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Molecular Mechanisms of Apoptosis in Neutrophil

Granulocytes Compared to Septic Granulocytes


Dr. med. Ladislav Mica
A little dog looking for broken bubbles with the tip of his nose.
2
Table of Contents:
Abbreviation Index 5
Authors contribution to this !or" #
$%& Introduction '
1.1 The Toll-like receptors 12
1.2 The MAP-Kinases 13
1.3 The IAP-proteins 15
1. The !aspases 1"
1.5 The Proteasome 2#
1." The $cl-2 Proteins 21
(%& Material and Methods (5
2.1 Patients 25
2.2 Isolation and !%lt%re o& 'e%trophil (ran%loc)tes 25
2.3 *%anti&ication o& Apoptosis 2"
(%) *lo+ Assisted Cytometry of Toll,li"e -eceptors ( and ) in .MN (/
(%5 Analysis of MA.,0inases in .MN (1
2.5.1 +,perimental Protocol 2-
2.5.2 .estern/lot Anal)sis o& 'e%trophil MAP-Kinases 2-
(%/ Analysis of cIA.( .rotein in .MN (#
2.".1 +,perimental Protocol 20
2.".2 !aspase-3 Activit) Meas%rement 21
2.".3 Detection o& cIAP2 m2'A /) 2T-P!2 21
2.". .estern/lot Anal)sis o& cIAP2 Protein 3#
(%1 Analysis of 2cl,( .roteins in .MN 3&
2.-.1 +,perimental Protocol 3#
3
2.-.2 Anal)sis o& PM' Apoptosis 31
2.-.3 .estern-$lot Anal)sis o& $cl-2 Proteins 32
2.-. Detection o& m2'A o& $cl-2 Proteins /) 2T-P!T 33
(%# Statistical Analysis 33
3%& -esults 35
3.#.1 Apoptosis is 2ed%ced in PM' &rom 3eptic Patients 35
3.1.1 +,pression o& TL2-2 and TL2- on 4reshl) Isolated PM' 35
3.1.2 Li5and $indin5 and 2eceptor +,pression 3"
3%( MA.,0inases and .MN Apoptosis 3#
3.2.1 +&&ect o& 6er/im)cin on 'e%trophil Apoptosis 30
3.2.2 Participation o& Phosphatases in the 2e5%lation o& PM' Apoptosis 31
3.2.3 +&&ect o& MAP-Kinase Inhi/itors on LP3 and I4'--Mediated PM'
Apoptosis 1
3.2. Detection o& Phosphor)lated +2K and p30 MAP-Kinase in PM' 3
3%3 cIA.( and Activity of Caspase,3 in Neutrophil Granulocytes )/
3.3.1 LP3 Ind%ces cIAP2 m2'A and Protein in PM' "
3.3.2 LP3 Ind%ces 7/i8%itination o& !aspase-3 in PM' -
3.3.3 2ed%ction o& 3pontaneo%s and !D15-Ind%ced Apoptosis /) LP3 1
3.3. LP3 2ed%ces !aspase-3-like Activit) 5#
3%) The Correlation of Mcl,$ +ith Apoptosis of .MN 5$
3..1 +,pression o& $cl-2 m2'A and Protein in PM' 51
3..2 +,pression o& Mcl-1 m2'A and Protein in PM' 53
3..3 +,pression o& $A9 m2'A and Protein in PM' 5
3.. +,pression o& $id m2'A and Protein in PM' 5"

)%& 4iscussion 5#
.1 !ell Mem/rane: The 'eedle +ar 51
.2 The Phosphate !ascades: *%ick 2es%scitation Action "1
.3 cIAP2: $lockin5 the 2oad to Death "3
. $cl-2 Proteins: The $alanced 3%icide Machiner) "5
.5 The 3trate5) "-
." 6)pothetical Molec%lar Tar5ets "0
.- .ork to /e done -#
5%& -eferences 13
/%& 2oo"s #1
1%& 4atabases #1
#%& Curriculum 5itae ##
'%& Authors .ublications '$
1.1 ;ral presentations 13
Certificate of Competency 1##
5
Abbreviation Index
AI4: Apoptosis Ind%cin5 4actor< endon%clease
APA!6+ II: Ac%te Ph)siolo5) and !hronic 6ealth +val%ation
ATP: Adenosin Tri-Phosphate
$A9: $!L2-Associated 9 protein
$cl-2: $-!ell Le%kemia T)pe 2 Protein
$6-Domain: $cl-6omolo5) Domain
$id: $63 Interactin5 Domain Death A5onist
$I2: $ac%loviral IAP 2epeat
!A2D: !aspase 2ecr%itment Domain
!aspase: !)stein)l-Aspartate 3peci&ic Protease
!D1: !l%ster o& Di&&erentiation 1< co-receptor o& TL2 &or LP3
!D15: 4as< !l%ster o& Di&&erentiation 15
c-m)c: Master5ene< re5%lates Transcription< protoonco5ene
!)to-c: !)tochrome-c< intercristar) space protein< proapoptotic in c)tosole
Dia/lo: Direct IAP-$indin5 Protein =ith Lo= pI
DI!: Disseminated Intravasal !oa5%lopath)
+1: 7/i8%itin Activatin5 +n>)me
+2: 7/i8%itin Trans&er +n>)me
+3: 7/i8%itin Li5ase
+-$o, proteins: 7/i8%itin Li5ation Associated Proteins
+2K: +,tracell%lar 3i5nal 2e5%lated Kinase
4ADD: 4as-Associated Death Domain< !D15 ?Associated Death Domain
(M-!34 : (ran%loc)te-Macropha5e !olon) 3tim%latin5 4actor
I-$: Inhi/itor) 4actor-$
IAP: Inhi/itor o& Apoptosis Protein
"
I!+ : Interle%kin-1 !onvertin5 +n>)me< !aspase-1
I4'-: Inter&eron-
I(4-1: Ins%lin-like (ro=th 4actor-1
I2AK: Interle%kin-1 2eceptor-Associated Kinase
LP3: Lipopol)saccharide
MALP2: Macropha5e Activatin5 Lipoprotein 2
MAP-Kinase: Mito5en Activated Protein Kinase
Mcl-1: Ind%ced M)eloid Le%kemia !ell Di&&erentiation Protein
M6!: Ma@or 6istocompa/ilit) !omple,
MKK: Mito5en-Activated Protein Kinase Kinase
M;D3: M%lti ;r5an D)s&%nktion 3)ndrome
M;4: M%lti ;r5an 4ail%re
mT;2: Mammalian Tar5et o& 2apam)cin
M)D00: M)eloic Di&&erentiation 4actor 00
'4-$: '%clear 4actor-$
p30: Mito5en-Activated Protein Kinase
PA3: Pha5ophore Assem/l) 3ite
P$M!: Peripheral $lood Monon%clear !ell
PM': Pol)morph '%clear !ells
2I'(: +2< 7/i8%itin li5ase s%/-domain o& a protein
3I23: 3)stemic In&lammator) 2esponse 3)ndrome
3mac: 3econd Mitochondria-Derived Activator o& !aspase
3TAT3: 3i5nal Transd%cer and Activator o& Transcription 3
TA$: A$!-!hannel &or oli5opeptides< +ndoplasmic retic%l%m
Toll: A(ermanB C DgreatD< Drosophila scientistEs iron)
-
T2A4: T'4 2eceptor Associated 4actor
7/c: +2< 7/i8%itin con@%5atin5 en>)me
7/l : +3< 7/i8%itin li5ase
7F2A(: 7F 2adiation 2esistance-Associated (ene Protein
0
Authors contribution to this +or"
4A!3 anal)sis =as partiall) per&ormed /) the a%thor and L. 6Grter PhD.
2T-P!2 anal)sis =as totall) per&ormed /) the a%thor.
.estern-$lot anal)sis =as partiall) per&ormed /) the a%thor and L. 6Grter PhD.
!aspase-3 Activit) assa) =as completel) per&ormed /) the a%thor.
!ell c%lt%re and harvestin5 =as partiall) per&ormed /) the a%thor an L. 6Grter PhD and 7.
3teckhol>er $3.
Ac8%isition o& samples &rom patients =as partiall) per&ormed /) the a%thor and L. 6Grter PhD
and 7. 3teckhol>er $3.
3tatistical anal)sis =as per&ormed /) the a%thor and L. 6Grter PhD =ith Sigma Stat.
1
$% Introduction
Despite o& increased sec%rit) in tra&&ic and civil li&e pol)tra%ma remains the most o&ten ca%se
o& death %nder the a5e o& # )ears H1-I. The /rain in@%r)< s%dden /lood loss and penetratin5
or /l%nt tra%ma are the leadin5 in@%ries ca%sin5 immediate death a&ter tra%ma. Direct
mechanical &orces on the or5anism ca%se primar) tiss%e dama5e. The dama5ed tiss%e s%&&ers
lo= s%ppl) =ith o,)5en leadin5 to dama5e increase and to %nspeci&ic stim%lation o&
ne%trophil 5ran%loc)tes HPM'I and monoc)tes HP$M!I /) released mediators &rom the tiss%e
H1-< "-1#I. ;nce< over helmin5 the or5anism =ith anti5enic load the cells o& the &irst line o&
de&ense< PM' and P$M!< are s)stemicall) activated leadin5 to s)stemic in&lammation. This
s)stemic in&lammation =as de&ined in 1111< thro%5h the consens%s con&erence o& the
American !olle5e o& !hest Ph)siciansJ3ociet) o& !ritical !are Medicine HA!!PJ3!!MI< as
s)stemic in&lammator) response s)ndrome H3I23I H5I HTa/le 1.I.
1#
Temperat%re K 30<0L! or M 3"<0L! 3.
'%m/er o& le%koc)tes K 12<###Jmm
3
or M ###Jmm
3
or 1#N @%venile ne%trophil 5ran%loc)tes
.
$reathin5 rate K 2#Jmin< respectivel)< 6)perventilation =ith decrease
o& the arterial !;
2
partial press%re HPa!;
2
I %nder 32 mm65.
2.
6eart rate K 1# /pm 1.
4or the de&inition o& 3I23< t=o or more parameters m%st /e &%l&iled. 3epsis is de&ined
as 3I23 =ith detection o& /acteremia or /acterial &oc%s. H5I
Table $: !linical parameters o& the s)stemic in&lammator) response s)ndrome H3I23I
Temperat%re K 30<0L! or M 3"<0L! 3.
'%m/er o& le%koc)tes K 12<###Jmm
3
or M ###Jmm
3
or 1#N @%venile ne%trophil 5ran%loc)tes
.
$reathin5 rate K 2#Jmin< respectivel)< 6)perventilation =ith decrease
o& the arterial !;
2
partial press%re HPa!;
2
I %nder 32 mm65.
2.
6eart rate K 1# /pm 1.
4or the de&inition o& 3I23< t=o or more parameters m%st /e &%l&iled. 3epsis is de&ined
as 3I23 =ith detection o& /acteremia or /acterial &oc%s. H5I
Table $: !linical parameters o& the s)stemic in&lammator) response s)ndrome H3I23I
'e%trophil 5ran%loc)tes are cells &rom the monopoetic cell line capa/le in pha5oc)tosis and
%nspeci&ic imm%ne response HInnate ImmunityI. $acterial and viral deca) prod%cts stim%late
speci&ic receptors on cell%lar s%r&ace a lead to pro-in&lammator) activation. D%rin5 3I23
PM' are acc%m%lated in di&&erent or5ans leadin5 to secondar) dama5e o& the or5ans /)
endothelial dama5e and disseminated intravasc%lar coa5%lopath) HDI!I< ca%sin5 necrosis and
apoptosis o& parench)mal cells /) de5ran%lation o& PM'. This h)per in&lammation ca%ses
M;D3 Hm%lti or5an d)s&%nction s)ndromeI and M;4 Hm%lti or5an &ail%reI H11-15I. $oth the
severit) o& tra%ma and the anti5enic load as =ell as the over &loodin5 /) pro-in&lammator)
c)tokines are leadin5 to this h)per stim%lation o& PM'.
*i6ure $: !ell%lar si5nalin5. A li5and /inds to a receptor. The interaction /et=een these t=o
proteins leads to con&ormational chan5es and the activation o& second messen5er s)stems
carr)in5 the messa5e H;rderI into the n%cle%s. In the n%cle%s the si5nal Hm%lti protein
comple,I res%lts in the activation o& transcription. The ne=l) s)nthetised proteins res%lt in an
ans=er o& the cell< A: intracell%lar chan5es< $: /ehavior.
11
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7nder ph)siolo5ic conditions PM' are distri/%ted in a circ%latin5 pool and in a mar5inal pool
adherin5 on veno%s =alls. 3tress hormones ca%se the set &ree o& the mar5inal pool o& the
veno%s =alls. 3tress-conditions also stim%late the set &ree o& immat%re ne%trophil
5ran%loc)tes &rom the /one marro= Hle&t shi&tI. The circ%latin5 PM' have onl) a short hal&
li&espan o& a/o%t 1" ho%rs< a/o%t "#N die a&ter this period o& time H1-I. The death o&
ne%trophil 5ran%loc)tes is a =ell controlled process called Apoptosis HGreek: OPQPRSTUV
falling leafI. This process =as &irst descri/ed /) Kerr and .)lie in the earl) 11-2 H1"I and
called shrinkage necrosis, descri/in5 the microsopical characteristics o& the cell d%rin5
apoptosis. The main characteristics descri/ed =ere cell%lar shrinka5e< &ormin5 o& /%//les in
the cell%lar mem/rane H/le//in5I and kariorrhe,is. The 5ro=in5 &ield o& molec%lar /iolo5)
led to a discover) o& a plent) o& &actors at the /e5innin5 o& 111#. The discover) o& death as a
=ell de&ined molec%lar process in the cell chan5ed the vie= on cell%lar li&e and centered the
process o& diein5 into the e,istence.
In this =ork =e =ill in an endoto,in model anal)>e the si5nalin5 thro%5h the Toll-like
receptors in the o%ter cell%lar mem/rane. This name =as 5iven to a 5ro%p o& proteins /) the
societ) o& the scientists o& the species Drosophila melanogaster. ;nce li5and /inds to a
receptor< the res%lts is an intracell%lar si5nal< activatin5 a second messen5er s)stems and
leadin5 to a cell%lar response. In the case o& ne%trophil 5ran%loc)tes the si5nalin5 path=a) o&
MAP-kinases HMito5en Activated PhosphatasesI =as anal)>ed. Di&&erent &actors are
deactivated /) the proteasome. The proteasome is the intracellular trash can! o& the cell.
All intracell%lar proteins< once ta55ed =ith 7/i8%itin< are destined to /e destro)ed /) the
proteasome. The interaction /et=een cIAP2 and !aspase-3 =as anal)>ed at the level o& the
proteasome. The role o& the most important mem/ers o& $cl-2 &amil) proteins =as anal)>ed.
The $cl-2 proteins consist o& a pro- and anti-apoptotic 5ro%p actin5 like the complementar)
s)stem a5ainst the o%ter mitochondrial mem/rane.
12
$%$ The Toll,li"e receptors
The &irst line o& de&ense in imm%nolo5) consists o& ne%trophil 5ran%loc)tes HPM'I and
monon%clear cells HP$M!I. The patho5ene reco5nition is initiated /) receptors o& the innate
imm%nit). These receptors =ere primar) reco5ni>ed in Drosophila melanogaster and =ere
called T;LL-receptors. 3imilar receptors =ith the same &%nction =ere discovered on h%man
pha5oc)tin5 cells and called Toll-like receptors H10-2#I. These receptors are like antennas
reco5ni>in5 patho5ens o& microor5anisms and vir%ses. ;nce activated /) a patho5en the
si5nal res%lts in prod%ction o& pro-in&lammator) c)tokines and the inhi/ition o& apoptosis in
PM'. To date %p to 1# di&&erent receptors have /een characteri>ed =ith di&&erent li5and
speci&ities H21< 22I. In this st%d) =e &oc%sed on the Toll-like receptor 2 and < /oth on the
s%r&ace o& PM'. The TL22 has /een &o%nd to si5nal &or the to,ins o& 5ram positive /acteria
like MALP2< macropha5e activatin5 lipoprotein 2 &rom "ycoplasma fermentans H23< 2I. The
TL2 to5ether =ith !D1 is activated /) LP3< lipopol)s)ccharide &rom 5ram ne5ative
/acteria like #scherichia coli H23< 2I.
A red%ced c)tokine response on repetitive stim%lation =ith LP3 has alread) /een sho=n. This
e&&ect called endoto,in tolerance H25I is kno=n &or man) )ears /%t the molec%lar mechanism
has not /een el%cidated )et. The h)pothesis o& do=n re5%lation o& TL2 d%rin5 endoto,in
tolerance =as s%pported /) a m%rine model. In contrast Medvede= reported incrased TL2
receptors on LP3 tolerant monoc)tes H2"I. 3eemin5l) a parado,on< the mechanisms to=ard
endoto,in tolerance seems not to ori5inate &rom cell%lar s%r&ace. There&ore =e investi5ated in
this st%d) the e,pression d)namics o& TL22 and TL2 %pon stim%lation =ith speci&ic li5ands.
.e =ill sho= an %pre5%lation o& TL22 and TL2 on PM' &rom health) individ%als %pon
stim%lation =ith LP3< MALP2 and patients =ith sepsis.
13
*i6ure (: The di&&erent Toll-like receptors are depicted on the top. This innate imm%nit)
s)stem reco5ni>es di&&erent /acterial and viral prod%cts. ;nce /o%nd to the receptor the
intracell%lar si5nal res%lts in a phosphor)lation o& M)D00< a si5nal transd%cer. M)D00 caries
the si5nal to I2AK1 and I2AK res%ltin5 in the activation o& the MAP kinases path=a) Hsee
1.2I. In this st%d) =e &oc%sed onl) on TL22 and TL2 =ith the li5ands MALP2 and LP3.
Hhttp:JJ===.5enome.@pJd/5et-/inJ===W/5etXpath=a)Yhsa#"2#I
$%( The MA.,"inases
The activation o& Toll-like receptors /) their li5ands Hin this st%d) LP3 &or TL22 and MALP2
&or TL2I leads to a si5nal transd%ction across the cell%lar mem/rane and the activation o&
M)D00 as the &irst pla)er in a hi5hl) diverse phosphor)lation cascade. This protein called
M)D00 represents a MAPK ver) %pstream o& an activation si5nal< in mammalian cells
%s%all) pro-in&lammator)< pro-mito5enic si5nals &rom c)tokine receptors< si5nals &rom
inte5rins via 2as and 3rc and si5nals via 2hoJ2ac s)stem. These si5nals normall) do not
occ%r as simple lonel) stim%li /%t the cell is normall) e,posed to an orchestra o& di&&erent
stim%li activatin5 these MAP-kinases. The =a) o& the activation o& MAP-kinase path=a) is
ver) diverse and leads via di&&erent steps Hsee &i5%rere 3I to the activation o& +2K Hp2JI<
p30 or Z'K Hc-Z%n terminal kinaseI H2"I. In this st%d) =e &oc%sed onl) on the +2K< p30 and
Z'K kinases representin5 the &inal path o& the MAP-kinase path=a). Altho%5h< the activation
o& Z'K %pon stim%lation =ith LP3 in PM'< has not /een sho=n )et H2-I.
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=ith its li5and the si5nal is /ein5 transd%ced via M)D00< I2AK< T2A4< TA$ and TAK to
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activated %nder apoptotic conditions H20I. The M+K1 activated +2K kinase has also /een
sho=n to re5%late cell%lar s%rvival in PM' &rom health) donors a&ter stim%lation =ith LP3.
6o=ever< onl) traces =ere &o%nd in the literat%re and the role o& MAP-kinases in PM'
remained to /e disc%ssed =ithin the disease o& sepsis.
$%3 The IA.,proteins
Inhi/itor o& Apoptosis proteins HIAPI =ere &irst identi&ied in /ac%lovir%ses. All IAP-proteins
contain several moti&s called $I2-domains< /ac%loviral IAP repeats. An IAP protein normall)
consists o& 1-3 $I2 domains. The $I2 domains allo= the IAP to /ind speci&icall) to proteases
and to inhi/it them. The $I2 domains o& some IAPs allo= them to /ind to caspases Hsee
chapter 1.I< that are the main e,ec%tioners in apoptotic cell death. The inhi/ition o& these
proteases< caspases< provides the simplest e,planation &or the inhi/ition o& cell death /) IAPs.
6o=ever< another domain has /een recentl) sho=n to participate in apoptotical death
re5%lation H31I. The 2I'(-domain is a/le to /ind mono%/i8%itin molec%les and to trans&er
these %/i8%itins to a s%/strate. ;nce covalentl) /o%nd to l)sine resid%es< the tar5et is destined
to /e de5radated /) the proteasome. 7/i8%itination is an active process cons%min5 ATP and
re8%irin5 the +-$o, proteins. The +-$o, proteins are s%/divided into three 5ro%ps. The +1
proteins provide the initial reaction /) activatin5 the %/i8%itin molec%le. The molec%le is
linked on the !-terminal 5l)cine car/o,)late to a 36-5ro%p o& the activatin5 en>)me< this step
is ATP cons%min5. In a transac)lation reaction< the 7/i8%itin is trans&erred &rom +1-7/ to a
c)steine 36 =ithin the active site o& the con@%5atin5 en>)me +2 and &orms +2-7/. The +3
en>)mes are the direct %/i8%itin li5ases H7/lI reco5ni>in5 the s%/strate and /rin5in5 +2 and
the s%/strate to5ether. This res%lts in an attachment o& a pol)%/i8%itin chain and s%/se8%ent
de5radation o& the tar5eted protein in the proteasomal path=a) Hsee chapter 1.5I. In this st%d)
the +2J+3 protein is represented /) the cIAP2 protein< =hich once activated har/ors an
%/i8%itin in the 2I'( domain< res%ltin5 in cIAP2-7/.
1"
*i6ure ): 7/i8%itination path=a)< e,ponential activation o& %/i8%itin-li5ases H7/lI /) less
%/i8%itin con@%5atin5 en>)mes H7/cI. Man) s%/strates H3I can /e %/i8%itinated /) one
%/i8%itin li5ase. ;n the ri5ht side the molec%le o& cIAP2: the 2I'( domain to5ether =ith the
!A2D domain are compara/le to +2 and +3 proteins. HKra%s< $iochemistr) o& 3i5nal
Transd%ctionI
A 2I'( domain trans&ers the %/i8%itin resid%es =itho%t additional reco5nition o& +3< this role
is pla)ed /) the $I2 domains. cIAP2 reco5ni>es =ith the $I2 domain the activated caspase-
3< =hich is one o& the main e,ec%tioners o& the apoptotic path=a)< a &inal common path.
1-
2I- 2I- 2I-
-ING
2I- 2I- 2I- CA-4
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2I- -ING
2I-
2I- 2I- 2I-
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2I- 2I- 2I- CA-4
-ING
2I- -ING
=IA.
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Survivin
Apollon
2I- 2I- 2I- 2I- 2I- 2I-
-ING -ING
2I- 2I- 2I- 2I- 2I- 2I- CA-4 CA-4
-ING -ING
2I- 2I- -ING -ING
2I- 2I-
2I- 2I- 2I- 2I- 2I- 2I-
2I- 2I-
2I- 2I- 2I- 2I- 2I- 2I- CA-4 CA-4
-ING -ING
2I- 2I- -ING -ING
=IA.
I7.(
cIA.$
cIA.(
M7,IA.
NAI.
Survivin
Apollon
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cIA.(
*i6ure 5: Di&&erent mem/ers o& h%man IAP protein &amil). 2I'(: 2I'( domain< %/i8%itin
li5ase. !A2D: !aspase recr%itment domain< /indin5 to !aspases. $I2: $ac%loviral IAP
repeats< en>)me inhi/ition. The di&&erent $I2 domains are a/le to inhi/it di&&erent en>)mes<
especiall) the caspases d%rin5 apoptosis. In this st%d) =e e,amined the e,pression pattern o&
cIAP2.
The activated caspase-3 is competitivel) inhi/ited /) $I23 and the 2I'( domain ma) attach
%/i8%itin molec%les to the activated caspase-3 modi&)in5 this en>)me to /e de5radated /) the
proteasomal path=a). There&ore< =e investi5ated the role o& cIAP2 in ne%trphil 5ran%loc)tes
thro=in5 a small li5ht to apoptotic resistance o& PM' %pon pro-apoptotic stim%li d%rin5
sepsis.
$%) The Caspases
10
The caspases Hc)stein)l-aspartate speci&ic proteaseI are directl) responsi/le &or the
/iochemical chan5es d%rin5 apoptosis in a d)in5 cell. The caspases /elon5 to a 5ro%p o&
intracell%lar en>)mes e,pressed as >)mo5enes. These en>)mes share the speci&it) &or
aspartate &rom =hich the name is derived &rom. D%rin5 apoptosis these >)mo5enes are
processed to active en>)mes /) an activation cascade and de5rade intracell%lar str%ct%res and
proteins H3I. The &irst discovered caspase< no=ada)s caspase-1< =as I!+ Hinterle%kin-1
convertin5 en>)meI in 1112 H33I. This en>)me =as responsi/le &or activatin5 limited
proteol)sis o& the pro-in&lammator) c)tokine interle%kin-1. 3everal other proteins =ere
se8%enced =ith I!+-sharin5 homolo5ies responsi/le &or the e,ec%tion o& cell death and
in&lammation. A/o%t 15 mem/ers o& the caspase &amil) =ere identi&ied %ntil no=\ caspase-1
seems to pla) onl) a role in the di&&erentiation o& keratinoc)tes H35I. !aspase-12 is e,pressed
in homo sapiens onl) as a catal)ticall) inactive &orm and is not represented in the list o&
h%man caspases. The implementation o& caspase-12 is disc%ssed in the in&lammator) path=a)
in a m%rine model H3"I.
All caspases consist o& a /i55er Hp2#I< smaller Hp1#I s%/%nit and a prodomain =itch varies in
si>e and &%nction. These >)mo5enes have to /e activated /) limited proteol)sis /et=een p2#
and p1#< normall) the prodomain is also removed. This activation res%lts in the &ormation o& a
heterotetramere H2p2# Y 2p1#I =ith t=o catal)ticall) active centers. The center speci&icall)
reco5ni>es the cleava5e site /) a se8%ence o& &o%r amino-acids. Aspartate is at the &irs
position< the =hole en>)me &amil) can also /e distin5%ished into 5ro%ps accordin5 to the
optimal cleava5e peptide se8%ence H3I.
T=o path=a)s o& the caspase-activation are kno=n< the e,trinsic receptor mediated path=a)
and the intrinsic mitochondrial path=a). The activation o& a death receptor leads to an
intracell%lar activation o& an initiator caspase H%s%all) caspase 0 or 1#I H3-I. The initiator
caspase activates do=nstream events leadin5 to the activation o& e&&ector caspases and the
e,ec%tion o& death. The intrinsic path=a) is characteri>ed /) the activation o& the pro-
11
apoptotic $cl-2 s)stem leadin5 to pore-&ormin5 in the o%ter mitochondrial mem/rane and the
leaka5e o& c)tochrome-c and other pro-apoptotic &actors Hsee chapter 1."I. !)tochrome-c
to5ether =ith Apa&-1 &orms a heteropentamere called the apoptosome H#I. The apoptosome
activates caspase-1 =hich leads do=nstream to the activation o& caspase-3 as the main
e&&ector caspase< and the e,ec%tion o& cell death.
In this st%d) =e &oc%s onl) on caspase-3 representin5 the &inal common path o& an possi/le
e,trinsic or intrinsic activation.
*i6ure /: Depicted are assorted h%man caspases involved in apoptotic process. All caspases
have a /i5 p2# and a small p1# s%/%nit< additional oli5omerisation %nits HD+D: Death
+&&ector Domain and !A2D: !aspase 2ecr%itment DomainI have onl) the initiator caspases.
Arro= indicates the cleava5e re5ion o& activation. The asterisk sho=s the appro,imate
position o& the active c)stein. H===.e,pas).chI
2#
6%man caspases in apoptosis
p$& p(& 4;4 4;4
Caspase,#
p$& p(& 4;4 4;4
Caspase,$&
p$& p(& CA-4
Caspase,'
p$& p(& CA-4
Caspase,(
p$& p(&
Caspase,3
p$& p(& Caspase,/
p$& p(& Caspase,1
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6%man caspases in apoptosis
p$& p(& p(& 4;4 4;4 4;4 4;4
Caspase,#
p$& p(& p(& 4;4 4;4 4;4 4;4
Caspase,$&
p$& p(& p(& CA-4 CA-4
Caspase,'
p$& p(& p(& CA-4 CA-4
Caspase,(
p$& p(& p(&
Caspase,3
p$& p(& p(& Caspase,/
p$& p(& p(& Caspase,1
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$%5 The .roteasome
The proteasome is a m%ltien>)mic comple, &ormin5 a t%nnel to allo= optimal access o& the
en>)mes to de5rade a pol)%/i8%itinated protein. A&ter the activation or the dama5e o& a
protein its tertiar) str%ct%re chan5es and opens a speci&ic %/i8%itination si5nal< the de5ron
se8%ence H30I. This se8%ence o& aminoacids is responsi/le &or the /indin5 o& an +2 protein
and the attachment o& a pol)%/i8%itin chain. The pol)%/i8%itinated protein is reco5ni>ed /)
the 113 re5%lator) domain o& the proteasome and /o%nd to it to allo= ATP-dependent
%n&oldin5 o& this protein. The %n&olded protein is dra=n into the proteasome H2#3 protease
comple,I and de5raded in aminoacids and oli5opeptides. The oli5opeptides are %sed to /e
presented on M6! I comple,es andJor to /e rec)cled. The proteasomal path=a) is the
dea5radation =a) &or intracell%lar proteins H30< Lodish< Molec%lar !ell $iolo5)I.
The proteasome< seemin5l) the intracell%lar trash can< is also involved in transcription
re5%lation. Di&&erent inhi/itors o& transcription &actors He.5. I$I H31I are %pon activation
de5raded /) the proteasome and the transcription &actor is set &ree to transd%ce into the
n%cle%s. In the n%cle%s the transcription is terminated /) de5radation o& the transcription
&actor /) the proteasomal path=a). The primaril) attached pol)%/i8%itin chain is
disconnected and rec)cled HLodish< Molec%lar !ell $iolo5)I.
The participation o& the proteasome has /een recentl) sho=n< =here the inhi/ition led to
inhi/ition o& the anti-apoptotic LP3-e&&ect< connectin5 this event =ith activation-inhi/ition o&
'4-$. 7s%all) I-$ is /ein5 pol)%/i8%itinated and de5raded /) the proteasome and '4-$
ma) translocate into the n%cle%s to activate transcription o& tar5et 5enes. I& the de5radation o&
I-$ is inhi/ited< the '4-$ inhi/ition persists and the transcription does not take place
H===.5enome.@pI. $oth< the initiation and the termination o& transcription are re5%lated /)
the proteasomal mechanism. In this st%d) =e scho= an ind%ction o& cIAP2 protein and
21
caspase-3-activit) dependin5 on the proteasome. In PM' onl) poor data are p%/lished to this
do%/le-ed5ed theme.
*i6ure 1: A pol)%/i8%itinated protein is reco5ni>ed /) the re5%lator) comple,. The protein is
%n&olded in an ATP dependent manner and dra=n into the proteasome. The m%ltimeric
comple, c%ts the protein into aminoacids and oli5opetides leadin5 to the deactivation o& the
protein. Hhttp:JJ===.5enome.@pJd/5et-/inJ===W/5etXpath=a)Yhsa#3#5#I
$%/ The 2cl,( proteins
The $cl-2 protein class is a =idel) spread class o& proteins and can /e s%/divided in t=o
s%/5ro%ps. The proaoptotic 5ro%p consists o& several mem/ers similar the anti-apoptotic
5ro%p< the cl%e o& the pro-apoptotic activit) is the a/sence o& the anti-apoptotic $6 domain
22
.rotein
.
r
o
t
e
i
n
L
i
d
L
i
d
113 2e5%lator) !omple,
2#3 Protease !omple,
2ec)clin5 o& %/i8%itin
monomeres
;li5opeptides
Pol)%/i8%itin chain
.rotein
.
r
o
t
e
i
n
L
i
d
L
i
d
113 2e5%lator) !omple,
2#3 Protease !omple,
2ec)clin5 o& %/i8%itin
monomeres
;li5opeptides
Pol)%/i8%itin chain
o& these proteins H#I. To 8%antitativel) reali>e the intracell%lar role o& these proteins =e
co%ld compare them to the complementar) s)stem o& /lood plasm. In the $cl-2 s)stem =e
have t=o di&&erent activatin5 =a)s. The e,trinsic path=a) is characteri>ed /) /indin5 o&
li5and to a receptor He.5. !D15L and !D15I initiatin5 limited proteol)sis He.5. $ID to t$IDI
to set a $63 domain &ree to start the pro-apoptotic $cl-2 process.
*i6ure #: $cl-2 proteins are s%/divided into t=o 5ro%ps. The $cl-2 mem/ers are anti-
apoptotic and have an o/li5ator) $6 domain H$cl-2< Mcl-1I. A mem/rane anchor is
&ac%ltative. The $63 proteins do not have a $6 domain< the proteins are characteri>ed /)
the pro-apoptotic $63 domain H$ID< $A9I. A mem/rane anchor is also here &ac%ltative.
3ome mem/ers have onl) the $63 domain and are called $63-onl) proteins H$imI.
H===.e,pas).chI
23
2A$ 2A( 2A3
Antiapototic
2A$ 2A( 2A3
.roapototic
Mem/rane anchor H&ac%ltativeI
2A3
2A) 2A$ 2A$ 2A( 2A3 2A3
Antiapototic
2A$ 2A$ 2A( 2A3 2A3
.roapototic
Mem/rane anchor H&ac%ltativeI
2A3 2A3
2A)
Antiapoptotic:
2cl,(
$cl-,l
$cl-=
DivaJ$oo
$&l-1
$okJMtd
Mcl,$
$ak
.roapoptotic:
6rk
$ad
$ik
'o,a
$cl-,3
$im
$'IP3
2id
'i,
2ax
P%ma
Antiapoptotic:
2cl,(
$cl-,l
$cl-=
DivaJ$oo
$&l-1
$okJMtd
Mcl,$
$ak
.roapoptotic:
6rk
$ad
$ik
'o,a
$cl-,3
$im
$'IP3
2id
'i,
2ax
P%ma
Table (: Depicted are the Mem/ers o& the $cl-2 protein &amil). +,amined proteins in this
st%d) are indicated in /old letters. ;n the le&t side is the anti-apoptotic 5ro%p and on the ri5ht
side is the pro-apoptotic 5ro%p. H===.e,pas).chI
This &ree $63 domain leads to the activation o& other do=n steam located pro-apoptotic $cl-2
mem/ers and to an assem/l) o& $A9 in the o%ter mitochondrial mem/rane.
*i6ure ': 3ho=n is the activation cascade o& the $cl-2 s)stem s%/divided into the e,trinsic
and the intrinsic path=a).
#$trinsic %athway: The /indin5 o& a li5and to a receptor res%lts in the activation o& caspases
Hcaspase 0 or 1#I< the activated caspases activate $ID /) limited proteol)sis to t$ID
Htr%ncated $IDI. t$ID activates $A9 =hich pol)merises %pon /indin5 to cardiolipin and
FDA( and &orms channels into the o%ter mitochondrial mem/rane. 'o=< di&&erent pro-
apoptotic &actors are released to enhance the apoptotic process.
2
!ell mem/rane
4ADD
!D15
Caspase,#B $&
$ID t$ID
2A=
2A=
2cl,(
AI*
Smac94iablo
Cyto,c
2im
2im
Mcl,$
Mitochondrion
Microt%/%li
;xtrinsic path+ay Intrinsic path+ay
!ell mem/rane
4ADD
!D15
Caspase,#B $& Caspase,#B $&
$ID t$ID
2A=
2A=
2cl,( 2cl,(
AI* AI*
Smac94iablo Smac94iablo
Cyto,c Cyto,c
2im
2im 2im
Mcl,$ Mcl,$
Mitochondrion
Microt%/%li
;xtrinsic path+ay Intrinsic path+ay
Intrinsic pathway: !ell%lar stress leads to /reak do=n o& microt%/%li or other intracell%lar
str%ct%res 5%arded /) $63-onl) proteins. These $63 proteins are no= released and inhi/it
the anti-apoptotic $cl-2 or Mcl-1. This inhi/ition shi&ts the cell to=ards apoptosis.
The oli5omerisation o& $A9 in the o%ter mitochondrial mem/rane leads to pore &ormin5 and
to set &ree o& components &rom the intercristar) space Hc)tochrome c< AI4< smac< Dia/loI.
These intercristar) &actors are responsi/le &or activation o& the apoptotic path=a) /) caspase
activation Hc)tochrome c< smac< Dia/loI H1I and D'A de5radation HAI4I H1I. The intrinsic
path=a) consists o& man) $63-onl) mem/ers co-associated =ith c)to-skeletal str%ct%res He.5.
microt%/%li< endoplasmic retic%la< 5ol5i apparat%s and n%clear mem/raneI.
These $63-onl) proteins He.5. /im< ni,< /ikI are set &ree /) dist%r/ances o& these internal
str%ct%res leadin5 to the activation o& $A9 H1I. In contrast the anti-apoptotic $cl-2 and Mcl-
1 inhi/it the assem/l) o& $A9 to &orm pores in the o%ter mitochondrial =all Hsee &i5%re 1I.
The speci&ic interaction /et=een the di&&erent $cl-2 mem/ers remains still %nclear. 6o= this
hi5hl) comple, intracell%lar complement system! precisel) =orks is s%/@ect o& &%rther
st%dies.
There&ore< =e anal)sed the e,pression pattern o& $id< $A9< $cl-2 and Mcl-1 in PM'. $id
stands &or receptor mediated He,trinsicI $cl-2 dependent apoptosis and Mcl-1 inhi/its $63-
onl) proteins &rom the intrinsic path=a).
25
(% Material and Methods
(%$ .atients
The st%d) pop%lation consisted patients admitted to the 3%r5ical Intensive !are 7nit o& the
7niversit) 6ospital ]^rich. The Ac%te Ph)siolo5) and !hronic 6ealth +val%ation HAPA!6+
III score at admission =as 23.- _ 5." Hran5e 15?3"I points H2I. 3evere sepsis =as dia5nosed
i& all criteria o& 3I23< evidence o& t=o or more or5an d)s&%nctions< and a proven septic &oc%s
=ere present H3I. In&ection =as d%e to pne%monia< peritonitis< menin5itis< or a/ort. Isolated
microor5anisms incl%ded mostl) 5ram-ne5ative /acteria< /%t also 5ram-positive /acteria in a
ratio 3:1. The overall mortalit) o& these septic patients =as 5N< these patients died d%e to
septic m%ltiple or5an &ail%re. The 5ro%p o& health) individ%als =as compara/le =ith that o&
patients =ith sepsis =ith re5ard to a5e and se,. All patients =ere enrolled into this st%d)
%nder in&ormed consent 5%idelines approved /) the 6%man +thical !ommittee o& the
7niversit) o& ]%rich.
(%( Isolation and Culture of Neutrophil Granulocytes
4resh heparini>ed /lood &rom health) individ%als HnC-I =as dil%ted 1:1 HvJvI =ith 6$33
HInvitro5en !orp.< Paisle)< 7nited Kin5domI< la)ered over 4icoll-6istopa8%e
`
H6istopa8%e
`
-
1#--< 3i5maI< and centri&%5ed at L!< 0## , 5 &or 2# min. The er)throc)teJ5ran%loc)te
containin5 pellet =as dil%ted in 1:1# HvJvI ammoni%m chloride-+DTA H155 mM '6

!l< 1#
mM 'a6!;
3
< 11 mM +DTA< p6 -."I and stored &or 3# min on ice &or the l)sis o&
er)throc)tes as descri/ed previo%sl).H-"I A&ter centri&%5ation the ne%trophils =ere
ad@%sted to a densit) o& 1 , 1#
"
cellsJmL in 2PMI 1"# HInvitro5en !orp.< Paisle)< 7nited
Kin5domI containin5 1# N 4!3< (entam)cin HInvitro5en !orp.I and (l%tama, HInvitro5en
2"
!orp.I H#.1 m5JmL eachI in pol)prop)lene 4alcon
`
t%/es H$ecton Dickinson $asel<
3=it>erlandI and c%lt%red at 3- L! and 5 N !;
2
&or the times indicated.
(%3 Cuantification of Apoptosis%
Determination o& apoptosis and secondar) necrosis %tili>es the hi5h a&&init) o& Anne,in-F &or
phosphatid)lserine =hich is e,posed on the s%r&ace o& apoptotic cells H-I. A&ter
inc%/ation period o& 1"-22 ho%rs PM' =ere =ashed =ith phosphate-/%&&ered saline<
res%spended in /indin5 /%&&er H1# mM 6+P+3J 'a;6< 1# mM 'a!l< 2.5 mM
!a!l
2
< p6 -.I and inc%/ated =ith #.25 m5JmL 4IT!-con@%5ated Anne,in-F and 1#
m5JmL PI. The mi,t%re =as kept on ice &or 5 min.< and the cell &l%orescence =as
meas%red /) t=o-parameter &lo= c)tometr) H4A!3 !ali/%r< $ecton Dickinson<
$asel< 3=it>erlandI H0I. .hen 5reen &l%orescence H4IT!I =as plotted a5ainst red
&l%orescence HPII three distinct cell pop%lations co%ld /e detected in a dotplot: via/le
cells H4IT!-JPI-I< apoptotic cells H4IT!YJPI-I and secondar) necrotic cells
H4IT!YJPIYI H0I.

A minim%m o& 1#<### events =as co%nted per sample and data
reported as the percenta5e o& apoptotic cells HAnne,in-F-4IT!YJPI-I.
(%) *lo+ Assisted Cytometry of Toll,li"e -eceptors ( and )
Isolated cells =ere maintained in 2PMI 1"#-medi%m =ith 1#N &etal cal& ser%m H4!3\ (i/co
$2LI s%pplemented =ith 1.5mmolJL L-(l%tama, H(i/co $2LI at a concentration o& 1,1#
"
ne%trophilsJmL in 2-=ell cell c%lt%re plates H!ostar !o.< !am/rid5e< MAI at 3-L! in a
h%midi&ied atmosphere H5N !;
2
I. !ells =ere inc%/ated &or and 1" ho%rs =ith or =itho%t
LP3 H15JmLI or MALP-2 H2nMI.
Meas%rement o& TL2-2 J - e,pression =ere carried o%t =ithin the &irst 2 ho%rs a&ter
dia5nosis o& sepsis< de&ined /) the &ollo=in5 clinical parameters Hde&ined septic &oc%s and
&%l&illment o& all criteria o& 3I23 H&ever< tach)cardia< tach)pnea or h)pocapnia< le%koc)tosisI
H1I. 4or meas%rement o& TL2 e,pression on h%man le%koc)tes ne%trophils H1 , 1#
"
JmL
2-
eachI =ere =ashed in sample /%&&er HP$3 containin5 5l%cose H15JLII at the end o&
e,periment. Ph)coer)thrine HP+I &l%orescence o& individ%al cells =as meas%red %sin5 a
4A!3-!ali/%r &lo= c)tometer H$ecton Dickinson A(< $asel< 3=it>erlandI< =hile 5atin5 on
ph)sical parameters to e,cl%de cell de/ris. A minim%m o& 1#<### events per 5ate =as co%nted
per sample. 2es%lts are reported as the mean &l%orescence corrected /) s%/tractin5 the
&l%orescence o& cells stained =ith the respective P+-la/eled isot)pe control anti/od)
He$ioscience< .em/le)< 7KI.
(%5 Analysis of MA.,0inases
(%5%$ ;xperimental .rotocol
Isolated ne%trophils =ere maintained in 2PMI 1"#-medi%m =ith 1# N &etal cal& ser%m
H4!3\ (i/co $2LI s%pplemented =ith 1.5 mmolJL L-(l%tama, H(i/co $2LI at a
concentration o& 1 , 1#
"
cellsJmL in 2-=ell cell c%lt%re plates H!ostar !o.< !am/rid5e< MAI
at 3- L! in a h%midi&ied atmosphere H5 N !;
2
I. !ells =ere preinc%/ated &or one ho%r =ith or
=itho%t her/im)cin H1 ) (5#) PD10#51 HAle,is LG%&el&in5en< !6I H5# ) (51)

and
3$2#350# HAle,is LG%&el&in5en< !6I H5 ) (52) &ollo=in5 stim%lation =ith or =itho%t LP3
H1 5JmLI or I4'- H/iolo5ical activit) 3.# , 1#
-
7Jm5< $oehrin5er-In5elheim< A%striaI H1#
n5JmLI. The concentrations o& I4'- =ere similar to those detected in the circ%lation o&
patients =ith severe sepsis H53I.

6er/im)cin and vanadate =ere %sed in concentrations =hich
have /een &o%nd to completel) inhi/it protein t)rosine kinase and protein-phosphot)rosine
phosphatase< respectivel) H5#< 5I.
(%5%( !esternblot Analysis of Neutrophil MA.,0inases
4or anal)sis o& phosphor)lated MAP kinases in ne%trophils< cells H1 a 1#
"
I =ere centri&%5ed
and &ro>en immediatel) in li8%id nitro5en at the end o& e,periment and =ere stored at b0#L!
20
%ntil &%rther processin5. 'e%trophils H1 a 1#
"
I =ere res%spended in 1## L o& Laemmli /%&&er
and =ere s%/se8%entl) /oiled &or 1# min. +8%al amo%nts o& =hole cell l)sate H1# LJlane
correspondin5 to 1 a 1#
5
cellsI =ere separated /) 3D3-PA(+ in 1#N pol)acr)lamide 5els in
a Mini Protean II cham/er H$io-2ad< 6erc%les< !AI. Proteins =ere s%/se8%entl)
electrotrans&erred onto a n)lon mem/rane HImmo/ilon P\ Millipore< $ed&ord< MAI in a Mini
Trans $lot trans&er cham/er H$io-2adI< and mem/ranes =ere /locked overni5ht at L! in
Tris-/%&&ered saline s%pplemented =ith #.1N T=een-2# and 2N milk dil%ent HKPL<
(aithers/%r5< MDI. The phosphor)lated MAP kinases +2K and p30 =ere detected %sin5
speci&ic anti/odies &rom 'e= +n5land $iola/s H$everl)< MAI and a pero,idase- co%pled 5oat
anti-ra//it secondar) anti/od) HDako< (lostr%p< DenmarkI. The total MAP kinases p2J
+2K and p30 protein =as detected %sin5 anti/odies &rom 3anta !r%> H3anta !r%>
$iotechnolo5)< 3anta !r%>< !AI and respective pero,idaseco%pled secondar) anti/odies
HDakoI. 3peci&ic /indin5 =as vis%ali>ed /) enhanced chemil%minescence HAmersham<
$%ckin5hamshire< 7KI &ollo=in5 the man%&act%rerEs recommendations. The molec%lar
=ei5ht o& the protein /ands =as determined /) %se o& prestained lo=-molec%lar-=ei5ht
markers H3i5ma !hemicalI on the same 5el.
(%/ Analysis of cIA.( .rotein
(%/%$ ;xperimental .rotocol
PM' =ere preinc%/ated either =ith or =itho%t LP3 H1 c5JmLI &or " ho%rs and then stim%lated
=ith an a5onistic a!D15 anti/od) H1## n5JmLI &or another 1" ho%rs or inc%/ated in medi%m
alone &or a total o& 22 ho%rs. The proteasome =as inhi/ited =ith the proteasome-inhi/itor
HP3I< 3# cM< A'-car/o/eno,)-L-isole%c)l-L-d-t-/%t)l-L-5l%tam)l-L-alan)l-L-le%cinalB
!al/iochemI. 1 ho%r prior to activation o& !D15 H31I. 4ollo=in5 inc%/ation cells =ere
harvested &or =estern/lot and caspase-3-activit) meas%rements< shock &ro>en in li8%id '
2
and
stored at ?0#L! %ntil &%rther %se. In parallel< ne%trophil apoptosis a&ter 22 ho%rs =as meas%red
21
/) &lo= c)tometr). The timepoints &or cIAP2 m2'A e,pression anal)sis =ere set at #< 1< 2
and ho%rs< either =ith or =itho%t LP3 inc%/ation. The e,pression o& cIAP2 protein =as
anal)>ed a&ter inc%/ation =ith medi%m< LP3 or a!D15 at #< 2 and ho%rs.
(%/%( Caspase,3,Activity Measurement%
3#
&aspase'( acti)ity was measured in cellular e$tracts from neutrophil samples. After
incubation, neutrophils were lysed by free*e'thaw procedure in hypotonic e$traction
buffer +,- m" .#%#S +'-2-h)dro,)eth)lpipera>ine-'e-2-ethanes%l&onic acidI, -
m" "g&l
,
, /.0 1 2riton'3 0//, p. 4.-, with addition of 0 m" %efablock
5
pepstatin,
leupeptin, and aprotinin +0 " each66, subse7uently centrifuged +0- min, 08,/// $ g,
8 &6, and the supernatant stored at 9:/ & until further used +8;6. 2he fluorometric
clea)age assay for caspase'('like acti)ity +D#<D'afc, &albiochem6 was carried out
in microtiter plates +Greiner, =uertingen, Germany6 according to the method
described by 2hornberry +((6 using the fluorometric plate reader <ictor', +>luostar,
Dr. Gurath Gmb., Germany6 with the e$citation wa)elength set at (:- nm and an
emission wa)elength of -/- nm +((6. 2he protein concentrations of the respecti)e
samples were measured with a commercially a)ailable kit +%ierce Assay, %ierce,
?nited @ingdom6, and caspase'( acti)ity was calculated as units +?6 per mg protein
with 0 ?Amg being e7ual to the clea)age of 0 Bmol 4'amino'8'
trifluromethylcoumarin +afc6 per mg protein and minute +8;6.
2.6.3 Detection of cIAP2 mRNA by RT-PCR.
31
4or detection o& speci&ic m2'A e,pression D'A =as isolated &rom ne%trophils /) Tri>ol
method &ollo=in5 standard proced%re. $rie&l)< ne%trophils H2.5 , 1#
-
I =ere
centri&%5ed a&ter the e,periments and &ro>en H-0#L!I %ntil &%rther %se. 4or isolation o&
m2'A cells =ere tha=ed in Tri>ol HInvitro5enI and total m2'A isolated /) T2I];L-
method &ollo=in5 man%&act%rers recommendation. A&ter D'Ase HD'ase I< 2ocheI
and 2'ase Inhi/itor H2'ase Inhi/itor< Invitro5enI treatment< m2'A =as transcri/ed
to cD'A /) 2T-reaction. The primer &or cIAP2 and -Actin =ere desi5ned =ith the
pro5ram Primer +,press HApplied $ios)stems< 4oster !it)< !AI %sin5 the cIAP2
se8%ence H'!$I accession n%m/er: 9MW##-15I as a template. 4or detection o& -
Actin the primers derived &rom the se8%ence H'!$I accession n%m/er: 9MW#"30-I
Hsee Ta/le 3.I =ere %sed. All primers =ere p%rchased &rom Micros)nth HMicros)nth<
$al5ach< 3=it>erlandI. A total o& 5 5 m2'A =as transcri/ed =ith reverse
transcriptase H3%perscript TM-II< Invitro5enI into cD'A. 4or the P!2 reaction #.1 5
cD'A =as ampli&ied =ith speci&ic primers< n%cleotides and pol)merase HTa8
Pol)merase< Invitro5enI &or a total o& 30 or 3 c)cles< respectivel). The ampli&ied
cD'A =as separated /) electrophoresis on a 1.0 N a5arose5el and vis%ali>ed %nder
7F a&ter ethidi%m/romide H#.5 NI stainin5.
2.6.4 Westernblot Analysis of cIAP2 Protein
32
Chole cell lysates +corresponding to 0.,- $ 0/
-
cells6 were loaded on 0/ 1 polyacrylamide
gels +"ini %rotean II, DioEad, .ercules, &A6 in Faemmli buffer +Sample Duffer
Faemmli, Sigma6. %roteins were separated at 8/ mA for -/ min and then transferred
onto %<D> membranes +DioEad6 for ;/ min at 0//<. "embranes were blocked in
2DS2 with /., 1 Do)ine Serum Albumin +Sigma6 o)ernight, and then incubated
either with polyclonal goat anti'human caspase'( +Santa &ru* Diotechnology, Santa
&ru*, &A6, polyclonal rabbit anti'human ?bi7uitin +Santa &ru*6 or polyclonal goat
anti'human cIA%, +Santa &ru*6 for 8 hours at room temperature, washed with 2DS2
and then e$posed to a secondary .E% conGugated antibody for 0 hour at room
temperature. &hemiluminescence was detected by #&F +Amersham Diosciences,
Duckinghamshire, ?nited @ingdom6 on a scientific imaging film +@odak 3'Hmat AE
>ilm, @odak, Fausanne, Swit*erland6.
(%1 Analysis of 2cl,(.roteins
(%1%$% ;xperimental .rotocol
6eparini>ed /lood H2# 7 heparinJmL\ heparin =as tested &or endoto,in: M 5 p5JmL heparinI
o/tained &rom the patients at da) o& sepsis dia5nosis< or &rom health) controls =as dil%ted 1:1
=ith 2PMI 1"# medi%m H(i/co $2L< Li&e Technolo5ies< Paisle)< 3cotlandI. 'e%trophils
=ere isolated /) densit) centri&%5ation in 6istopa8%e-1#-- H3i5ma !hemical !o.I &ollo=ed
/) t=o =ashin5 steps in phosphate /%&&ered saline HP$3I as previo%sl) descri/ed H5"-50I.
L)sis o& resid%al er)throc)tes =as per&ormed %sin5 nine vol%mes o& an ice-cold isotonic
ammoni%m chloride sol%tion H'6

!l 155 mM< K6!;


3
1# mM< +DTA #.1 mMI to one
vol%me o& cell pellet at # L! &or 2# min%tes. The &inal ne%trophil preparation contained K 15N
ne%trophils as =as determined /) &lo= c)tometr) anal)sis %sin5 &l%orescein isothioc)anate-
la/eled monoclonal anti/od) anti-!D15 H!o%lter< 6ialeah< 4LI. !ell via/ilit) =as K 10N as
determined /) tr)pan /l%e e,cl%sion. The cell loss in ne%trophil c%lt%res =as M 5N
33
irrespective o& the e,perimental desi5n or the added proteins %sin5 tr)pan /l%e e,cl%sion and
microscopic cell co%ntin5.
Isolated ne%trophils =ere maintained in 2PMI 1"#-medi%m =ith 1#N &etal cal& ser%m H4!3\
(i/co $2LI s%pplemented =ith 1.5 mmolJL L-(l%tama, H(i/co $2LI at a concentration o& 1
, 1#
"
cellsJmL in 2-=ell cell c%lt%re plates H!ostar !o.< !am/rid5e< MAI at 3- L! in a
h%midi&ied atmosphere H5N !;
2
I. !ells =ere stim%lated =ith or =itho%t LP3 H1 5JmLI or
H a!D15I H1## n5JmLI &or the times indicated.
(%1%( Analysis of .MN Apoptosis
4or meas%rement o& D'A &ra5mentation ne%trophils H1 , 1#
"
JmLI =ere =ashed in sample
/%&&er HP$3 containin5 5l%cose H1 5JLII at the end o& e,periment. !ells =ere &i,ed in 1 mL o&
-#N ethanol over 12 ho%rs at L!. 4i,ed cells =ere inc%/ated in 1 mL propidi%m iodide HPII
stainin5 sol%tion Hsample /%&&er =ith 5# f5Jml propidi%m iodide and 1## 7Jml 2'ase A
H$oehrin5erII at room temperat%re. Propidi%m iodide &l%orescence o& individ%al cells =as
meas%red %sin5 an 4A!3-!ali/%r &lo= c)tometer H$ecton DickinsonI< =hile 5atin5 on
ph)sical parameters to e,cl%de cell de/ris. A minim%m o& 1#<### events =as co%nted per
sample. 2es%lts are reported as the percenta5e o& h)podiploid H&ra5mentedI n%clei re&lectin5
the relative proportion o& apoptotic cells H55-50I.
(%1%3 !estern,2lot Analysis of 2cl,( .roteins
4or anal)sis o& $cl-2 proteins in ne%trophils< cells H1 , 1#
"
I =ere res%spended in l)sis /%&&er
HP$3 s%pplemented =ith A+$34 H1 mMI and Le%peptin H1 m5JmLII< centri&%5ed and &ro>en
immediatel) in li8%id nitro5en at the end o& e,periment and stored at ?0# L! %ntil &%rther
processin5. 'e%trophils H1 , 1#
"
I =ere res%spended in LGmmli /%&&er H1## lI and
s%/se8%entl) /oiled &or 1# min. +8%al amo%nts o& =hole cell l)sate H1# lJlane correspondin5
to 1 , 1#
5
cellsI =ere separated /) 3D3-PA(+ in 1# N pol)acr)lamide 5els in a Mini Protean
3
II cham/er H$io2ad< 6erc%les< !AI. Proteins =ere s%/se8%entl) electrotrans&erred onto a
n)lon mem/rane HImmo/ilon P< Millipore< $ed&ord< MAI in a Mini Trans $lot trans&er
cham/er H$io2adI and mem/ranes =ere /locked overni5ht at L! in T$3 s%pplemented =ith
#.1 N T=een-2# and #.2 N $3A H3i5maI. The proteins $cl-2 and $A9 =ere detected %sin5
speci&ic anti/odies &rom 'e= +n5land $iola/s H$everl)< MAI and a pero,idase co%pled 5oat-
anti-ra//it secondar) anti/od) HDako< (lostr%p< DenmarkI. The $id protein =as detected
%sin5 anti/od) &rom 3anta !r%> H3anta !r%>< !A< 73AI and Mcl-1 /) an anti/od) &rom
Trevi5en Inc. H(aithers/%r5< MD< 73AI and their respective pero,idase co%pled secondar)
anti/odies HDako< DenmarkI. 3peci&ic /indin5 =as vis%ali>ed /) enhanced
chemil%minescence HAmersham< $%ckin5hamshire< 7KI on 9-ra) &ilm HKodakI &ollo=in5
man%&act%rers recommendations. The molec%lar =ei5ht o& the protein /ands =ere determined
/) %se o& prestained lo=-molec%lar-=ei5ht markers H3i5maI on the same 5el. The amo%nt o&
speci&ic protein =as 8%anti&ied /) densitometr). 3tainin5 o& 9-ra) &ilm =as meas%red =ith an
Ima5in5 s)stem HAlphaInnotech< 3an Leandro< !AI and the relative densit) o& /ands is 5iven
in mean _ 3D HNI o& the # ho%r val%e Hset at 1## NI a&ter s%/traction o& the speci&ic
/ack5ro%nd.
(%1%) 4etection of m-NA of 2cl,( .roteins by -T,.C-
4or detection o& speci&ic m2'A e,pression D'A =as isolated &rom ne%trophils /) Tri>ol
method &ollo=in5 standard proced%re. $rie&l)< ne%trophils H2.5 , 1#
-
I =ere centri&%5ed a&ter
the e,periments and &ro>en H-0#L!I. !ells =ere tha=ed in Tri>ol HInvitro5enI. Total m2'A
=as isolated &rom =hole cells /) T2I];L method and transcri/ed to cD'A a&ter D'Ase
treatment /) 2T-reaction. P!2 =as carried o%t =ith primers speci&ic &or $cl-2< $A9< $id and
Mcl-1 Hsee Ta/le 3.I< and vis%ali>ed =ith ethidi%m/romide a&ter electrophoresis on a 1.0 N
35
a5arose 5el. The primer &or $cl-2< $A9< $id and Mcl-1 =ere desi5ned =ith the pro5ram
Primer +,press HApplied $ios)stemsI and the p%/lished se8%ence HM1-5< 'MW130-"1<
'MW##111"< and 'MW#211"# respectivel)I. A total o& 5 5 m2'A =as transcri/ed =ith
reverse Transcriptase H3%perscript TM-II< Invitro5enI into cD'A. 4or the P!2 reaction #.1
5 cD'A =ere inc%/ated =ith speci&ic primers and pol)merase HTa8 Pol)merase< Invitro5enI
&or 35 H3I c)cles. The ampli&ied cD'A< to5ether =ith an ampli&ied ho%se-keepin5 5ene -
Actin =as separated /) a5arose electrophoresis and vis%ali>ed %nder 7F a&ter
ethidi%m/romide stainin5. Detection limit o& P!2 =as de&ined as the lo=est c)clen%m/er that
)ielded a clear positive /and &or the positive control sample He.5. T6P-1 cellsI.
(%# Statistical Analysis
Data are presented as mean _ 3+M. Mean val%es =ere compared %sin5 3t%dent t=o-tailed t-
test &or independent means. Di&&erences =ere re5arded as si5ni&icant< i& p M #.#5.
3"
5E-!TT!!(TAATTA((AA!!T(-3E
5E-!TT(!ATATAAT(AA(T(AA-3E
'MW#211"# Mcl-1
5E-AA(TT!!TA!!A!T(T(!AAT(-3E
5E-!AA(TA(AT(A(((AA!T((!-3E
9MW##-15 cIAP2
5g-AT((A!T(T(A((T!AA!-3g
5g-A(T!!AT!!!ATTT!T((-3g
'MW##111" $ID
5E-A(AT(T!!A((!A(!T(!A!-3E
5E-T(TT(A!TT!A!TT(T((!!-3E
M1-5 $cl-2
5E-(A!!!((T(!!T!A((A-3E
5g-AT((T!A!((T!T(!!A-3g
'MW130-"1 $A9
5E-A(!(((AAAT (T(!AT(-3E
5E-!A(((TA!!T((T((T(!!-3E
9MW#"30- -Actin
4or=ard primer
2everse primer
'!$I accession n%m/er Protein
5E-!TT!!(TAATTA((AA!!T(-3E
5E-!TT(!ATATAAT(AA(T(AA-3E
'MW#211"# Mcl-1
5E-AA(TT!!TA!!A!T(T(!AAT(-3E
5E-!AA(TA(AT(A(((AA!T((!-3E
9MW##-15 cIAP2
5g-AT((A!T(T(A((T!AA!-3g
5g-A(T!!AT!!!ATTT!T((-3g
'MW##111" $ID
5E-A(AT(T!!A((!A(!T(!A!-3E
5E-T(TT(A!TT!A!TT(T((!!-3E
M1-5 $cl-2
5E-(A!!!((T(!!T!A((A-3E
5g-AT((T!A!((T!T(!!A-3g
'MW130-"1 $A9
5E-A(!(((AAAT (T(!AT(-3E
5E-!A(((TA!!T((T((T(!!-3E
9MW#"30- -Actin
4or=ard primer
2everse primer
'!$I accession n%m/er Protein
Table 3: The List o& %sed primers in o%r st%d). Depicted is the name o& the protein< the '!$I
accession n%m/er and the &or=ard and reverse primer.
3-
3%& -esults
3%&%$ Apoptosis is -educed in .MN from Septic .atients
3pontaneo%s apoptosis in ne%trophils &rom patients =ith sepsis H20.0N _ .1NI =as
si5ni&icantl) lo=er a&ter 1" ho%rs o& inc%/ation compared =ith that in controls H".#N _
2.0NI. Inc%/ation =ith endoto,in HLP3< 1 5JmLI &%rther red%ced apoptosis in ne%trophils
&rom patients H11.5N _ .NI and controls H35.1N _ 3.2NI. In contrast< inc%/ation o&
ne%trophils &rom septic patients =ith the a5onistic anti-!D15 anti/od) H1## n5JmLI
completel) restored the li&e span o& those cells to levels seen in %nstim%lated controls H".3N
_ ".0NI. 3tim%lation o& ne%trophils &rom health) controls =ith the a5onistic anti-!D15
anti/od) si5ni&icantl) Hp M #.#5I enhanced apoptosis a&ter 1" ho%rs to 12.1N _ #.0N.
3%$%$ ;xpression of T7-,( and T7-,) on *reshly Isolated 7eu"ocytes
In comparison =ith cells &rom health) controls< le%koc)tes &rom patients e,pressed
si5ni&icantl) Hp M #.#5I increased amo%nts o& TL2-2 and TL2- receptors. TL2-speci&ic
&l%orescence =as calc%lated /) s%/traction o& &l%orescence &rom the respective isot)pe control
HI5(I &rom speci&ic TL2-2 or TL2- &l%orescence. 'o di&&erences /et=een patients and
controls =ere seen &or mean &l%orescence o& isot)pe I5( control on PM' H1#.3 _ 1.1 and 1#.#
_ 1.5I or monoc)tes H1#.0 _ 3.2 and 0." _ #.-I. ;n &reshl) isolated PM' &rom patients =ith
sepsis< the mean &l%orescence =as -0.# _ 10." &or TL2-2 and 11. _ 2.3 &or TL2-< =hereas
on control PM' the mean &l%orescence =as 12.0 _ 2.2 &or TL2-2 and 2.3 _ #. &or TL2-.
3%/5ro%p anal)sis o& s%rvivors Hn C 1I vers%s nons%rvivors Hn C -I =ithin the patient cohort
revealed no si5ni&icant di&&erences in TL2-2 H-1. _ 25.- vs. -5.1 _ 22." in s%rvivors vers%s
nons%rvivorsI or TL2- H1.0 _ 2.5 vs. 1.1 _ .1< respectivel)I e,pression.
30
*i6ure $&: +,pression o& TL2-2 and TL2- receptors on PM'. 4reshl) isolated h%man
ne%trophil 5ran%loc)tes HPM'I &rom a health) control Hn C 12I HAI or a patient =ith sepsis Hn
C 21I H$I =ere inc%/ated =ith P+-la/eled anti-I5( HI5(I< anti-TL2-2< or anti-TL2-
anti/odies. 3peci&ic &l%orescence =as meas%red in 4A!3. 2epresentative P+-&l%orescence
histo5rams &or each 5ro%p are sho=n. +,pression o& TL2-2 and TL2- receptors on &reshl)
isolated PM' H!I. Mean &l%orescence =as determined /) s%/traction o& nonspeci&ic P+-
&l%orescence &rom TL2 speci&ic &l%orescence. hp M #.#5< patients vers%s controls.
3%$%(% 7i6and 2indin6 and -eceptor ;xpression
The increased TL2-2 and TL2- e,pression on le%koc)tes &rom patients =ith sepsis mi5ht /e
a res%lt o& previo%s contact =ith endoto,ins. There&ore< the e,pression o& TL2-2 and TL2-
on PM' =as investi5ated a&ter inc%/ation =ith their respective li5ands< MALP-2 and LP3.
Isolated PM' H1 a 1#
"
JmLI =ere inc%/ated =ith either medi%m< MALP-2 H2 nMI or LP3 H1
5JmLI &or and 1" h< and TL2-2 and TL2- e,pression =as meas%red in 4A!3.
31
I6G
T7-,) T7-,(
I6G
T7-,)
T7-,(
A
2
Controls
.atients
T7-,( T7-,)
m
e
a
n
f
l
u
o
r
e
s
c
e
n
c
e
&
(&
)&
/&
#&
$&&
*
*
C
I6G
T7-,) T7-,(
I6G
T7-,)
T7-,(
A
2
Controls
.atients
T7-,( T7-,)
m
e
a
n
f
l
u
o
r
e
s
c
e
n
c
e
&
(&
)&
/&
#&
$&&
*
*
Controls
.atients
T7-,( T7-,)
m
e
a
n
f
l
u
o
r
e
s
c
e
n
c
e
&
(&
)&
/&
#&
$&&
*
**
C
*i6ure $$: TL2-2 and TL2- receptor e,pression on inc%/ated PM'. Isolated ne%trophil
5ran%loc)tes HPM'I &rom health) controls HiI or patients =ith sepsis HjI =ere inc%/ated =ith
medi%m< LP3 H1 c5JmLI< or MALP-2 H2 nMI &or and 1" h. Mean &l%orescence =as
determined /) s%/traction o& nonspeci&ic P+-&l%orescence &rom TL2-2- or TL2--speci&ic
&l%orescence. hp M #.#5< patients vers%s controls.
In all cells the e,pression o& TL2s increased a&ter h< independent o& the stim%l%s and cell
pop%lation< =ith a stron5er increase in cells &rom patients than in cells &rom controls.
6o=ever< in le%koc)tes &rom patients =ith sepsis< a non-si5ni&icant decline o& TL2
e,pression =as seen /et=een h and 1" h o& inc%/ation on PM'. !ompared =ith medi%m
alone< inc%/ation =ith the TL2-2 li5and MALP-2 H2 nMI or TL2- li5and LP3 H1 5JmLI
had no e&&ect on e,pression o& TL2-2 or TL2- on PM'. This =as seen not onl) in cells &rom
#
Medium
& ( ) / # $& $( $) $/
m
e
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hours
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T7-,(
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*
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*
Medium
& ( ) / # $& $( $) $/
&
(&
)&
/&
#&
$&&
7.S
& ( ) / # $& $( $) $/
&
(&
)&
/&
#&
$&&
MA7.,(
hours
& ( ) / # $& $( $) $/
&
(&
)&
/&
#&
$&&
T7-,)
*
*
*
*
*
*
*
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Medium
& ( ) / # $& $( $) $/
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Medium
& ( ) / # $& $( $) $/
&
(&
)&
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7.S
& ( ) / # $& $( $) $/
&
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$&&
MA7.,(
hours
& ( ) / # $& $( $) $/
&
(&
)&
/&
#&
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T7-,)
*
*
*
*
*
*
*
*
*
controls /%t also in cells &rom patients =ith sepsis a&ter h as =ell as 1" h o& inc%/ation. This
indicates that li5and /indin5 ma) not /e responsi/le &or %p-re5%lation or do=nre5%lation o&
respective TL2s on le%koc)tes in patients =ith sepsis.
3%( MA.,0inases and .MN Apoptosis
3%(%$ ;ffect of Aerbimycin on .MN apoptosis
The in&l%ence o& t)rosine kinase /lockade in ne%trophil apoptosis =as investi5ated %sin5 the
kinase inhi/itor her/im)cin. 3pontaneo%s ne%trophil apoptosis in patients =ith sepsis H3".#N
_ 3.1NI =as si5ni&icantl) lo=er than in ne%trophils &rom health) individ%als H-2.1N _ 2.5NI
a&ter 1" h o& inc%/ation. #$ )i)o stim%lation o& ne%trophils =ith LP3 H1 5JmLI &or 1"h
si5ni&icantl) red%ced ne%trophil D'A &ra5mentation in controls &rom -2.1N _ 2.5N to 31.1N
_ 3.1N and in patients &rom 3".#N _ 3.1N to 2#.0N _ 2.0N. 'e%trophils inc%/ated =ith I4'-
H1# n5JmLI &or 1" h sho=ed a si5ni&icant red%ction Hp M #.#5I o& spontaneo%s ne%trophil
D'A &ra5mentation to 2".2N _ 1.0N in health) controls and to 15.N _ 1.1N in patients
=ith sepsis. Preinc%/ation o& cells =ith her/im)cin H1 MI a/ro5ated Hp M #.#5I the LP3
H-#.1N _ 2.0NI or I4'- H"#.3N _ 3.5NI e&&ect on ne%trophil D'A &ra5mentation in
ne%trophils &rom health) controls and reconstit%ted apoptosis in ne%trophils &rom patients
=ith sepsis to the level o& spontaneo%s apoptosis H#.-N _ 3.-N and 23.1N _ 2.2N<
respectivel)I. 6o=ever< her/im)cin &ailed to &%ll) reconstit%te the rate o& D'A &ra5mentation
to levels seen in health) controls< indicatin5 that other mechanisms mi5ht /e involved in
patients =ith sepsis. 4%rthermore< the anti-apoptotic e&&ect o& I4'- co%ld not /e &%ll)
a/ro5ated /) the %se o& her/im)cin< indicatin5 that the LP3- and I4'--mediated e&&ect mi5ht
/e re5%lated /) di&&erent kinases. 6er/im)cin itsel& had no e&&ect on spontaneo%s ne%trophil
apoptosis.
1
*i6ure $(: In&l%ence o& LP3 and I4'- on apoptosis o& ne%trophil 5ran%loc)tes. $oth< LP3
and I4'-< Inhi/it spontaneo%s apoptosis o& PM' in health) individ%als HcontrolsI< patients
=ith 3I23 and patients =ith sepsis. Data are 5iven as Mean _ 3+M<hp M #.#5 spontaneo%s vs.
stim%l%s< D p M #.#5 controls vs. patients
3%(%( .articipation of .hosphatases in the -e6ulation of .MN apoptosis
Phosphatases have /een sho=n to re5%late the activit) o& protein kinases H2-I. To investi5ate
=hether phosphatases in&l%ence the activit) o& kinases in the si5nal transd%ction o&
spontaneo%s and endoto,in-mediated apoptosis< ne%trophils =ere preinc%/ated =ith the
phosphatase inhi/itor vanadate H5 MI &or 1 h /e&ore stim%lation =ith or =itho%t LP3 H1
5JmLI &or %p to 1" h. 6o=ever< vanadate had no e&&ect on spontaneo%s or endoto,in-
mediated ne%trophil apoptosis in health) controls as =ell as in patients =ith sepsis.
2
Medium
7.S
I*N,
Apoptosis
EF .IG
controls
nH'
SI-S
nH'
sepsis
nH'
&
(&
)&
/&
#&
$&&
D
*
*
*
*
*
D
Medium
7.S
I*N,
Apoptosis
EF .IG
controls
nH'
SI-S
nH'
sepsis
nH'
&
(&
)&
/&
#&
$&&
D
*
*
*
*
*
D
*i6ure $3: In&l%ence o& her/im)cin H1 cMJmLI on apoptosis o& ne%trophils &rom health)
individ%als Hcontrols< n C 1#I and patients =ith sepsis Hpatients< n C 10I in the a/sence or
presence o& LP3 H1 c5JmL< AI or I4'- H1# n5JmL< $I. D'A &ra5mentation =as anal)>ed /)
&lo= c)tometr) a&ter stainin5 =ith PI. 2es%lts are depicted as mean _ 3+M. hp M #.#5<
stim%l%s vs. medi%m control\ kp M #.#5< her/im)cin vs. stim%l%s\ lp M #.#5< patient vs.
respective control.
3
.atients Controls
A
p
o
p
t
o
s
i
s

E
F

.
I
G
&
(&
)&
/&
#&
$&&
I
I
D
&
(&
)&
/&
#&
$&&
Medium
Aerbimycin
I*N, I*N, , ,
&
(&
)&
/&
#&
$&&
&
(&
)&
/&
#&
$&&
D
A
p
o
p
t
o
s
i
s

E
F

.
I
G
I
D
I
D
7.S 7.S , ,
J
J
J
J
A
2
.atients Controls
A
p
o
p
t
o
s
i
s

E
F

.
I
G
&
(&
)&
/&
#&
$&&
I
I
D
&
(&
)&
/&
#&
$&&
Medium
Aerbimycin
I*N, I*N, , ,
&
(&
)&
/&
#&
$&&
&
(&
)&
/&
#&
$&&
D
A
p
o
p
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i
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F

.
I
G
I
D
I
D
7.S 7.S , ,
J
J
J
J
A
2
3%(%3 ;ffect of MA.,0inase Inhibitors on 7.S and I*N,,Mediated .MN Apoptosis
To &%rther anal)>e the involvement o& MAP kinases in the re5%lation o& ne%trophil apoptosis<
cells &rom 1# patients =ere preinc%/ated &or 1 h =ith the +2K kinase inhi/itor PD10#51 H5#
MI H11I or the p30 MAPK inhi/itor 3$2#350# H5MI H"#I /e&ore stim%lation =ith LP3 H1
5JmLI or I4'- H1#n5JmLI. In health) controls< the LP3-red%ced apoptosis H33.1N _ 3.3NI
=as reconstit%ted H52."N _ 3.-NI a&ter inc%/ation =ith PD10#51 Hp M #.#5I< /%t not =ith the
p30 inhi/itor 3$2#350# H35.3N _ ".5NI. In cells inc%/ated =ith I4'- H2.-N _ 3.5NI<
PD10#51 onl) sli5htl) increased apoptosis H3.1N _ 3.1NI< /%t inhi/ition o& p30 =ith
3$2#350# si5ni&icantl) Hp M #.#5I increased apoptosis H51.3N _ -.-NI. .hereas PD10#51
alone H"#.2N _ 2.NI had no e&&ect on spontaneo%s ne%trophil apoptosis< inc%/ation =ith
3$2#350# increased spontaneo%s apoptosis in ne%trophils to 02.N _ 3.3N. In ne%trophils
&rom patients =ith sepsis< inc%/ation =ith LP3 H1".1N _ 2.3NI red%ced spontaneo%s
apoptosis H2-.2N _ 3.1NI /) 3-.1N and inc%/ation =ith I4'- H13.1N _ 2.#NI /) 0.1N.
Inhi/ition o& +2K =ith PD10#51 &%ll) restored the LP3-ind%ced H2".N _ 3.5NI< /%t not the
I4'--ind%ced H10.N _ 3.1NI red%ction o& ne%trophil apoptosis. The p30 MAPK inhi/itor
3$2#350# alone enhanced spontaneo%s apoptosis H2.#N _ 5.0NI< and sli5htl) decreased
LP3-ind%ced apoptosis to 12.5N _ 2.1N. 3imilar to controls< the I4'-- mediated red%ction
o& apoptosis =as a/ro5ated H25."N _ 5.0NI /) inc%/ation =ith 3$2#350#. 6o=ever< neither
PD10#51 nor 3$2#350# restored red%ced apoptosis in ne%trophil &rom patients =ith sepsis to
levels seen in controls.

*i6ure $): In&l%ence o& MAP kinase inhi/itors PD10#51 H5# cMI and 3$2#350# H5 cMI on
apoptosis o& ne%trophils &rom health) individ%als Hcontrols< n C 1#<I and patients =ith sepsis
Hpatients< n C 1#<I inc%/ated =ith medi%m Hmedi%mI< LP3 H1 c5JmLI< or I4'- H1# n5JmLI.
D'A &ra5mentation =as anal)>ed /) &lo= c)tometr) a&ter stainin5 =ith PI. Data are depicted
5
A
p
o
p
t
o
s
i
s

E
F

.
I
G
-
7.S I*N,
&
(&
)&
/&
#&
$&&
I
I
I
I
Controls
I
I
I
I
I
&
(&
)&
/&
#&
$&&
-
7.S I*N,
A
p
o
p
t
o
s
i
s

E
F

.
I
G
.atients
D
D
D
D
D
Medium
.4'#&5'
S2(&35#&
A
p
o
p
t
o
s
i
s

E
F

.
I
G
-
7.S I*N,
&
(&
)&
/&
#&
$&&
I
I
I
I
Controls
I
I
I
I
I
&
(&
)&
/&
#&
$&&
-
7.S I*N,
A
p
o
p
t
o
s
i
s

E
F

.
I
G
.atients
D
D
D
D
D
Medium
.4'#&5'
S2(&35#&
as mean _ 3+M. hp M #.#5< stim%l%s vs. medi%m control\ kp M #.#5< inhi/itor vs. respective
control.
3%(%) 4etection of .hosphorylated ;-0 and p3# MA.,0inases in .MN
Phosphor)lation o& the MAP kinase p2J +2K and p30 =as anal)>ed /) .estern /lot in
ne%trophil cell l)sates separated /) 3D3-PA(+. As a loadin5 control< the same samples r%n
in parallel =ere pro/ed =ith anti/odies speci&ic &or total p2J +2K or p30 protein. The
phosphor)lated &orm o& p2J +2K and p30 MAP kinases =ere /oth &o%nd in ne%trophils
&reshl) isolated &rom patients =ith sepsis as =ell as &rom controls< =ith phosphor)lation o&
p30 and +2K /ein5 more intense in ne%trophils &rom patients than &rom controls. In contrast
to medi%m alone< inc%/ation =ith LP3 led to phosphor)lation o& p2J +2K and p30 /oth in
patients and controls< =hereas I4'- did not. The inhi/ition o& p2J +2K /) PD10#51 =as
clearl) seen< as PD10#51 inhi/its M+K1 kinase and th%s prevents phosphor)lation o& p2J
+2K. 'o red%ction o& p30 phosphor)lation =as seen in cells inc%/ated =ith 3$2#350#<
=hich is to /e e,pected< as 3$2#350# inhi/its p30 kinase activit)< /%t not p30
phosphor)lation H"1I. Interestin5l)< in ne%trophils &rom patients< /%t not in controls<
phosphor)lation o& p2J +2K and p30 =as seen a&ter pretreatment =ith 3$2#350# and
s%/se8%ent stim%lation =ith I4'-.
"
*i6ure $5: Phosphor)lation o& p2J +2K in ne%trophils &rom health) vol%nteers HcontrolI
or patients =ith sepsis HpatientI. PM' H1 a 1#
"
I =ere preinc%/ated &or 1 h =ith DM3; H#.1N<
AI< PD10#51 H1# cM< $I< or 3$2#350# H5 cM< !I. 3%/se8%entl)< cells =ere stim%lated &or 15
min =ith medi%m alone Hmedi%mI< LP3 H1 c5JmLI< or I4'- H1## n5JmLI. +8%al amo%nts o&
protein &rom =hole-cell l)sates H5 a 1#
5
cellsJlaneI =ere separated /) 3D3-PA(+ H12NI< and
-
)(
total ;-0
.hosphorylated,;-0
M!
K"4aL
)(
;-0$
;-0(
A 2 & C A 2 C A 2 C
7.S I*N, Medium
;-0$
;-0(
Control
total ;-0
.hosphorylated,;-0
.atient
A 2 & C A 2 C A 2 C
7.S I*N, Medium
;-0$
;-0(
;-0$
;-0(
)(
)(
A: Medium
2: .4'#&5'
C: S2(&35#&
)(
total ;-0
.hosphorylated,;-0
M!
K"4aL
)(
;-0$
;-0(
A 2 & C A 2 C A 2 C
7.S I*N, Medium
;-0$
;-0(
Control
total ;-0
.hosphorylated,;-0
.atient
A 2 & C A 2 C A 2 C
7.S I*N, Medium
;-0$
;-0(
;-0$
;-0(
)(
)(
A: Medium
2: .4'#&5'
C: S2(&35#&
A: Medium
2: .4'#&5'
C: S2(&35#&
the phosphor)lated H%pper panelI and total +2K kinase Hlo=er panelI =ere detected /)
imm%no/lottin5. The molec%lar =ei5ht HM.I is indicated on the ri5ht.
*i6ure $/: Phosphor)lation o& p30 in ne%trophils &rom health) vol%nteers HcontrolI or
patients =ith sepsis HpatientsI. PM' H1 a 1#
"
I =ere preinc%/ated &or 1 h =ith DM3; H#.1N<
AI< PD10#51 H1# cM< $I< or 3$2#350# H5 cM< !I. 3%/se8%entl)< cells =ere stim%lated &or 15
0
.hosphorylated,p3#
M!
K"4aL
p3#
p3#
total p3#
)(
)(
A 2 & C A 2 C A 2 C
7.S I*N, Medium
Control
7.S I*N, Medium
.hosphorylated,p3#
M!
K"4aL
p3#
p3#
total p3#
A 2 & C A 2 C A 2 C
)(
)(
.atient
.hosphorylated,p3#
M!
K"4aL
p3#
p3#
total p3#
)(
)(
A 2 & C A 2 C A 2 C
7.S I*N, Medium
Control
.hosphorylated,p3#
M!
K"4aL
p3#
p3#
total p3#
)(
)(
A 2 & C A 2 C A 2 C
7.S I*N, Medium
Control
7.S I*N, Medium
.hosphorylated,p3#
M!
K"4aL
p3#
p3#
total p3#
A 2 & C A 2 C A 2 C
)(
)(
.atient
7.S I*N, Medium
.hosphorylated,p3#
M!
K"4aL
p3#
p3#
total p3#
A 2 & C A 2 C A 2 C
)(
)(
.atient
min =ith medi%m alone Hmedi%mI< LP3 H1 c5JmLI< or I4'- H1## n5JmLI. +8%al amo%nts o&
protein &rom =hole-cell l)sates H5 a 1#
5
cellsJlaneI =ere separated /) 3D3-PA(+ H12NI< and
the phosphor)lated H%pper panelI and total p30 kinase Hlo=er panelI =ere detected /)
imm%no/lottin5. The molec%lar =ei5ht HM.I is indicated on the ri5ht.
3%3 cIA.( and Activity of Caspase,3 in Neutrophil Ganulocytes
3%3%$ 7.S Induces cIA.( m-NA and .rotein in .MN
Inc%/ation o& ne%trophils H1 , 1#
"
J mLI =ith LP3 H1 5JmLI ind%ced %pre5%lation o& cIAP2
m2'A =ithin one ho%r and %p to &o%r ho%rs. In contrast< no cIAP2 m2'A =as detected in
&reshl) isolated ne%trophils< or =hen cells =ere inc%/ated =ith medi%m alone. A&ter &o%r
ho%rs inc%/ation =ith LP3 the e,pression o& cIAP2 m2'A seemed to decline.
*i6ure $1: +,perimental protocol. 'e%trophil 5ran%loc)tes H1 , 1#
"
JmLI =ere stim%lated
=ith and =itho%t LP3 H1 5JmLI &or " ho%rs< &ollo=ed /) sim%lation =ith or =itho%t an
a5onistic a!D15 anti/od) H< 1## n5JmLI &or another 1" ho%rs< res%ltin5 in a total o& 22 ho%rs
inc%/ation. ;ne ho%r /e&ore stim%lation =ith a5onistic a!D15 anti/od) the speci&ic
proteasome inhi/itor P3I H3# MI =as added.

*i6ure $#: LP3 ind%ces cIAP2 m2'A e,pression. PM' H1 , 1#
"
JmLI =ere inc%/ated either
in medi%m or =ith LP3 H1 5JmLI at 3- L! &or 1< 2 or ho%rs and cells =ere harvested
1
7.S
&,h
.SI
5,h
aC4'5
/,h ((,h
7.S
&,h
.SI
5,h
aC4'5
/,h ((,h
cIA.,(
,Actin
M &h 7.S C
$h
<
,
(h
<
,
)h
<
,
cIA.,(
,Actin
M &h 7.S C
$h
<
,
(h
<
,
)h
<
,
s%/se8%entl). Total m2'A =as isolated and transcri/ed in cD'A and 5 5Jml cD'A %sed as
a template &or P!2. cIAP2 P!2 =as r%n =ith 30 c)cles and -Actin P!2 r%n =ith 35 c)cles
in parallel. Ampli&ied cD'A =as separated on 1.0 N a5arose 5el and vis%ali>ed %nder 7F
li5ht a&ter ethidi%m/romide stainin5. M: Marker< !: Positive control. 2epresentative /lot o&
si, separate e,periments.
4or detection o& cIAP2 protein e,pression correspondin5 samples =ere anal)>ed /)
imm%no/lottin5. 3peci&ic =estern/lots sho=ed cIAP2-protein in &reshl) isolated cells =ith an
increased e,pression a&ter t=o to &o%r ho%rs inc%/ation =ith LP3. 6o=ever< a red%ced level
o& cIAP2-protein =as detected =hen cells =ere inc%/ated =ith the a5onistic a!D15 anti/od)
as =ell as in PM' inc%/ated =ith medi%m alone indicatin5 a possi/le de5radation or deca) o&
cIAP2 protein.
*i6ure $': Alterations in cIAP2 protein e,pression a&ter LP3 or !D15 stim%lation. PM' H1 ,
1#
"
JmLI =ere inc%/ated either in medi%m< LP3 H1 5JmLI or =ith an a5onistic a!D15
anti/od) H< 1## n5JmLI at 3-L! &or 2 or ho%rs. At the end o& e,periment cells =ere
harvested and l)sed immediatel). .hole cell l)sate correspondin5 to 5 , 1#
5
cells =ere
separated /) 3D3-PA(+ H1# NI< /lotted onto PFD4 mem/rane and inc%/ated =ith speci&ic
anti/odies. cIAP2 speci&ic /ands =ere vis%ali>ed /) +!L a&ter inc%/ation =ith secondar)
62P-co%pled anti/odies. 7nspeci&ic /ands =ere taken as loadin5-control. H2-I
3%3%( 7.S Induces >biMuitination of Caspase,3 in .MN
IAPs have /een sho=n to possess %/i8%itination activit)< =hich =o%ld destine the tar5et
protein &or de5radation /) the proteasome. There&ore %/i8%itination o& caspase-3 =as
5#
,
7.S aC4'5
cIA.,(
7oadin6
control
(h )h
,
7.S aC4'5
&h
,
7.S aC4'5
cIA.,(
7oadin6
control
(h )h
,
7.S aC4'5
&h
anal)>ed in ne%trophils /) =estern/lot a&ter inc%/ation =ith LP3 or a!D15. !omparison o&
%/i8%itin- and caspase-3-speci&ic =estern/lots o& =hole cell l)sates &rom PM' inc%/ated =ith
LP3 revealed a parallel acc%m%lation o& a caspase-3-anti/od) positive /and =ith same si>e
like %/i8%itin-anti/od) positive /and indicatin5 that caspase-3 mi5ht /e %/i8%itinated a&ter
inc%/ation =ith LP3. Preinc%/ation =ith LP3 and s%/se8%ent stim%lation =ith a!D15 also
revealed an acc%m%lation o& %/i8%itinated caspase-3.
*i6ure (&: 7/i8%itination o& caspase-3 a&ter stim%lation o& !D15 in ne%trophils. Isolated
PM' H1 , 1#
"
JmLI =ere preinc%/ated =ith LP3 H1 5JmLI &or 5 ho%rs< &ollo=ed /) P3I H3#
MI &or another ho%r /e&ore stim%lation o& PM' =ith or =itho%t an a5onistic a!D15
anti/od) H1## n5JmLI &or 1" ho%rs. .hole cell l)sate =as separated /) 1# N pol)acr)lamide
3D3-PA(+ and s%/se8%entl) /lotted onto PFD4 mem/rane. .estern/lots =ere anal)>ed &or
%/i8%itin HAI or caspase-3 H$I. Procaspase-3 and activated caspase-3 =ere detected at app.
31." kDa and 2# kDa and %/i8%itinated proteins at 05 kDa. 7nspeci&ic /ands =ere taken as
loadin5-control. H2-I Depicted =estern/lot represents one &rom si, independent anal)ses.
The caspase-3-speci&ic =estern/lot allo=ed discrimination o& the pro-caspase-3 and caspase-3
/ands. Inc%/ation =ith LP3 led to an increase o& procaspase-3 =itho%t an increase o& the
51
A
2
.rocaspase,3
Caspase,3
7oadin6
control
>biMuitinated
Caspase,3
7oadin6
control
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.SI
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.SI
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caspase-3 /and. In contrast< a prominent increase o& the caspase-3 /and =as seen in cells
inc%/ated =ith a!D15. 7/i8%itination o& proteins ind%ces de5radation /) the proteasome<
there&ore cells =ere inc%/ated =ith the proteasome inhi/itor P3I H3# mMI one ho%r prior to
stim%lation =ith a!D15. .hereas P3I had no e&&ect on LP3-treated samples< inhi/ition o& the
proteasome red%ced the procaspase-3 and %/i8%itinated caspase-3 /and in a!D15 treated
samples preinc%/ated =ith LP3. 3ho=n is one o& si, /lots per&ormed.
3%3%3 -eduction of Spontaneous and C4'5,Induced Apoptosis by 7.S
The e&&ect o& P3I indicates an involvement o& the proteasome in re5%lation o& ne%trophil
apoptosis. There&ore< ne%trophil apoptosis =as meas%red in 4A!3. Inc%/ation o& PM'
H1,1#
"
JmLI =ith LP3 H1 5JmLI alone si5ni&icantl) decreased H2.0 _ .0 N apoptotic cells<
p M #.#5I spontaneo%s apoptosis H"".1 _ 2.3 N apoptotic cells< p M #.#5I a&ter 22 ho%rs.
*i6ure ($: LP3 red%ces spontaneo%s and !D15-ind%ced PM' apoptosis. Isolated PM' H1 ,
1#
"
JmLI =ere preinc%/ated =ith LP3 H1 5JmLI &or 5 ho%rs< &ollo=ed /) P3I H3# MI &or
another ho%r /e&ore stim%lation o& PM' =ith or =itho%t a5onistic a!D15 anti/od) H1##
n5JmLI &or 1" ho%rs. PM' apoptosis =as detected /) &lo= c)tometr) H4A!3I< a&ter stainin5
=ith Anne,in-F and PI. Data are 5iven as mean _ 3+M o& Anne,in-F positive and propidi%m
iodide positive cells o& si, e,periments. 3tatistical si5ni&icance =as eval%ated =ith st%dents-t-
52
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test. hp M #.#5 =ith vs. =itho%t LP3< kp M #.#5 =ith vs. =itho%t P3I< lp M #.#5 =ith vs.
=itho%t a!D15.
Additionall)< preinc%/ation =ith LP3 &or " ho%rs si5ni&icantl) inhi/ited the a!D15-ind%ced
apoptosis H".3 _ .2 N vers%s 1#.0 _ #.1 N< p M #.#5I. .hereas preinc%/ation =ith the
speci&ic proteasome inhi/itor HP3II alone had no e&&ect H5. _ 2.- NI on spontaneo%s
ne%trophil apoptosis H"".1 _ 2.3 NI< P3I a/olished the endoto,in ind%ced inhi/ition o&
spontaneo%s apoptosis H52." _ 2. NI and the endoto,in ind%ced inhi/ition in a!D15-ind%ced
apoptosis H00.- _ 2." N< p M #.#5I &%rther stren5thenin5 the involvement o& the proteasome in
re5%lation o& ne%trophil apoptosis.
3%3%) 7.S -educes Caspase,3,7i"e Activity
Parallel to apoptosis caspase-3-like activit) =as meas%red in ne%trophils. The /asal caspase-
3-like activit) o& #.5 _ #.1 7 increased a&ter 22 ho%rs inc%/ation =ith medi%m alone H11.2 _
3.2 7I. Inc%/ation =ith LP3 si5ni&icantl) red%ced caspase-3-like activit) in PM' H5.0 _ 1.1
7< p M #.#5I. Inhi/ition o& the proteasome /) P3I had no e&&ect on caspase-3-like activit) in
PM' inc%/ated =ith medi%m alone H1#.0 _ 2.- 7I. 6o=ever< parallel to apoptosis< inc%/ation
=ith P3I completel) a/olished the LP3-ind%ced inhi/ition o& caspase-3-like activit) H12.5 _
1.5 7I. In addition< preinc%/ation =ith LP3 red%ced the a!D15-ind%ced increase o& caspase-
3-like activit) H1.0 _ 2.5 7 vers%s 11.1 I 2." 7I< al/eit onl) moderatel). Inhi/ition o& the
proteasome =ith P3I completel) a/olished the LP3 e&&ect on the a!D15-ind%ced caspase-3-
like activit) H15.0 _ 2.1 7< p M #.#5I.
53
*i6ure ((: LP3 red%ces caspase-3 activit) in PM'. Isolated PM' H1 , 1#
"
JmLI =ere
preinc%/ated =ith LP3 H1 5JmLI &or 5 ho%rs< &ollo=ed /) P3I H3# MI &or another ho%r
/e&ore stim%lation o& PM' =ith or =itho%t a5onistic a!D15 anti/od) H1## n5JmLI &or 1"
ho%rs. !aspase-3-like activit) =as meas%red in =hole cell l)sate /) D+FD-a&c-cleava5e
assa). ;ne 7nit A7B =as de&ined as fmol cleaved D+FD-a&c per m5 protein per min%te. Data
are 5iven as mean _ 3+M o& si, separate e,periments. 3tatistical si5ni&icance =as eval%ated
=ith st%dents-t-test. hp M #.#5 =ith vs. =itho%t LP3< kp M #.#5 =ith vs. =itho%t P3I.
3%) The Correlation of Mcl,$ +ith Apoptosis of .MN
3%)%$ ;xpression of 2cl,( m-NA and .rotein in .MN
The e,pression o& the anti-apoptotic $cl-2 protein and m2'A =as determined in ne%trophils
&rom patients and health) controls. 'o $cl-2 protein =as detected in ne%trophils &rom /oth
5ro%ps< either in &reshl) isolated cells or in cells inc%/ated =ith medi%m< LP3< or a5onistic
anti-!D15 anti/odies a&ter 1" ho%rs as compared =ith a monoc)te sample. Densitometric
5
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anal)sis o& three separate .estern /lots &or each 5ro%p )ielded val%es &or $cl-2 protein
e,pression o& 1.0N _ 1.2N compared =ith a monoc)te sample H1##NI &or controls and 1.2N
_ 1.#N &or samples &rom patients =ith sepsis inc%/ated &or 1" ho%rs. Inc%/ation =ith LP3
H1.5N _ 1.N in controls and 2.5N _ 1."N in patientsI or a5onistic !D15 anti/od) H2.3N _
2.2N in controls and 3.-N _ 2.2N in patientsI had no si5ni&icant e&&ect on $cl-2 protein
e,pression. $cl-2 m2'A =as monitored /) 2T-P!2 =ith speci&ic primers &or 35 c)cles. In
contrast to T6P-1 cells Hpositive controlI no m2'A &or $cl-2 =as detected in ne%trophils
&rom either 5ro%p. All samples e,pressed e8%al amo%nts o& -actin m2'A Hdata not sho=nI.
*i6ure (3: Anal)sis o& $cl-2 m2'A /) 2T-P!2. PM' H1 , 1#
"
JmLI &rom controls or
patients =ere stim%lated =ith medi%m< LP3 H1 5JmL< LP3I or =ith an a5onistic !D15
anti/od) H1## n5JmL< a!D15I &or 1" ho%rs. +,tracted m2'A =as transcri/ed and tested in
P!2 =ith speci&ic primers &or 35 c)cles. $ands =ere vis%ali>ed %nder 7F a&ter
electrophoresis on a 1.5 N a5arose 5el and ethidi%m/romide stainin5.
55
Control
.atient
< & h $/ h 7.S NC4'5 M
Control
.atient
< & h $/ h 7.S NC4'5 M
Control .atient
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*i6ure (): $cl-2 .estern/lot o& control and patient PM'. !ells H1 , 1#
"
JmLI =ere inc%/ated
&or # or 1" ho%rs =ith Medi%m H1"-hI< LP3 H1 5JmL< LP3I< or =ith an a5onistic !D15
anti/od) H1## n5JmL< a!D15I. +8%al protein o& =hole cells l)sate =as separated /) 3D3-
PA(+ on a 15N 5el and imm%nostained =ith anti-$cl-2 anti/odies and respective secondar)
anti/odies. $cl-2 protein< detected in monoc)tes< monoc)tes H1 , 1#
"
JmLI preparation =as
%sed as positive control HYI. Depicted is one o& three =estern/lots &or each 5ro%p.
3%)%( ;xpression of Mcl,$ m-NA and .rotein in .MN
$oth protein and m2'A o& the anti-apoptotic protein Mcl-1 =ere &o%nd in ne%trophils &rom
patients and controls. 4reshl) isolated PM' &rom patients =ith sepsis contained sli5htl)
hi5her levels o& Mcl-1 H110.5N _ 55.0NI compared =ith ne%trophils &rom controls Hset at
1##NI\ /%t a&ter 1" ho%rs o& inc%/ation Mcl-1 protein decreased si5ni&icantl) in ne%trophils
&rom patients H2-.3N _ 13.0N< p M #.#5 vers%s initial val%eI< =ith mar5inall) hi5her levels
seen in cells stim%lated =ith LP3 H31.-N _ 12.0N< p M #.#5 vers%s initial val%eI than =ith
anti-!D15 H3#.N _ 1.1N< p M #.#5 vers%s initial val%eI. In contrast< PM' &rom controls
e,pressed %nchan5ed amo%nts o& Mcl-1 protein a&ter 1" ho%rs inc%/ation =ith medi%m
H11-.5N _ 0.1NI< LP3 H1#".#N _ 13.2NI< or a5onistic !D15 anti/od) H11#.1N _ 1-.2NI.
*i6ure (5: Anal)sis o& Mcl-1 m2'A /) 2T-P!2. PM' H1 , 1#
"
JmLI &rom controls or
patients =ere stim%lated =ith LP3 H1 m5JmL< LP3I< or =ith an a5onistic !D15 anti/od) H1##
n5JmL< a!D15I &or 1" ho%rs. +,tracted m2'A =as transcri/ed and tested in P!2 =ith
speci&ic primers &or 3 c)cles. $ands =ere vis%ali>ed %nder 7F a&ter electrophoresis on a
1.5N a5arose 5el and ethidi%m/romide stainin5.
5"
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7
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7
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.atient Control
+,pression level o& Mcl-1 m2'A =as monitored /) 2T-P!2 =ith speci&ic primers &or 3
c)cles. In contrast to protein levels m2'A =as not &o%nd in samples &rom controls inc%/ated
&or 1" ho%rs\ /%t e8%al amo%nts o& Mcl-1 m2'A =ere &o%nd in all samples &rom patients =ith
sepsis< indicatin5 an increased e,pression o& Mcl-1 m2'A in PM' &rom patients =ith sepsis.
All samples e,pressed e8%al amo%nts o& -actin m2'A Hdata not sho=nI.
*i6ure (/: Mcl-1 =estern/lot o& control and patient PM'. !ells H1,1#
"
JmLI =ere inc%/ated
&or 1" ho%rs =ith Medi%m H# h< 1" hI< LP3 H1 5JmL< LP3I< or =ith an a5onistic !D15
anti/od) H1## n5JmL< a!D15I. +8%al protein o& =hole cells l)sate =as separated /) 3D3-
PA(+ on a 1# N 5el and imm%nostained =ith anti-Mcl-1 anti/odies and respective secondar)
anti/odies. Depicted is one o& three =estern/lots &or each 5ro%p.
3%)%3 ;xpression of 2A= m-NA and .rotein in .MN
The e,pression o& the pro-apoptotic $A9 protein and m2'A =as determined in ne%trophils
&rom patients and health) controls. In contrast to $cl-2< $A9 protein =as detected in all
samples &rom /oth 5ro%ps. 3li5ht di&&erences =ere detected /et=een di&&erent treatments /%t
these =ere not si5ni&icant. Densitometric anal)sis o& .estern /lots )ielded val%es &or
inc%/ation =ith medi%m H-.2N _ 1".1N and -".-N _ 1-.3NI< =ith LP3 H"1.N _ "."N and
53.1N _ 10.NI< or a5onistic!D15 anti/od) H02.3N _ 1.2N and 51."N _ 21.2NI &or controls
5-
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Control .atient
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and patients< respectivel). Fal%es =ere derived &rom three separate .estern /lots &rom each
5ro%p. +,pression level o& $A9 m2'A =as %nchan5ed in ne%trophils &rom those o& controls\
in PM' &rom patients sli5ht red%ction in $A9 m2'A levels =as seen a&ter stim%lation =ith
LP3 compared =ith medi%m alone.
*i6ure (1: Anal)sis o& $A9 m2'A /) 2T-P!2. PM' H1 , 1#
"
JmLI &rom controls or
patients =ere stim%lated =ith LP3 H1 5JmL< LP3I< or =ith an a5onistic !D15 anti/od) H1##
n5JmLI Ha!D15I &or 1" ho%rs. +,tracted m2'A =as transcri/ed and tested in P!2 =ith
speci&ic primers &or 35 c)cles. $ands =ere vis%ali>ed %nder 7F a&ter electrophoresis on a 1.5
N a5arose 5el and ethidi%m/romide stainin5.
50
Control
&

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7
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Control
.atient
*i6ure (#: $A9 =estern/lot o& control and patient PM'. !ells H1 , 1#
"
JmLI =ere inc%/ated
&or 1" ho%rs =ith Medi%m H# h< 1" hI< LP3 H1 5JmL< LP3I< or =ith an a5onistic !D15
anti/od) H1## n5JmL< a!D15I. +8%al protein o& =hole cells l)sate =as separated /) 3D3-
PA(+ on a 15 N 5el and imm%nostained =ith anti-$A9 anti/odies and respective secondar)
anti/odies. Depicted is one o& three =estern/lots &or each 5ro%p.
3%)%) ;xpression of 2id m-NA and .rotein in .MN
In contrast to $A9< $id protein =as not distri/%ted e8%all) in all samples. In &reshl) isolated
cells &rom patients sli5htl) hi5her levels o& $id protein =ere &o%nd compared =ith those in
controls. Lo=er levels =ere seen in cells &rom controls< =hich corresponded to hi5h rates o&
apoptosis< a&ter 1" ho%rs inc%/ation =ith medi%m H5.3N _ 1.NI or anti-!D15 anti/od)
H1".N _ 0."NI. In contrast inc%/ation =ith LP3 res%lted in a sli5htl) hi5her $id protein
content H55.5N _ 1#.3NI. Preinc%/ation o& PM' &or 1 ho%r =ith the pan-caspase inhi/itor >-
FAD-&mk H2# molJLI inhi/ited the decrease o& $id in PM' inc%/ated =ith a5onistic !D15
anti/od) H50.2N _ 1.2NI< indicatin5 that $id is tr%ncated /) caspases. In parallel samples
&rom patients =ith sepsis levels o& $id protein =ere #.#N _ 1#.3N &or medi%m< ".0N _
1#.5N &or LP3< and 2".0N _ 1".3N &or !D15 stim%lation< inversel) re&lectin5 the level o&
apoptosis. 'o $id m2'A =as &o%nd in samples &rom patients< either in &reshl) isolated cells
or in cells stim%lated &or 1" ho%rs.
51
&
h
$
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$
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h
7
.
S
&
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4
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5
.atient Control
M &
h
$
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$
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7
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4
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.atient Control
M
*i6ure (': Anal)sis o& $id m2'A /) 2T-P!2. PM' H1 , 1#
"
JmLI &rom controls or patients
=ere stim%lated =ith LP3 H1 5JmL< LP3I< or =ith an a5onistic !D15 anti/od) H1## n5JmL<
a!D15I &or 1" ho%rs. +,tracted m2'A =as transcri/ed and tested in P!2 =ith speci&ic
primers &or 35 c)cles. $ands =ere vis%ali>ed %nder 7F a&ter electrophoresis on a 1.5N
a5arose 5el and ethidi%m/romide stainin5.
In contrast $id m2'A =as detected in PM' &rom health) controls< /%t onl) in &reshl)
isolated cells and not in cells stim%lated therea&ter. All m2'A isolates e,pressed e8%al
amo%nts o& -actin m2'A Hdata not sho=nI.
*i6ure 3&: $id =estern-/lot o& control and Patient PM'. !ells H1 , 1#
"
JmLI =ere pre-
inc%/ated =ith medi%m or >-FAD-&mk H>FAD< 2# MI &or one ho%r and then inc%/ated &or
15 ho%rs =ith Medi%m H# h< 1" hI< LP3 H1 5JmL< LP3I< or =ith an a5onistic !D15 anti/od)
H1## n5JmL< a!D15I. +8%al protein o& =hole cells l)sate =as separated /) 3D3-PA(+ on a
15 N 5el and imm%nostained =ith anti-$id anti/odies and respective secondar) anti/odies.
Depicted is one o& three =estern-/lots &or each 5ro%p.
"#
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"1
)%& 4iscussion
Tra%ma is most o&ten ca%se o& death in ind%strial nations in the pop%lation %nder # )ears H1-
I. This &act points to the socio-str%ct%ral importance o& tra%ma s%r5er) d%e to an over-a5in5
o& o%r nations. 4orced initial investment in a pol)tra%mati>ed patient sells a /etter o%tcome
and minimi>es &ollo= costs &or inte5ration. Modern medical proced%res =ith modern dr%5s let
develop ne= %nkno=n sit%ations &or the h%man or5anism. ;ptimi>ed dr%5 application and
s%r5ical treatment co%ld ameliorate the o%tcome o& severel) in@%red patients. This 5ro%p o&
patients is at a hi5h risk to develop 3I23 and sepsis accompanied =ith M;D3 HM%lti 8r5an
4)s&%nction S)ndromeI and M;4 HM%lti 8r5an *ail%reI. The anti5enic &lood o& crashed
tiss%e and e,ternal &orei5n anti5ens stim%late directl) and indirectl) hostEs &irst line o&
de&ense. ;ver-activation o& PM' and P$M! leads to a set &ree o& pro-in&lammator) c)tokines
and h)perstim%lation o& pro5enitor cells in the /one marro= leadin5 to an ampli&)in5 loop
and a &lood o& PM' and P$M!. These cells de5ran%late and in&iltrate parench)mate%s or5ans
at the local site o& in@%r) and in %nin@%red or5ans. This mmetastaticD /ehavior is the ca%se &or
M;D3 and M;4. 3im%ltaneo%sl) compensator) mechanisms are started H!A23\
Compensator) Anti-in&lammator) -esponse S)ndromeI to s%ppress the over-activation o&
PM'. 3tartin5 !A23 occ%rs %s%all) too late and leads to an imm%nos%pression endan5erin5
patientEs li&e HTimin5 o& s%r5ical proced%res: Ta/le I. T=o e,treme poles in pol)tra%ma
disease =hich< =hen re5%lated co%ld improve patientsE o%tcome HD)namics o& disease: Ta/le
5I.
"2
)%$% Cell membrane: The Needle ;ar
Intracell%lar anti5ens ma) mimicr) proper li5ands o& the toll like receptors and activate those
intracell%lar si5nalin5 cascades. The massive attack o& lo= molec%lar =ei5ht peptides and
oli5osaccharides &rom the crashed tiss%e and the e,ternal =orld occ%pies &ree receptors ver)
8%ickl). +ven i& there is a ver) hi5h /indin5 constante< the concentration makes this
di&&erence disappear.

Table ): Decision makin5 and strate5) in a pol)tra%mati>ed patient. A55ressive initial
treatment leads to anti5enic control and a red%ced h)perin&lammation. !A23 makes s%r5er)
li&e threatenin5 and deletar) in&ections pro/a/le a&ter appro,imatel) the da) 1#. HPro&. Dr.
med. ;tmar Trent>I.
The Toll-like receptors are the primar) and ver) &ast sensors o& an or5anism to detect &orei5n
or5anism in a /od) and to set it in a de&ensive position. This s)stem seems to /e
"3
Surgical inter)ention 2iming
7ife savin6 sur6ery
O
P4ama6e controlP Day 1
;arly total care
PSecond loo"PB onlyQ Day 2-3
Scheduled definitive
sur6ery
Day -1!
No s"r#ery$
Secondary reconstructive
sur6ery
Wee% 3
%hysiological status
6)per- in&lammation
Imm%nos%ppression
2ecover)
2esponse to
res%scitation:
&Win'o( of
o))ort"nity&
<
Surgical inter)ention 2iming
7ife savin6 sur6ery
O
P4ama6e controlP Day 1
;arly total care
PSecond loo"PB onlyQ Day 2-3
Scheduled definitive
sur6ery
Day -1!
No s"r#ery$
Secondary reconstructive
sur6ery
Wee% 3
%hysiological status
6)per- in&lammation
Imm%nos%ppression
2ecover)
2esponse to
res%scitation:
&Win'o( of
o))ort"nity&
<
ph)lo5eneticall) ver) old resem/lin5 the &irst imm%nit) s)stem. The Toll-like receptor s)stem
has /een sho=n to /e e,pressed on 'e%trophil (ran%loc)tes< TL2 2 as =ell as TL2 H"2<"3I.
+ndoto,in tolerance =as descri/ed several )ears /e&ore and =as controversel) disc%ssed H25I.
Table 5: The D)namics o& 3I23 and !A23 in time H01I. The initial h)perin&lammation is
&ollo=ed /) an overinhi/ition H!A23I o& the imm%ne response. ;nl) the t=o e,tremes are
sho=n< /oth take place at the same time.
Instead o& a loss o& Toll-like receptors on cell%lar s%r&ace< there is an increase o& these
receptors accompanied =ith a red%ced cell%lar response %pon stim%lation =ith LP3 or
MALP2. Interestin5l) the same cells inc%/ated e$ )i)o =ith the speci&ic li5ands &or TL22
HMALP2I and &or TL2 HLP3I revealed neither a receptor %p-re5%lation nor a do=n-
re5%lation. !ells anal)>ed directl) &rom septic patients revealed a si5ni&icant increase in
receptor concentration on cell%lar s%r&ace =ith a similar stim%lation response o& MALP2 and
LP3< /%t on a lo=er level. Internal control anal)sis o& IL0 and T'4 in the s%pernatant o& the
"
Inflammatory
Anti,Inflammatory
SI-S
CA-S
Infection
7ate M84S
;arly M84S
InRury
EtimeG
Sepsis
Inflammatory
Anti,Inflammatory
SI-S
CA-S
Infection
7ate M84S
;arly M84S
InRury
EtimeG
Sepsis
inc%/ation medi%m revealed a s%&&icient response to the speci&ic stim%li Hdata not sho=nI.
This &indin5s e,cl%de a proced%ral phenomenon o& a/sent TL2 ind%ction in an e$ )i)o assa).
There&ore< the receptor concentration cannot /e responsi/le &or di&&erent stim%lation ans=ers
in PM' &rom health) individ%als and PM' &rom septic patients. I&< the tolerance on
endoto,in does not root in the desen>itation o& Toll-like receptors< then other intra or
e,tracell%lar mechanisms m%st /e responsi/le &or this e&&ect. The m)eloid di&&erentiation
&actor HM)D00I is recr%ited to the c)toplasmic domain o& TL22 and TL2. M)D00 /inds to
I2AK HIL-1 receptor associated kinaseI a&ter stim%lation H"I. I2AK coassociates =ith IKK
Hinhi/itor) $-kinaseI =itch leads to phosphor)lation o& I-$ and se8%ent pol)%/i8%itination
and proteasomal de5radation o& I-$. The &indin5 that M)D00 de&icient mice cannot
transd%ce the MALP2 or LP3 si5nal and are there&ore endoto,in resistent. This co%ld center
M)D00 in the scienti&ic interest /%t =e co%ld not &ind an) di&&erences on the level o& m2'A
in health) individ%als and septic patients Hdata not sho=nI. The kinase path=a) is not linear
/%t a =idespread cascade-like si5nalin5 e&&ect involvin5 a lot o& other &actors s%/-5ro%ped as
MAP-Kinases.
)%( The .hosphate Cascades: Cuic" -esuscitation Action%
Diversi&)in5 the si5nal o& M)D00 the phosphate resid%es are translocated to a plent) o&
di&&erent MAPHnIK %pon /indin5 o& LP3 or I4' to its receptor. The si5nal is shi&ted &rom
MAPHnIK to MAPHn-1IK ver) 8%ickl)< =ithin min%tes. The M)D00 dependent path=a)
terminates in the activation o& Z'K< p30 and p2J +2K. These three kinases are the
activators o& the transcription &actor AP-1 Hhttp:JJ===.5enome.@p\ hsa/8;,/I. .e
demonstrated in this st%d) the anti-apoptotic e&&ect o& LP3 and I4' in PM'. The LP3
ind%ced anti-apototic e&&ect seems to /e predominantl) re5%lated /) the MAP-kinase p2J
+2K< o/served in /oth health) vol%nteers and patients =ith sepsis. 3peci&ic inhi/ition o&
"5
p2J +2K a/olished the anti-apoptotic e&&ect o& LP3 and the speci&ic inhi/ition o& p30
inhi/ited the I4' ind%ced anti-apoptotic e&&ect in PM'. These res%lts point onto a central
role o& +2K and p30 in LP3 and I4'- si5nalin5. It has /een sho=n that pro-in&lammator)
c)tokines pla) a pivotal role in anti-apoptotic si5nalin5 in P'M H"5< ""I. 2eco5nition o& these
li5ands /) their speci&ic inhi/itors initiates protein phosphor)lation transmittin5 the si5nal on
intracell%lar e&&ector proteins H"-I. .e sho=ed in o%r in )itro in&lammation model that the
stim%lation o& PM' =ith LP3 or I4' leads to activation o& protein t)rosine kinases< and their
inhi/ition a/ro5ates the anti-apoptotic e&&ect o& LP3 and I4'. In the control 5ro%ps inc%/ated
onl) =ith the speci&ic inhi/itors< =e co%ld not &ind an) si5ni&icant di&&erences in the apoptotic
rate compared to the control 5ro%p< respectivel). This data s%pport the thesis that the MAP-
kinases /ehave passivel) and are activated onl) %pon contact =ith proin&lammator) stim%li.
This thesis is s%pported /) t=o &indin5s: no e&&ect on spontaneo%s apoptosis /) inhi/ition o&
MAP kinases and no e,pos%re o& activit) in %nstim%lated cells. The speci&icit) o& MAP-
kinase inhi/ition /) PD10#51 and 3$2#350# has /een sho=n else=here H"0< "1I. Altho%5h
the anti-apoptotic e&&ect o& LP3 and I4' has /een completel) a/ro5ated /) 6er/im)cin< the
LP3 mediated anti-apoptotic e&&ect seems to /e mediated /) p2J +2K and the I4' e&&ect
/) p30. This indicates indirectl) the involvement o& '4-$ in the LP3 mediated s%rvival
Hhttp:JJ===.5enome.@p\ hsa/8;,/I H-#I.
6o=ever< the &%ll inhi/ition o& MAP-kinase activation in PM' /) 6er/im)cin co%ld not &%ll)
restore the apoptotic rate in septic PM'. ;ther mechanism /esides MAP kinase si5nalin5
m%st e,ist that promotes PM' s%rvival %nder in&lammator) conditions.
)%3 cIA.(: 2loc"in6 the -oad to 4eath
""
In ph)lo5eneticall) ancient times or5anisms inte5rated /ac%loviral D'A. The repetitive
in&ection let develop a s)m/iosis =ith inte5ration in o%r 5enome. cIAP2 is a mem/er o& the
$I2 Hbac%loviral IAP repeatsI protein 4amil) and re5%lates the activit) o& intracell%lar
proteases. The proteinEs rin5 &in5er domain translocates %/i8%itin resid%es /) an isopetid)l
/onda5e and destines the protease to proteasomal de5radation.
In this st%d) =e have chosen an e$ )i)o stim%lation protocol =ith a preinc%/ation time =ith
LP3 o& 5 ho%rs< to allo= the cIAP2 ind%ction. cIAP2 has the a/ilit) to /ind and inhi/it
caspase?3 and the 2I'(-domain can transd%ce %/i8%itin resid%es to the inhi/ited caspase-3
to /e de5raded /) the proteasomal path=a). The $lockade o& the proteasome inhi/its the
caspase-3 de5radation %pon stim%lation =ith a!D15.
In the e, vivo e,periment =e shoved a h)pothetical protection mechanism to randoml)
activated caspases< in this case caspase-3. PM' taken &rom the septic patient have a
comm%nication histor) =ith patientEs /lood. The transcription &actors are activated and
transcription takes place< this co%ld e,plain the partial caspase resistance %pon !D15
activation. 'olan H31I and co=orkers sho=ed that an earl) inhi/ition o& the proteasome led to
a red%ction o& LP3-mediated s%rvival< s%55estin5 an involvement o& transcription &actors like
'4-n$ d%e to lack o& I$ inactivation H31I. There&ore< =e have chosen in o%r e,periments a
lon5er preinc%/ation period %p to 5 ho%rs =ith LP3 to allo= 5ene transcription and protein
e,pression =ith a possi/le cIAP2 ind%ction< mimickin5 PM' histor) in patientEs /lood. As
=e co%ld sho=< cIAP2 m2'A and Protein =as e,pressed d%rin5 this time period. To test the
participation o& the proteasome in this mechanism< =e %sed the speci&ic proteasome inhi/itor
P3I one ho%r prior to stim%lation =ith a!D15. This time point =as chosen to di&&erentiate
/et=een a direct e&&ect o& proteasomal inhi/ition on '4-n$ re5%lated 5ene transcription /)
inhi/ition o& I-n$ de5radation and the later e&&ect o& proteasomal de5radation o& %/i8%itinated
proteins H-1I. Activation o& the !D15 cascade activates caspase-3< =hich then can /e
%/i8%itinated /) IAPs and destined &or de5radation /) the proteasomal path=a). It has /een
"-
sho=n previo%sl) that IAP-protein class mem/ers are a/le to inhi/it and %/i8%itinate caspase-
3.H-2<-3I D%rin5 this process the IAPs are /ein5 cleaved and de5raded /) activated caspase-3<
/%t also a %/i8%itin molec%le is /ein5 attached to caspase-3 =hich event%all) leads the
%/i8%itinated caspase-3 to=ards proteasomal de5radation. ;%r data sho= that the
preinc%/ation o& PM' =ith LP3 leads to an earl) ind%ction o& cIAP2 m2'A and protein< and
that cIAP2-mediated caspase-3 %/i8%itination takes place %pon caspase-3 activation H-5I s%ch
as a&ter stim%lation o& !D15. In parallel< =e o/served in =estern/lot a red%ction o& cIAP2
protein a&ter t=o to &o%r ho%rs stim%lation o& !D15< &%rther s%pportin5 this h)pothesis. The
/indin5 o& IAPs to caspase-3 is mediated /) the di&&erent $I2-domains o& the cIAP2 protein.
The $I2-domain &its e,actl) into the active center o& caspase-3 and inhi/its their activit) H-I<
in parallel the rin5-&in5er domain o& the IAP molec%le< responsi/le &or %/i8%itination comes
in close contact =ith caspase-3 and attaches one %/i8%itin molec%le to the activated caspase-3.
This process takes place repetitivel) leadin5 to a pol)%/i8%itin chain on the activated caspase-
3. A pol)%/i8%itinated protein< like caspase-3< is destined &or proteasomal de5radation.
6o=ever< as the cIAP2 protein itsel& =as cleaved and de5raded as earl) as &o%r ho%rs a&ter
!D15 stim%lation< the cIAP2-mediated inhi/ition o& apoptosis co%ld /e inhi/ited /) a
caspase-3-mediated &eed/ack loop< =hich mi5ht also e,plain the resid%al apoptosis seen in
!D15-stim%lated PM' preinc%/ated =ith LP3.
Inhi/ition o& the proteasome /) P3I< in t%rn< seemin5l) increased the amo%nt o& %/i8%itinated
caspase-3 a&ter LP3-stim%lation d%e to lack o& proteasomal de5radation. 6o=ever< inhi/ition
o& the proteasome ma) also elevate the level o& cIAP2 protein =hich d%e its
a%to%/i8%itination a/ilit) =o%ld /e like=ise de5raded H-3I. These cIAP2 molec%les co%ld
shi&t the /alance to a red%ction o& caspase-3 activit) /eca%se o& the direct cIAP2-dependent
caspase-3 inhi/ition H-"I. 6o=ever< the remainin5 caspase-3 activit) and apoptosis rate in
cells treated =ith P3I s%55ests that %/i8%itin has no direct inhi/itor) e&&ect on activated
caspase-3< /esides tar5etin5 caspase-3 &or proteasomal de5radation. The LP3-e&&ect on !D15-
"0
ind%ced apoptosis and caspase-3 activit) =as completel) a/olished a&ter inhi/ition o& the
proteasome indicatin5 that the %/i8%itinated caspase-3 retains its activit).
In this short anal)sis o& the inhi/ition o& caspase-3 %pon stim%lation =ith LP3 =e
demonstrated the central role o& the proteasome in protein-protein interaction in the
c)toplasm. The proteasome is a m%lti-phased str%ct%re involved in the s%rvival and death o&
the cell. All seems to depend on the point o& time the interaction takes place H30I.
)%) 2cl,( .roteins: The 2alanced Suicide Machinery
;ther &actors re5%latin5 the e,ec%tion o& apoptosis are the $cl-2 proteins. A plent) o&
mem/ers o& these proteins has /een identi&ied %ntil no= Hsee Ta/le 2.I. This s)stem o& pro-
apoptotic and anti-apoptotic $cl-2 proteins /%ilds a protection and o&&ensive line to the o%ter
mitochondrial mem/rane. A 5ood model ima5ination is an intracell%lar complementar)
s)stem =hich is al=a)s r%nnin5 and apoptosis takes onl) place =hen the /alance is shi&ted to
the advanta5e o& the pro-apoptotic $cl-2 mem/ers. ;%r 5ro%p has sho=n that the spontaneo%s
apoptotic cell death o& PM' is not caspase-dependent< in contrast to receptor mediated
apoptosis H-0I. ;%r res%lts con&irm the data o& other scienti&ic 5ro%ps &or $cl-2 H-0I< $A9
H-1I< or /oth H0#< 01I. The ver) small amo%nts o& $cl-2 in PM' l)sates mi5ht /e e,plained /)
tin) P$M! contamination d%rin5 preparation H02I. This &indin5 is s%pported /) densitometric
anal)sis o& these .estern-/lots compared to 1##N P$M! preparation.
At the level o& $A9-protein =e =ere not a/le to &ind an) si5ni&icant di&&erences in PM' &rom
health) individ%als or septic patients. A small di&&erence in the e,pression pattern o& $A9-
m2'A =as &o%nd. The lo=er m2'A e,pression in septic PM' co%ld /e e,plained /) their
a5e. 3eptic PM' are m%ch older d%e to their increased li&espan d%rin5 sepsis and th%s mi5ht
e,press lo=er 5ene-activit) in 5eneral. The hi5h e,pression o& $A9-protein in /oth 5ro%ps
ma) /e attri/%ted to its hi5h sta/ilit) despite the lack o& m2'A H03I.
"1
In contrast to $cl-2 and $A9 =e o/served di&&erences in $id-protein levels in PM' &rom
health) individ%als and septic patients. A si5ni&icant decrease o& the $id-protein =as al=a)s
o/served in e,perimental 5ro%ps =ith a hi5h apoptotic rate. This sho=s that $id-protein mi5ht
inversel) re&lect the level o& apoptosis. This thesis =as en&orced /) the o/servation that $id-
protein red%ction occ%rred also in septic PM' s%/@ected to receptor mediated apoptosis. This
red%ction =as a/olished /) the pancaspase inhi/itor >FA4-&mk H03I. Levels o& m2'A
remained %nchan5ed in /oth 5ro%ps. These &indin5 let $id participate apoptotic proced%res
/%t make it %nlikel) to re5%late it.
In contrast to $cl-2 =e o/served the most prominent di&&erences &or Mcl-1 protein and
m2'A. Previo%s o/servations co%ld /e con&irmed /) o%r &indin5s H01< 0I. In contrast to
other (ro%ps H0I =e &o%nd a red%ction o& 5#N o& Mcl-1 protein in PM' &rom health)
individ%als inc%/ated =ith medi%m< LP3 or a!D15. This =as ca%sed =ith a hi5h pro/a/ilit)
/) the di&&erent inc%/ation time H1"h vs. 2#hI. The hi5h apoptotic rate o& PM' co%ld not have
le&t man) non non-apoptotic cells a&ter 2# ho%rs. The stim%lation =ith a!D15 led not to a
decrease o& Mcl-1 protein s%55estin5 a caspase-independent path=a) o& Mcl-1 =aste< or a
shorter inc%/ation protocol. 6ere =e =ere a/le to detect m2'A o& Mcl-1 onl) in septic PM'
despite lo= Mcl-1 protein. 6o=ever< ho= the Mcl-1 t%rnover takes place remains to /e
el%cidated. Interestin5l)< the inc%/ation o& PM' =ith LP3 led not to an ind%ction o& Mcl-1
m2'A in o%r e,periments. This point in the re5%lation o& the Mcl-1 5ene d%rin5 sepsis sho=s
that PM' stim%lation =ith LP3 ma) not represent a 5eneral model o& sepsis.
Di&&erent ind%ctors o& Mcl-1 have /een &o%nd< IL-" H05I and I(4-1 H0"I controlled /) 3TAT3
H0-I. A model &or the s%rvival o& PM' d%rin5 sepsis has alread) /een proposed H01I. The
red%ction o& apoptosis d%rin5 sepsis is achieved /) a m%lti-c)tokine orchestra and the
ind%ction o& short-lived anti-apoptotic proteins s%ch as Mcl-1 H00I. This ind%ction shi&ts the
cell to=ards s%rvival and anta5oni>es the lon5-lived &actors like $id and $A9. These &indin5s
-#
are s%pported /) o%r res%lts< /%t &%rther investi5ation and anal)sis has to /e done to
%nderstand the &ine net=ork o& the $cl-2 s)stem.
)%5 The Strate6y
D%rin5 tra%ma health) tiss%e is /ein5 cr%shed and intracell%lar as =ell as e,ternal patho5ens
ma) 5ain access to /lood circ%lation. The &irst line o& de&ense HPM'I< reco5ni>e several
moti&s /) their TL2 receptors leadin5 to activation o& these cells =ith a si5ni&icantl)
prolon5ed s%rvival. I& the s%ppl) o& patho5ens persists and the cells are over-activated< the)
&lood the or5anism and acc%m%late in parench)mateo%s or5ans. The earl) phase =e call
M;D3 in this phase the or5an dama5e is pontetiall) reversi/le. Persistin5 patho5enic load
and there&ore< s%stained stim%lation let the cells de5ran%late< and dama5es the site o&
de5ran%lation HM;4I. The or5ans s%&&er irreversi/le dama5e and the patient dies in a
septicall) ind%ced M;4.
In o%r st%d) =e anal)sed di&&erent possi/le mechanisms o& spontaneo%s apoptosis and their
inhi/ition /) LP3. The Toll-like receptors =ere %p-re5%lated d%rin5 sepsis and not ind%ci/le
/) LP3 or MALP2 indicatin5 not an a%to-re5%lation /%t a pol)-&actorial event. $indin5 o&
LP3 or MALP2 led to activation o& MAP-kinases and to an increased s%rvival rate o& PM'.
$acterial patho5ens are most likel) to si5nal /) p2J +2K and I4'- /) p30< the inhi/ition
o& these kinases restored the e&&ect o& the stim%li. The intracell%lar mtrash canD< the
proteasome< =as alread) sho=n to re5%late apoptosis /) the inhitition o& the actvation o& '4-
$ H31I. 6o=ever< the e&&ect o& the proteasome-inhi/ition ma) /e a /i-c%ttin5 ed5e. ;n the
one hand the inhi/ition ma) ind%ce apoptosis and on the other hand it ma) inhi/it apoptosis.
.e co%ld sho= the ind%ction o& cIAP2 and the %/i8%itination o& caspase-3 %pon activation.
The caspase remained active =hen the proteasome =as /locked. This possi/l) opens a tin)
=indo= on caspase resistance in PM'. The anal)sis o& $cl-2 s)stem revealed not directl)
concl%sive data< /%t Mcl-1 seems to /e a plat&orm o& the $cl-2-s)stem d%rin5 sepsis. The
-1
ind%ction o& Mcl-1 m2'A and the a/sence o& Mcl-1 protein can /e e,plained /) its
de5radation d%rin5 the apoptotic process< th%s inhi/itin5 this process. The clinical strate5)
=ill still consist o& s%r5ical intervention and hemodial)sis to red%ce the anti5enic load.
4re8%ent second looks and dail) dressin5 chan5es =ill red%ce the anti5enic load at the side o&
in@%r). Dial)sis red%ces the anti5enic load s)stemicall). $oth strate5ies lead o&ten to s%ccess
in patients =ith severe sepsis. The cl%e approach remains: mKeep the receptors tid)D.
)%/ Aypothetical Molecular Tar6ets
Insistin5 on ne= h)pothetical molec%lar tar5ets &or imm%no mod%lation ma) lead to /lindness
&or principall) simple pro/lems. LetEs s=itch to an other point o& vie=< and consider a septic
ne%trophil as a t%mor cell and not an)more as an in&lammator) cell. These cells onl) tr) to
s%rvive and to escape their apoptotic destin). The cl%e 8%estion is =here to /lock and to
do=n-re5%late to achieve a smart imm%no mod%lation in the sense o& s%rvival. At the level o&
cell%lar mem/rane =e have o/served an ind%ction o& TL2-receptors< /%t this does not e,plain
the endoto,in tolerance o& septic PM'. mKeep the receptors tid)D is the act%al therap) in
severe sepsis. The myes or no! si5nalin5 =a) o& receptors is not s%ita/le &or mod%lation< there
is the mmaybe! missin5. At the level o& second messen5ers HMAPKI h)pothetical
phosphor)lation controllin5 co%ld slo= do=n the endoto,in ind%ced s%rvival cascade Hsee
chap. .2< .5I. MAPKs are %/i8%ito%s si5nalin5 components< there&ore< an inhi/ition co%ld
ca%se massive side e&&ects. !aspases as e,ec%tioners o& apoptosis &ail as tar5ets d%e to an
overall apoptotic cell death %pon s)stemical activation. ;nl) t=o components o& the apoptotic
net=ork remain the IAP and $cl-2 proteins. These proteins are involved in a &ine net=ork
inside the s%icide machiner) and co%ld represent possi/le molec%lar tar5ets &or imm%no
mod%lation. 2ecent p%/lications H1#I sho= a possi/le role &or triphen)l%rea and derivates to
inhi/it $I2-domains in IAP proteins. The imm%no mod%lative e&&ect =as sho=n &or 9IAP
and pancreatic cell carcinoma H11I. The positive e&&ect co%ld /e linked to the tar5etin5 o& a
-2
defensi)e! molec%lar str%ct%re. This tar5et< in this case 9IAP< comes to action onl) =hen
caspases are activated. $I2 HIAPI inhi/ition leads to an e&&ect onl) =hen $I2 is needed. In
the case o& a septic PM' co%ld it restore the apoptotic rate and ameliorate the septic o%tcome
Hsee !hap 3.3I. 3)stemic e,periments still have to /e done.
Lessons learned &rom !LL Hchronic l)mphatic le%kemiaI H12I point to the $cl-2 s)stem. 4or
the didactical reason the pro-apoptotic $cl-2 s)stem =ill /e called $63-s)stem in this section<
d%e to its pro-apoptotic activit). $63 proteins Hsee !hap. 3.I /elon5 to the pro-apoptotic
5ro%p o& $cl-2 proteins. +speciall) the $63 onl) proteins H$id and $imI once activated co-
associate =ith $ak and $A9 leadin5 to pore &ormation in o%ter mitochondrial mem/rane and
conse8%entl) to apoptosis. In man) hematolo5ical t%mors< le%kemias< are the anti-apoptotic
$cl-2 proteins ind%ced. That is also the reason =here $cl-2 has 5ot its name &rom H$-!ell
Le%kemia T)pe 2I. The conse8%ence o& ind%ction o& s%rvival /) $cl-2 is more or less a
resistant cell to apoptotic stim%li. Mimicin5 the $63-e&&ect /) recentl) introd%ced s%/stances<
A$T--3- and A$T-2"3 H13I< revealed %nder e,perimental conditions inhi/ition o& t%mor
5ro=th in )itro. The cell death =as normali>ed ad detected as mitochondrial apoptosis.
+,perimental proced%res in 3!ID mice =ith transplanted 4D!-P1 t%mor cells and application
o& A$T--3- led to complete t%mor re5ression H13I. &'myc driven $-cell l)mphomas in mice
co%ld /e inhi/ited /) the application o& A$T--3- H1I. The plat&orm o& the &inal common
path co%ld /e Mcl-1 indicated /) si2'A screens in 611" 3!L! cells H1I. As in o%r =ork the
Mcl-1 =as in septic PM' al=a)s ind%ced Hsee !hap. 3..2I. Application o& $63 mimics
co%ld restore the spontaneo%s apoptosis o& septic PM' =itho%t severe side e&&ects< d%e to
tar5etin5 o& a mpassiveD molec%le. The cell%lar si5nalin5 and apoptotic mechanism =o%ld not
/e dist%r/ed.
-3
*i6ure 3$:
Theoreticall)< these t=o molec%lar tar5ets ma) provide a &ield &or &%t%re sepsis therapies.
)%1 !or" to be done
'o one kno=s ho= li&e appeared on the +arth< the step=ise evol%tion &rom simple to comple,
is ver) pro/a/le. I& =e ass%me prokar)otic li&e =itho%t mitochondria< mechanisms &or
en>)matic di5estin5 o& pha5oc)tosed material m%st have e,isted. These s)stems ma) still
persist to hi5her e%cariotes onl) sli5htl) chan5ed in &%nction. ;n the search &or alternative
apoptotic path=a)s a lot o& =ork has /een done. Models o& interaction o& $A9 =ith
l)sosomal mem/rane and the res%ltin5 a%topha5oc)tic cell death /) the released l)sosomal
material have /een developed H1-I. These models are not applica/le &or ever) cell t)pe and
have to /e revised. Alternative 5enes have /een characteri>ed and called AT( 5enes mainl)
in )east /%t also in mammals H15< 1"I. These &actors can co-assem/l) to a m%lti&actor
comple, and constr%ct a%todi5estive machiner). In other =ords the res%lt =o%ld /e the same
like in apoptotic cell death. ;ther h)pothetical &%nction co%ld /e the protection a5ainst
intracell%lar patho5ens s%ch as Shigella spp. and "ycobacteria spp. This co%ld have a pro-
s%rvivin5 e&&ect rather than a pro-apoptotic e&&ect. A lot o& research has to /e done to
-
A$T--3-< chemical compo%nd mimicin5
$63-domain activit). Abbott Gmb. J &o.<
L%d=i5saha&en< (erman). Application o&
A$T--3- restored apoptotic activit) in !LL<
and led to t%mor re5ression H12I.
characteri>e the di&&erent &actors in mammalian cells and to t)pe o%t their &%nctions. The
alternative cell death pro5ramme in ne%trophil 5ran%loc)tes has not /een &o%nd< )et. 6ence
the additive molec%lar tar5ets &or imm%nolo5ical mod%lation /eside $63 and $I2 are still to
/e e,pected. The ac8%isition o& m%ltiple possi/le tar5ets co%ld lead to a possi/le therap)
model o& /alanced imm%no mod%lation< individ%all) &itted &or each sit%ation. The &lo= =ill
al=a)s 5o in the same direction it depends onl) ho= &ast this happens. As ph)sicians =e =ont
/e a/le save patientEs live< =e 5ain onl) time. ;nl) e,perimental =ork in )i)o and in )itro
will sho= i& =e precede the ri5ht direction. Al=a)s /e critical to a nice /attle plan and do not
loose )o%r primar) options o& therap): m'o /attle plan s%rvives the &irst &ive min%tes o&
contact =ith the enem)mH(enenar&eldmarschall 6elm%th Karl $ernhard (ra& von Moltke<
h2".1#.10## - k2.#.1011I
-5
.AS
7C3,II
ATG5,ATG$(B
ATG$/7
2eclin,$
5.S3)
5.S$5
2C7(
2C7,=7
>5-AG
!I.I
mT8-
>70
ATG'
A>T8.AAG?
.AS
7C3,II
ATG5,ATG$(B
ATG$/7
2eclin,$
5.S3)
5.S$5
2C7(
2C7,=7
>5-AG
!I.I
mT8-
>70
ATG'
A>T8.AAG?
*i6ure 3(: A%topha5ic cell death: Assem/l) o& a m%ltimeric protein comple, at PA3
Hpagophore assembly siteI. AT( related 5enes =ere identi&ied in oeast and Drosophila. Poor
correlation to mamalian h)pothetical a%topha5osome H15< 1"I. This comple, has pro/a/l) a
pros%rvivin5 role rather than a pro-apoptototic role.
-"
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0
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$ak. Shock 2##2\1-:-.
0-
0#. Mo%ldin5 DA< Ak5%l !< Dero%et M< et al. $cl-2 &amil) e,pression in h%man ne%trophils
d%rin5 dela)ed and accelerated apoptosis. K Feukoc Diol 2##1\-#5:-03.
01. Ak5%l !< Mo%ldin5 DA< +d=ards 3.. Molec%lar control o& ne%trophil apoptosis. >#DS
Fett 2##1\0-:310.
02. Mo%ldin5 DA< *%a)le ZA< 6art !A< +d=ards 3.. Mcl-1 e,pression in h%man
ne%trophils: re5%lation /) c)tokines and correlation =ith cell s%rvival. Dlood 1110\12:215.
03. (ross A< oin 9M< .an5 K< et al. !aspase cleaved $ID tar5ets mitochondria and is
re8%ired &or c)tochrome-c release< =hile $!L-9L prevents this release /%t not t%mor necrosis
&actor-21J 4as death. K Diol &hem 1111\2-:115".
0. Leuenroth S), *rutkoski +S, ,"ala ,, Si--s HH. .he loss o Mcl-% e/pression in hu-an
pol"-orphonuclear leukoc"tes pro-otes apoptosis. J Leukoc Biol #$$$&0('%5(.
(5. Zo%rdan M< De Fos Z< Mechti '< Klein $. 2e5%lation o& $cl-2- &amil) proteins in
m)eloma cells /) three m)eloma s%rvival &actors: interle%kin-"< inter&eron-alpha and ins%lin-
like 5ro=th &actor 1. &ell Death Differ 2###\-:12.
0". Derenne 3< Monia $< Dean 'M< Ta)lor ZK< 2app MZ< 6aro%ssea% ZL< $ataille 2< Amiot
M. Antisense strate5) sho=s that Mcl-1 rather than $cl-2 or $cl-,HLI is an essential s%rvival
protein o& h%man m)eloma cells. Dlood 2##2\1##:11.
00
0-. +plin5-$%rnette PK< Li% Z6< !atlett-4alcone 2< et al. Inhi/ition o& 3TAT3 si5nalin5 leads
to apoptosis o& le%kemic lar5e 5ran%lar l)mphoc)tes and decreased Mcl-1 e,pression. K &lin
In)est 2##1\1#-:351.
00. Ak5%l !< T%rner P!< .hite M2< +d=ards 3.. 4%nctional anal)sis o& the h%man Mcl-1
5ene. !ell Mol Li&e 3ci 2###\5-:"0.
01. Mannick ZA< 2odrick ML< Lederer ZA. The imm%nolo5ic response to in@%r). K Am &oll
Surg. 2##1\113H3I:23-.
1#. .an5 ]< !%dd) M< 3am%el T< .elsh K< 3chimmer A< 6anaii 4< 6o%5hten 2< Pinilla !<
2eed Z!. !ell%lar< /iochemical< and 5enetic anal)sis o& mechanism o& small molec%le IAP
inhi/itors. K Diol &hem. 2## 12\2-1H"I:01"0--".
11. Karikari !A< 2o) I< Tr)55estad +< 4eldmann (< Pinilla !< .elsh K< 2eed Z!< Armo%r +P<
.on5 Z< 6erman Z< 2akhe@a D< Maitra A. Tar5etin5 the apoptotic machiner) in pancreatic
cancers %sin5 small-molec%le anta5onists o& the 9-linked inhi/itor o& apoptosis protein ."ol
&ancer 2her. 2##- Mar\"H3I:15--"".
12. .)nham 6< .ilson AT< Levine AM< D%nleav) K< Krivoshik AP< 6a5e) A+. A phase
1J2a st%d) eval%atin5 the sa&et)< pharmacokinetics< and e&&icac) o& A$T-2"3 in s%/@ects =ith
re&ractor) or repsed l)mphoid mali5nancies. 3ession T)pe: Poster 3ession< $oard no. 525-1.
Dlood< 2##-\11#\11.
01
13. La/i F< (respi 4< $a%m5artner 4< Fill%n5er A. Tar5etin5 the $cl-2-re5%lated apoptosis
path=a) /) $63 mimetics: a /reakthro%5h in anticancer therap)X &ell Death and Diff
2##0\15: 1---10-.
1. Lin 9< Mor5an-Lappe 3< 6%an5 9< Li L< ]ak%la DM< Fernetti LA. m3eedD anl)sis o& o&&-
tar5et si2'As reveals an essential role &or Mcl-1 in resistance to the small-molec%le $cl-
2J$cl-9L inhi/itor A$T--3-. Hncogene 2##-\2":31-2-31-1.
15. Mi>%shima< '. A%topha5): process and &%nction. Genes De). 2##-\21< 20"1?20-3.
1". De5terev A< o%an Z. +,pansion an evol%tion o& cell death pro5rames. =at rew mol cell
biol 2##0\1:3-0-31#.
1-. ZGGttelG M< !andq !< Kroemer (. L)sosomes and mitochondria in the commitment to
apoptosis: a potential role &or cathepsin D and AI4 &ell Death and Differentiation< 2##\11
H2I< 135-13".
1#
/%& 2oo"s:
Zane=a) !A<Travers P< .alport M< !apra ZD. Immunobiolo6yB The Immune System in
Aealth and 4isease% 4o%rth +dition. Garland. &hurchill Fi)ingstone. 1111 I3$': #-0153-
321--3
Lodish 6< $erk A< Mats%daira P< Kaiser !A< Krie5er M< 3cott MP< ]ip%rsk) 3L< Darnell Z.
Molecular Cell 2iolo6y% 4i&th +dition. >reeman 2## I3$': #--1"--3""-3
Kra%ss (. 2iochemistry of Si6nal Transduction and -e6ulation% Third +dition. Ciley'
<&. 2##5 I3$'-13:1-0-3-52--3#511-1
(omperts $D< Kramer IM< Tatham P+2. Si6nal Transduction% Academic %ress 2##3 I3$':
#-12-201"31-1
3tr)er L. 2iochemistry% 4o%rth +dition. >reeman 1115 I3$': #--1"--2##1-
.atson ZD<.itko=ski Z< (ilman M< ]oller M. -ecombinant 4NA% 3econd +dition.
Scientific American Dooks 2##1 I3$': #--1"--111-#
1%& 4atabases:
National Center for 2iotechnolo6y Information: http:JJ===.nc/i.nlm.nih.5ovJ
Genome Net+or": http:JJ===.5enome.@pJ
The ;x.ASy H;xpert .rotein Anal)sis SystemI: http:JJ===.e,pas).chJ
11
#%& Curriculum vitae:
Dr. med. Ladislav Mica
4eldstrasse 15
!6-"3## ]%5
$(%&#%$'1) 2orn in 2rno Hr332I
Mother: Pavla Mica-Mil%ska< Dr.med. h1#.12.11
4ather: Ladislav Mica< Dr.med h2".12.11"
A%5%st 11-- - A%5%st 110# Kinder5arden 6erstkova in $rno
#1.#1.110# - 31.#".1103 Primar) school 6erstkova 11 in $rno .
*"ly 1+,3 -mi#ration to .(it/erlan'.
A%5%st 1103-A%5%st 110- Primar) school in 3=it>erland
1.#1.110- - #".#".111 ()mnasi%m: Instit%t Dr. P&ister in ;/erG5eri. H](< !6I
&/%&/%$'') Maturity Typus 2 HLatinI at the Instit%t Dr. P&ister in
;/erG5eri< ](< 3=it>erland
21.1#.111 - #2.1#.2### 3t%dies o& h%man medicine in ]^rich.
111--2##1 !oncomitant e,perimental doctoral st%d)
in the la/orator) o& Pro&. Dr. med. .. +rtel: uIn )i)o
st%dies o& hepatic microcirc%lation in a m%rine model.m
&(%$&%(&&& *inishin6 exams at the >niversity of @Srich
Curriculum laboris:
#".11.2### - 31.#3.2##1 !linical =ork at department o& Tra%ma 3%r5er) at the
7niversit) 6ospital o& ]^rich H!hie&: Pro&. Dr. med. ;.
Trent>I.
#1.#.2##1 - 31.#3.2##2 Post5rad%ate !o%rse o& +,perimental Medicine H6ead:
Pro&. Dr. med. Z. ]ap&< sponsorin5: 3=iss 'ational
4o%ndationI.
&1%&3%(&&( .romotion to 4octor of Auman Medicine K>niversity
of @SrichL
12
#1.#.2##2 ? 31.#3.2##3 3cienti&ic =ork at the Department o& Tra%ma 3%r5er)
H!hie&: Pro&. Dr. med. ;. Trent>I.
#1.#.2##3 ? 3#.#1.2## !linical =ork at the department o& Tra%ma 3%r5er)
7niversit) 6ospital o& ]^rich H!hie&: Pro&. Dr. med. ;.
Trent>I.
#1.1#.2## ? 3#.#.2##5 4%rther +d%cation in Intensive !are Medicine<
7niversit) 6ospital o& ]^rich H!hie&: Pro&. Dr.med. 2.
3tockerI
&5%$$%(&&5 *irst exams in 6eneral sur6ery K*MAB >oederatio
"edicorum .el)eticorumL
#1.#5.2##5 - 31.#0.2##" !linical =ork at the department o& Tra%ma 3%r5er)
7niversit) 6ospital o& ]^rich H!hie&: Pro&. Dr. med. ;.
Trent>I.
#1.#1.2##" ? 31.12.2##0 !linical =ork at the clinic &or 5eneral s%r5er) in
Kreisspital MGnnedor& H]^rich< !6I H!hie&s: Pro&.
Dr.med. A. 6ollin5er v Dr.med. A. Follen=eiderI
($%$$%(&&# Specialist in Sur6ery *MA
#1.#1.2##1 ? 31.12.2##1 !linical =ork at the department o& Tra%ma 3%r5er)
7niversit) 6ospital o& ]^rich H!hie&: Pro&. Dr. med. 6.P.
3immenI
#1.#1.2#1#- Teamleader at the at the department o& Tra%ma 3%r5er)
7niversit) 6ospital o& ]^rich H!hie&: Pro&. Dr. med. 6.P.
3immenI
*urther ;ducation:
Co"rses0 Micros%r5er) H]^rich< !6I 21-25.#1.1110
A;-!o%rse Davos H!6I 1-11.12.2##3
Fasc%lar International H!6I 21-2.#1.2##"
A/dominal 3%r5er)< Davos< H!6I
#-1#.#3.2##"
ATL3 H]^rich< !6I 23.#1.2##
.iener 6andchr%r5ie HAI 2#-25.#5.2##-
13
;xperimental experience:
1olec"lar 2iolo#y0
D=A: Isolation< 3o%thern $lot< 2estriction mappin5<
Trans&ectionJTrans&ormation< P!2.
E=A: Isolation< 2T-P!2< si2'A.
%roteins: !oncentration< .estern $lot< 2D-$lot< +n>)me- Kinetics
"embranes: Transporter activities
Animal e3)eriments0
Intravital microscop)
M%rine micros%r5er) and anesthesia.
Cell c"lt"res0 6%man cells: 4i/ro/lasts< (ran%loc)tes< T6P-1< Z%rkat
Additional Mualifications:
4in#"istic s%ills0 CTech Hmother lan5%a5eI
German Hmother lan5%a5eI
;n6lish< &l%entl).
*rench< 5ood kno=led5e
7atin< te,t onl)
Italian< passive comprehension.
International Cooperations:
Active participation HPD Dr.med. M. Keel< Dr.med. L.
MicaI in: A multi,centerB randomiTedB double,blindB
parallel 6roupB placebo controlled trial to evaluate the
efficacy and safety of activated recombinant factor
5II Kr*5IIa9NovoSeven
U
9NiaStase
U
L in severely
inRured trauma patients +ith bleedin6 refractory to
standard treatment% =o)o=ordisk< 3tart 7niversit)
6ospital o& ]^rich #1.11.2##5 ? 11.#".2##0.
1
'%& .ublications
.anner (A< Mica L< .anner-3chmid +< 6ent>e 6< Kol/ 3< Trent> ;< +rtel .. Inhi/ition o&
caspase activit) prevents !D15-mediated hepatic microvasc%lar per&%sion &ail%re and restores
K%pp&er cell clearance capacit). 5A.-2 *< 13:1231-120< 1111
6Grter L< Mica L< 3tocker 2< Trent> ;< Keel M. Mcl-1 !orrelates =ith 2ed%ced Apoptosis in
'e%trophils &rom Patients =ith 3epsis. *o"rnal of American Colle#e of ."r#eons 1"-1-3<
2##3.
Mica L< 6Grter L< Trent> ;< Keel M. +ndoto,in red%ces !D15-Ind%ced ne%trophil apoptosis
/) cIAP2-mediated !aspase-3 De5radation. *o"rnal of American Colle#e of ."r#eons 111
HI 515-"#2< 2##.
6Grter L< Mica L< 3tocker 2< Trent> ;< Keel M. Increased e,pression o& toll-like receptor-2
and ? on le%koc)tes &rom patients =ith sepsis. .c6oc% 22 H5I: #3-#1< 2## IM.ACT: (%#&#
Keel M< Mica L< 3tover Z< 3tocker 2< Trent> ;< 6Grter L. Thiopental-ind%ced apoptosis in
l)mphoc)tes is independent o& !D15 activation. Anest6esiolo#y 1#3 H3I: 5-"-50. 2##5
La/ler L< .edler F< Mica L< Trent> ;. +ntrapment o& the anterior ti/ial arter) in a distal ti/ial
&ract%re a&ter intramed%llar) nailin5. Der 7nfallc6ir"r# 1#1H2I:15"-1. 2##" A(ermanB.
Fon KGnel 2< 6epp 7< $%dde/er5 !< Keel M< Mica L< Asch/acher K< 3chn)der 7. Altered
$lood !oa5%lation in Patients =ith Posttra%matic 3tress Disorder. Psyc6osomatic 1e'icine
2##" in press. IM.ACT: 3B#51
La/ler L< Mica L< 6Grter L< Trent> ;< Keel M. +in&l%ss der F.A.!. Therapie a%& >)tokine %nd
.achst%ms&aktoren in tra%matischen .%nden. 8entralblatt f9r C6ir"r#ie. 131: "2-"-. 2##"
Fon KGnel 2< 6epp 7< Kraemer $< Tra/er 2< Keel M< Mica L< 3chn)der 7. +vidence &or
lo=-5rade s)stemic proin&lammator) activit) in patients =ith posttra%matic stress disorder. *
Psyc6iat Res< 1H1I:--52.
15
Fon KGnel 2< 6epp 7\ Tra/er 2< Kraemer $< Mica L< Keel M< Ma%s/ach $T< 3chn)der 7.
Meas%res o& endothelial d)s&%nction in plasma o& patients =ith posttra%matic stress
Disorder. * Psyc6iat Res< 15\150H3I:3"3-3-3. 2##0
Mica L< (ianom D< $ode $< Zaklin P< 6ollin5er A. 2are !a%se o& D)spha5): (iant Pol)poid
+sopha5eal .ell-Di&&erentiated Liposarcoma. Case Re) :astroenterol 1: --1. 2##-
Fac%%m-assisted clos%re therap) increases local interle%kin-0 and vasc%lar endothelial 5ro=th
&actor levels in tra%matic =o%nds. La/ler L< 2ancan M< Mica L< 6Grter L< Mihic-Pro/st D<
Keel M. * Tra"ma. 2##1 Mar\""H3I:-1-5-.
+arl) ser%m procalcitonin< interle%kin-"< and 2-ho%r lactate clearance: %se&%l indicators o&
septic in&ections in severel) tra%mati>ed patients. $illeter A< T%rina M< 3ei&ert $< Mica L<
3tocker 2< Keel M. Worl' * ."r#. 2##1 Mar\33H3I:550-"".
Av%lsion o& the 6amstrin5 M%scle (ro%p: A 4ollo=-7p o& " Ad%lt 'on-Athletes =ith +arl)
;perative Treatment: A $rie& 2eport. Mica L< 3ch=aller A< 3to%pis !< Penka I< Fomela Z<
Follen=eider A. Worl' * ."r#. 2##1\ 33: 1"#5-1"1#.
The 3everit) o& In@%r) and the +,tent o& 6emorrha5ic 3hock Predict the Incidence o&
In&ectio%s !omplications in Tra%ma Patients. T. L%sten/er5er< M. T%rina< $. 3ei&ert< L.Mica
and M. Keel -"ro)ean *o"rnal of Tra"ma an' -mer#ency ."r#ery< Fol%me 35H"I: 530-
5".
3%sanne 6a/elt< Adrian 3ch=aller< Al/ert 6ollin5er< Ladislav Mica. 3eptic pol)arthritis
ca%sed /) Streptococcus pneumoniae: primar) pne%mococcal pne%monia as a risk &actor in
older patientsX A case report 21* Case Re)orts 2##1 Adoi:1#.113"J/cr.#2.2##1.1"#B
Ladislav Mica< Falentin 'e%ha%s< +nrico Pwschmann< Dilek Kwn^-Le/le/icio5l%< 7rs
3ch=ar>< (%ido A .anner< !lqment ML .erner< 6ans-Peter 3immen. .ydrocephalus
communicans a&ter tra%matic %pper cervical spine in@%r) =ith a cere/rospinal &l%id &ist%la: a
rare complication. 21* Case Re)orts 2#1#: p%/lished online 15 Z%l) 2#1#<
doi:1#.113"J/cr.#2.2#1#.2-31
1"
'%$ 8ral .resenations
.anner (A< Mica L< Trent> ;< +rtel .. Anti-4as anti/od) ind%ces hepatic microvasc%lar
per&%sion &ail%re and decreases K%pp&er cell clearance capacit). ."r# 5or"m 1:10-105<
1110
.anner (A< Mica L< 6ent>e 6< Trent> ;< +rtel .. 6emm%n5 der !aspase-AktivitGt
verhindert das 4as-vermittelte Mikro>irk%lationsversa5en der Le/er %nd die red%ktion der
!learance-4%nktion der K%pp&er>ellen. 4an#enbec%s Arc6 C6ir 3%ppl I: 11-21<1111
.anner (A< Mica L< Trent> ;< +rtel .. Inhi/ition o& caspase activit) atten%ates endoto,in-
mediated hepatic microvasc%lar per&%sion &ail%re and le%koc)te response. ."r# 5or"m 5#<: 3-
< 1111
Mica L< .anner (A< Trent> ;< +rtel .. Activation o& apoptosis /) a5onistic anti-!D15
anti/odies ind%ces hepatic microvasc%lar per&%sion &ail%re and decrease K%pp&er cell
clearance capacit). -"r ."r# Res 31 Hs%ppl 1I.2< 1111
Mica L< .anner (A< 6ent>e 6< Trent> ;< +rtel .. ]onal hetero5enit) o& !D15-mediated
hepatic microvasc%lar per&%sion &ail%re-role o& caspases. .6oc% 11 Hs%ppl 1I. 1< 1111
.anner (A< Mica L< 6ent>e 6< K^nstle (< Kol/ 3< Trent> ;< +rtel .. 2olle des !D15
2e>eptors %nd der Kaspasen-AktivitGt &^r die +ndoto,in-asso>iierte 6epatoto,i>itGt %nd
LetalitGt. C6ir"r#isc6es 5or"m 21< 5#1-511< 2###
.anner (A< Mica L< Kol/ 3< Trent> ;< +rtel .. Inhi/ition o& caspase activit) prevents
hepatic microvasc%lar per&%sion &ail%re and le%koc)te acc%m%lation a&ter ischemia and
reper&%sion. ."r# 5or"m 52< 0-5#< 2##1
Keel M< Mica L< +id K< Trent> ;< +rtel .. Die $ede%t%n5 der !rash-
LaparotomieJThorakotomie /ei sch=erverlet>ten Patienten im hGmorrha5ischen 3hock. ;efte
/" 'er 7nfallc6ir"r#< =irsc6ner>.t9rmer H6rs5.I< 3prin5er-Ferla5 $erlin 6eidel/er5 3#-
31< 2##1
1-
Mica L< 6Grter L< Trent> ;< Keel M. $id or $cl-2 do not re5%late ne%trophil apoptosis in
patients =ith 3epsis. ."r# 5or"m 115\ 33< 3-1-30#< 2##2
6Grter L< Mica L< Trent> ;< Keel M. Toll-like receptor-2 and ? on le%koc)tes &rom patients
=ith sepsis. ."r# 5or"m 115\ 33< 303-30 2##2.
Mica L.< 6Grter L.< Trent> ;.< Keel M. $id %nd $cl-2 re5%lieren nicht die Apoptose
ne%trophiler (ran%lo>)ten /eim Patienten mit 3epsis. In: ;efte /" 'er 7nfallc6ir"r#<
Re6m>.t9rmer>Pro%o) H6rs5.I< pp. 3-3-3-< 3prin5er-Ferla5: $erlin 6eidel/er5 2##2.
Keel M< Mica L< Trent> ;< 6Grter L. +rhwhte e,pression der Toll-like 2e>eptoren-2%nd ?
a%& Le%ko>)ten von Patienten mit 3epsis. In: ;efte /" 'er 7nfallc6ir"r#<
Re6m>.t9rmer>Pro%o) H6rs5.I< pp. 3-"-3--< 3prin5er-Ferla5: $erlin 6eidel/er5 2##2.
.anner (A< Mica L< Kol/ 3< Trent> ;< +rtel .. $ede%t%n5 der KaspasenaktivitGt &^r das
hepatische Mikro>irk%lationsversa5en nach IschGmie %nd 2eper&%sion. C6ir"r#isc6es 5or"m
31: 333-335< 2##2.
Mica L< 6Grter L< Trent> ;< Keel M. $eschle%ni5te %/i8%itinier%n5 %nd de5radation der
aktivierten caspase-3 in ne%trophilen 5ran%lo>)ten von patienten mit sepsis. C6ir"r#isc6es
5or"m 32< 13-15< 2##3.
Mica L< 6Grter L< Trent> ;< Keel M. +ndoto,in-vermittelte 6emm%n5 der Apoptose
ne%trophiler (ran%lo>)ten ist Proteasom< @edoch nicht '4-n$ a/hGn5i5. .(iss ."r#ery
."))lement"m 1< 5< 2##3.
Mica L< 6Grter L< Trent> ;< Keel M. Das Proteasom re5%liert die LP3-vermittelte 2ed%ktion
der spontanen Apoptose ne%trophiler (ran%lo>)ten %na/hGn5i5 von '4-k$. In: 6?.
*a6resta#"n# 'er 'e"tsc6en :esellsc6aft f9r 7nfallc6ir"r#ie > ,+. Ta#"n# 'er 'e"tsc6en
#esellsc6aft f9r @rt6o)A'isc6e C6ir"r#ie > 44. Ta#"n# 'es 2er"fsBerban'es 'er 5ac6Ar/te
f9r @rt6o)A'ie. ===.e5ms.deJenJmeetin5sJd5%2##3J#3d5%#3".shtml< 2##3
10
Mica L< 6Grter L< Trent> ;< Keel M. +ndoto,in 2ed%ces !D15-Ind%ced 'e%trophil
Apoptosis /) cIAP2-Mediated !aspase-3 De5radation% ."r#ical 5or"m 11- 33< 33--330<
2##3
Mica L< 6Grter L< Trent> ;< Keel M. 3TAT-3 re5%liert die verminderte Apoptose ne%trophiler
(ran%lo>)ten /eim Patienten mit 3epsis. C6ir"r#isc6es 5or"m 33< 21-251< 2##.
Mica L< 6Grter L< Trent> ;< Keel M. 3TAT-3 re5%lates the red%ced apoptosis in ne%trophils
&rom patients =ith sepsis. 3
r'
Day of Clinical Researc6< 7niversit) 6ospital o& ]^rich< -3<
2##.
6Grter L< Mica L< Trent> ;< Keel M. 7pre5%lation o& Toll-like receptors in ne%trophils is
re5%lated /) 3TAT-3. 3
r'
Day of Clinical Researc6< 7niversit) 6ospital o& ]^rich< 23< 2##.
Mica L.< 6Grter L.< Trent> ;.< Keel M. 3TAT-3 re5%lates the red%ced apoptosis in ne%trophils
&rom patients =ith sepsis. -"ro)ean * Tra"ma< 3%pplement 1< -"\201< 2##
6Grter L.< Mica L.< Trent> ;.< Keel M. I4'-5-ind%ced %pre5%lation o& toll-like receptors is
re5%lated /) 3TAT-3 in ne%trophils. -"ro)ean * Tra"ma< 3%pplement 1< -"\200< 2##
La/ler L.< Mica L.< 6Grter L.< Keel M. FA!
`
-therap) ind%ces a local release o& interle%kin-0
and vasc%lar endothelial 5ro=th &actor. -"ro)ean * Tra"ma< 3%pplement 1< \150< 2##
Mica L< 6Grter L< Trent> ;< Keel M. 2e5%lation o& ne%trophil apoptosis in patients =ith
sepsis /) 3TAT 3. .(iss =nife< .)ecial -'ition 0< 2##
La/ler L< L%sten/er5er T< L^thi 3< Mica L< Trent> ;< Keel M. 6emorrha5ic shock increases
in&ections o& closed and open &ract%res. 6t6 -"ro)ean Con#ress of Tra"ma an' -mer#ency
."r#ery. 2otterdam 2##
L%sten/er5er T< L^thi 3< La/ler L< Mica L< Trent> ;< Keel M. The pre-hospital dela)
in&l%ences the posttra%matic mor/idit). 6t6 -"ro)ean Con#ress of Tra"ma an' -mer#ency
."r#ery. 2otterdam 2##
11
L^thi 3< L%sten/er5er T< La/ler L< Mica L< 3tocker 2< Trent> ;< Keel M. !raniotom) a&ter
head in@%r) in&l%ences incidence o& s)stemic in&lammation. 6t6 -"ro)ean Con#ress of
Tra"ma an' -mer#ency ."r#ery. 2otterdam 2##
Mica L< L%sten/er5er T< L^thi 3< La/ler L< Trent> ;< Keel M. The 3everit) o& In@%r) and
6emorrha5ic 3hock !orrelates =ith the Incidence o& Posttra%matic In&ectio%s !omplications.
6t6 -"ro)ean Con#ress of Tra"ma an' -mer#ency ."r#ery. 2otterdam 2##
Mica L< 6Grter L< Trent> ;< Keel M. +ndoto,in red%ces !D15-ind%ced 'e%trophil apoptosis
/) cIAP2- mediated caspase-3 de5radation. 3
r'
.(iss A)o)tosis 1eetin# 1"-1-. 3eptem/er
$ern 2##
6Grter L< Mica L< Trent> ;< Keel M. 3TAT-3 participates in endoto,in-ind%ced s%rvival in
ne%trophils &rom patients =ith sepsis. 3
r'
.(iss A)o)tosis 1eetin# 1"-1-. 3eptem/er $ern
2##.
Mica L< 6Grter L< Trent> ;< Keel M. '4-$ re5%liert die LP3-ind%>ierte ])tokin&reiset>%n5<
nicht a/er die 2ed%ktion der Apoptose in ne%trophilen (ran%lo>)ten von Patienten
mit 3epsis. Chirur6isches *orum (8, ,//-
Mica L< 6Grter L< Trent> ;< Keel M. The I4'-5-ind%ced %pre5%lation o& Toll-like receptors is
re5%lated /) 3TAT-3. .(iss =nife< .)ecial -'ition 32< 1.#< 2##5.
Keel M< La/ler L< L%sten/er5er T< Mica L< 3tocker 2< Trent> ;. ;%tcome a&ter mDama5e
!ontrolD or m+arl) Total !areD Mana5ement in "22 3everel) In@%red Patients. .(iss =nife<
.)ecial -'ition 1< 1.#2< 2##5.
Keel M< La/ler L< L%sten/er5er T< Mica L< Trent> ;. Da)-;ne-3%r5er) in "1" 3everel)
In@%red Patients /) (eneral Tra%ma 3%r5eons. .(iss =nife< .)ecial -'ition 1#< 1.#"< 2##5.
Mica L< 6Grter L< Trent> ;< Keel M. 3TAT-3 re5%liert die I4'--ind%>ierte +,pression der
Toll-like 2e>eptoren -2 %nd - in Le%ko>)ten. In: 1. :emeinsamer =on#ress @rt6o)A'ie "n'
7nfallc6ir"r#ie ===.a/stractserver.deJa/stractsJo%2##5Ja/##030.htm< 2##5.
1##
La/ler L< Mica L< 6Grter L< Trent> ;< Keel M. +rhwhte Interle%kin-0 %nd F+(4 3pie5el in
FA!-/ehandelten .%nden von Patienten nach Tra%ma. In: 1. :emeinsamer =on#ress
@rt6o)A'ie "n' 7nfallc6ir"r#ie ===.a/stractserver.deJa/stractsJo%2##5Ja/##032.htm<
2##5.
Keel M< L%sten/er5er T< Mica L< L^thi 3< La/ler L< Trent> ;.

Der 3ch=ere5rad der
Ferlet>%n5en %nd des haemorrha5ischen 3chocks korrelieren mit der In>iden> von
In&ektionen %nd septischen KomplikationenIn: 1. :emeinsamer =on#ress @rt6o)A'ie "n'
7nfallc6ir"r#ie ===.a/stractserver.deJa/stractsJo%2##5Ja/##0-.htm< 2##5.
Mica L< La/ler L< L%sten/er5er T< Trent> ;< Keel M. ;%tcome o& pol)tra%mati>ed elder
patients: a retrospective st%d) o& -0# patients. -"r * Tra"ma< 3%pplement 1: 3-< 2##".
La/ler L< Mica L< Trent> ;< Imho& 6(. Mild tra%matic /reain in@%r) in elder) patients. -"r *
Tra"ma< 3%pplement 1: 31< 2##".
6Grter L< Mica L< Trent> ;< Keel M. 7p-re5%lation o& Toll-like receptors on monoc)tes
d%rino sepsis. -"r * Tra"ma< 3%pplement 1: 230< 2##".
L. Mica< L. La/ler< ;. Trent>< L. 6Grter< M. Keel. Increased s%rvival o& ne%trophil
5ran%loc)tes in FA!-treated compared to +pi5ard-treated =o%nds. .(iss =nife .)ecial
-'ition 2##"\35-3"< 1-.#"
L. Mica< L. 6Grter< M. Keel< ;. Trent>. LP3 prevents l)sosomal deca) d%rin5 spontaneo%s
apoptosis in ne%trophil 5ran%loc)tes. .(iss =nife .)ecial -'ition 2##"\ 3"< 1-.#0
2. von Kaenel< 7. 6epp< 2. Tra/er< $. Kraemer< L. Mica< M. Keel< 7 3chn)der. +vidence &or
endothelial d)s&%nction in posttra%matic stress disorder. American Psyc6osomatic .ociety A-
5< 2##"
Mica L< La/ler L< 6Grter L< Trent> ;< Keel M. Increased s%rvival o& ne%trophil 5ran%loc)tes
in FA!-treated compared to +pi5ard-treated .o%nds. 6
t6
Day of Clinical Researc6<
7niversit) 6ospital o& ]^rich< March< 2##-.
1#1
6Grter L< La/ler L< Mica L< Trent> ;< Keel M. FA!-therap) ind%ces local activation o&
ne%trophil 5ran%loc)tes in tra%matic =o%nds. 6
t6
Day of Clinical Researc6< 7niversit)
6ospital o& ]^rich< March< 2##-.
T. L%sten/er5er< L. Mica< M. T%rina< ;. Trent>< M. Keel. 3evere 6emorha5ic 3hock
Increases Mortalit) in Patients =ith Tra%matic $rain In@%r). .(iss =nife .)ecial -'ition
2##-\53< 21.1"
M. T%rina< A. $iletter< L. Mica< T. L%sten/er5er< ;. Trent>< M. Keel. 3er%m Procalcitonin
and Interle%kin-" !orrelate =ith In&ectio%s !omplikations in 1#-1 3everel) Tra%mati>ed
Patients< =ith stron5est !orrelation ;/served in 3%/se8%entl) 3eptic Patients. .(iss =nife
.)ecial -'ition 2##-\5< 2.3
L. Mica< L. La/ler< ;. Trent>< L. 6Grter and M. Keel Increased s%rvival o& ne%trophil
5ran%loc)tes in FA!
`
-treated compared to +pi5ard
`
-treated =o%nds. -AT-. (ra> 2##-
T. L%sten/er5er< L. Mica< M. T%rina< ;. Trent> and M. Keel Tra%matic /rain in@%r) increases
mortalit) and mor/idit) in patients =ith hemorrha5ic shock. -AT-. (ra> 2##-
P.M. Len>lin5er< T. L%sten/er5er< L. Mica< M. Keel. 3evere !hest In@%r) in Pol)tra%ma.
-"ro)ean *o"rnal of Tra"ma an' emer#ency ."r#ery 2##0\3 H3%pp. II< 5
L. Mica< M. 2ancan< L. 6Grter< T. L%sten/er5er< M. Keel. Increased (-!34 in .o%nd 4l%id
&rom FA!-Treated .o%nds is not 2esponsi/le &or Increased 'e%trophil 3%rvival. -"ro)ean
*o"rnal of Tra"ma an' emer#ency ."r#ery 2##0\3 H3%pp. II< "3
L. Mica< D. (ianom< $. $ode< P. Zaklin< A. 6ollin5er. 2are ca%se o& d)spha5): =ell
di&&erentiated esopha5eal =all liposarcoma. .(iss =nife 2##0\ special edition< -3
L. Mica< A. 3ch=aller< !. 3to%pis< I. Penka< Z. Fomela< A. Follen=eider. Pelvis-near av%lsion
o& the hamstin5 m%scle 5ro%p. .(iss =nife .)ecial -'ition 2##1\ 3"
1#2
M#33 ? A. 4rischknecht< T. L%sten/er5er< M. $%k%r< M. T%rina< A. $illeter< L. Mica< M.
Keel. Dama5e control in severel) in@%red tra%ma patients. A ten-)ear e,perience. -.T-.
2#1# $r%,elles
1#3
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