Antimicrobial peptides isolated from ovine blood neutrophils

255 Pages·2010·6.56 MB·English

by  Anderson, Rachel C

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ANTIMICORBIAL PEPTIDESI SOALTED FROM OVINE
BLOOD NEUTROPHILS
A thepsrisese ntienpd a rtfuilafimlle notft hree quirefomr entdhteesg roefe
DoctoofPr h ilpohsyion B oitencolhogy
atM assUenyi vteyrP,sam ilersNtoornt Nhe,wZ eanlda.
RacheCl Anderson
2005
Abstarct
ABSTRACT
The aoifmt her esearpcrhe senitnet dh itsh eswiass t oi nvestitghaept reo pertoife sta hnet imicrobial
peptidfeosu nidn o vinbel ooidn,o rdetroa ssetshse piort entaisaa lh igh-vaplruoed ucDtu.e t o the
largneu mbeorf l ambasn ds heetph aatr es laughtNeerweZ de alan(adp proxim2a5tm eillyl iloanm b
and m5i llisohne eppe ry eart)h,e raer ec onsidervaoblluem eosfo vine balvoaoidl afbolrpe r ocessing
(approxima4t0e mliyl liloint rpeesry ear)C.u rrentthliybs l ooids d rieadn ds olads a lowv alue
producTth.e f irosbtj ectoifvt eh irse searwcahst op uriafyn dc haractetrhieas net imicrpoebpitaild es
isolaftreodm o vinnee utprhoilsA. n umber porfo line/argipneipntei-draiescsw h,e lals t wos mall
fragmenotfsl argperro teitnhsa,dt i splayaendt imicroabcitailv wietrye i dentifiTehde.s econd
objectoifvt eh irse searwcahst oi nvestitghaemt eec haniosfma ctioofno vinaen timicrpoebpitaild es.
Fort hiisn vestigtahtrieooenv ,i npee ptidae-sh,e liScMaAlP 29 apnrdo line/argiOnaiBnaec-5rmiicnhi
andO aBac7.5mwienries, y nthesiOsfet dh.e sSeM,A P29w ast hem ostp otenTth.e t hrepee ptidaelsl
bounGdr am-negabtaicvteae lrL iPSa ndc ausetdh eo utemre mbranteob ep ermeabiliSsMeAdP.2 9
causesdi gnificdaenpto larisoaftt ihoen cytoplmaesmmbirca nteh alte dt oc ellly sis. Howtehvee r,
othetrw op eptidoensl cya usesdl igdhetp olarisoaftt ihoen cytoplmaesmmbirca nweh,i cihn dicates
thatth ey probably pastsheemd e mtbhrraonueg ht o iwnitttehhr eia ncnte cre llucloanertn tsT.h et hird
objectoifvt eh irse searwcahst oi nvestitghaemt oer phologcihcaanlg teosb acterciealll s inbdyu ced
theo vinaen timicropbeipatli deTsr.a nsmisseiloenc trmoinc roscoapnyda tomifco rcmei croscopy
confirmtehdaS tM AP29c ausesdi gnificdaanmta gteo t hem embraneosfb actercieallla sn d induced
cellly siwsh;e reaOsa,B ac5micnaiu semdi noarl terattioot nhseb actermieamlb ranebsu td idn ot
induccee llly sisT.h e fourtohb jectoifvt eh irse searwcahs t od etermitnhee effoefc tth e
environmecnotnadli tioontn hse a ctivoiftt yh ep eptidTehse.p eptidweesr ev ersyt abolvee ar r ange
ofp H valueasn dw henh eatetdot emperatuuprt eos8 0°C.T hea ctivoiftt yh ep eptiddeesc reased
slighitnlt yh ep resenocfme o novalceantti oannsd w asi nhibibtyet dh ep resenocfde i valceantti ons.
Thep eptidweesr es ignificmaonrteal cyt iivnec ombinattihoanin n dividuaanldtl hye,wy e res trongly
synergistipco lwyimtyhxB i,an p eptiadnet ibioTthiefc i.n oablj ectoiftv he irse searwcahst od evelop
a pilot-secxatlrea ctpiroonc efsosr t hei solatoifoa nni tmicrobpieaplt idferso mo vinbel oodT.h e
laboratoryp-rsoccaelses swiamsp lifainedda daptetdo d esigan p rocestsh acto ulbde used
industriTahlelc yr.u dpei lot-plantw aesxa tcrtiaavcgeta inas btr oad-raonfgf eo opda thogeannsd
diseacsaeu sionrgg anisTmhse.a ntimicrpoebpitaild feosun idn o vinbel oohda vet he potetnobt ei al
useads b iopreservfaotrci hvielsll eadm bp roducotrsi ,na t opiccarle afmo rc utasn dg razetsh;e refore
iti sr ecommendtehda ftu rthreers earicsch a rrioeudtt oi nvestitghaeat beo vaep plicatainodni.sf
successtfhuefl e,a siboiflc iotmmye rcialitshietn egc hnology.
iii
Acknowledgements
ACKNOWLEDGEMENTS
First faonrde moIsw to ulldi ke to mtyh saunpke rvisOorrP sa.k -LaYmu ,t hank yotua kfioanr g
keeni nteriensm ty projeacntdf ort eachimneg a lIln eedt ok nowt ob ea successrfeusle archer.
OrB riaWni lkinstohna,n yko uf orb einagr ounwdh enI n eedetdh aetx trbai otf h elopr a dvice.
Profes[saonrM addo,xt hank fyoourj oinitnhget eamt oh elmpe witthh ep reapratioofnt his
manuscript.
Iw ouladl sloi kteo t hanPkr ofesRsoobre rHta ncocakn dh ist eamf ora llowimneg t ov istihte ir
laborataottr hyeD epartmoefnM ti crobioalnodgI ym munoloUgnyi,v ersoifBt ryi tiCsohl umbia,
fort hremeo nthsa,n df ors upervisainndag s sistwiintghm y bactermieamlb rane interaction
experiments.
Thisw orkw asm adep ossibblye t hefi nancisauplp orIt r eceivferdo mM eata ndW oolN ew
Zeala(nfdo rmeMrelayt NZi)n,t hef ormo fb otah d octosrcahlo larasnhdip pr ojefcutn ding. The
projewcats a lspoa rtiaflulnyd ebdy t heM asseUyn iversRietsye arFcuhn d( MURF)a,n d my
reseatrrcihtp oU BC wasf undebdyt heC .A lmaB akeTrr ust.
Thiwso rkw asm ade eabsyih eerl [pr eceivferdo mn umeropueso ple inctlhueId TiEnt ge chnical
stafefs,p eciAanlnley- MarJiaec ksaonnd M ikeS ahayaamn,d t hes taofffF eildiLnamgb P ackers,
who collecttheeds heep blfooormd y experimen[tasl.s roe ceivveadl uabhleel pfr omt he
undergradaunadft oer eign-isnttuedrentn htasat s sisotnev da riopuasr tosft hipsr ojeicntc,l uding
DaviHdo uldi(nlga boraetxotrrtyai copnr ocesAsd)i,S ugiar(tRoP -HPLCM)a,r iBeo uri(nc rude
extraMcItC s)a ndA ndreLwi st(epri lot-esxctarlaec tioIn rse)c.e ivaesds istafnrcoemA aron
Hick(sI nstiotfuV teet erinAanriym,a aln dB iomediSccaile ncteosp )r epatrheeT EM samples,
HortReseatroic mha gteh eT EM sampleasn,dA ssociPartoef esRsiocrh aHradv erkatmopi mage
theA FM samples.
Final[lw yo,u lldi kteot hanfka milaynd f rienwdhso h elpekde epm e santeh roughtohuiwtsh ole
procesOst.h epro stgraedssp,e ciCarlaliySg t,e pheRno,l anadn dA nnai,ta lwayhse lpetdok now
thatth erwee reo thewrhso s hartehd es ameo,r w orsed,i fficu-lGtoioedls u ctko y oua llR.e gan,
thanyko uf orc arinegn ougtho w adet hroutghhi tsh estiosf intdh es pellainndgg rammatical
mistak-easb esftr iewnhdo d oublaessa profo-readwehra,tm orec oulIda skf or?D adI,w ould
nevehra vme adei tt hifsa wri thotuhtes uppoorfty oua nd" TheA ndersTornu s-t"I t hinIkw as
theb esfte du ndergradsutautdeei nntt own.A ndf inally, thPeertaeer ren ,o tw ordtso d escribe
howm uchI a ppreciyaotu-e I l oofko rwatrodt hef utuwreew ilslp entdo gether.
I dedicattheit sh estiosm y mothewrh,o [ k noww ouldh aveb eenp roudH.e re ncouragement,
suppoarntdl ovwei lble w itmhe always.
v
TabloefC onetnts
TABLEO F CONTENTS
Abstract iii
........................... ............................................. ........................ ....................... .........
Acknwoledgeme.n.t.s. .....................................................................................v... ....... .............
Tabloef c onte.n.t.s. .........................................................................................v..i.. ..................
Lisotf fi gur.es.. ................................................................................................x..i..i.. ..............
Lisotf t abl.e..s. ................................................................................................x..v..i. ..............
Lisotf a bbrevia.t.i.o.ns.. ................................................................................x...v..i.i.. ... .........
i
Lisotf public.a.t.io.n..s.. ................................................................................................x...x .. .
CHAPTER1
PROJECITN TRODUCTION
1.1R easofno tr her esearch. . . .. . . 1
........ ..................... .......... .......... .............. . ............... ..... .....
1.2 Prjoecotb jectives . . . . ..2
................. ............. ............ ...................................... .......... ...... ...
CHAPTE2R
ANTIMIOCBRIAPLE PTIDELSIE TRATURE REVIWE
2.1I ntrotdiuocn . .. 4
..................... .......................... . ...................... ........................................
2.2A ntimiocbriaple ptides 5
.................................................... .............................................
2.2.A1ni mal antimicrobial .pep.tides . 5
.......... ..... ......................................... ............... ....
2.2.P2la natn timicrpoebpitaild es . ..... 11
................................................ ...... ...... ..............
2.2.M3ic robainatli microbial peptides . 15
.......................................................... ..............
2.3T hea nimailm mune system . 18
.................................. ........................................ .............
2.3.I1nn ate immunity . 19
............... .................................. ................. ............................. ..
2.3.A2da ptive immunity 24
............................................................................... ..............
2.3.R3ol e of antimpiecprtoibdiienaas nl i mailm munsey stems . 26
.................... ......... ....
2.4P otentaipapll icnasto ifao ntimicrpoebpitaild es 28
..........................................................
2.4.A1pp licatfiooran nst imibciraople ptides 29
.............................. .................................
2.4.P2os sibalpep licatfiooron vsi nbelo oda ntimibciraople ptides 31
...............................
2.5P urificatainodcn h aracitseartioofn animal antipmeipctriodbeisa l 35
..............................
2.5.T1ec hniquepsu rtioafy n dc hararcitseaen timicrpoebpitali des 36
................ ..............
2.5.L2iv estbolcookd a ntimibcirapole ptides . 39
.................. .......................... ..................
2.5.O3vi naen timicrpoebpitaild es . . 45
.......................... ............................. .....................
2.6M echaniosfma ctioofna nimaaln tiimcrobpieaplt ides 46
...............................................
2.6.T1ec hniqtuoed se termimneec hanisomfas c tioofna ntimicrpoebpitaild es 47
...........
vi
TabloefC ontents
2.6M.e2c hanisomfas c ti..o...n... ................................................................................. 48
2.6M.e3c haniosfma ctioofno vinaen itmicropbeipatli .d.es. .............5..1.. ................ ......
2.7M orphologcihcaanlgt eosm icrobcieallil nsd ucbeyda nimaanl timicrpoebpitail.d. 5es1
2.17 T.echniqtuoie nsv estimgoartpehl ooigcaclha nge...s... .........................................5 2
2.7M.o2r phologcihcaanlg. .e...s... ........... .................................................................... 53
2.7M.o3r phologcihacnaglei sn ducbeydo vien antimicrpoebpitail.d e.s. .......5..3.. ........ ..
2.8E ffecotf e nivronmenctoadnlii tosno na ctivoifat nyi maanlt imicrpoebpitail.d.. e....s. 5 4
2.18 T.echniqtuode est eirnmeeff ect enovfi ronmecnotnadli t.i...o...n....s.. .................. 54
2.8E.ff2e ctosfe nvoinrmetnaclo ndiiotn...s... .............................................................. 55
2.8E.ff3ec tso fe nvironmecnotnadli toinoo nvsi naeni tmicropbeipatli .d...e...s... ........ 56
2.9P il-ocstaleex tracotfia onni maanl timiclrp oebpitai.de.s. ..................5..7.. ......................
21.0C onclounss. i........................................................................ ...................................... 57
CHAPTE3R
MATERIALASN D METOHDS
3.1M ateriaanldms e thoudsse fdo rp eptipduer ciafiito.n.... .............................................. 59
31..C1r udeex tcrtaoin.. ............................................................................................... 59
3.21 G.ele lectroph..o...r...e..s...i...s... ......................................... ........................... ...... 60
3.31 G.elfi ltra.t..i....o...n... .. ..................... ................................................................... 61
3.41 C.ationeixcc-hancgherm oatogra.p...h..y... ........................................................... 61
31.5. Peptipduer ificautsiionHngP LC. ....................................................................... 62
3.61 R.adiadlifu fsiopnal teas sa.y. .........................6.2. ...................................................
31.7. Radidaffuils ionp lataes say MmIeCt ho...d... ...................................................... 63
3..18M asss pectros...c...o...p..y... ....................................... ......................................... 64
3.91 N.- termisneaqlu einnc.g... .................................... ................................................ 65
31.1.0Pe pitdceh aratceriisoa.n.t. ..................................................... .............................. 65
31..1A1 nalysoifps r oilne/ianirnge-rsiecqhu en..c..e...s... .............................................. 66
32. Matreiaalnsdm ethoduss efdo rm echanisomfa ctiotne s.t...s... ..................................6 6
32.1. Peptisdyen the...s..i...s... ........................ .............................. ............................... 66
32..2M icro-rbotdhil utiMoInC m etho...d... ........................................................... ...... 67
32..3Ci rcluadrci hroisspme ctros.c..o.p.y ..............................6..8.. ................................. .
32..4LP Sb indiansgs a...y.. .........................................................................................6 9
32..5Ou temre mbranpee rmeialbaitsio.n. .........................................6.9. .........................
32..6Cy toplasmmeimcbr andee polari..s..a..t....i...o...n.. ............... ............................... 70
vii
TabloefC ontents
32..7O ptidceanlsa intvdyi acbelcleo lu notvset mrie . .................7..1.. ............ ................
32..8P eptideb-iDnNd.Ai. n.g. ...................................7.1. ................................................
33. Materainamdle sto hduss etdoi nvestbiagcattecere imlaollr h poolgciaclh an.g..e.s7. 2. ..
3.13 T.rasnmisseiloencrrU tcrroons c.o.p.y.. ....................................7...2... .................... ...
3.3A.t2o rrUfcor crrUec rosc.o.p.y. ..............................7.3. ................................................
34. Materainamdl ehsto duss etdoa sstehsesefe fcotfc onidoinsot np eptaicdtei .v.i7.t4.y . ..
34.1. Satel ffec.t.s. ..........................................................................7...4... .......................
3.4C.a2t ieoffenc .t.s. ............................................74. ......................................................
34..3p He ffec.ts.. ......................................................7..5.. ..............................................
3.4T.e4m peraetffuerce.t s. ................................................................7..5.. ............... .......
34..5S ynergiefsfecttibsce twteeepsnet p t.i.d.e...s.. .....................................7..5... ...........
3.4S.y6en rgiesffetcitbces t wteeepsnet p taindcdeo smm ona ntib.i.o.t.i..c..s... ..7...6. . .....
3.5M atieasrl amnedt houdsesfd ort hpei lote-xstccratnalio oefa ntimicpreopbtiifraodlem s
oveib nlo.o.d.. ...............................................................................7..6... ........................
3.15 C.ruedxet rapcrtoico.en.s .s. .................................................7...6... ........................ ..
35..2M iniummi nhtiobrciyoc netnrtaio.n.s.. ..................................7...8... ..................... ...
3.5T.r3as nmisseeilcotnrm oinc ros.c.o..p..y... ..................................7..9.. ................... ....
3.45 Y.eilcda llcaiuton.s. ......................................................7...9... .......................... .......
CHAPTE4R
ISOLTAIOANN D CHARACTERITSIAOONF A NTIMCIROBAIL PTEIPDEFSRO M
OVINNEE UTORPHISL
41. Introd.u,..c . t.i.on.. ...................................................................................8..0.. ...............
4.2E xtractcirouandn eto ifm icsroolbu.it..ai.lo. n. ......................................8...1... ................
4.3P urificoafat niitomni crpoebpitauilsd iegsne gfil tl ratainodn - RHPPL..C.. ...8.3. ...........
4.4C haracatiteoronifO s aBaacn5vd a ri.a.n.t.s.. ................................8..7.. ..........................
4.5C haracitoeonrft i rsuantcOaatBea7dc.. 5. .......................................8...9... .......................
4.6C haractieoronifO saaBtalcl tarnudn c.a.t.e.s. ........................9...2.. ............ ..................
4.7M inuimmi hnibtioyrc ocnenttinrosa. ...............................................9...3... .....................
4.8S equeanncael oyfps riosel /riagnininpee-prti.ic.dh.e .s.. ..............................9..4.. ............
4.9P uriicifaotonfa ntiombiicparelp tuisdeicsna gt ieoxncihca cnhgmreao tgorapahnyRd P-
HPLC 98
................... ......................................................................................................
41.0O thperre diccattlehiidecd i.n.s. .................................1...3.0. ........................ ...................
4.1C1o cnlus.io.n.s. .........................................................................1...0...4. ...................... ...
viii
TabloefC onetnts
CHATPER5
SPECTRUMO FA CTIVIATNY DB ACTEIRALM EMBRANEI NTEARCTIONOSF
SYNTHTEICO VNIEC ATHELlCIDINS
5.1 Itnroduction 106
.............................................................................................................
5.2 Minuimmi nihbtiorcyo nctertanions 108
.........................................................................
5.3 Cricudlciahrri ossmp ectroscopy 111
..............................................................................
5.4L PSb indaisnsga y 112
....................................................................................................
5.5O utmeerm brpaenrem beialiiosna t 116
............................................................................
5.6 Cytoplmaesmmbircda enepl oariiosna t 119
.....................................................................
5.7 Kiclulr ves 123
...............................................................................................................
5.8 DNAb inindg . 124
................................................. .........................................................
5.9 Cocnulsiso n 126
.............................................................................................................
CHAPTERS
MORPOHLOGYO FB ACTERIACLE LLS TREATEDW ITHS YNTHTEICOV INE
CATHELlCIDINS
6.1 Itnrodiuocnt . 128
............................................................................................................
6.2 E.c oTlEiM r esults . .128
................................................................................................
6.3S .a ureTuEsMr esults. 129
............. ................................................................................
6.4 aureAuFsMm ethdoedvo eplemnt 132
S. .......................................................................
6.5S .a ureAuFMs r esu.lts . 135
... ................................. ........................................................
6.6 E. coAlFiM m ethdoedv eleonpptrm oblems 139
............................................................
6.7 Cocnulsions . . 141
................................................................................................ ...........
CHAPTE7R
FACTORASFF ECTINTGH EA NTIMIOCBRAILA CTIVIOTFY SNYTHTEICO VNIE
CATHELIlDICNASG AINTS E.C OLl0157:H7
71. Itnroduction 143
.............................................................................................................
7.2 Effecto fs alt . 144
...........................................................................................................
7.3 Effecto f meintosa.l 145
.................................................................................................
7.4 Efefcotfp H . .148
......................................................................................................... ..
7.5 Effectt eopmfe rature 150
...............................................................................................
7.6 Sy nerbgeyt wpeeepnt ides 151
........................................................................................
7.7 Syenrgbye twpeeenp taindkden so wannt ibiotics 153
.....................................................
ix
TabloefC onetnts
7.8C onclusions . .. 145
..........................................................................................................
CHAPTE8R
PILO-TSC ALEE XTRCATIONO FA NTIMRIOCBIAPLE PTIDEFSRO MO VINE
BLOOD
8.1I tnrodiuocnt 175
.............................................................................................................
8.2C ruedxet ractio.n. . . 185
...................................................................................................
8.3M inuimmi nhtioybrc ionecnttriaon.s . 161
.......................................................................
8.4T rasnmisseiloencm tirocosrnc o.p.y. ...........................1..36.. .........................................
8.5Y eilcda lcul.a.t.i..o..n.. ...........................................................1..56. ...............................
8.6I dnustriaplr-oscceaslse . .1 66
..........................................................................................
8.7C onclusions . 196
............................................................................................................
CHAPTE9R
CONCULSIONASND RECOMMENDATIONS
9.1S ummaorfry e secaroccnlhu s.i.on.s. ......................................................1...17... ........... .
92. Recomdmaetnifoornfu st urrees e.ar.c.h. ...................................1.. .47... .........................
9.3F niaclo nlcusn.i .o...........................................................................1..67. .......................
Reefrences 177
.........................................................................................................................
APPENDIX A1
RAW DATAA NDC ALCUALTIONFSR OM CHARACTERISTAIOSNT UDIES
A1 .M1a sSsp ecotftr hape u irfiHePdL Cp ea.k.s .....................................1.79. .......................
A1 2. Exacmpallec ulofac toiniofdne innctee rfrvoma plalsta es sraaywd at.a. .....2 .0.3. ..........
A1.3R awd atcaa,l actueMldI sC a9n5d% c onfideinnctee rfovrta hles MoIftC ehsp uriiefd
pepti.des .. . . 205
...............................................................................................................
APPENDAI2X
RAW DATAA NDC ALCULATIONFSRO M MECHANISMO FA CTIOSNT UDIES
A2.1R awd atfraom thmei crroo-tdbhi luMtIiCom ne th.o.d. ........................2..0...9... ............
A2.2E xampclael culoaftt himeoe naM nI Ca ndc onfidenicnet erfvorta ehlm se afrno m the
radw at.a. ....................................................2.1.1 ...........................................................
A2.3R awD atfraom thLeP Sb indaisnsg.a .y. ....................................2.....1... 2... ...................
A2.4C alculoafmtaIixao nnId o sf roLmP Sb indaisnsgra aydw a t.a. ........2.1.5. .......................
A2.5R awd atfrao m tohuteem re mbrpaenrea mbeiisliaotans s.a.y. ...................2...1...7. .........
x
Tableof C onetnts
A2.6A nalyosfvi asr iaonfcNNeP u pktead at.a.. .......................................2...1...9.. ...............
A2.7R awd atfrao m tohueti enrn meerm brdaenepl oarisaasts.iao.yn. ..........2.2..0.. .......... .....
A2.8A nalysos fvi ariaonfcDSeiC 3r5e ledaast..ea. . ..................2.2.2. ....................................
APPENDXI A3
RAW DATA CAANLDC ULATINOSF ROM EFFCETO FC ONDITOINSS TUDIES
A3.1R awd atoafM ICastd ieffresnatcl ontc enttirso.a n..................................2...2..3.. ..... .....
A3.2R awd atoafM ICastd fiefrecnatt cioocnne nttiroa.n.s. ..................2...2...4 . ....................
A3.3R awd atoafM ICastd ieffrepnHtv al.u.e...s.. ....................................2...2...6... .......... ....
A3.4R awd atoafM ICasft ehre iantgd ifterfoe nt taetmu.pre.ers.. ..............2.2.7. ....................
A3.5E xampclael culoaftt himeoe naM nI Ca ncd ofindenicnet erfvorta hlmese afrno mt he
radwa ta 228
...................................................................................................................
A3.6A nalyosfvi asr iaonfMc IeCd atfrao md fifreencto ndi.t.io.n.s. .....2.2.9. ........................
APPENDIAX4
RAWD ATA ACNAD LCLUATIONFSR OMP ILOT -SCALAEC TEIXOTSNRT UDIES
A4.1R awd atfrao mt hmei cro-dbirloutMthIi Cmo ent h.o.d. ..........2.31. ................................
A4.2E xampclael culoaftt himeoe naM nI Ca ndc ofindenicnet erfovrta hlmese afrno mt he
raw d.a.t.a. .......................................................................................2...3...2.. ................
A4.3C allcautoifto hnse e ltitvnegol cteiiso ff fdreientty poefbs l ocoedl .l.s.. ...........2.3.3. .....
APPEDNXIA5
PEE-RREVWIEEDP UBILCTAIONS
A5.1O viannet imicpreopbtiinadelewp s r:o dufrcotmas n agien-doul.sd.t .r.y... ........2...3..6. .
A5.2I slaotiaonndc haraacittoeonrfp i rsonle/iareg-icrnhcii anetilhcipdeipnt ifrdoemos v ine
neuotprh.i.l.s ........................................................................2..4...4.. ........ .....................
A5.3A ntimicraocbtiiavalin tdyb actemreimablar nei ntcetorina ofo vi-ndeerived
ctahelis.c .i.d.i.n...........................................................................2...5...4.... ...................
A5.I4vn estigoaftm ioornpl hoogicchaanlg etso aureiunsd ucbeydo vnie-derived
S.
antiimcropbeipatlui sdieTnsEg M aAnFdM. .....................................2.....5. .9.. .............
A5.5F actaoffercstg ti hnaen itmicraocbtiiaovlfoi vtiyn ee-ddce aretilhividciangsa iEn.cs otl i
0157:7. H............................................................................................26.6. ...................
xi