felix R (tt197) Ala,~netic fluid h',lwahermiu fMFItt in sciemOi,r aud clinical appli. ci+t6ms +q'mtt,++ne+ir ,e,ar,rie,rs t l"l~it'cli U t'/al, edn) Pleutuu Press, New- Ytu'k, 56~.)-5t)5