23rd Meeting of the Austrian Society of Transplantation

23rd Meeting of the Austrian Society of Transplantation,Transfusion and Genetics

Seefeld, October 21–23, 2009

Guest Editor:Walter Mark, Innsbruck, Austria

Mit freundlicher Unterstützung von

Eur Surg � Vol. 41 � Supplement Nr. 231 � 2009

Supplement 231

23rd Meeting of the Austrian Society of Transplantation,Transfusion and Genetics

Seefeld, October 21–23, 2009

Guest Editor:Walter Mark, Innsbruck, Austria

01Viral load predicts outcome of hepatitisC patients after liver transplantation

I. Graziadei, H. Zoller, K. Nachbaur, W. Mark,R. Margreiter, W. Vogel

Department of Internal Medicine II, Center of Internal Medicine,Medical University of Innsbruck, Innsbruck, Austria

Background. Recurrent hepatitis C (HCV) infection is almost

ubiquitous after liver transplantation (LT). Risk factors associated

with HCV recurrence include donor, recipient and viral param-

eters. Conflicting data have been reported regarding viral load

and severity of recurrent HCV disease. The aim of this study was

to analyse the impact of viral load within the first year after LT on

severity of recurrent HCV infection.

Methods. Between 1980 and 2006, 175 patients were

transplanted due to HCV-cirrhosis at our institution. Only

patients (n¼ 130, age: 56.8 � 8.9 years; 31 females, 99 males)

who survived more than 6 months and with histological as-

sessment of recurrent HCV infection were included in this

study. Non of the excluded patients died due to HCV recur-

rence. Viral loads were measured at week 2, month 3, 6 and

12 post LT, using the bDNA HCV RNA 3.0 assay (Bayer Diag-

nostics). Thirty-four patients (18.4%) developed either a

cholestatic type of HCV recurrence (n¼ 16; 8.6%) and/or a

rapid progression to advanced fibrosis/cirrhosis (n¼ 23,

12.4%). The overall follow-up was 6.1 years.

Results. The actuarial patient survival of all patients at 1-,

5- and 10 years were 87%, 75% and 59%. Patients with

cholestatic type recurrence and advanced fibrosis had signifi-

cantly decreased survival rates at 1-, 5- and 10 years with

90%, 65% and 45%, compared to patients with mild/moderate

or no recurrent disease with 98%, 81% and 71%. Cox regres-

sion analysis showed that the development of a cholestatic

recurrence, patients’ age and viral load at week 2 were associ-

ated with poor patient survival. Although higher viral loads

were seen in patients with a severe recurrence at any time,

multivariate binary regression analysis showed that only vire-

mia at week 2 and donor age were predictive factors for the

cholestatic HCV recurrence in contrast to viral loads at

months 3 and 6 together with recipient age and cold ischemic

time for the rapid development to advanced fibrosis. Geno-

type, immunosuppression and several other parameters did

not reach statistical significance.

Conclusions. Our study shows that viral load is an important

parameter for the development of severe recurrent disease and

recipient survival after LT. Whereas early viremia is highly

predictive for the cholestatic type, high viral loads between

month 3 and 6 are associated with advanced fibrosis/cirrhosis

of the liver allograft.

02Predictive factors for the outcomeof patients with acute on chronicliver failure

H. Nagel, I. Graziadei, K. Nachbaur, W. Vogel

Department of Internal Medicine II, Center of Internal Medicine,Medical University of Innsbruck, Innsbruck, Austria

Background. Acute on chronic liver failure (AoCLF) carries a

high risk of mortality mainly due to multiorgan dysfunction.

Several prognostic models have been discussed to predict the

outcome of these patients. The aim of this study was to

determine the natural history of patients with AoCLF referred

to a tertiary center and to analyze predictive parameters for

patient survival and the prognostic accuracy of the currently used

models.

Methods and results. Eighty-four consecutive patients with

decompensated liver cirrhosis who required intensive monitoring

and/or could not be treated outside an intermediate care unit

were included in this study. The mean age was 55 years (43%

female, 63% male). The major underlying liver diseases were

alcoholic or non-alcoholic fatty liver disease (50%) and viral

cirrhosis (27.4%). At admission the mean MELD, MELD Na and

SOFA score were 26.6, 29.6 and 9.4. Infections, in particular SBP

and pneumonia, were the major precipitating events for

decompensation.

The cumulative mortality rate of all AoCLF patients was

76.2%, the median survival 1.58 (95% CI: 0.84–2.32) months

and the median liver transplant (LT) free survival 1.28 (95% CI:

0.89–1.68) months. Sixty (71.4%) patients were evaluated and 45

(53.6%) patients actually listed for liver transplantation (LT).

Severe sepsis and acute alcoholic hepatitis were the main reasons

Eur Surg � Vol. 41 � Supplement Nr. 231 � 2009 1

Supplement 231

not to consider LT. Out of the 45 patients listed for LT 15 could be

successfully transplanted. Sepsis and multiorgan failure were

the main causes of death on the waiting list. Univariate cox-

regression analysis showed that sepsis, hepatorenal syndrome

(HRS) type 1, renal (hemofiltration) and respiratory insufficiency

(intubation), multiorgan failure (3 or more) and a SOFA score

>8 were negative predictive factors. MELD, MELD Na and Child-

Pugh score did not reach statistical significance. On multivariate

analysis sepsis, HRS 1, multiorgan failure and respiratory insuffi-

ciency remained significant. LT was the only positive predictive

factor.

Conclusions. Our study confirms the poor prognosis of

patients with AoCLF. LT, the only curative treatment, could be

performed successfully in only 18% of patients. Sepsis, HRS 1,

intubation and multiorgan failure were negative predictive fac-

tors for survival.

03Outcome of patients with recurrenthepatocellular carcinoma after livertransplantation

A. Finkenstedt1, I. Graziadei1, K. Nachbaur1,H. Zoller1, W. Mark2, R. Margreiter2, W. Vogel1

1Department Internal Medicine II (Gastroenterologyand Hepatology), Center of Internal Medicine, Medical Universityof Innsbruck, Innsbruck, Austria; 2Department of Visceral,Transplant and Thoracic Surgery, Center of Operative Medicine,Medical University of Innsbruck, Innsbruck, Austria

Background. Liver transplantation (LT) is the only cura-

tive therapeutic option for patients with hepatocellular carci-

noma (HCC). Although HCC recurrence after LT has

significantly decreased over the last years, recent studies have

still reported a recurrence rate around 15%. So far, very few

studies have addressed the issue of natural history and ther-

apeutic options for patients with recurrent HCC. The aim of

our study was to evaluate (1) the outcome of patients with

recurrent HCC and (2) the effect of different therapeutic

options.

Methods and results. 202 patients (26 f/176 m; median age

60) were included in this study. The major underlying liver dis-

eases were viral cirrhosis (512%) and alcoholic or non-alcoholic

fatty liver disease (33%). According to Child Pugh classification

40% of patients presented with stage A, 46% stage B and 14%

stage C. PreLT TACE was performed in 138, RFA in 8 and a

combination of both in 10 patients. In 15 patients the HCC was

found incidentally in the explanted liver. The mean follow-up

was 4.6 � 3.7 years.

Mean overall patient survival was 9.0 (0–15) years; 36

patients (18%) developed a HCC recurrence with a median

time of 1.2 (0.2–7.4) years after LT. Twenty four patients died

due to recurrent HCC. The median overall survival after re-

currence was 0.8 (0.1–5.5) years. Recurrence rate was not

significantly different between patients with and without

pre LT therapy. However, patients with a complete response

after pre LT therapy had a significantly lower risk of recur-

rence in comparison to patients with partial (HR¼ 10.6) or no

response (HR¼ 20.5). In patients with TACE, the risk of re-

currence increased significantly in those with 2–3 (HR¼ 4.0)

or more than 3 sessions (HR¼ 10.3). Univariate analysis

showed that poor differentiation (HR¼ 8.9), vascular invasion

(HR¼ 4.4), advanced UICC stage and increasing tumor size

were negative predictive factors for HCC recurrence and pa-

tient survival. Median survival was significantly better in

patients treated with surgical and/or local ablative therapy

compared to patients without therapy (3.6 vs. 0.4 years,

p¼ 0.005).

Conclusions. HCC recurrence significantly shortens the

survival after LT. Patients suitable for surgical or loco-ablative

therapy, however, have an excellent prognosis. Therefore,

these treatments should be intended in patients with

recurrent HCC.

04Single center experience with pancreasretransplantation – is it worth it?

M. Linecker, M. Biebl, M. Maglione, R. Öllinger,C. Holzknecht, Y. Ricardo-Pupo, F. Aigner,R. Margreiter, W. Mark

Department of Visceral, Transplant and Thoracic Surgery,Center of Operative Medicine, Medical University Innsbruck,Innsbruck, Austria

Background. Pancreas transplantation is an established

treatment for diabetes mellitus with end-stage renal disease,

however graft loss occurs more frequently than in other

organs.

Methods. Thirty-nine pancreas retransplantations (28

[71.7%] PAK, 7 [17.9%] SPK, 4 [10.2%] PA, 3 re-retransplanta-

tions) were included in this retrospective study. Before 1996,

immunosuppression comprised steroids, cyclosporine and

azathioprine, thereafter induction therapy (anti-thymocyte

globuline 78%, alemtuzumab 16%, OKT3 3%, IL 2 antagonist

3%), followed by a triple regimen of steroids, calcineurin inhi-

bitos and MMF. Data are reported as mean � standard devia-

tion, or total numbers (%).

Results. Of 39 patients (51.3% female, mean age 43.9 � 9

years), 37 (94.9%) were type I diabetics. Retransplant was per-

formed 67.0 � 67.3 months after the first transplant with the

non-functioning pancreas being removed simultaneously

(71.8%), previously (18.7%) or left in situ (9.3%). Operation

time was 258.9 � 92.8min, packed red blood cells were used

in 36.1%. Severe adhesions were noted in 17.9%. Aterial anas-

tomosis was performed to the common (77.1%) or external

iliac artery (11.4%), the previous pancreas’ conduit (8.5%), or

the artery of an old renal transplant (2.8%). Venous anastomo-

sis was performed using the inferior vena cava (68.5%), the

iliac vein (19.9%), the first pancreas’ portal vein (8.5%), the

superior mesenteric vein (2.8%), or the stump of a previous

renal transplant (2.8%). Early in this series, bladder (13.1%) or

external (5.2%) drainage was used, later on only enteric drain-

age (81.5%). Length of stay was 34.4 � 22.8 days, ICU stay 3.2

� 3.9 days. Of 21 infectious episodes (56.7%), 10 (27.8%) were

intraperitoneal. Morbidity, rejection and reoperation rate were

59.0%, 33.3% and 43.6%, respectively. Time to full endocrine

function was 7 � 8.2 days. After a follow-up of 37.2 � 39.0

months, graft loss occurred in 34.3%.

Conclusions. While complication rates are higher after pan-

creas retransplantation compared to first-time transplantation,

mid-term results are satisfactory.

2 Eur Surg � Vol. 41 � Supplement Nr. 231 � 2009

23rd Meeting of the Austrian Society of Transplantation

05Pädiatrische Patienten mit terminalerNiereninsuffizienz zwischen 1965und 2007 – eine retrospektive Analysein Österreich

J. Falger1, A. Schneider1, B. Izay2, Ch. Aufricht3,R. Kramar4

1Kinder- und Jugendabteilung, Landesklinikum WeinviertelMistelbach, Österreich; 2Universitätsklinik für Chirurgie,Medizinische Universität Wien, Wien, Österreich; 3KinderdialyseWien, Universitätsklinik für Kinder- und Jugendheilkunde Wien,Medizinische Universität Wien, Wien, Österreich;4Österreichisches Dialyse- und Transplantationsregister, ÖDTR

Grundlagen. Nierenersatztherapie der padiatrischen Patien-

tenpopulation in Osterreich hat sich in den letzten Jahren durch

das Aufkommen von neuen Behandlungsmoglichkeiten sehr

verandert.

Methodik. In dieser retrospektiven Analyse wurden 680

padiatrischen Patienten aus dem Osterreichischen Dialyse- und

Transplantationsregister (ODTR) der Osterreichischen Gesell-

schaft fur Nephrologie, die ihre erste Nierenersatztherapie

zwischen 1965–2007 erhielten, bezuglich der Veranderung der

Nierenersatztherapien und deren Einfluss auf die Mortalitat

untersucht.

Ergebnisse. Die Geschlechtsverteilung lag bei 382 (56,2%)

mannlichen und 298 (43,8%) weiblichen Patienten, das

Alter bei Beginn der Nierenersatztherapie war durchschnittlich

13,3 � 5,5 Jahre. Die durchschnittliche Beobachtungszeit

betrug 9,5 (IQR: 14,9) Jahren. Die Anzahl an Neupatienten

mit Nierenersatztherapie pro Jahr war in den letzten 20

Jahren stabil (12–26, n.s.). Jedoch hat sich die Art der ersten

Nierenersatztherapie geandert. Die Dialyse als erste

Nierenersatztherapie ist gesunken: 97,6% (bis 1983), 90,2%

(1984–96), 80,6% (1997–00), 75,0% (2001–07) (p<0,001, s.).

Die Anzahl der Transplantationen hat zugenommen:

2,4% (bis 1983), 9,8% (1984–96), 19,4% (1997–00), 25,0%

(2001–07). Das Alter bei Ersttransplantation ist gesunken:

14,6 � 4,4 Jahre (bis 1983) vs. 12,3 � 6,5 Jahre (2001–07)

(p<0,001). Die Anzahl der padiatrischen Patienten mit Nie-

rentransplantation lag durchschnittlich bei 16 pro Jahr

(IQR: 12). Die Dauer zwischen erster Nierenersatztherapie

(HD/PD) und erster Nierentransplantation hat sich verkurzt:

2 Jahre (bis 1983) auf 1 Jahr (2001–07). Die Transplantatuber-

lebensrate ist signifikant angestiegen (p<0,001): 31,9% (bis

1983), 46,5% (1984–96), 80,6% (1997–00), 93,4% (2001–07);

die Mortalitat nach Transplantation ist signifikant

gefallen (p<0,001): 50,0% (bis 1983), 40,9% (1984–96), 5,3%

(1997–00), 3,8% (2001–07), ebenso hat sich die Gesamtmorta-

litat bei padiatrischen Patienten mit Nierenersatztherapie

signifikant vermindert (p<0,001): 53,6% (bis 1983), 24,7%

(1984–96), 16,1% (1997–2000), 3,7% (2001–07).

Schlussfolgerungen. Durch das Auftreten neuer Behand-

lungsstrategien konnten die Transplantatabstoßung und die

Patientenmortalitat bei terminalem Nierenversagen und nach

Nierentransplantation signifikant vermindert werden.

06Akzeptable Langzeitergebnisse nachNierentransplantation bei über70-jährigen Empfängern

C. Bösmüller, St. Scheidl, M. Maglione, Ch. Margreiter,R. Öllinger, St. Schneeberger, W. Mark, R. Margreiter

Universitätsklinik für Visceral-, Transplantations- undThoraxchirurgie, Department Operative Medizin,Medizinische Universität Innsbruck, Innsbruck, Österreich

Grundlagen. Wir analysierten retrospektiv das Patienten-

und Transplantatuberleben, die Transplantatfunktion und

schwere Komplikationen bei >70-jahrigen Nierentransplantat-

empfangern im Rahmen des ET-Senior-Programms.

Methodik. Von insgesamt 84 Nierentransplantatempfan-

gern im Rahmen des ET-Senior-Programms zwischen 5/1999

und 1/2009 an unserem Zentrum waren 19 Patienten uber

70 Jahre, im Mittel 72,7 Jahre alt. Die Anzahl an Mismatches

betrugen im AB-Lokus 2,5 und im DR-Lokus 1,2, die

mittlere kalte Ischamiezeit 12:05 Stunden. Nach initialer

CNI-freier Immunosuppression mit einem IL-II-Blocker

MMFþCortison, wurde nach Stabilisierung der Nierenfunktion

Cyclosporin A (im Mittel an Tag 9,8) oder Tacrolimus (im Mittel

an Tag 4,7) begonnen.

Ergebnisse. Alle 7 Falle mit initialer Nichtfunktion haben

sich erholt und 3 akute Abstoßungen waren reversibel. Zwei

Transplantate gingen an chronischer Abstoßungsreaktion im

Monat 32 bzw. 49 verloren. Vier Patienten verstarben

wahrend der gesamten Beobachtungszeit an Herzversagen,

2 an Pneumonie, je einer an Sepsis und intrazerebraler

Blutung. 6/8 Patienten (75%) verstarben mit funktionieren-

dem Transplantat. Das Patienten- bzw. Transplantatuberle-

ben nach einem Jahr betrug je 89%, nach 5-Jahren 66%/

50%. Schwere Komplikationen waren Vorhofflimmern (n¼ 4),

kardiale Dekompensation (n¼ 3), intrazerebrale Blutung

(n¼ 2), je 1 Fall an uberlebtem Herzstillstand und

Myokardinfarkt, je ein Fall von Bradykardie mit Schrittma-

cherimplantation, Mitralklappenersatz, multiplen Knochen-

frakturen, Huftkopfnekrose mit Prothesenimplantation,

Sigmoidperforation, Ileus, Mesenterialarterienthrombose.

Es traten je ein Pankreas-, Magen-, Prostatakarzinom, weiters

4 Basaliome und 2 Plattenepithelkarzinome der Haut auf.

Das mittlere Serum-Kreatinin im Jahr 1/5/9 betrug 1,6/1,8/

1,7 mg/dl.

Schlussfolgerungen. Nach Nierentransplantation an

>70-jahrigen Empfangern konnen exzellente Kurz- und

akzeptable Langzeitergebnisse erzielt werden. Die meisten

Todesfalle und Komplikationen resultierten aus altersbeding-

ten Komorbiditaten.

07Durch Erythropoetin erhöhteHämoglobinwerte über 12,5 g/dl sindbei Nierentransplantatempfängern miterhöhter Sterblichkeit assoziiert

G. Heinze1, A. Kainz2;3, W. H. Hörl2, R. Oberbauer2;3;4


Supplement 231

1Core Unit of Medical Statistics and Informatics, MedizinischeUniversität Wien, Wien, Österreich; 2Klinische Abteilungfür Nephrologie und Dialyse, Universitätsklinik für Interne MedizinIII, Medizinische Universität Wien, Wien, Österreich; 3Abteilungfür InterneMedizin III, Nephrologie, Krankenhaus der Elisabethinen,Linz, Österreich; 4Österreichisches Dialyse- und Transplantregister

Die Pravalenz der Anamie nach Nierentransplantationen

betragt etwa 40%. Ungefahr 20% von den anamischen Patienten

werden mit Erythropoetin stimulierenden Wirkstoffen (ESA)

behandelt. Der Effekt dieser Behandlung auf die Uberlebensrate

ist jedoch nicht bekannt. Neueste Daten weisen darauf hin, dass

eine Verwendung von ESA die Sterblichkeit unter Umstanden

erhohen kann.

Wir haben in dieser Studie die Assoziation von ESA,

Hamoglobin und Sterblichkeitsrate bei 1794 nierentransplantier-

ten Patienten, die im Osterreichischen Dialyse- und Transplant-

register in der Zeit vom 1.1.1992 bis 31.12.2004 eingetragen sind

und mindestens 3 Monate nach Transplantation gelebt haben,

analysiert. Mit Hilfe der Cox-Regressionsanalyse haben wir fur

mehrere Kovariablen adjustiert. Das Modell wurde zeitabhangig

berechnet. Die Variablenauswahl erfolgte mit Hilfe des ,,Purpose-

ful Selection‘‘-Algorithmus.

Die Pravalenz der ESA-Verwendung erhohte sich in den letz-

ten 15 Jahren auf 25%. Die nicht adjustierte erweiterte Kaplan-

Meier Analyse ergibt eine hohere Mortalitat fur die Patienten, die

ESA verwenden. 78% von non-ESA-Patienten uberlebten 10

Jahre, aber nur 57% von ESA-Patienten (p<0,001). Die absolute

Todesrate war 5.4/100 PYR (person years at risk) in der ESA-

Gruppe und 2.6 bei non-ESA-Patienten (p<0,001).

Werden ,,Confounding by indication‘‘, andere Krankheiten

und Ko-Medikation sowie Laborwerte in die Analyse mit einbe-

zogen, haben Hamoglobinwerte uber 12,5g/dl zu einer erhohten

Mortalitat bei Verwendung von ESA gefuhrt (Hazard Ratio (HR)

von 14g/dl vs. 12,5 g/dl Hamoglobin: 2,8; 95% CI: 1,0–7,9), nicht

aber bei Patienten ohne ESA-Verwendung (HR: 0,7; 0,4–1,5).

Bei 14,7g/dl betragt das Risikoverhaltnis signifikant erhoht

(3,0; 1,0–9,4). Folglich lasst sich ableiten, dass eine ESA-Verwen-

dung bei erhohten Hamoglobinwerten uber 14g/dl in nieren-

transplantierten Patienten mit einer erhohten Mortalitat

verbunden ist.

08Biomarkervalidierung für akuteNierentransplantatschädigungin mikrodissezierten Nierenbiopsien

J. Wilflingseder1;2, R. Korbély1;3, A. Kainz1;2,P. Perco2;4, R. Langer3, B. Mayer4, R. Oberbauer1;2

1Abteilung für Nephrologie, Krankenhaus der Elisabethinen,Linz, Österreich; 2Klinische Abteilung für Nephrologie,Universitätsklinik für Innere Medizin III, Medizinische UniversitätWien, Wien, Österreich; 3Abteilung für Transplantationund Chirurgie, Semmelweis Universität, Budapest, Ungarn;4Emergentec Biodevelopment GmbH, Wien, Österreich

Grundlagen. Die Analyse von Nierenbiopsien basiert zurzeit

auf histologischer Erkennung von typischen morphologischen

Strukturen und immunhistologischer Detektion von Protein-

Expressionsanderungen. Durch die Quantifizierung geeigneter

Biomarker kann die Diagnose und Prognose von Nierenerkran-

kungen moglicherweise optimiert werden. In dieser Kohorten-

Studie vergleichen wir die Genexpression von 12 moglichen

Biomarkern in mikrodissezierten Nierenbiopsien um das prog-

nostische Potenzial fur akute Nierentransplantatschadigung

(delayed graft function, DGF) zu evaluieren.

Methodik. 34 Nadelbiopsien von hirntoten Organspendern

und 9 Nephrektomieproben, die als Kontrolle fungierten, wurden

durch Mikrodissektion in Tubulointerstitium und Glomeruli

getrennt. Nach RNA-Isolierung und Pre-Amplifikation wurde

der Genexpressionlevel mittels real-time PCR bestimmt. Fur die

Validierung wurde das prognostische Potenzial der einzelnen

Biomarker und deren Kombination zwischen DGF und normal

funktionierenden Transplantaten (primary function, PF) in den

beiden Kompartimenten mittels logistischer Regression berech-

net und durch die receiver operating curve (ROC) dargestellt.

Ergebnisse. Vier Biomarker waren signifikant aufreguliert in

der DGF Gruppe im Tubulointerstitium (CCL19 p¼ 0,05, LCN2

p¼ 0,04, HAVCR1 p¼ 0,03 und GZMA p¼ 0,05). Drei weitere Bio-

marker waren signifikant unterschiedlich im glomerularen Kom-

partiment (NRP1 p¼ 0,02, LCN2 p¼ 0,01 und CYR61 p¼ 0,05).

LCN2 und CYR61 zeigten einen hoheren Expressionswert und

NRP1 einen niedrigeren Expressionswert in der DGF-Gruppe.

Die Kombination von drei Biomarkern im Tubulointerstitium

ergab eine ROC-AUC von 0.80 (CCL19, LCN2 und GZMA) und

in den Glomeruli eine AUC von 0.87 (NRP1, LCN2 und CYR61).

Schlussfolgerungen. Genexpressionsmessung von poten-

ziellen Biomarkern in mikrodissezierten Nierenbiopsien kann

eine gute Erganzung zu der klassischen histologischen Unter-

suchung sein. Basierend auf den Genexpressionswerten von

drei Markerproteinen kann DGF in 80 bis 87% der Falle vo-

rausgesagt werden.

09Herz-Retransplantation (Re-HTX): Zahltsich der Aufwand wirklich aus?

F. Eskandary, M. Grömmer, A. Aliabadi, S. Mahr,D. Zimpfer, D. Dunkler, G. Laufer, A. Zuckermann

Klinische Abteilung für Herz-Thoraxchirurgie, Universitätsklinikfür Chirurgie, Medizinische Universität Wien, Wien, Österreich

Grundlagen. Nur etwa 1% der HTX weltweit sind Re-HTX.

Aufgrund der ethischen Problematik und der in der Literatur

beschriebenen Ergebnisse wird die Re-HTX hinterfragt. Ziel der

Analyse ist es, die Ergebnisse der Re-HTX in Wien innerhalb der

letzten 25 Jahre zu evaluieren.

Methodik. In Wien wurden zwischen 1984–2008 1150 HTX

durchgefuhrt. 39 waren Re-HTX (3,5%; inkl 2 Re-Re-HTX). Indi-

kationsstellungen waren akutes Spenderherzversagen (PGD),

akute Abstoßung (AR) und Graft-Vaskulopathie (GV). Mittels ret-

rospektiver Analyse wurden Demographik, Indikation und Ergeb-

nisse untersucht.

Ergebnisse. Geschlecht (m: 87,5%) und Alter (48,45 � 12,13)

waren gleich wie bei HTX. Indikationen waren PGD (30,9%,) AR

(17,9%), GV (51,2%). 1990–99 wurden 69% und 2000–08 wurden

31% Re-HTX durchgefuhrt. Die Indikationen waren unterschied-

lich 90–99: PGD (41%), CAV (37%), AR (22%); 00–08 PGD (8%),

CAV (84%), AR (8%). Vor Re-HTX waren 48,7% intensivpflichtig

(IP), und je 25,6% hospitalisiert oder zu Hause. Von diesen litten

58% an PGD, und je 21% an GV oder AR. Die 30-Tagesmortalitat

war 40%. Bei PGD war die perioperative Mortalitat am hochsten

(75%) gefolgt von AR (43%) und GV (20%). Patienten die vor Re-



HTX IP waren, hatten ebenfalls eine hohe perioperative Mortali-

tat (74%). Gesamt-ein-, funf-, und zehn-Jahresuberleben betrug

56%, 48% und 48%. Die 1- und 5-Jahresuberlebensraten waren

unterschiedlich in den 3 Indikationsgruppen: PGD (1a: 25%, 5a:

25%), AR (1a: 57%, 5a: 43%), CAV (1a: 75%, 5a: 59%). Das 1-

Jahresuberleben war 1990–99 (52%) niedriger als 2000–08 (75%).

Haupttodesursachen waren Infekte (43,4%), perioperatives

Rechtsherzversagen (26%), CAV (8,6%), intraoperative Blutung

(8,6%), zerebrale Komplikation (8,6%) und Tumor (4,4%).

Schlussfolgerungen. Bei GV stellt die Re-HTX eine geeignete

Indikation dar, sofern eine restriktive Patientenselektion durch-

gefuhrt wird. PGD und die AR sind heute obsolete Indikationen.

Bei PGD ergibt der Einsatz der ECMO akzeptable Ergebnisse und

bei AR stehen heute bessere Therapieoptionen zur Auswahl, die

eine Re-HTX nicht notwendig machen.

10Therapie des Herzversagens nachHerztransplantation mit dem Levitronix-Centrimag-System

N. Reiss, U. Schulz, L. Kizner, L. Arusoglu,K. Hakim-Meibodi, J. Gummert

Klinik für Thorax- und Kardiovaskularchirurgie, HerzzentrumNordrhein-Westfalen, Ruhr-Universität Bochum, Bad Oeynhausen

Haufig kann eine hamodynamische Stabilisierung beim pri-

maren Graftversagen, beim Rechtsherzversagen sowie beim Low-

output-Syndrom im Rahmen einer akuten Abstoßung nach Herz-

transplantation nur durch die Implantation eines mechanischen

Kreislaufunterstutzungssystems erreicht werden.

Das Levitronix-Centrimag-System, eine neue optimierte

Zentrifugalpumpe mit frei schwebendem Impeller, scheint

aufgrund seiner Eigenschaften ein außerst geeignetes

Unterstutzungssystem in diesen Situationen zu sein.

In unserer Einrichtung wurde das Levitronix-Centrimag-Sys-

tem bei 12 Herztransplantierten (Durchschnittsalter 53 Jahre)

implantiert. Indikation fur die Implantation war bei 7 Patienten

eine akute Abstoßung, bei 1 Patienten ein primares Graftversagen

und bei 4 Patienten ein akutes Rechtsherzversagen. Acht der 12

Patienten waren bereits vor der Herztransplantation mit einem

mechanischen Kreislaufunterstutzungssystem behandelt worden

(CardioWest n¼ 5, DuraHeart n¼ 1, CorAide n¼ 1, Thoratec

LVAD n¼ 1). Bei 7 Patienten wurde das Centrimag-System als

femoro-femoraler Bypass implantiert, bei 4 Patienten als

Rechtsherzunterstutzungssystem und bei 1 Patienten als bivent-

rikulares Support-System. Die mittlere Unterstutzungszeit

betrug 9 Tage. Letztendlich konnten 9 Patienten entwohnt wer-

den, 1 Patient wurde retransplantiert. Funf der 12 Patienten sind

Langzeituberlebende.

Nach unserer Erfahrung kann das Levitronix-Centrimag-Sys-

tem sicher und effektiv bei der Behandlung des Herzversagens

nach Herztransplantation eingesetzt werden. In allen Fallen wur-

den suffiziente Kreislaufverhaltnisse sowie eine adaquate Entlas-

tung des Herzens erreicht. Das System kann sowohl beim

isolierten Rechtsherzversagen als auch beim biventrikularen

Graftversagen als Bridge-to-recovery oder als Bridge-to-retrans-

plant eingesetzt werden. Jedoch sind Patienten, die nach einer

Herztransplantation einer mechanischen Kreislaufunterstutzung

bedurfen, trotz dieser maximalen Therapie mit einer hohen Kom-

plikations- und Letalitatsrate behaftet.

11Five years single-center experiencewith Everolimus in cardiac transplantation

M. Schweiger, P. Stiegler, A. Wasler, G. Prenner,M. Sereinigg, K. H. Tscheliessnigg

Division for Transplantation Surgery, Department of Surgery,Medical University of Graz, Graz, Austria

Background. Long-term results for cardiac transplant reci-

pients receiving Everolimus (EvA) especially outside major trials

are limited.

Methods. Between 2004 and 2009 44 patients (34 male,

10 female), mean age 63 years, were switched to an EvA based

immunosuppressive regime. Indications were: de-novo (3), renal

insufficiency (18), cardiac allograft vasculopathy (14), recurrent

rejection (7), others (2). Initial combination were either EvA along

with Cyclosporine A [34], Mycophenolate Mofetil [8], or others

[2]. In all cases Aprednislon was part of the regime. Dead, biopsy

proven acute rejections (BPAR), renal function, infections and

switch of combination were evaluated retrospectively.

Results. 5 patients died, four of them still on EvA treatment

at the time of death (intracerebral bleeding [1], embolism [1],

cardiac arrest [2], unknown [1]). EvA was discontinued in four

patients due to severe side effects (Edema [1], gastrointestinale

[1], x[]). Currently combinations are EvA, MMF, A (14), EvA,

Cyclo, A (19), others (3). There were four life threatening infec-

tions (all pneumonia; [3 months to 4 ys. post switch]) resulting in

a complete recovery. In all cases EvA was paused during the

infection. 4 patients are on dialysis, one patient underwent kid-

ney transplantation (2 patients out of these 5 were prior to EvA

on dialysis). No patient had to be switched to an alternative

regime because of acute rejection; no higher rejection than grad

2 (new ISHLT grading) were diagnosed; nevertheless all patients

receiving EvA because of recurrent rejection had to be combined

with a Calcineurin Inhibitor. In the EvA group combined with

MMF initially more mild rejection were observed.

Conclusions. EvA proved to be safe as in combination with

either Cyclo A or MMF and is becoming the third most used

immunosuppressant besides Aprednislon and Cyclo. Younger

patients were more favoured to receive EvA.

12Testosterone replacement in cardiactransplant patients on topof bisphosphonate therapy exerts multiplebenefits on bone mass, libido and sexualactivity: a 5-year prospective study

D. Wagner2, H. Dobnig1, K. H. Tscheliessnigg2,M. Schweiger2, G. Prenner2, A. Wasler2, D. Kniepeiss2,J. C. Piswanger-Sölkner1, A. Fahrleitner-Pammer1

1Division of Endocrinology and Nuclear Medicine,Department of Internal Medicine, Medical University of Graz,Graz, Austria; 2Division of Transplantation Surgery,Department of Surgery, Medical University of Graz,Graz, Austria


Supplement 231

Background. Hypogonadism is frequently encountered in

cardiac transplant patients (CTX) with immunosuppressive treat-

ment regimens and exerts negative effects on bone mass,

libido and quality of sex life. Aim of the study was to investigate

whether testosterone replacement therapy (TRT) of hypogonadal

CTX recipients on top of intravenous bisphosphonate treatment

confers positive effects on bone mass and quality of sex life

compared to untreated hypogonadal and eugonadal CTX

patients.

Methods. Parenteral ibandronate (2mg every 3 months)

as well as 1200mg calcium and 880 IU vitamin D3 was

given to all patients. Fourteen out of 31 hypogonadal

patients (45%) were treated with testosterone enanthate

(250mg intramuscularly every 3–5 weeks) or daily 50mg

testosterone gel.

Results. At baseline 77% of the hypogonadal patients

admitted loss of libido (compared to 27% of eugonadal men,

P¼ 0.005) and reported an average 7 � 6 annual sexual activities

(as compared to 15� 14 of eugonadal men, P¼ 0.005). Hypogo-

nadal men at baseline had also markedly lower Z-score values at

the femoral neck (�1.54 vs. 0.15), and total hip (�1.34 vs. 0.01)

(all P¼ 0.0001) as well as a higher percentage of prevalent verte-

bral fractures (63.3%; vs. 13.6%; P¼ 0.0003). After 1 and 5 years of

treatment BMD had significantly increased in hypogonadal

patients with TRT (neck 12.4 and 16.4%, total hip 9.2 and

12.4%, respectively, all P<0.001) as compared to eugonadal

patients (neck 2.9 and 3.4%, total hip 3.7 and 4.4%). A similarly

low BMD increase was found in unreplaced hypogonadal

patients (neck 2.3 and 3%, total hip 3.2 and 4.2%). Compared

to baseline annual sexual activities had increased in patients with

TRT after 1 year (29 � 8; p<0.0001) and 5 years (25 � 9;

p<0.0005) and had remained unchanged in unreplaced hypogo-

nadal (5 � 4 after 5 years) as well as eugonadal patients (16 � 9

and 14� 8). At 1 and 5 years the number of annual sexual

activities in replaced hypogonadal patients was also higher when

compared to eugonadal CTX patients.

Conclusions. Intravenous IBN therapy increases hip BMD in

CTX patients on continuing immunosuppressive treatment.

Hypogonadal patients clearly benefit from additional TRT over

at least 5 years with respect to bone mass changes and a better

quality of sex life.

13ECMO als Support bei primäremGraftversagen (PGV) nachHerztransplantation

M. Grömmer, A. Aliabadi, F. Eskandary, St. Mahr,D. Zimpfer, D. Hutschala, S. Taghavi, A. Zuckermann

Klinische Abteilung für Herz-Thorax-Chirurgie,Universitätsklinik für Chirurgie, Medizinische Universität Wien,Wien, Österreich

Grundlagen. PGV ist eine der Hauptursachen fur Morbiditat

und Mortalitat in der perioperativen Phase nach Herztransplan-

tation. Die extrakorporale Membranoxigenierung (ECMO) ist ein

Unterstutzungssystem, welches den Kreislauf im Falle von aku-

tem Spenderherzversagen unterstutzen kann. Ziel dieser Unter-

suchung war die Evaluation der ECMO bei PGV.

Methodik und Ergebnisse. PGV wurde durch rechts-,

links-, oder bi-ventrikulares Versagen des Spenderherzens

und somit die Notwendigkeit einer mechanischen Unterstut-

zung zum Entwohnen von der Herz-Lungenmaschine, defi-

niert. Zwischen 1.1.2000 und 1.7.2009 trat bei 64 von 448

(14,3%) der Patienten PGV auf. Wir analysierten die Uberle-

bensrate, die Wahrscheinlichkeit auf Erholung und das Auf-

treten von Komplikationen. Nach 100-wochigem Follow up

lag die Uberlebensrate bei 41%. 44 (74,6%) der Patienten

konnten von der ECMO entwohnt werden; von diesen uber-

lebten 55%. Dauer der Unterstutzung lag bei 5,25 � 4,15

Tagen. Fruhe Erfahrungen (2000–03) mit der ECMO zeigten

signifikant schlechtere Uberlebensraten (23% vs. 50%;

p¼ 0,000) als spatere (2004–07/2009). Die Komplikationsrate

lag bei 64,1%. Die am haufigsten beobachteten Komplikationen

waren Blutungen (N¼ 16, 27%; Kannulierungsgebiet [n¼ 9] und

Hamatothorax [n¼ 7]) und Infektionen (N¼ 11, 18%). Die

Dauer des ECMO-Supports hatte keinen Einfluss auf das

Uberleben der Patienten (bei 66% zeigten sich Probleme

wahrend des Weanings, 40% konnten nicht geweant werden,

bei 30% trat ein plotzliches GV im Rahmen des ICU-

Aufenthalts auf).

Schlussfolgerungen. Die ECMO ist ein wertvolles Unterstut-

zungssystem um PGV nach der Herztransplantation zu uber-

brucken. Durch zunehmende Erfahrung konnten die Ergebnisse

signifikant verbessert werden. Trotzdem treten nach wie

vor Probleme auf, deren richtiges Management von großter

Wichtigkeit ist.

14Does ischemia time have an impacton postoperative outcome after bilaterallung transplantation?

H. B. Hangler, E. Ruttmann-Ulmer, S. Semsroth,Ch. Geltner, L. Ch. Müller

Department of Heart Surgery, Center of Operative Medicine,Medical University of Innsbruck, Innsbruck, Austria

Background. Prolonged cold ischemia time has been

purposed to be a risk factor for poor outcome after lung trans-

plantation. Aim of our study was to investigate whether pro-

longed ischemia time has an impact on postoperative outcome

after bilateral lung transplantation.

Methods. All 115 patients receiving first bilateral lung trans-

plantation from 1993 to 2009 at the University Hospital of

Innsbruck were studied retrospectively.

Results. Prolonged ischemic time (defined as total ischemia

time of more then 330 minutes) was highly correlative with

prolonged postoperative ventilation time (27.1 � 22.9 hours vs.

63.4� 69.3 hours, p¼ 0.006), but did not have an influence on

perioperative mortality (p¼ 0.74, n.s.). Concerning long term out-

come after bilateral lung transplantation, prolonged ischemia

time did not have an impact on long term survival (1-year-sur-

vival: 75.1% vs. � 85.9%, 3-year survival: 68.0% vs. 68.9%, 5 year-

survival: 54.3% vs. 66.4%, log-rank: p¼ 0.256), however there was

a trend towards higher long term survival in patients with pro-

longed ischemia time. Regarding long term freedom from bron-

chiolitis obliterans syndrome (BOS), there was no significant

different influence of prolonged ischemia time in BLTX (1-year

freedom: 92.4% vs. 94.7%, 3-year freedom: 83.5% vs. 80.8%,

5-year freedom: 72% vs. 74.0%; log-rank: p¼ 0.66). Concerning

long-term freedom from acute rejection episodes, patients with

prolonged ischemia time did not have a higher risk for acute



rejection episodes compared to patients with ischemia times

shorter then 330min (log-rank: p¼ 0.30).

Conclusions. Prolonged ischemia time after bilateral lung

transplantation is highly correlated with prolonged postoperative

intubation time, but does not have an impact on perioperative

survival or later outcome parameters.

15Myeloperoxidase and carbonyl proteinsas serum markers for non-invasivemonitoring of graft rejection in patientsafter heart transplantation

P. Stiegler1, M. Schweiger1, S. Köstenbauer1,V. Stadlbauer2, U. Mayrhauser1, B. Leber1, A. Wasler1,S. Zelzer3, T. Stojakovic3, M. Scarpatetti4,J. Greilberger5, K. H. Tscheliessnigg1

1Division of Transplantation Surgery, Department of Surgery,Medical University of Graz, Graz, Austria; 2Department of InternalMedicine, Medical University of Graz, Graz, Austria; 3ClinicalInstitute of Medical and Chemical Laboratory Diagnostics, MedicalUniversity of Graz, Graz, Austria; 4Institute of Pathology, MedicalUniversity of Graz, Graz, Austria; 5Institute of PhysiologicalChemistry, Center for Physiological Medicine,Medical University of Graz, Graz, Austria

Background. After heart transplantation (HTX) endomyocar-

dial biopsy (EMB) is currently the standard method to diagnose

acute graft rejection. A non-invasive marker of rejection would be

desirable as an alternative or to permit more selective use of the

costly and wearing EMB.

Methods. In this retrospective study, outcomes of rou-

tinely taken EMBs were used to select 28 HTX patients

(EMB grade 0R, 1R or 2R). For these patients myeloperoxi-

dase (MPO) and carbonyl proteins (CP) in plasma were mea-

sured using ELISA.

Results. MPO and CP levels in HTX patients with rejec-

tion grade 2R were significantly elevated at the time of rejec-

tion but not one month before rejection occurred. The best

cut-off point for MPO was identified as 37.5 [mg/l] using the

ROC curve (AUC¼ 0.8977; specificity¼ 100%; sensitivity

50%). The best cut-off point for CP was identified as 222.5

[pmol/mg] using the ROC curve (AUC¼ 0.9625; specificity

100%; sensitivity¼ 87.5%). If the optimal cut off levels of

MPO were used before EMBs were performed, we would have

correctly biopsied all 11 patients with rejection grade 2R.

Because of a false positive result of the test, 9 patients with

no rejection would have been biopsied. If the optimal cut off

level of CP were used, all patients with 2R were correctly

biopsied. Only 3 patients have been biopsied due to a false

positive result. These results indicated that MPO and CP

levels predict rejection grade 2R.

Conclusions. These biochemical markers seem to be useful

to monitor rejection events non-invasive and to minimize the

application of EMBs after HTX.

16Lung transplantation and sequentialcardiac surgery – 20 years of experienceby a single centre

L. Hatos-Agyi, M. A. Hoda, B. Ghanim, B. Jaradat,P. Jaksch, W. Klepetko

Division of Cardiothoracic Surgery, Department of Surgery,Medical University of Vienna, Vienna, Austria

Background. The impact of sequential cardiac surgery on

the overall outcome after lung transplantation is still discussed

in literature. In 2001, the use of extracorporeal membrane

oxygenation (ECMO) was established as standard of intra- and

perioperative support in lung transplantation at our centre.

Methods. We retrospectively reviewed all 33 cases of lung

transplantation with concomitant cardiac surgery performed at

our centre between November 1989 and August 2009. Clinical

and demographical data and postoperative outcome were ana-

lysed. Eighteen patients (54.5%) underwent the combined

intervention before 2001 (early group) and 15 patients (45.5%,

recent group) were operated after 2001.

Results. During the observation period, 33 of 929 lung

transplant recipients (13 male, 20 female; mean age 37.1 �15.2 years; 3 single and 30 bilateral lung transplantation) un-

derwent sequential cardiac surgery. The underlying diseases

for transplantation were pulmonary hypertension (n¼ 15),

Eisenmenger-reaction (n¼ 8), pulmonary fibrosis (n¼ 4), cys-

tic fibrosis (n¼ 2) and others (n¼ 4). The cardiac procedures

were for atrial septal defect (n¼ 20), ventricular septal defect

(n¼ 3), persistent ductus arteriosus (n¼ 3), coronary bypass

(n¼ 2), mitral valve reconstruction (n¼ 2) and others (n¼ 3).

The mean overall survival was 1310 � 1776.7 days (range 1–

6173). The 1-year and 5-year survival were 62.3% and 50.2%,

respectively. The overall survival of the 33 patients does not

differ significantly from the cohort of patients that underwent

lung transplantation without cardiac surgery during the obser-

vation period (log rank p¼ 0.297). However, the mortality was

significantly lower in the recent group compared with the early

group (log rank p¼ 0.032).

Conclusions. Lung transplantation and sequential cardi-

ac surgery are not a standard procedure and should be re-

served for a carefully selected pool of patients. Growing

experience in surgery and better hemodynamic management

in the perioperative period have significant impact on the

overall survival.

17Improvement of outcome in patientswith chronic myeloid leukaemia afterallogeneic stem cell transplantation

A. Boehm, B. Walcherberger, W. R. Sperr,S. Wöhrer, K. Lechner, W. Hinterberger,K. Dieckmann, A. Rosenmayr, G. Fischer,N. Worel, G. Mitterbauer, I. Schwarzinger,H. T. Greinix, M. Mitterbauer, O. A. Haas,P. Valent, W. Rabitsch, P. Kalhs


Supplement 231

1Bone Marrow Transplantation Unit, Department of InternalMedicine I, Medical University of Vienna, Vienna, Austria; 2Divisionof Hematology and Hemostaseology, Department of InternalMedicine I, Medical University of Vienna, Vienna, Austria;3Department for Blood Group Serology and Transfusion Medicine,Medical University of Vienna, Vienna, Austria; 4Departmentof Radiotherapy, Medical University of Vienna, Vienna, Austria;5Department of Medical and Chemical Laboratory Diagnostics;Medical University of Vienna, Austria; 6St. Anna Children'sHospital, Vienna, Austria; 7Second Department of InternalMedicine, Danube Hospital, Vienna, Austria

Background. Although imatinib has become the standard

first-line therapy in patients with chronic myeloid leukemia

(CML), allogeneic stem cell transplantation (SCT) still remains

an important treatment option with curative potential for

patients with loss of response to tyrosine kinase inhibitors or

advanced phase of the disease.

Methods and results. We analyzed 175 adult patients with

CML, who underwent allogeneic stem cell transplantation (SCT)

at our institution between 1983 and 2007 in a retrospective study.

Ninety-four patients had a sibling donor, 80 patients had an

unrelated donor and one patient was transplanted with a

haploidentical donor. Thirty-two patients received imatinib prior

to SCT, 165 patients had myeloablative conditioning, and 10

patients had reduced-intensity conditioning. With a median

follow-up of 5 years we examined the clinical outcome of all

patients and potential prognostic variables. The probability of

overall survival (OS) for all patients was 62 % with significantly

improvement from 50% for transplantations performed in the

first decade (1983–1994) to 76% for transplantations performed

in the third decade (2000–2007). Post-transplant-relapse

occurred in 30 patients, and 31 patients died from treatment-

related complications. In multivariate analysis we revealed acute

and chronic graft versus host disease as independent prognostic

variables concerning overall survival.

Conclusions. Based on our data and similar published stud-

ies we confirm that allogeneic SCT is still an important and

successful treatment option for patients with CML and under-

lines the existing need for defining the optimal strategy for trans-

plantation performance.

18Einfluss vonMinorhistokompatibilitätsantigen-Inkompatibilität auf das Ansprechennach humaner Nieren-und Stammzelltransplantation

B. Kircher1, P. Schumacher1, H. Schennach2,R. Margreiter3, D. Nachbaur1

1Immunbiologie und Stammzelllabor, Universitätsklinikfür Innere Medizin V – Hämatologie und Onkologie, MedizinischeUniversität Innsbruck, Innsbruck, Österreich; 2Zentralinstitutfür Bluttransfusion und Immunologische Abteilung, Landeskran-kenhaus Innsbruck, Innsbruck, Österreich; 3Universitätsklinikfür Visceral-, Transplantations- und Thoraxchirurgie, DepartmentOperative Medizin, Medizinische Universität Innsbruck,Innsbruck, Österreich

Grundlagen. Spender-T-Zellen, die polymorphe Selbst-Pep-

tide, Minorhistokompatibilitatsantigene (mHA), eines humanen

Leukozyten-Antigen (HLA)-identen Patienten erkennen und die

mHA-prasentierenden Zellen zerstoren, sind hauptsachlich fur

die Graft-versus-host-Erkrankung (GvHD), eine schwere Kompli-

kation nach Stammzelltransplantation, verantwortlich. Zahl-

reiche Studien weisen darauf hin, dass mHA auch eine Rolle in

der Entstehung von Komplikationen nach humaner Nierentrans-

plantation spielen konnen.

Methodik. Zweck dieser Studie war daher, einen mogli-

chen Einfluss des Geschlechts-spezifischen mHA HY und der

autosomal kodierten mHA HA-1, HA-2, HA-3 und HA-8 auf das

Transplantat-Uberleben und Komplikationen von 50 Patienten

mit Nieren- und 130 Patienten mit Stammzelltransplantation

mittels Polymerase-Ketten-Reaktion zu untersuchen. Die

Anzahl der mHA-Unterschiede war in beiden Studienkohorten

ahnlich, es wurden lediglich mehr Patienten mit einem HY-

Unterschied stammzelltransplantiert (21%) als nierentrans-

plantiert (12%).

Ergebnisse. Das Ansprechen der Patienten auf die HLA-

idente Nierentransplantation war sehr gut, sodass nur wenig

akute Abstoßungsepisoden oder Transplantatverluste zu ver-

zeichnen waren. Die Inzidenz fur diese Komplikationen war

jedoch erhoht, wenn das Patienten/Spenderpaar eine Nichtuber-

einstimmung in den mHA HA-1 oder HA-8 aufwies. Jedoch

waren erhohte Panel-reaktive Autoantikorper vor und das Beno-

tigen einer Dialyse nach Nierentransplantation ebenso ein Indiz

fur diese Komplikationen. Alle Patienten mit einer HA-2-Inkom-

patibilitat zu ihrem Spender entwickelten eine akute GvHD.

Diese Patienten und mannliche Patienten mit einem weiblichen

Spender hatten ein geringeres Uberleben. Im Gegensatz dazu

zeigten Patienten ohne HA-1- und ohne HA-8-Mismatch eine

hohere Relapsrate nach Stammzelltransplantation. Unterschiede

im mHA HA-3 waren weder fur Patienten mit Nieren- noch

mit Stammzelltransplantation ein pradiktiver Marker fur das

Ansprechen.

Schlussfolgerungen. Die Daten dieser Studie zeigen, dass

der positive Effekt von HA-1- und HA-8- Inkompatibilitat in der

Stammzelltransplantationskohorte einem negativen Effekt auf

die nierentransplantierten Patienten gegenuber steht. Allerdings

sollten diese Ergebnisse in großeren Studien bestatigt werden.

19Österreichische Erfahrung mit Plerixaforzur Stammzellmobilisierungin Kombination mit G-CSF bei Patientenmit Mobilisierungsversagen nachChemotherapie und G-CSF

N. Worel, H. T. Greinix, B. Pribitzer, P. Neumeister,D. Nachbauer, H. Kasparu, E. Schlögl, N. Zojer,F. Keil, G. Russ, P. Kalhs1Klinische Abteilung für Transfusionsmedizin und Universitätsklinikfür Innere Medizin I, Medizinische Universität Wien, Wien,Österreich; 2Klinische Abteilung für Hämatologie, Universitätsklinikfür Innere Medizin, Medizinische Universität Graz, Graz, Österreich;3Universitätsklinik für Innere Medizin V (Hämatologie undOnkologie), Medizinische Universität Innsbruck, Innsbruck,Österreich; 4Interne Abteilung, Krankenhaus der Elisabethinen,Linz, Österreich; 5Hanusch Krankenhaus, Wien, Österreich;6I. Medizinische Abteilung,Wilhelminenspital der StadtWien,Wien,Österreich; 7Klinische Abteilung für Hämatologie, KrankenhausLeoben, Österreich; 8St. Johanns Spital, Salzburg, Österreich



Die autologe Blutstammzelltransplantation (PBSZT) ist

eine etablierte Therapieoption fur Patienten mit hamato-

onkologischen Erkrankungen. Bei einigen Patienten (,,Poor Mobi-

lizer‘‘) konnen nach Chemotherapie (CHT) und G-CSF nicht

ausreichend Stammzellen (SZ) fur eine erfolgreiche SZ-

Sammlung mobilisiert werden. Als Risiko dafur gelten: hoheres

Alter, Knochenmarkbefall, Strahlentherapie und intensive

Chemotherapie. Seit kurzem wird Plerixafor zur Verbesserung

der SZ-Mobilisierung bei ,,Poor Mobilizer‘‘ Patienten mit Multi-

plem Myelom, Non Hodgkin- und Hodgkins Lymphom, einge-

setzt. Plerixafor fuhrt zu einer reversiblen Blockade der Bindung

zwischen dem stromal derived factor 1 (SDF1) und dem Chemo-

kinerezeptor 4 (CXCR4) und bewirkt dadurch eine Ausschwem-

mung von SZ ins periphere Blut. Osterreichweit wurde Plerixafor

bei 24 Patienten (8 M, 16 F) mit einem median Alter von 56

Jahren (21–65) eingesetzt. Die Patienten erhielten 2�5mg/kg/Tag G-CSF s.c. fur 4 Tag gefolgt von 240mg/kg Plerixafor s.c.

am Tag 4 abends. Am Tag 5, 10–11 Stunden nach der Plerixafor-

gabe wurde die SZ-Sammlung durchgefuhrt. Bei drei Patienten

waren 2 Mobilisierungszyklen notwendig.

Die Patienten hatten im Median 8 (3–27) CHT-Zyklen

vor einem erfolglosen Mobilisierungsversuch erhalten. Nach G-

CSF und Plerixafor waren am Tag 5 eine mediane Anzahl von 8,1

(0–75) CD34þ/ml messbar und es wurden 2,0 (0,1–6,8)�106

CD34þ/kg gesammelt. Am 2. Pheresetag konnten 1,12 (0,14–

2,47), am 3. Pheresetag 0,5 (0,08–1,56) und am 4. Pheresetag

1,01�106/kg gesammelt werden. Bei 19 Patienten wurden 2,

bei 5 Patienten 3 und bei 1 Patient insgesamt 4 SZ-Pheresen

durchgefuhrt. Ein Patient mobilisierte trotz Plerixafor keine SZ.

Bei 18 Patienten (75%) konnten ausreichend CD34þ fur eine

PBSZT gesammelt werden. Neun Patienten wurden bisher trans-

plantiert und zeigten ein rasches Engraftment mit >500 Granu-

lozyten/ml nach median 11 (9–16) Tagen und >20.000

Thrombozyten/ml nach median 14 (9–38) Tagen.

Bei etwa 75% der Patienten, die als Poor Mobilizer gelten,

konnen durch Plerixafor und G-CSF ausreichend SZ fur eine

suffiziente SZ-Sammlung mobilisiert werden. Nach Transplanta-

tion dieser SZ kommt es zu einer raschen und dauerhaften

hamatologischen Regeneration.

20Haploidente Stammzelltransplantationbei Kindern und jungen Erwachsenen

W. Schwinger1, P. Sovinz1, H. Lackner1, M. Benesch1,A. Moser1, S. Sipurzynski2, C. Urban1

1Klinische Abteilung für Pädiatrische Hämatologie/Onkologie,Universitätsklinik für Kinder und Jugendheilkunde, MedizinischeUniversität Graz, Graz, Österreich; 2Universitätsklinikfür Blutgruppenserologie und Transfusionsmedizin, MedizinischeUniversität Graz, Graz, Österreich

Grundlagen. Die Verwendung haploidenter Stammzellspen-

der (Eltern) ist bei Fehlen eines HLA-identen Spenders eine sinn-

volle Alternative wegen der uneingeschrankten Verfugbarkeit von

Stammzellen und Donorlymphozyten.

Methodik und Ergebnisse. Seit 1997 wurden 17 Patienten

(12 weiblich, 5 mannlich) mit einem medianen Alter von 12,4

Jahren (3 Monate – 24 Jahre) einer haploidenten Stammzelltrans-

plantation unterzogen (eine Patienten wurde retransplantiert).

Fur alle Patienten wurde die dringende Indikation zur Stamm-

zelltransplantation gestellt, fur keinen der Patienten wurde ein

geeigneter Fremdspender identifiziert. Die Diagnosen waren bei

8 Patienten hamatologische Erkrankungen: refraktare AML

(n¼ 2), HR-ALL (n¼ 2), SCID (n¼ 1), SAA (n¼ 1), ALPS (n¼ 1),

Mb. Hodgkin-Rezidiv (n¼ 1); bei 8 Patienten fortgeschrittene/

relapsierte solide Tumore: Neuroblastom IV (n¼ 3), Osteosarkom

IV (n¼ 2), Ewing’s Sarkom (n¼ 2), Wilms-Tumor (n¼ 1); bei

einer Patientin eine Stoffwechselerkrankung (Mb. Krabbe). Alle

Patienten mit malignen Erkrankungen waren durchwegs intensiv

vorbehandelt, einschließlich Hochdosischemotherapien mit

autologem Stammzellsupport (n¼ 6). Die Konditionierungsche-

motherapie war Fludarabin-basiert bei 15 Patienten (zusatzlich

TBI n¼ 1, TLI n¼ 1), ein Patient erhielt BU/Cy, die SCID-

Patientin wurde ohne Chemotherapie transplantiert. Stammzell-

spender waren Mutter n¼ 8, Vater n¼ 8, Bruder n¼ 1, Schwester

n¼ 1. In 6 Spender-Empfanger-Konstellationen wurde ein KIR-

mismatch in GvH-Richtung identifiziert. Stammzellquelle war

Knochenmark (n¼ 2) sowie periphere Stammzellen (n¼ 16).

Knochenmark wurde unmanipuliert verabreicht, die peripheren

Stammzellen wurden CD34-positiv selektioniert (n¼ 4) CD3/19

depletiert (n¼ 1) oder einer kombinierten CD3/19 Depletion

und CD34-positiv Selektion (n¼ 11) unterzogen. Die mediane

Anzahl an transplantiertenCD34-Zellen war 11,9�106/kg (2,46–

139�106/kg). Alle Patienten zeigten ein komplettes 3-Linien-

Engraftment, die Patientin mit Mb. Krabbe hatte am Tag þ33

eine Transplantatabstoßung und wurde von einem anderen

Elternteil retransplantiert. 7 Patienten mit malignen Grunder-

krankungen erhielten eine adoptive Immuntherapie mit Donor-

lymphozyteninfusionen (n¼ 1–94). 12 Patienten uberleben

median 21 Monate (4–73) (CR n¼ 7; SD n¼ 2; PR n¼ 3).

5 Patienten verstarben an PD n¼ 1, GvHD n¼ 1, Sepsis n¼ 1,

MOF n¼ 3.

Schlussfolgerungen. Die haploidente Stammzelltransplanta-

tion ist durch moderne Stammzellmanipulationstechniken und

immunsuppressive Therapien eine anwendbare Alternative bei

Patienten mit einer dringlichen Transpolantationsindikation

ohne geeigneten HLA-identen Spender. Die zukunftige Rolle

der Graft versus Tumor/Leukamie-Komponente dieser Technik

wird durch zahlreiche Studien gepruft.

21Long-term cultured human MSCs revealvariations in genomic stability

K. Schallmoser1;2, E. Rohde1;2, A. C. Obenauf3,A. Reinisch1;4, Ch. Bartmann1;4, G. Lanzer2,W. Linkesch4, D. Strunk1;4

1Stem Cell Research Unit, Medical University of Graz, Graz,Austria; 2University Clinic of Blood Group Serology andTransfusion Medicine, Medical University of Graz, Graz, Austria;3Institute of Human Genetics, Medical University of Graz, Graz,Austria; 4University Clinic of Internal Medicine, Departmentof Hematology, Medical University of Graz, Graz, Austria

Human multipotent mesenchymal stromal cells (MSCs)

are currently tested in clinical trials for immunomodulatory

and regenerative therapy. We and others have recently estab-

lished MSC propagation with pooled human platelet lysate

(pHPL) substituting fetal bovine serum (FBS). As doubts re-

garding the use of MSCs cultured with FBS and their possible

genomic instability have arisen, we investigated safety

aspects of short- and long-term cultures with FBS compared

to pHPL.


Supplement 231

Bone marrow aspirates were seeded in Alpha-MEM with

pHPL. Clinical-scale expanded MSCs were harvested after prima-

ry culture (short-term). Representative cultures were continued

for long-term expansions each with directly comparing pHPL

and FBS stimulation until proliferation ceased (46–51 population

doublings). Comparative genome hybridization (array-CGH) was

carried out with short- and long-term expanded MSCs using a

whole genome microarray platform.

pHPL is highly efficient in stimulating MSC expansion result-

ing in 780 � 150 million MSCs after one passage. Flow cytometry

revealed >95% viability, >95% CD73/90/105 reactivity and <2%

hematopoietic contamination. We could show adipo/osteo/

chondrogenic differentiation potential, endotoxin levels

<0.025EU/mL and negative bacterial/fungal/mycoplasma test-

ing. In all short-term MSC products, array-CGH revealed bal-

anced genomic profiles. We detected several small copy

number variations previously found in healthy individuals not

associated with phenotype changes. In contrast, after long-term

culture with FBS as well as pHPL, MSCs showed de novo copy

number amplifications previously not reported in the database of

genomic variants.

Despite a high proliferation rate in the short-term pHPL-

driven culture, MSCs showed genomic stability according to ar-

ray-CGH results. These data support our earlier findings that

MSCs expanded under humanized conditions did not form

tumors in vivo in animal experiments. It is not clear whether in

vitro genomic variations in long-term propagated MSCs under

humanized as well as xenogeneic culture conditions may be

associated with a risk of malignant transformation rather than

representing replicative senescence. Therefore safety concerns

have to be vigilantly addressed parallel to the clinical use

of MSCs.

22Modifiziertes Campath-BEAM Protokollals Konditionierung für allogenehämatopoetischeStammzelltransplantation beirefraktärem M. Hodgkin

S. Eder, W. Rabitsch, P. Kalhs, Z. Kuzmina,A. Schulenburg, C. Zielinski, H. Greinix

Knochenmarktransplantation, Universitätsklinik für InnereMedizin I, Medizinische Universität Wien, Wien, Österreich

Grundlagen. Bekanntermaßen haben PatientInnen mit

therapie-refraktarem M. Hodgkin eine schlechte Prognose. Sie

konnen von einer allogenen hamatopoetischen Stammzelltrans-

plantation (SZT) profitieren, jedoch sind myeloablative Konditio-

nierungen oft mit einer hohen transplant-assoziierten Mortalitat

(TRM) vergesellschaftet. Seit dem Einsatz von dosisreduzierten

Konditionierungen konnen allogene SZT nicht nur bei alteren

PatientInnen oder solchen mit Komorbiditaten, sondern auch

Hodgkin-PatientInnen mit geringerer TRM durchgefuhrt werden.

Bisher erhielten letztere im Rahmen der Konditionierung meist

100mg Campath-1H zur Immunsuppression. Um schwere Kom-

plikationen der prolongierten Immundefizienz zu vermeiden,

fuhrten wir eine klinische Studie mit reduzierter Campath-1H-

Dosierung durch.

Methodik. Elf PatientInnen (8 Manner, 3 Frauen) mit einem

mittleren Alter von 35 (24–44) Jahrenmit refraktaremM. Hodgkin

wurden an unserem Zentrum einer allogenen SZT nach

median 43 (13–100) Monaten ab Diagnosestellung unterzogen.

Sieben PatientInnen hatten zuvor eine autologe SZT. Die dosis-

reduzierte Konditionierung erfolgte nach dem BEAM-Protokoll,

Campath-1H (Alemtuzumab) wurde an den Tagen -5 bis -1 in

einer Dosis von je 10mg verabreicht. Danach wurden im Mittel

6.3�106 CD34þ Blutstammzellen/kg KG (3.7–8.7) eines HLA-

identen verwandten (n¼ 7) bzw. unverwandten (n¼ 4) Spenders

transplantiert. Die Graft-versus-Host-Prophylaxe bestand aus

Cyclosporin A.

Ergebnisse. Wahrend 10 PatientInnen eine rasche hamato-

logische Regeneration hatten, erlitt 1 Patient eine Abstoßung und

verstarb nach 17 Monaten am progredienten M. Hodgkin. Bestes

Therapieansprechen nach allogener SZT war eine komplette Re-

mission (CR) in 7 von 11 Fallen, sowie eine partielle in 2. Ein

Patient (9%) starb transplant-assoziiert 146 Tage nach SZT an

einem Multiorgan-Versagen. Drei der restlichen 10 PatientInnen

hatten eine akute Graft-versus-Host-Erkrankung (GvHD), und

weitere 3 eine chronische GvHD. Rund 3 Monate nach SZT hat-

ten nur 5 PatientInnen einen kompletten Spenderchimarismus.

Von den weiteren 5 PatientInnen rezidivierte einer und starb, 4

PatientInnen befinden sich in CR inklusive 2 nach Gabe von

Spenderlymphozyten. Ein Rezidiv bzw. eine Progression der

Grunderkrankung wurde in 4 von 11 PatientInnen (36%) median

181 (50–356) Tage nach SZT beobachtet, davon erreichte 1 Pa-

tient eine CR nach Gabe von Spenderlymphozyten. Derzeit sind 7

von 11 PatientInnen (64%) nach im median 33 (10–58) Monaten

nach SZT in kompletter Remission am Leben, die Kaplan-Meier-

Wahrscheinlichkeit des 5-Jahres-Uberleben betragt 62%.

Schlussfolgerungen. Die allogene SZT mit dosisreduzierter

Konditionierung nach dem modifizierten Campath-BEAM Proto-

koll stellt eine erfolgversprechende kurative Therapie-option fur

PatientInnen mit refraktarem M. Hodgkin dar. Im Posttrans-

plant-Verlauf kann durch die Gabe von Spenderlymphozyten

ein kompletter Spenderchimarismus induziert sowie das Behand-

lungsergebnis verbessert werden, um krankheitsfreies Langzeit-

uberleben zu erreichen.

23Safety of stem cell transplantationto treat cardiovascular diseases: foamcell formation of human bloodand marrow-derived cells as a risk factorof therapeutic angiogenesis?

E. Rohde1;2, K. Schallmoser1;2, A. Reinisch1;3,N. A. Hofmann1;3, Th. Pfeifer4, E. Froehlich5,G. Rechberger6, G. Lanzer2, D. Kratky4, W. Linkesch3,D. Strunk1;3

1Stem Cell Research Unit, Medical University of Graz, Graz,Austria; 2University Clinic of Blood Group Serologyand Transfusion Medicine, Medical University of Graz, Graz,Austria; 3Department of Hematology and Stem CellTransplantation, Department of Internal Medicine,Medical University of Graz, Graz, Austria; 4Institute of MolecularBiology and Biochemistry, Medical University of Graz, Graz,Austria; 5Center of Medical Research, Institute of MolecularBiosciences, Medical University of Graz, Graz, Austria

Clinical trials for therapeutic angiogenesis use blood- or mar-

row-derived transplants containing endothelial progenitor cells



(EPCs), monocytes and mesenchymal stromal cells (MSCs) to

support vascular regeneration. Safety concerns have emerged

since all three cell types could possibly contribute to atheroscle-

rosis via lipid uptake and lipid droplet (LD) storage consequently

leading to typical foam cell formation. We therefore examined

the foam cell potential of these cells as a surrogate marker for

potential pro-atherogenic side effects.

Foam cell development was tested in vitro by exposure of

human EPCs, monocytes and MSCs to acetylated low-density-

lipoprotein (acLDL). The impact of an initial 3-day pro-angiogen-

ic induction on subsequent foam cell formation was studied to

mimic a relevant setting for clinical applications. Intracellular

lipid accumulation in LDs was detected with fluorescence la-

ser-scanning microscopy, flow cytometry and cholesterol mea-

surement. Additionally the activation state of various intracellular

signaling molecules indicating response to stress-induced stimuli

was determined.

Characteristic foam cells developed from monocytes with

significantly increased lipid accumulation after pro-angiogenic

culture conditions imitating those applied in clinical trial proto-

cols. Compared to growth factor free conditions the phosphory-

lation state of the p38 mitogen activated protein kinase related to

foam cell development and stress induced stimuli was also sig-

nificantly enhanced in monocytes after exposure to pro-angio-

genic culture conditions. EPCs and MSCs did not accumulate

lipids or form LDs following pro-angiogenic conditioning and

acLDL exposure.

These data raise serious concerns that cellular therapy with

monocyte-containing hematopoietic cell preparations may be

counterproductive or even aggravate atheroma formation in

patients with cardiovascular disease if underlying pathologic

conditions are not appropriately reverted. We therefore support

previous arguments that the role of transplanted cells in the

various aspects of vascular homeostasis, regeneration and

therapeutic angiogenesis must be re-examined prior to further

clinical trials.

24Stammzellapheresen bei Kindern unter20 kg Körpergewicht: 9 Jahre Erfahrungan der Transfusionsmedizin Graz

K. Rosskopf1, S. Sipurzynski1, T. Wagner1,W. Schwinger2, C. Urban2, G. Lanzer1

1Universitätsklinik für Blutgruppenserologie undTransfusionsmedizin, LKH-KlinikumGraz,Medizinische UniversitätGraz, Graz, Österreich; 2Klinische Abteilung für PädiatrischeHämatoonkologie, Universitätskinderklinik, LKH-Klinikum Graz,Medizinische Universität Graz, Graz, Österreich

Grundlagen. Kleinkinder unter 20kg Korpergewicht stellen

eine Besonderheit hinsichtlich der Vorbereitung und der Durch-

fuhrung einer autologen Stammzellapherese im Vergleich zu

alteren Patienten dar. Wir berichten uber unsere Erfahrungen

mit Kleinkindapheresen aus einem Zeitraum von 9 Jahren

(2000–2009) hinsichtlich Durchfuhrbarkeit, Sicherheit und

Effizienz.

Methodik. Das Schlauchset des Zellseparators (Cobe

Spectra, manuelles Programm) wurde jeweils mit einem

Erythrozytenkonzentrat-Plasma-Gemisch vorgefullt. Die

Patienten waren mit einem doppellumigen zentralen Venen-

katheter (ZVK) ausgestattet und wurden von einem Elternteil

und einem Kinderarzt begleitet und pulsoxymetrisch

uberwacht.

Ergebnisse. 137 Apheresen bei 37 Patienten (25m, 12w)

wurden ausgewertet. Indikationen waren Neuroblastom (n¼ 19),

Medulloblastom (n¼ 6), Ewingsarkom (n¼ 2), Ependymoblas-

tom (n¼ 2), andere (n¼ 8). Antikoagulation war ACD (1:12,

n¼ 91) oder Heparin-ACD (1:18, n¼ 46). Im Median (min–max)

wurden 4 (2–7) Apheresen pro Pat. durchgefuhrt. Bei einem

medianen Alter von 2,4 (0,7–6,5) Jahren und 13 (8–20) kg

Korpergewicht wurde das Blutvolumen im Median 3,4 fach

(1,1–5,9) innerhalb von 164 Minuten (70–240) mit einem Blut-

fluss von 23 (8–45) ml/min apheresiert. 3/137 (2,2%) Apheresen

wurden wegen ZVK-Komplikationen ohne Ergebnis abgebrochen

(Paravasat n¼ 2, kein Fluss n¼ 1), aber an den Folgetagen

nachgeholt. 6 weitere Apheresen (4,4%) wurden wegen ZVK-

Problemen fruhzeitig beendet. Es entstanden 134Konzentrate

mit 5,5 (0,5–75) und in Summe pro Pat. 23,5 (1,9–146)�106/kg

CD34. Nach Apherese sanken immedian Hb um 0,7g/dl, Throm-

bozyten um 23�109/l sowie ion. Calcium um 0,27mmol/l. Die

Substitutionen erfolgten in der Regel auf der Kinderstation.

Schlussfolgerungen. Die autologe Stammzellapherese bei

Kleinkindern unter 20kg stellt – verbunden mit isovolumet-

rischer Sammlung und erhohter Patientenbetreuung – eine

sichere und effiziente Methode bei geringer Komplikationsrate

(meist im Zusammenhang mit ZVK) dar. Der teilweise hohe

Stammzellertrag war erforderlich fur anschließende Selektions-

verfahren und Tandemtransplantationen.

25Primary outcomes from a randomized,phase iii study of belatacept vs.cyclosporine in kidney transplantrecipients (BENEFIT study)

T. Wekerle1, F. Vincenti, J. Grinyó, B. Charpentier,J. Medina Pestana, L. Rostaing, Y. Vanrenterghem,G. Di Russo, P. Garg, C.-S. Lin, C. Larsen1Department of Surgery, Medical University of Vienna, Vienna,Austria; 2UCSF, San Francisco, CA, USA; 3University Hospitalof Bellvitge, Barcelona, Spain; 4Bicetre Hospital, Kremlin Bicetre,France; 5Hospital do Rim e Hipertens~ao Unifesp, Sao Paulo, Brazil;6University Hospital, Toulouse, France; 7University HospitalLeuven, Leuven, Belgium; 8Bristol-Myers Squibb, Princeton, NJ;9Emory University School of Medicine, Atlanta, GA, USA

Background. Belatacept, a co-stimulation blocker, is being

developed as an immunosuppressant for kidney transplant reci-

pients to avoid the renal and extra-renal toxicities of calcineurin

inhibitors (CNIs) that impact long-term patient/graft survival.

BENEFIT assessed belatacept-based regimens vs. a cyclospor-

ine-based regimen (CsA) in kidney transplant recipients.

Methods. BENEFIT is a 3-year, randomized, phase III study

in adults receiving a kidney transplant from a living or deceased

donor with an anticipated cold ischemia time <24 hours.

Patients were randomized 1:1:1 to a more intensive (MI) or less

intensive (LI) regimen of belatacept or CsA; all patients received

basiliximab induction, MMF, and corticosteroids. Co-primary

endpoints were composite patient/graft survival, composite renal

function (measured GFR (mGFR) <60mL/min/1.73m2 at month

12 or a decrease in mGFR �10mL/min/1.73m2 from month 3 to

month 12), and incidence of acute rejection (AR).


Supplement 231

Results. Six hundred and sixty-six patients were randomized

and transplanted. 58% received living donor transplants; 42%

from deceased donors. Patient/graft survival with belatacept

regimens was non-inferior to CsA (95.4% MI; 96.5% LI; 92.8%

CsA) at month 12. Renal function was superior in belatacept vs.

CsA patients as shown by the proportion meeting the composite

renal endpoint (55% MI; 54% LI; 78% CsA; p<0.0001 MI or LI vs.

CsA) and the mGFR at Month 12 (65mL/min MI, 63mL/min LI,

and 50mL/min CsA; p<0.0001 MI or LI vs. CsA). The incidence

of AR was 22%, 17%, and 7% in the MI, LI, and CsA groups; the LI

regimen was non-inferior to CsA. AR in belatacept patients had

limited impact on graft survival and the relative renal benefit of

belatacept. Infection and overall malignancy rates were similar

across arms; PTLD was observed in 1 (0.5%), 2 (0.9%), and

1 (0.5%) patients in the MI, LI, and CsA groups in the first

12 months.

Conclusions. At 12 months, belatacept regimens demon-

strated superior renal function and similar patient/graft survival

vs. CsA, despite an increase in AR in the early post-transplant

period. Belatacept represents a promising, non-nephrotoxic

therapy option in kidney transplant patients.

26Primary outcomes from a randomized,phase III study of belatacept vs.cyclosporine in ECD kidney transplants(BENEFIT-EXT study)

F. Mühlbacher1, A. Durrbach, C. Larsen,J. Medina Pestana, Y. Vanrenterghem, F. Vincenti,S. Florman, A. Block, P. Garg, K. Sen, J. Grinyó1Division of Transplantation, Department of Surgery, MedicalUniversity of Vienna, Vienna, Austria; 2Bicetre Hospital, KremlinBicetre, France; 3University School of Medicine, Atlanta, GA, USA;4Hospital do Rim e Hipertens~ao Unifesp, Sao Paulo, Brazil;5University Hospital Leuven, Leuven Belgium; 6UCSF,San Francisco, CA, USA; 7Tulane School of Medicine,New Orleans, LA, USA; 8Bristol-Myers Squibb, Princeton,NJ, USA; 9University Hospital of Bellvitge, Barcelona, Spain

Background. Belatacept, a selective co-stimulation blocker,

is being evaluated as an immunosuppressant in renal allograft

recipients to avoid the renal and extra-renal toxicities of

calcineurin inhibitors. As recipients of extended criteria donor

(ECD) kidneys are at elevated risk of graft dysfunction and loss,

they may particularly benefit from a non-nephrotoxic option

such as belatacept.

Methods. BENEFIT-EXT is a 3-year, randomized, Phase III

study in adults receiving an ECD kidney transplant. Patients were

randomized 1:1:1 to receive a more intensive (MI) or less

intensive (LI) regimen of belatacept or CsA; all patients received

basiliximab induction, MMF, and corticosteroids. The two co-

primary endpoints were: composite patient/graft survival at 12

months and composite renal function (measured GFR <60mL/

min/1.73m2 at month 12 or a decrease in measured GFR

�10mL/min/1.73m2 from month 3 to month 12. Secondary

endpoints included the incidence of acute rejection (AR).

Results. Five hundred and forty-three patients were random-

ized and transplanted. Patient/graft survival with belatacept was

non-inferior to CsA (86% MI, 88% LI, 85% CsA) at month 12.

Renal function was superior in belatacept MI vs. CsA patients

as shown by fewer patients reaching the composite renal end

point (71% MI, 76% LI, and 85% CsA; p¼ 0.002 MI vs. CsA;

p¼ 0.06 LI vs. CsA) and by the measured GFR at Month 12

(52mL/min MI, 50mL/min LI, and 45mL/min CsA; p¼ 0.008

MI vs. CsA; p¼ 0.10 LI vs. CsA). The prevalence of AR was 17%,

18%, and 14% in the MI, LI, and CsA groups. The overall rates of

infection and malignancy were comparable between groups.

PTLD was observed in 1 (0.5%) and 2 (0.9%) patients in the MI

and LI groups and in none in the CsA group in the first 12

months.

Conclusions. Belatacept regimens demonstrated better renal

function, with comparable patient/graft survival and AR

compared to a CsA-based regimen in ECD kidney transplant

recipients. Belatacept represents a promising immunosuppres-

sant therapy in ECD kidney transplant recipients.

27Renal benefit of belatacept vs.cyclosporine in kidney transplant patientsis not impacted by acute rejection(BENEFIT study)

B. Watschinger1, C. Larsen, F. Vincenti, J. Grinyó,B. Charpentier, G. Di Russo, P. Garg, Y. Dong1Department of Internal Medicine III, Medical University of Vienna,Vienna, Austria; 2Emory Univ. School of Medicine, Atlanta, GA,USA; 3UCSF, San Francisco, CA, USA; 4University Hospitalof Bellvitge, Barcelona, Spain; 5Bicetre Hospital, Kremlin Bicetre,France; 6Bristol-Myers Squibb, Princeton, NJ, USA

Background. Belatacept-based immunosuppression is

associated with superior renal function and comparable

patient/graft survival in two phase III trials in kidney trans-

plant recipients vs. cyclosporine (CsA). In one trial (BENEFIT;

non-ECD recipients), there was a higher incidence of acute

rejection (AR) with belatacept-based regimens vs. CsA. This

descriptive analysis characterizes AR and its impact on 1-year

outcomes in BENEFIT.

Methods. BENEFIT is a 3-year, randomized, phase III study

in adults receiving a kidney transplant from a living or deceased

donor (anticipated cold ischemia time <24 hours). Patients were

randomized to a more intensive (MI) or less intensive (LI) regi-

men of belatacept or CsA; all received basiliximab induction,

MMF, and corticosteroids.

Results. In the ITT analysis at 12 months, 22% (MI), 17%

(LI), and 7% (CsA) exhibited AR by month 12. 10% of patients

(MI), 5% (LI), and 1% (CsA) group had Banff grade �IIb AR.

Most AR occurred within the first 3 months. Few patients

experienced >1 episode of AR (2.7%, 1.7%, and 0.9% for MI,

LI, and CsA) and sub-clinical rejection was observed infre-

quently (4%–5% across groups) at the week 52 biopsy. The

most common AR treatment was corticosteroids; 6% (MI),

4% (LI), and 0% (CsA) of patients were resistant. Initial T-cell

depletion was given in 6% (MI), 4% (LI), and 1% (CsA) of

patients. Approximately 50% of patients with AR in the bela-

tacept groups remained on belatacept. Few patients (n¼ 1 MI,

n¼ 2 LI, n¼ 2 CsA) were adjudicated to have graft loss due to

AR. Of the patients with AR, 94%, 92%, and 94% in the MI, LI,

and CsA group survived with a functioning graft, compared to

96%, 97%, and 93% without AR. Among AR patients at

month 12, measured GFR (mGFR) was 62ml/min/1.73m2



(MI), 61ml/min/1.73m2 (LI), and 48ml/min/1.73m2 (CsA); for

patients without AR, mGFR was 66ml/min/1.73m2 (MI),

65ml/min/1.73m2 (LI), and 51ml/min/1.73m2 (CsA).

Conclusions. The relative benefit of belatacept regimens

on renal function was maintained vs. CsA in patients who

had AR, despite higher rates and grades of AR in living/

deceased kidney transplant recipients. The impact of rejec-

tion on graft function and survival at 12 months was limited

and longer-term effects will continue to be assessed over the

3-year trials.

28Stabilization of renal function in a CNI-Everolimus based immunosuppressiveregime after lung transplantation

B. Zweytick, B. Ghanim, M. Winter, L. Hatos,G. Boehmig, P. Jaksch, W. Klepetko

Division of Cardiothoracic Surgery, Department of Surgery,University of Vienna, Vienna, Austria

Background. Benefits of calcineurin inhibitors (CNI) are

limited by nephrotoxiticity. Protective effects of Everolimus, a

proliferation inhibitor, on kidney function were achieved in heart

and renal transplanted patients, but no data exist in lung trans-

planted patients (LUTX), whose immunosuppressive therapy is

the highest of all. The aim of the study was to evaluate the impact

of a CNI reduced, Everolimus (EV), Mycofenolat Mofotil (MMF)

and steroid based immunosuppression in LUTX with chronic

renal failure.

Methods. In 43 LUTX (24 m/19 f; mean age: 51.9 � 11.9

years, median after TX: 1.9 years) with deterioration in renal

function (mean GFR: 33.1 � 10.4ml/min) CNI (CYA: 16 pts/

Tacrolimus: 27 pts) was stepwise halved and EV was titrated

to an average trough level between 5–8 ng/ml (CNI-EV). The

maintenance EV dose was between 0.75–3mg twice daily.

Routine laboratory values, GFR, CNI-EV trough levels were

monitored monthly before CNIEV, at time of conversion

and 3, 6, 9, and 12 months after CNI-EV switch. We also

evaluated on safety, side effects and biopsy proven rejections

retrospectively.

Results. A positive stabilizing effect was achieved in all 43

LUTX (GFR pre switch: 36.6 � 15.3ml/min; GFR post switch:

36.7 � 16.0ml/min; p¼n.s.). Only in patients with GFR

>40ml/min at time of onset of CNI-EV therapy, deterioration

of renal function could be stabilized, in all other LUTX GFR

deteriorated in the follow up period. 3 LUTX (7%) died and 1

LUTX underwent dialysis. Withdrawal of EV was indicated

in 3 LUTX (7%-1 peripheral edema, 1 Quincke edema, 1 leg

weakness). There were no significant changes in cholesterol

and blood counts after CNI-EV switch, but a significant

increase of triglycerides (p¼ 0.05). No clinical relevant

rejection episodes were detected.

Conclusions. CNI-EV immunosuppressive regimen is a safe

feasible therapy (rejection/side effects) to stabilize renal function.

The best protective renal effect can be achieved in patients with

GFR greater than 40ml/min.

29Side effects in a Calcineurin Everolimusbased immunosuppressive regimein lung transplanted recipients

B. Zweytick, B. Ghanim, M. Winter, L. Hatos,G. Boehmig, P. Jaksch, W. Klepetko


Background. Everolimus, a proliferation inhibitor, is a safe

and well tolerated immunosuppressor in kidney and heart

transplanted recipients. But less experience exist in patients

(pts) after lung transplantation (LUTX). The aim of this study

was to evaluate the safety of Everolimus in lung recipients.

Methods. Sixty patients (29 m/31 f; mean age: 52þ 12 years)

were switched from a Calcineurin inhibitor (CNI), Mycofenolat

Mofetil, Methylprednisolone to a quadruple therapy in which

CNI was reduced to about 50% of baseline levels and Everolimus

(EV) added and titrated to an average EV trough level between 5–

8ng/ml. 50 LUTX (83.3%) were switched because of renal deteri-

oration (median years after LUTX: 1.9) and 10 pts (16.7%) be-

cause of a postoperative CNI-EV based immunosuppressive

study regimen 3 months after LUTX. All pts were followed up

for 4.7 years in mean.

Results. Withdrawal of Everolimus was indicated because

of side effects in 9 pts (15.0%). Reasons for withdrawal were:

Quincke edema (1 pts), exanthema (1 pt), mental disorder

(1 pt), headache (1 pt), diarrhea/nausea (2 pts), peripheral

edemas (1 pt), liver failure (1 pt) and others (1 pt). All adverse

effects were reversible after EV withdrawal. The most

frequent side effects under the quadruple therapy were

leucopenia in 13 pts (21.7%), anemia in 4 pts (6.7%), throm-

bocytopenia in 6 pts (10.0%), hypercholesterinemia in 49 pts

(81.4%), hypertriglyceridemia in 54 pts (88.4%) and Qunicke

edema in combination with an ACE inhibitor therapy in

3 pts (5.0%).

Conclusions. A CNI reduced immunosuppressive therapy

based on Everolimus for preserving renal function is a safe and

well tolerated immunosuppressive therapy, associated with less

side effects in all lung transplant recipients after anastomoses

and wound healing is finished.

30The influence of mTOR inhibitors on CMVrecurrence after whole organtransplantation

P. Stiegler1, M. Schweiger1, M. Haid1, V. Stadlbauer2,A. Wasler1, S. Schaffellner1, F. Iberer1,K. H. Tscheliessnigg1

1Division of Transplantation Surgery, Department of Surgery,Medical University of Graz, Graz, Austria; 2Department of InternalMedicine, Medical University of Graz, Graz, Austria

Background. Cytomegalovirus (CMV), a member of the �-

herpes-virus group, is one of the most important infections after


Supplement 231

solid organ transplantation. mTOR inhibitors are a new group of

immunosuppressive drugs used in solid organ transplantation,

represented by Sirolimus and its derivate Everolimus. Our main

intent was to evaluate if there is a difference in occurrence of

CMV infection in recipients treated either with Sirolimus or

Everolimus.

Methods. Patients after orthotopic heart transplantation

(HTX) and after orthotopic liver transplantation (LTX) trans-

planted between 1983 and December 2008 at the Medical

University Graz, Austria, Department for Surgery, Division

of Transplantation Surgery were included in this study.

Receiving either Everolimus or Sirolimus in their immuno-

suppressive regimen was the inclusion criteria. Data about

CMV infections, CMV donor status and through levels of

immunosuppression was collected retrospectively and anal-

ysed statistically.

Results. 2 HTX recipients (5%) and 20 LTX recipients (29%)

developed CMV infection in this period. From CMV infected

recipients 9.1% received Everolimus and 90.9% received Siroli-

mus. In Sirolimus treated patientd CMV infection occurred sig-

nificantly more often than in Everolimus treated (p¼ 0.003).

Overall, significantly more LTX patients developed CMV infection

than HTX patients did. CMV serostatus of the donor or the

recipients prior to transplantation had no influence on the

occurrence of CMV infection.

Conclusions. Because there are no previous studies compar-

ing Sirolimus and Everolimus related to their effect on the occur-

rence of CMV infection, we could not compare our results with

others. Comparing our infection rates with others, the data for

Everolimus treated recipients is similar, data for Sirolimus treated

does not correlate. Infection rate in our HTX and LTX recipients

is different to other studies. There could also be a bias in our

study due to the fact that most of our Sirolimus treated patients

where LTX recipients and most of HTX patients received

Everolimus. Anyway, it is difficult to compare our results with

others, because of different immunosuppressive regimens and

different study designs. Besides the size of our study group is

too small to see a definite trend, so further investigations have to

be performed.

31Renal function in cardiac recipientsreceiving Everolimus

M. Schweiger, P. Stiegler, A. Wasler, G. Prenner,M. Sereinigg, K. H. Tscheliessnigg

Division of Transplantation Surgery, Department of Surgery,Medical University of Graz, Graz, Austria

Background. Aim of this study was to compare the effect on

renal function of cardiac transplant recipients receiving either

reduced-dose Cyclosporine A plus Everolimus (EvA) or EvA

combined with MMF.

Methods. Thirty one patients (25 male, 6 female) post HTX

were switched to an EvA based immunosuppressive regime.

Study population data may be seen in Table 1. Indications were

cardiac allograft vasculopathy [45%], chronic renal failure [25%],

acute rejection [20%], and other reasons [10%]. Group A (n¼ 12)

received EvA, MMF, Aprednislon and group B (n¼ 16) received

EvA, Low-dose Cyclo. A, Aprednislon. Additionally all patients

received statins and antihypertensive medication. Everolimus,

Cyclosporine A (CsA) through levels and laboratory values were

controlled monthly. Drug administration was adapted to EvA

through levels between 3–8ng/mL and for group B CsA trough

levels between 50ng/ml and 80ng/ml. Death, safety, side effects,

biopsy-proven acute rejection, laboratory values and through

levels were evaluated over a follow-up period of 18 months ret-

rospectively.

Results. Although recipients in group had a higher baseline

creatinine level (2.73mg/dl) compared to group b (1.75mg/dl) a

tendency towards decreasing creatinine levels was seen in group

A (1.53mg/dl) with two patients who had to undergo dialysis

compared to group B (1.73mg/dl) with one patient on dialysis

(p¼n.s.). No difference was seen on mortality, infections, side

effects and EvA trough levels. Biopsy proven acute rejection

showed a higher tendency in group a without significant

differences.

Conclusions. EvA in combination with MMF is well tolerated

and might be a good alternative in patients with impaired renal

function. Limitation of this study is that patients with impaired

renal function were more likely to receive EVA without Calcineur-

ininhibitors.

32Spendervorbehandlung mit Steroid kannnicht gestörten Metabolismusim Transplantat normalisieren

J. Wilflingseder1;2, A. Kainz1;2, I. Mühlberger3,P. Perco3, B. Mayer3, R. Oberbauer1;2

1Abteilung für Nephrologie, Krankenhaus der Elisabethinen,Linz, Österreich; 2Klinische Abteilung für Nephrologie,Universitätsklinik für Innere Medizin III, Medizinische UniversitätWien, Wien, Österreich; 3Emergentec Biodevelopment GmbH,Wien, Österreich

Grundlagen. Wir haben kurzlich in einer randomisierten

kontrollierten Studie gezeigt, dass die Behandlung von hirntoten

Organspendern mit 1000mg Methylprednisolon zu einer

Hemmung der Inflammation im Spenderorgan, aber nicht zu

einer Reduzierung der Inzidenz von akutem Nierentransplantat-

versagen (delayed graft function, DGF) fuhrt. Die hier prasen-

tierte Analyse soll molekulare Risikofaktoren fur DGF außer

Inflammation in steroidvorbehandelten hirntoten Organspen-

dern identifizieren.

Methodik. Zwanzig Nierenbiopsien von steroidvorbehandel-

ten Spendern wurden auf der Ebene genomweiter Genexpression

und Protein–Protein Interaktion analysiert, um eine genetische

Signatur fur DGF zu erhalten, die unabhangig von der Inflamma-

tion im Spenderorgan ist.

Ergebnisse. SAM (Significance Analysis of Microarrays)

identifizierte 63 signifikant supprimierte Transkripte, die mit

DGF assoziiert sind. Diese Gene konnen gemaß PANTHER Onto-

logies in zwei biologische Hauptprozesse kategorisiert werden:

Transport (p<0,001) und Metabolismus (p<0,001), die im

Rahmen der Hypoxie wahrend der Spenderkonditionierung

supprimiert werden

Schlussfolgerungen. Unsere Daten zeigen, dass moleku-

lare Signalwege, die durch Ischamie beeinflusst werden wie

z. B. Transport und Metabolismus, mit DGF assoziiert

sind. Potenzielle Behandlungsstrategien basierend auf

diesen Ergebnissen konnen PPAR-Agonisten oder Caspase

Inhibitoren sein.



33The hPan hanging drop technique – a new3D model system to study hypoxiaassociated cell stress/death in humanpancreatic cells

O. Nussbaumer1, M. Hermann1, K. Pansi1,A. Deutschmann1, A. Draxl1, J. Troppmair2,R. Margreiter1;2, P. Hengster1

1KMT Laboratory, Department of Visceral, Transplantand Thoracic Surgery, Center of Operative Medicine,Medical University of Innsbruck, Innsbruck, Austria; 2DanielSwarovski Research Laboratory, Department of Visceral,Transplant and Thoracic Surgery, Center of Operative Medicine,Medical University of Innsbruck, Innsbruck, Austria

Besides allograft rejection, several other factors have been

described to contribute to poor engraftment and primary non-

function of transplanted islets. Stress factors such as hypoxia

play a crucial role during the transport as well as the early

posttransplant phase in which revascularization of the islet

grafts has not been completed. Near anoxic conditions in the

core regions especially of the bigger islets inevitably leads to

apoptosis/necrosis, which contributes to early graft loss. Cel-

lular changes and the response to hypoxia associated cell

stress are difficult to monitor by conventional methods. In

addition, methods such as classical immunohistochemistry

are often time consuming and not well suited to document

subtle tissue/cell damage caused by hypoxia.

Herein we describe a new model system which allows a fast

and detailed live analysis of hypoxia associated damage in 3D

clusters of human pancreatic cells. For this purpose, we used the

human pancreatic cell line hPan and seeded the cells into single

drops onto cell culture dishes (fractions with 15000 cells per 35mlRPMI medium), which were inverted and incubated for 24 hours

under cell culture conditions. Under such conditions hPan cells

form vital cell–cell clusters (organoids) reminding in size and

shape human islets. The average diameter of the hPan cell aggre-

gates was 40� 15mm (mean � SD).

Applying real time live confocal microscopy, organoid mor-

phology, cell viability, and organoid architecture of aggregated

hPan cells grown under normoxic conditions was compared to

those cultured under hypoxic conditions. The following live

stains were used: Tetramethylrhodamine methyl ester, sytox,

propidium iodide and fluorescent wheat germ agglutinin allow-

ing for the visualization of time-dependent mitochondrial mem-

brane potentials, total number of cell nuclei, cell death and

organoid/cell morphology. Cell clusters growing under normal

conditions showed intact mitochondria, no Rhod-2 staining and

high cell viability (>95%). Switch to hypoxic conditions was char-

acterized by cell death which was preceded by obvious signs of

cell stress such as enhanced positivity for Rhod-2, changes in

mitochondrial potential/morphology as well as organoid

dissociation.

In summary, the hPan hanging drop technique presented

here is simple, economical and cost effective in terms of the time

taken and the reagents required and is applicable to other studies

in which specific media modifications are tested for their protec-

tive efficacy against hypoxia related cellular damage. In addition,

the increased surface area to volume ratio in the hanging drop

results in an rapid equilibration with changes in the cellular

environment.

34Etablierung eines Hirntod-Modellsin der Maus

G. Pomper1, K. Trescher 1;2, D. Santer1, M. Hasun1,A. Baumgartner1, M. Inci1, K. Adelmann1, W. Dietl1,A. O. Zuckermann3, B. K. Podesser1;2

1Ludwig Boltzmann Cluster für kardiovaskuläre Forschung,Medizinische Universität Wien, Wien, Österreich; 2Abteilungfür Herzchirurgie, Landesklinikum St. Pölten, Österreich;3Klinische Abteilung für Herz-Thoraxchirurgie, Universitätsklinikfür Chirurgie, Medizinische Universität Wien, Wien, Österreich

Grundlagen. Experimentelle Hirntodmodelle, bei welchen

es durch Steigerung des intrakraniellen Druckes (ICP) mittels

eines intrakraniell platzierten Ballonkatheters zum Eintritt

des Hirntodes kommt, sind bestens etabliert und werden im

Rahmen transplantationsrelevanter Fragestellungen seit Jahr-

zehnten angewandt. Unsere Intention war es, ein Hirntodmo-

dell an der Maus zu entwickeln, sowie die explosive und

graduelle Hirntodinduktion unter ICP-Monitoring miteinan-

der zu vergleichen.

Methodik. Als Versuchstiere dienten weibliche OF-1

Mause, die nach Randomisierung den Hirntod-Gruppen,

explosive [BD ex, n¼ 14] und graduelle Hirntodinduktion

[BD grad, n¼ 9], und einer Kontroll-Gruppe [Control, n¼ 7]

zugeteilt wurden. Der Hirndruck wurde durch Inflation des

Ballonkatheters mit 20 ml [BD grad] und 40 ml [BD ex] saliner

Losung alle 5 Minuten unter Elektroenzephalogramm (EEG)-

und ICP-Monitoring bis zum Eintritt des Hirntodes gesteigert.

Das Hauptkriterium hierfur war ein Null-Linien-EEG, der

Ausfall der Spontanatmung und weite, lichtstarre Pupillen.

Der intrakranielle Druck, die zerebrale Aktivitat und die

Herzfrequenz (HF) wurden wahrend der 6stundigen

Beobachtungsphase kontinuierlich aufgezeichnet. In der

Kontrollgruppe wurde der Schadel lediglich trepaniert, aller-

dings kein Ballonkatheter platziert. Funf Tiere mussten

aufgrund von technischen und chirurgischen Problemen

exkludiert werden.

Ergebnisse. Zum Zeitpunkt des Eintrittes des Hirntodes

zeigten beide Hirntod-Gruppen gleiche intrakranielle Druckni-

veaus, sowie 15 Minuten danach erhohte Herzfrequenzen, und

einen Abfall nach 30 Minuten, 402 � 29bpm (beats per minute)

[BD ex] und 409 � 26bpm [BD grad] (n.s. vs. Control). Wahrend

sich die Werte der Gruppe mit gradueller Hirntodinduktion

stabilisierten und im weiteren Verlauf des Experiments auf dem

Niveau der Kontrollgruppe blieben, konnte in der Gruppe mit

explosiver Hirntodinduktion ein progressiver Abfall der HF-

Werte ab 210 Minuten nach Hirntodeintritt beobachtet

werden, 271 � 44bpm (p<0,05 vs. Control).

Schlussfolgerungen. Die Maus als Versuchstier eroffnet

durch das breite Spektrum an Genotypen und Knock-out-

Varianten ein weites Feld moglicher Fragestellungen. Das

Hirntodmodell an der Maus stellt zwar eine chirurgische

Herausforderung dar, ist aber dennoch standardisiert

durchfuhrbar.


Supplement 231

35Tetrahydrobiopterin but not vitamin Cdonor pre-treatment leads to long termsurvival in a murine graft pancreatitismodel

B. Cardini1, R. Oberhuber1, M. Hermann2, P. Obrist3,P. Hengster1, G. Werner-Felmayer4, E. R. Werner4,R. Margreiter1, G. Brandacher1, M. Maglione1

1Department of Visceral, Transplant and Thoracic Surgery,Center of Operative Medicine, Medical University of Innsbruck,Innsbruck, Austria; 2Department for Islet Cell Transplantation,Medical University of Innsbruck, Innsbruck, Austria;3Institute of Pathology, St. Vinzenz Hospital, Zams, Austria;4Division of Biological Chemistry, Biocenter,Medical University of Innsbruck, Innsbruck, Austria

Background. Ischemia-reperfusion injury (IRI) is a major

cause for graft pancreatitis following pancreas transplantation.

Recent findings showed dramatically reduced IRI early after mu-

rine pancreatic graft reperfusion if donors were pre-treated with

tetrahydrobiopterin, a strong antioxidant and essential cofactor

of nitric oxide synthases. In this study we analyzed if tetrahydro-

biopterin supplementation was also able to prolong recipient

survival, since occurrence of graft pancreatitis in this model

was found to be lethal, and compared its efficacy with the anti-

oxidant vitamin C.

Methods. Male syngenic C57Bl6 (H-2b) mice were used as

size-matched donor and recipient pairs. Murine cervical pancre-

as transplantation was performed with a modified no-touch tech-

nique. To induce pancreatitis grafts were subjected to 16h

prolonged cold ischemia time (CIT) as well as to 45min warm

ischemia time. Different treatment regimens were applied: un-

treated animals (I), tetrahydrobiopterin 50mg/kg i.m. (II) and

vitamin C 350mg/kg i.m. (III), both prior to organ retrieval. Me-

dian survival of the different groups was analyzed. Intravital fluo-

rescence microscopy was used for graft microcirculation analysis

(functional capillary analysis – FCD). Parenchymal damage was

analyzed by histology and by determination of peroxynitrite for-

mation.

Results. Following prolonged CIT only pancreatic grafts

treated with tetrahydrobiopterin displayed markedly higher

values of FCD compared to non-treated animals (p<0.01).

In contrast, vitamin C did not improve microcirculation.

Compared to non-treated animals application of both com-

pounds significantly attenuated peroxynitrite formation

(p<0.05) but reduction of early parenchymal damage in

pancreatic grafts was more pronounced following tetrahydro-

biopterin pre-treatment (p<0.05). Pancreatic graft recipients

treated with tetrahydrobiopterin prior to organ explantation

showed substantially longer survival compared to non-

treated animals (p<0.0001). In contrast, pretreatment of

donor animals with vitamin C did not improve recipient

survival.

Conclusions. Tetrahydrobiopterin attenuates IRI related

graft pancreatitis and significantly improves recipient survival

rate and might therefore be a novel promising agent in preven-

tion of graft pancreatitis.

36Irradiated cultured apoptotic peripheralblood mononuclear cells regenerateinfarcted myocardium

M. Lichtenauer1, K. Hoetzenecker1, W. Dietl6,A. Soleiman2, M. Wolfsberger3, C. Gerner4, S. Hacker1,M. Mildner5, B. K. Podesser6, H. J. Ankersmit1

1Department of Surgery, Medical University of Vienna, Vienna,Austria; 2Pathology, Medical University of Vienna, Vienna, Austria;3Paediatrics, Medical University of Vienna, Vienna, Austria;4Tumor Biology, Medical University of Vienna, Vienna, Austria;5Dermatology, Medical University of Vienna, Vienna, Austria;6Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna,Austria

Background. Acute myocardial infarction (AMI) followed by

cardiac remodeling is a major cause of congestive heart failure

and death. Of clinical relevance are reports that demonstrated

that infusion of apoptotic cells lead to allogeneic hematopoietic

cell engraftment in transplantation models and to a delay of

lethal acute graft-versus-host disease by initiating immunesup-

pressive mechanisms.

Methods. Immunemodulatory function of irradiated apo-

ptotic peripheral blood mononuclear cells (PBMC) was evalu-

ated by mixed-lymphocyte reaction and co-culture assays

using LPS stimulated cells in vitro. Reverse transcription poly-

merase chain reaction and enzyme-linked immunosorbent

assay were utilized to quantify pro-angiogenic Interleukin-8,

Vascular endothelial growth factor and Matrix metalloprotei-

nase-9 in cell culture supernatants obtained from viable and

apoptotic PBMC. Cell suspensions of viable and irradiated

apoptotic PBMC were infused in an experimental rat AMI

model. Immunehistological analysis was performed to detect

inflammatory and pro-angiogenic cells within 72 hours after

myocardial infarction. Cardiac function was analyzed by

echocardiography and determination of infarction size was

conducted by planimetry after six weeks.

Results. Irradiated apoptotic PBMC attenuated immune

reactivity and evidenced secretion of pro-angiogenic Interleu-

kin-8 and Matrix metalloproteinase-9 in vitro. AMI rats that were

infused with irradiated apoptotic PBMC cell suspensions showed

enhanced homing of endothelial progenitor cells within 72 hours

as compared to controls (medium alone, viable PBMC). Echocar-

diography and planimetric analysis showed a significant reduc-

tion of infarction size and improvement of post AMI remodeling

as evidenced by an attenuated loss of ejection fraction (p<0.05

and p<0.001).

Conclusions. These data indicate that infusion of irradiated

apoptotic PBMC suspensions in experimental AMI circumvented

inflammation, caused preferential homing of regenerative EPC

and preserved cardiac function.

37Role of lipocalin 2/lcn2 in liverregeneration

K. Kienzl1, A. R.Moschen2, S. Geiger2, G. Brandacher1, F.Aigner1, R. Margreiter1, H. Tilg2



1Department of Visceral, Transplant and Thoracic Surgery,Center of Operative Medicine, Medical University of Innsbruck,Innsbruck, Austria; 2Department of Internal Medicine II(Gastroenterology and Hepatology), Center of Internal Medicine,Medical University of Innsbruck, Innsbruck, Austria

Background. Small-for-size syndrome is a limiting factor in

living donor and split liver transplantation. Maximization of liver

regeneration represents a promising strategy to overcome the risk

of liver failure due to insufficient liver mass in either the donor or

the split graft recipient. Growing evidence suggests that lipocalin

2 has a role in regenerative processes.

Methods. To establish the role of lipocalin 2 in liver regener-

ation lcn2þ/þ , lcn2þ /– and lcn2–/– mice were subjected to 2/3

partial hepatectomy. Hepatic proliferation was measured by

BrdU and PCNA immunohistochemistry. Hepatic lcn2 expression

was analyzed by qRT-PCR and western blots. Serum levels of

lcn2, Il-6, and TNF-alpha were determined by ELISA.

Results. Hepatic regeneration in lcn2þ/þ mice was

analyzed at 24, 48, 72 and 96h after partial hepatectomy. The

peak of hepatic proliferation as indicated by the number of BrdU-

and PCNA-positive cells was confirmed to be at 48h post surgery.

Analysis of hepatic lcn2 expression showed a 140-fold up-regula-

tion only 24h after liver resection in lcn2þ/þ animals with a

stepwise reduction during the observation period (48h 15.7-fold,

72h 5.5-fold, 96h 5.8-fold). Western blots confirmed significant

lcn2 protein over-expression 24h after partial hepatectomy. Also,

serum lcn2 levels were significantly elevated upon liver resection.

To determine the biological relevance of lcn2 induction on liver

regeneration, hepatocyte proliferation was analyzed in lcn2þ/–and lcn2–/– mice 48h after partial hepatectomy. The number of

BrdU- and PCNA-positive cells did not differ significantly be-

tween the groups. However, lcn2–/– animals exhibited a signifi-

cantly elevated baseline liver regeneration (6.6-fold lcn2–/– vs.

lcn2þ/þ , p<0.05).

Conclusions. Up-regulation of lipocalin 2 after murine par-

tial hepatectomy is striking but without significant impact on

hepatocyte proliferation. Our results imply that lcn2 induction

upon liver resection either constitutes a redundant pathway or

simply displays an epiphenomenon.

38A novel cellular biomarker for predictionof response to therapy of chronicgraft-versus-host disease

Z. Kuzmina1, H. Greinix1, R. Weigl1, S. Eder1,R. Knobler2, U. Körmöczi3, A. Rottal3, Ch. Zielinski1,W. Pickl3

1Department of Internal Medicine I, Bone Marrow Transplantation,Medical University of Vienna, Vienna, Austria; 2Instituteof Immunology, Medical University of Vienna, Vienna, Austria;3Institute of Immunology, Medical University of Vienna,Vienna, Austria

B-lymphocytes are increasingly assigned an important role in

the pathogenesis of auto-and alloimmune diseases including

chronic graft-versus-host disease (cGVHD) where abnormalities

of B-cell homeostasis have been observed in patients with active

disease. Numerous immunosuppressive agents have been used

for therapy of steroid-refractory cGVHD achieving a wide range of

responses. Due to the side effects of long-term immunosuppres-

sion biomarkers for prediction of response would be highly desir-

able. Therefore, we investigated B-lymphocyte subpopulations in

peripheral blood (PB) of 74 patients given different systemic ther-

apies including cyclosporine A (CSA n¼ 24), tacrolimus (n¼ 13),

sirolimus (n¼ 18) and ECP (n¼ 19) for moderate (n¼ 29) or se-

vere (n¼ 45) cGVHD according to the NIH Consensus. cGVHD

manifestations at study entry were skin in 45 patients (61%), oral

mucosa in 50 (68%), eyes in 28 (38%), liver in 22 (30%) and lungs

in 23 (31%). Multiparameter flow cytometry was investigated

for analysis of PB leukocytes after staining for CD19, CD27,

CD21 and surface I g. Overall, 45 patients (61%) responded to

therapy including 20/24 (83%) given CSA, 6/13 (46%) on tacroli-

mus, 7/18 (39%) on sirolimus, and 12/19 (63%) given ECP,

respectively. Prior to start of immunosuppressive therapy non-

responders had significantly (p¼ 0.03) higher proportions of im-

mature CD19þCD21-B-cells with a mean of 19.4% compared

with a mean of 13.3% in responders. Significantly higher propor-

tions of immature CD19þCD21-B-lymphocytes were observed in

non-responders compared to responders to either sirolimus

(mean of 23% vs. 9.3%, p¼ 0.02), tacrolimus (mean of 17.9% vs.

8.6%, p¼ 0.02), or ECP (mean of 22% vs. 13.7%, p¼ 0.04), respec-

tively. After 6 months of immunosuppressive therapy all respond-

ing patients had a significant (p¼ 0.01) decrease of immature

CD19þCD21-B-cells from a mean of 13.3% (range, 1.3–54) prior

to therapy to a mean of 8.2% (range, 1.1–30) 6 months later. In

summary, CD19þCD21-B-lymphocytes could serve as a novel

biomarker for predicting response and measuring disease activity

of cGVHD quantitatively and objectively.

39Die Rolle des Cyclin-abhängigen Kinase(CDK) Inhibitors p21 in der renalenIschämie und Reperfusion

J. M. Huber, A. Tagwerker, D. Heininger, G. Mayer,A. R. Rosenkranz, K. Eller

Universitätsklinik für Innere Medizin IV (Nephrologieund Hypertensiologie), Medizinische Universität Innsbruck,Innsbruck, Österreich

Grundlagen. Cyclin-abhangige Kinase (CDK) Inhibitoren,

wie p21, spielen eine Rolle in Prozessen, die wahrend der renalen

Ischamie und Reperfusion (I/R) von großer Bedeutung sind

(Apoptose, Zell-Zyklus, Inflammation). Mit einer in vivo Trans-

fektions-Methode soll die genaue Funktion von p21 in der Patho-

genese der I/R ermittelt werden.

Methodik. In unserem Mausmodell der renalen I/R induzie-

ren wir eine Ischamie von 30min, welche von einer Reperfusions-

phase von 24 oder 48 Stunden gefolgt ist. Fur die in vivo

Transfektion wurde den Mausen 12 Stunden praoperativ entwe-

der ein Expressionsvektor, welcher die cDNA des humanen p21

(pcDNA3.1–p21) enthalt, oder der Vektor ohne Insert (,,Vehikel‘‘,

pcDNA3.1) als Polyethylenimin-Komplex intravenos verabreicht.

Die Transfektionseffizienz wurde mittels realtime-PCR und Wes-

tern Blot evaluiert. Es wurde das Serum-Kreatinin, die Tubulus-

Nekrose sowie die Th1 und Th2 spezifischenMarker (t-bet, gata-3)

quantifiziert. Fur die Lokalisation und Quantifizierung der

apoptotischen und infiltrierenden Zellen wurden immunhisto-

chemische Farbungen fur Cleaved Caspase 3, CD4, CD8 und

F4/80 herangezogen.

Ergebnisse. Die erhohte Expression von p21 konnte sowohl

auf Transkriptions- als auch auf Translationsebene nachgewiesen


Supplement 231

werden. Mause, in denen p21 hochexprimiert wurde, zeigten eine

signifikante Verringerung des Serum-Kreatinins verglichen mit

den Kontroll-Tieren 24 und 48h nach Induktion der I/R. Dieser

protektive Effekt ist nicht durch eine Inhibition der Inflammation

zu erklaren. Weder die Infiltration von Entzundungszellen noch

die Th1- und Th2-Marker t-bet und gata-3 waren unterschiedlich

reguliert in den p21 transfizierten Tieren. Ebenso war die tubu-

lare Nekrose nicht verandert. Im Gegensatz dazu zeigte sich eine

Verringerung der apoptotischen tubularen Zellen in den p21-

transfizierten Mausen im Vergleich zu den Kontrolltieren. Dies

wurde mittels Immunhistochemie der Cleaved Caspase 3 und

TUNEL-Farbungen nachgewiesen.

Schlussfolgerungen. In der prasentierten Arbeit konnten wir

erfolgreich p21 in murinen Nieren uberexprimieren. Dies fuhrte

zu einer signifikanten Erniedrigung des Serum-Kreatinins nach

Induktion der renalen I/R durch Inhibition von Apoptose in

renalen Tubuluszellen.

40Die Therapie des Transplantates mitBilirubin verbessert den kardialenIschämie-Reperfusionsschaden

F. Bösch1, M. Thomas2, P. Kogler1, A. Kostron1,S. Son1, A. Kaiser1, D. Wiedemann3, J. Troppmair1,R. Margreiter1, R. Öllinger1

1Universitätsklinik für Visceral-, Transplantations- und Thorax-chirurgie, Department Operative Medizin, Medizinische UniversitätInnsbruck, Innsbruck, Österreich; 2Universitätsklinik für Chirurgie,Grosshadern, Medizinische Universität München, München,Deutschland; 3Universitätsklinik für Herzchirurgie, MedizinischeUniversität Innsbruck, Innsbruck, Österreich

Grundlagen. Ziel der Studie war es, die optimale Art der

Administration des Antioxidants Bilirubin zu finden. Es wurde

entweder der Spender oder der Empfanger oder das Transplantat

therapiert. Der Effekt wurde anhand des Aktivierungsgrads der

Mitogen aktivierten Proteinkinasen (MAPK), der Transplantat-

funktion und der Apoptoserate evaluiert.

Methodik. Herzen von C57Bl/6 Mausen wurden isogen

heterotop nach 12 bzw. 18 Stunden kalter Ischamie transplan-

tiert. Die Transplantate wurden vor Reperfusion mit Ringer

Laktat (RL), unkonjugiertem Bilirubin (UCB) [125 mM] oder

konjugiertem in RL gelostem Bilirubin [125 mM] gespult. Wei-

ters wurde dem Spender und/oder dem Empfanger eine Dosis

UCB (17,5mg/kg) iv. verabreicht. 15 Minuten bzw. 12 Stunden

nach Transplantation wurden die Herzen explantiert und

untersucht. Western Blots fur die totale und die phosphory-

lierte Form von MAPKs als auch fur Caspase 9 wurden ange-

fertigt. Serum-Werte von CK-MB wurden 12 Stunden nach

Transplantation bestimmt. Apoptose wurde mittels einer

TUNEL-Farbung und die RNA-Konzentration der Ham Oxyge-

nase-1 mittels RT-PCR quantifiziert, die Herzfunktion durch

Palpation uberwacht.

Ergebnisse. Die Kontrollherzen zeigten nach 15 Minuten

Reperfusion einen signifikanten Anstieg in der Aktivierung der

MAPK und nach 12 Stunden einen Anstieg der Caspase 9 cleava-

ge. Eine dreifache Therapie (Spender, Transplantat, Empfanger)

mit UCB inhibierte die Aktivierung der MAPK signifikant. Dies

konnte auch bei einer alleinigen UCB-Therapie des Transplan-

tates gezeigt werden, jedoch nicht, wenn die konjugierte Form

von Bilirubin verwendet wurde. Die Transplantat-Therapie mit

UCB fuhrte nach 12 Stunden zu einer besseren Transplantatfunk-

tion, erhohter HO-1-Konzentration und einem niedrigeren

Serum CK-MB Level verglichen mit den Kontrollen (p<0,05).

Weiters waren in den therapierten Herzen auch signifikant weni-

ger apoptotische Zellen nachzuweisen und die Caspase 9 cleava-

ge geringer.

Schlussfolgerungen. Die Therapie von Maus-Herztransplan-

taten mit UCB, nicht jedoch mit konjugiertem Bilirubin, bewahrt

vor MAPK-Aktivierung, verbessert das Outcome, reduziert die

Apoptoserate und konnte ein viel versprechender Ansatz sein

den Ischamie-Reperfusionsschaden zu minimieren.

41Creation of a prevascularized sitefor cell transplantation in rats

P. Stiegler1, V. Matzi4, E. Pierer1, O. Hauser5,S. Schaffellner1, H. Renner4, J. Greilberger3, R. Aigner7,A. Maier4, C. Lackner6, F. Iberer1, F. M. Smolle-Jüttner4,V. Stadlbauer2, K. H. Tscheliessnigg1

1Division of Transplantation Surgery, Department of Surgery,Medical University of Graz, Graz, Austria; 2Divisionof Gastroenterology and Hepatology, Department of InternalMedicine, Medical University of Graz, Graz, Austria; 3Instituteof Physiological Chemistry, Centre for Physiological Medicine,Medical University of Graz, Graz, Austria; 4Division ofThoracic Surgery and Hyperbaric Medicine, Departmentof Surgery, Medical University of Graz, Graz, Austria; 5Ziel Bio-pharma Ltd, Ireland; 6Institute of Pathology, MedicalUniversity of Graz, Graz, Austria; 7Division of NuclearMedicine, Department of Radiology, Medical University of Graz,Graz, Austria

Background. After transplantation cells, especially islet

cells are likely to become apoptotic due to local hypoxia lead-

ing to graft dysfunction. Isolated pancreatic islet cells depend

on diffusion of oxygen from the surrounding tissue; therefore

access to sufficient oxygen supply is beneficial, particularly

when microcapsules are used for immunoisolation. The aim

of this study was to create a prevasularized site for cell

transplantation in rats and test its effectiveness with micro-

encapsulated HEK293 cells.

Methods. The combination of implantation of a foam

dressing, vacuum-assisted wound closure (foamþVAC) and

hyperbaric oxygenation (HBO) was used in 40 Sprague-Dawley

rats. Blood flow and VEGF levels were determined. Sodium

cellulose sulphate (SCS) microencapsulated HEK293 cells were

transplanted into the foam dressing in rats pre-treated with HBO

and angiogenesis and apoptosis were assessed.

Results. Vessel ingrowth and VEGF levels increased depend-

ing on the duration of HBO treatment. The area containing the

foam was significantly better perfused in experimental groups as

compared to controls. Only a small amount of apoptosis occurs

in SCS microencapsulated HEK293 cells after transplantation.

Conclusions. As ischemia damaged cells are likely to under-

go cell death or loose functionality due to hypoxia, therefore

leading to graft dysfunction, the combination foamþVAC and

HBO might be a promising method to create a prevascularised

site to achieve better results in cell transplantation. Further

experiments with microencapsulated porcine islet cells will be

carried out to evaluate the positive impact of the combination

foamþVAC and HBO in islet cell transplantation.



42Impact of cold ischemia on chronicrejection and mitochondrial injury aftercardiac transplantation

St. Schneeberger1;2, A. Amberger3, J. Heitzinger3,Th. Hautz1, O. Renz1, P. Obrist4, H. Meusburger1,G. Brandacher1;2, W. Mark1, D. Strobl1, J. Troppmair1,R. Margreiter1, A. V. Kuznetsov1;5

1Daniel Swarovski Research Laboratory, Department of Visceral,Transplant and Thoracic Surgery, Center of Operative Medicine,Medical University of Innsbruck, Innsbruck, Austria;2Division of Plastic Surgery, Department of Surgery, UPMC,Pittsburgh, and Department of Surgery, VA, University Drive,Pittsburgh, PA, USA; 3Tyrolean Cancer Research Institute,Medical University of Innsbruck, Innsbruck, Austria;4Department of Pathology, St. Vinzenz Hospital Zams,Zams, Austria; 5Cardiac Surgery Research Laboratory,Department of Cardiac Surgery, Center of Operative Medicine,Medical University of Innsbruck, Innsbruck, Austria

Chronic rejection (CR) remains an unsolved hurdle for long-

term heart transplant survival. In addition to alloimmune-medi-

ated mechanisms, several non-immunologic risk factors such as

cold ischemia (CI) contribute to the development of CR. The

effects of CI on progression of CR and the mechanisms resulting

in functional deficit were investigated by studying cardiac con-

tractile performance, gene expression, cardiac vasculopathy, mi-

tochondrial respiratory function and activities of several marker

enzymes.

Allogeneic (Lew!F344) and syngeneic (Lew!Lew) rat

heart transplantations were performed with or without 10 h

of CI. After evaluation of myocardial contraction, hearts were

excised at 2, 10, 40 and 60 days for investigation of vasculo-

pathy, gene expression, enzymatic activities and mitochondri-

al respiration.

Gene expression studies identified a gene cluster coding

for subunits of the mitochondrial electron transport chain

regulated in response to CI and CR. Myocardial performance

(cardiac score), mitochondrial function (respiration with

various substrates) and mitochondrial marker enzyme activ-

ities (citrate synthase and respiratory chain complexes I, II

and IV) declined in all allografts with time after transplan-

tation. These declines were more rapid and severe in CI

allografts (CR-CI) and correlated well with progression of

vasculopathy, intimal thickening (smooth muscle cell prolif-

eration) and myocardial fibrosis. General loss of mitochon-

drial functional capacity was found to be involved in the

mechanisms associated with CR, in contrast with more spe-

cific damages of mitochondrial respiratory complexes in CI.

A significant reduction in mitochondrial respiratory capacity

in response to cold ischemia and chronic rejection suggests

an important role of these organelles in the loss of organ

function.

We provide evidence here that mitochondria related gene

expression and mitochondrial function are substantially

compromised with progression of CR and show that CI

significantly impacts on progression, gene profile and

mitochondrial function of CR. Monitoring mitochondrial

respiratory function and enzyme activities provide a way for

earlier and sensitive detection of CR progression and cardiac

allograft dysfunction.

43Wahl des Analyseverfahrens beider Bestimmung sedierenderMedikamente in Serum: Konsequenzenfür die Hirntoddiagnostik

R. W. Schmid, St. Unterweger, J. Ebner

Klinisches Institut für Medizinische und ChemischeLabordiagnostik, Medizinische Universität Wien, Wien,Österreich

Grundlagen. Vor Beginn einer neurologischen Hirntod-

diagnostik bei potentiellen Spenderpatienten muss ein phar-

makologischer Einfluss von sedierenden Medikamenten, wie

Benzodiazepine oder Barbiturate, ausgeschlossen werden. In

einem Kapitel der Empfehlungen zur Durchfuhrungen der

Hirntoddiagnostik des OBIG aus 2005 sind Vorgehensweisen

fur den Nachweis zentral nervos wirkender Substanzen im Blut

diskutiert.

Methodik. Eine schnelle und robuste HPLC Methodik

wurde zur gleichzeitigen empfindlichen Bestimmung

der Benzodiazepine Midazolam und dessen beiden Haupt-

Hydroxy-Metaboliten in Serum Proben (mit LOQ <25ng/ml)

sowie der Barbiturate Pentobarbital, Thiopental und

Methohexital (250ng/ml LOQ) fur routinemaßige Messungen

im Rahmen der Hirntoddiagnostik am Allgemeinen Kranken-

haus in Wien entwickelt. In Ermangelung klarer Regelungen in

Osterreich wurde eine Entscheidungsgrenze von 50ng/ml als

Midazolam-positives Ergebnis gewahlt. Entsprechend der

Empfehlungen des OBIG wurde auch ein modifiziertes

immunologisches Messverfahren (CEDIA) zur Messung von

Benzodiazepinen im Harn fur die Messung von Midazolam in

Serum untersucht.

Ergebnisse. HPLC-Messungen von mehr als 100 Patien-

tenproben der letzten 3 Jahre wurden mittels der CEDIA

Immunoassay-Methodik wiederholt. Dabei konnte eine

betrachtliche Diskrepanz der quantitativen Ergebnisse mittels

HPLC und Immunoassay festgestellt werden, wobei ca. 1/3

der HPLC-negativen Ergebnisse bei dem CEDIA-Assay (teil-

weise weit) uber dem Cut-Off von 50 ng/ml lagen. Diese

Diskrepanz war nicht unerwartet, da festgestellt werden

konnte, dass der Immunoassay nicht nur auf Midazolam

empfindlich reagiert, sondern auch mit den Haupt-Hydroxy-

Metaboliten und den entsprechenden glukuronierten

Metaboliten kreuz-reagiert.

Schlussfolgerungen. 1) Fur die Analytik von Patienten-

proben im Rahmen der Hirntoddiagnostik muss die Spezi-

fitat der Messverfahren festgelegt werden, inwieweit neben

der Ausgangsverbindung Midazolam auch die entsprechen-

den Metaboliten miterfasst werden: Sonst muss, wie in die-

ser Studie gezeigt werden konnte, mit ,,falsch-positiven‘‘

Ergebnissen gerechnet werden, die die neurologische Hirn-

toddiagnostik verzogern oder erschweren konnen. 2) Im

Rahmen der Hirntoddiagnostik sind klare und verbindliche

Richtlinien bezuglich der Entscheidungsgrenzen dringend

erforderlich. 3) Dies gilt auch fur alle zu beobachtenden

Midazolam-Metaboliten, deren (moglichen) Einflusses auf

die neurologische Hirntoddiagnostik genauer definiert wer-

den sollte, wobei, wenn erforderlich, ebenso entsprechende

verbindliche Entscheidungs-Grenzwerte festgelegt werden

mussen.


Supplement 231

44Minimization of oxidative stressin a porcine kidney transplant model

P. Stiegler1, M. Schweiger1, M. Sereinigg1,A. Puntschart1, D. Kniepeiss1, T. Marko2,J. Greilberger3, N. Dandachi4, F. Iberer1,K. H. Tscheliessnigg1

1Division of Transplantation Surgery, Department of Surgery,Medical University of Graz, Graz, Austria; 2Departmentof Anaesthesiology, Medical University of Graz, Graz, Austria;3Clinical Institute of Medical and Chemical Laboratory Diagnostics,Medical University of Graz, Graz, Austria; 4Division of Oncology,Department of Internal Medicine, Medical University of Graz, Graz,Austria

Background. Ischemia reperfusion damage still causes late

graft function or even graft dysfunction and failure in kidney

transplantation. Currently used preservation methods can not

guarantee to totally prevent ischemia reperfusion injury. The

aim of this study was to test a newly developed preservation

solution in a porcine kidney transplant model in terms of organ

quality and function.

Methods. 10 pigs served as multi organ donors and were

explanted according to routine procedures. Organs of 5 animals

were preserved in UW solution, 5 donor organs were preserved

with a new preservation solution based upon alpha glutaric acid

and 5-MMF. 10 pigs served as recipients and bilateral nephrec-

tomy was performed prior to kidney transplantation after 24h of

ischemia time. The recipients survived 7 days and immunosup-

pression was performed using MMF, Tacrolimus and Apredni-

slon. Blood and urinary samples were taken on a daily basis and

after sacrification, the explanted organ was analysed using his-

tology, immunhistochemistry and PCR. ATP levels were moni-

tored during preservation, reperfusion and after sacrification.

Results. All animals survived the whole period of time. Ani-

mals transplanted with organs stored in the new preservation

solution showed less oxidative stress during reperfusion, higher

ATP levels in the explanted liver and creatinine levels normalized

within 3 days after transplantation. Markers of oxidative stress

were significantly higher in organs stored in UW, during preser-

vation and reperfusion as well as after explantation.

Conclusions. It is feasible to reduce oxidative stress in trans-

planted kidneys by storing them in a preservation solution based

on alpha ketoglutaric acid and 5-MMF. However, 5 days after

transplantation, both groups showed similar results in terms of

kidney function. Further investigations will help us to optimize

this preservation method and to understand more clearly pro-

cesses during ischemia and reperfusion injury.

45Donor T cells trigger rejection of donorbone marrow independent of NK cells

K. Hock, N. Pilat, U. Baranyi, Ch. Klaus, M. Gattringer,H. Ramsey, F. Muehlbacher, Th. Wekerle

Division of Transplantation, Department of Surgery, MedicalUniversity of Vienna, Vienna, Austria

Background.Mature donor T cells have pleiotropic effects in

relation to allogeneic BMT, enhancing BM engraftment, mediat-

ing GVL effects and improving protective immunity, but also

causing GVHD. Co-transplantation of high amounts of donor

T cells together with donor BM causes rejection of donor BM

despite costimulation-blockade. The mechanism of this

paradoxical phenomenon remains unclear. In this study we

investigate whether NK cells are essential for mediating BM

rejection and whether a dose of donor T cells can be determined

that has an engraftment-enhancing effect without causing GVHD

or BM rejection.

Methods. Recipients (C57BL/6) were treated with 3Gy TBI

(d-1) and received approximately 20�106 fully mismatched

Balb/c BMC (d0), costimulation-blockers anti-CD154 mAb

(1mg; d0) and CTLA4I g (0.5mg; dþ 2). Mature T cells were

isolated from Balb/c spleen and lymph nodes by MACS

separation and different doses (25�106, 8�106, 4�106) were

administered together with donor BM with or without anti-

NK1.1 (0.5mg; d� 1, 0.25mg; dþ 2, þ4). Multilineage chimerism

was followed by flow cytometry.

Results. Additional treatment with 25�106 mature donor

T cells completely abrogated chimerism (0/3 chimeras) under

co-stimulation-blockade in this otherwise successful protocol

(3/3 chimeras). NK depletion of the recipient was not able to

prevent the detrimental effect of donor T-cell administration

(0/4 chimeras) suggesting a T cell mediated BM rejection. Trans-

plantation of lower amounts of mature T cells (4�106, 8�106)

did not result in BM rejection, but did not have a detectable

chimerism-enhancing effect either (5/5, 4/4 chimeras; mean

B-cell chimerism: 4�106¼ 57%, 8�106¼ 67%, p¼ 0,002 at

13 wks post BMT). The addition of mature T cells did not

significantly increase the chimerism-level.

Conclusions. The abrogation of the BM engraftment

through co-transplantation of donor T-cells is not NK cell-

dependent but seems to be T cell-mediated. Thus, donor

T-cells activate recipient T-cells despite costimulation-blockade,

revealing a potentially important mechanism of costimulation

blockade-resistant rejection. In this particular model the negative

effect of donor T-cells could not be separated from a potential

positive effect.

46Prolongation of rat limb allograft survivalby targeting selectins after weaningsystemic immunosuppression

T. Hautz1, J. Grahammer1, C. Krapf1, B. Zelger2,G. Brandacher1;4, M. P. Schön3, W. P. A. Lee4,R. Margreiter1, S. Schneeberger1;4

1Department of Visceral, Transplant and Thoracic Surgery,Center of Operative Medicine, Medical University of Innsbruck,Innsbruck, Austria; 2Department of Pathology, Medical Universityof Innsbruck, Innsbruck, Austria; 3Department of Dermatology,Georg-August-University-Göttingen, Göttingen, Germany;4Division of Plastic Surgery, University of Pittsburgh MedicalCenter, Pittsburgh, PA, USA

Background. Skin rejection is still the major hurdle to

overcome after composite tissue allotransplantation. Among

a battery of chemokines and immune cells involved in the

mechanism of skin rejection also several adhesion molecules

can be found. We therefore analyzed the expression of E-þP-



selectin in skin biopsies of human hand allografts and the local

effect of efomycine-M, a special inhibitor of selectins, in a rat

limb transplant model.

Methods. 150 skin biopsies from three bilateral hand

transplants were assessed by H&E-histology and immunohis-

tochemistry (anti-E-þP-selectin-antibody). In an orthotopic

rat hind limb allotransplant model (BN-LEW) efomycine-M

was investigated for its local effect on skin rejection. Animals

received either efomycine-M alone (5mg/kg/weekly s.c. into

the graft) or in combination with 50 days of systemic

immunosuppression (ALS 0.5ml on pod 0þ 3 and tacrolimus

0.3mg/kg/day). Efomycine M was continued until pod 100.

Untreated animals and animals receiving ALSþ tacrolimus

alone served as controls. Skin rejection was assessed clinically

(grade 0-IV) and H&E-histology.

Results. Upon rejection, E-and P-Selectin expression in the

vascular endothelium was significantly upregulated and correlat-

ed well with severity of rejection in human hand allografts. In the

experimental trial animals of the control group showed grade III

rejection on pod 61� 1 after weaning systemic tacrolimus on pod

50. Histology displayed necrosis and massive infiltration of lym-

phocytes in all tissues by then. Additional treatment with local

efomycine-M resulted in long term (150 days) allograft survival.

Histology on day 150 showed a lymphocytic infiltrate in the

dermis and a dermal-epidermal interphase reaction consistent

with grade II rejection. However, Efomycine-M treatment alone

had no effect on skin rejection. Allografts showed grade III rejec-

tion by day 9� 1, which was similar to untreated animals.

Conclusions. Selectins are upregulated upon skin rejection

after human hand transplantation. Local administration of a

selectin blocker results in significant prolongation of graft

survival after total withdrawl of systemic immunosuppression,

but doesn’t prevent chronic rejection in a rat limb transplant

model.

47Real time live confocal‘‘biopsychronology’’ – A novel approachfor the study of ischemia-reperfusioninjury

M. Hermann1, M. I. Ashraf2, R. Margreiter1;2,P. Hengster1, J. Troppmair2

1KMT Laboratory, Department of Visceral-, Transplant- andThoracic Surgery, Center of Operative Medicine, MedicalUniversity of Innsbruck, Innsbruck, Austria; 2Daniel SwarovskiResearch Laboratory, Department of Visceral-, Transplant-and Thoracic Surgery, Center of Operative Medicine,Medical University of Innsbruck, Innsbruck, Austria

Ischemia (I) and reperfusion (R) trigger a series of events,

which culminate in severe injuries (IRI) to transplanted

organs. Not only graft failure but also delayed and inferior

graft function constitutes a clinically relevant problem re-

quiring novel therapeutic options. At present, the mechan-

isms underlying the pathophysiological changes associated

with ischemia/reperfusion injury in transplantation are in-

completely understood. Animal studies as well as in vitro

approaches are essential to gain further insights and require

novel technical approaches. The response to IRI is usually

monitored through measurement of kidney function and by

histological examination of the affected tissue. Also the anal-

ysis of the effects of important players in ischemia/reperfu-

sion injury like of reactive oxygen species (ROS) is limited to

the study of marker molecules. The goal of the study pre-

sented here was to device a more informative method for the

investigation of IR, which allows for the study of the molec-

ular mechanisms involved but also for a first testing of novel

therapeutic approaches.

Here we established the conditions for the transient cul-

ture of kidney biopsies for the analysis of cellular changes

associated with ischemia and reperfusion. Kidneys were re-

moved from Wistar rats and biopsies were taken using a

Super-Core Semi-automatic Biopsy Instrument. Biopsy mate-

rial was analysed either immediately after taking the biopsy

or after 24 hours of culture. Transport as well as culture

medium was DMEM/F12. Culture of the biopsy was per-

formed in a rotating manner on an inclined plane in a hu-

midified tissue culture incubator at 37. The biopsies were

analysed applying real time live confocal microscopy after

staining with the following dyes: Tetramethylrhodamine

methyl ester, sytox, propidium iodide and fluorescent wheat

germ agglutinin. In combination they were used to assess

and visualize time-dependent mitochondrial membrane

potentials, total number of cell nuclei, cell death and tis-

sue/cell morphology.

Cell viability, tissue integrity and mitochondrial status

within different localizations of the kidney biopsy can be im-

aged in an unprecedented precision. In addition, staining as

well as analysis of the living tissue biopsies can be performed

in less than 30 minutes. Quantification of cell viability in spe-

cialized structures such as the glomeruli can be performed by

comparing sytox and PI positive nuclei. Our analyses demon-

strated that biopsy quality remained intact over 24 hours,

thereby allowing follow-up analyses.

In summary, we describe a novel method, which may be

generally suitable for the ex vivo assessment of organ

function, not only in the context of transplantation. In

addition, such an approach promises to be also helpful in

the in vitro testing of therapeutic approaches using tissue

biopsy samples.

48Activation of ERK 1/2 and p38 MAPKduring human and rat kidneytransplantation

R. Sucher, M. Biebl, G. Rumberg, P. Kogler,C. Margreiter, C. Bösmüller, W. Mark, R. Margreiter,R. Öllinger

Department of Visceral, Transplant and Thoracic Surgery,Center of Operative Medicine, Medical University of Innsbruck,Innsbruck, Austria

Background. Mitogen activated protein kinases (MAPK) are

crucial in cell survival and death. MAPKs are rapidly activated

upon external stress via phosphorylation by upstream kinases.

Blockade thereof has been shown to modulate ischemia reperfu-

sion injury (IRI). Purpose of the study was to determine whether

the MAPKs ERK 1/2 and p38 are being activated during reperfu-

sion in a rat model of kidney transplantation and whether the

results would correlate to MAPK activation in human kidney

transplantation.


Supplement 231

Methods. Isogenic heterotopic kidney transplantation was

carried out in Lewis rats (n¼ 6) after a 12 h period of cold

ischemia time (CIT). During CIT kidneys were stored in UW

solution at 4C. Rat kidney grafts were collected at the end

of CIT or 15min after reperfusion. Further, core needle

biopsies of 27 consecutive human cadaveric kidney grafts

transplanted at our center (CIT 14 h 22min � 5 h 8min) were

taken at the end of cold ischemia and 15 minutes after

reperfusion in the recipient. Western blot analysis was

carried using antibodies directed against total and phosphor-

ylated ERK 1/2 and p38 MAPK. Western blots were quantified

using ImageJ. Statistical analysis was carried out using

students t-test.

Results. In the rat model, reperfused grafts showed a 7.3 fold

increase in ERK 1/2 phosphorylation and a 3.9 fold increase in

p38 MAPK phosphorylation when compared to the kidneys col-

lected at the end of CIT. Reperfusion in human kidney allografts

led to a 7.2 fold increase in ERK 1/2 phosphorylation but no

significant changes in p38 MAPK phosphorylation were observed

(p¼ 0.62).

Conclusions. Activation of p38 MAPK found during reper-

fusion in rat kidneys was not seen in human kidney grafts. ERK

1/2 activation occurs during reperfusion in human kidney

grafts, similarly as in a rat model of kidney transplantation.

Modulation thereof may be a promising strategy to improve

early graft function.

49Heart and skin graft tolerance throughTreg treatment and rapamycin in a non-cytotoxic murine mixed chimerism model

N. Pilat1, U. Baranyi1, Ch. Klaus1, E. Jaeckel2, F. Wrba3,R. Oberhuber4, G. Brandacher4, F. Muehlbacher1,Th. Wekerle1

1Division of Transplantation, Department of Surgery, MedicalUniversity of Vienna, Vienna, Austria; 2Departmentof Gastroenterology, Hepatology and Endocrinology, HannoverMedical School, Hannover, Germany; 3Institute of ClinicalPathology, Medical University of Vienna, Vienna, Austria;4Daniel Swarovski Research Laboratory (DSL), Department ofVisceral, Transplant and Thoracic Surgery, Center ofOperative Medicine, Medical University of Innsbruck, Innsbruck,Austria

Background. The mixed chimerism approach is a promis-

ing strategy for the induction of transplantation tolerance but

non-cytotoxic bone marrow (BM) transplantation protocols

allowing routine clinical application are still an unmet need.

We explored whether the therapeutic use of regulatory T-cells

(Tregs) leads to permanent engraftment of clinically realistic

doses of allogeneic BM and donor-specific tolerance without

irradiation or cytotoxic drugs. Since natural occurring Tregs

(CD4þCD25þFoxP3þ) are a rather rare population we also

examined the potency of polyclonal FoxP3-transduced Tregs

and TGFbeta-induced Tregs.

Methods. Non-irradiated C57BL/6 recipients received

20�106 fully mismatched Balb/c BM cells (d0) under the

cover of costimulation blockade (anti-CD154 mAb d0,

CTLA4Ig dþ 2) and short-course rapamycin (d� 1/0/þ 2)

with or without 0.5–5�106 Tregs (d0) which were either

transduced to express FoxP3 (FoxP3-Tregs) or in vitro culti-

vated in the presence of TGFbeta (iTregs) to induce suppres-

sor phenotype. Multilineage chimerism was followed by flow-

cytometry. Tolerance was assessed by skin grafts and vascu-

larized heart grafts.

Results. Therapeutic Treg treatment led to the induction of

long-term multilineage chimerism and donor-specific toler-

ance in this otherwise unsuccessful regimen. Treg-treated

recipients showed long term survival of donor-specific skin

(>100 days 7/7 for FoxP3 Tregs, p<0.0002; 5/6 for iTregs

p<0.0043) and heart (>100 days) allografts. Recipients receiv-

ing Tregs without rapamycin uniformly failed to develop chi-

merism and tolerance (8/8 chimeras with rapamycin; 0/8

without rapamycin; p¼ 0.0002). FoxP3-Tregs were shown to

be effective at low doses (3/3 chimeras with 1.5�106, 2/2

chimeras with 0.5�106).

Conclusions. These results demonstrate that polyclonal

Tregs lead to engraftment of conventional doses of BM with

unique potency, obviating the need for cytoreductive recipi-

ent treatment. Lasting mixed chimerism and heart and skin-

graft tolerance are achieved. Rapamycin is an indispensable

part of the regimen, acting in synergy with Tregs. These

findings are an important step towards the development

of non-toxic chimerism-based tolerance protocols for

clinical use.

50Mixed chimerism and tolerance withclinically approved therapeutics –

one step away from clinical application

Ch. Klaus1, N. Pilat1, U. Baranyi1, M. Gattringer1,H. Ramsey1, R. Oberhuber2, G. Brandacher2,F. Muehlbacher1, Th. Wekerle1

1Division of Transplantation, Department of Surgery, MedicalUniversity of Vienna, Vienna, Austria; 2Department of Surgery,Medical University of Innsbruck, Innsbruck, Austria

Background. Establishment of mixed hematopoietic chi-

merism through transplantation of donor bone marrow (BM)

is a potent tolerance strategy. Costimulation blockers as part

of BM transplantation protocols allowed the least toxic rodent

protocols to be developed to date. For more than one decade,

anti-CD40L, a highly effective costimulation blocker, has been

universally used in all non-myeloablative mixed chimerism

protocols. However, due to severe side effects in humans,

anti-CD40L is clinically not available. We thus wanted to de-

velop a protocol employing only clinically approved drugs that

allows mixed chimerism and tolerance without anti-CD40L

treatment.

Methods. Recipient mice (C57BL/6) received mild total

body irradiation (2Gy; d� 1), 20�106 fully mismatched

Balb/c BM cells (d0), CTLA4Ig (Abatacept, Orencia+; 0.5mg,

dþ 2, þ 4), rapamycin (5mg/kg/d; d� 1, 0, þ2) and anti-LFA-1

(clone M17/4; corresponding to the anti-humanLFA drug efa-

lizumab, Raptiva+, 0.5mg, d� 1, þ2). Five mice were grafted

with vascularized hearts (Balb/c donor) 3 weeks post BMT. As

control, 1 mice received a 3rd party graft (C3H). Multi lineage

chimerism and deletion of donor reactive T-cells was followed

by flow cytometry. Recipients were checked daily for donor

heart beating.

Results.Mice treated with this protocol showed stable mixed

chimerism in myeloid and lymphoid lineages (i.e. 43% CD4, 22%



CD8, 58% B cell and 88% myeloid chimerism at 12 wks

post BMT) which persisted until the end of follow-up (>4 months

post-BMT). Donor heart grafts survived more than 100 days (5/5;

histological results pending). A 3rd party heart graft was rejected

18 days after transplantation.

Conclusions.We developed a novel protocol for induction of

mixed chimerism and donor-specific heart graft tolerance

through combined treatment with anti-LFA-1, CTLA4Ig and

rapamycin. Such a tolerance protocol, employing only compo-

nents approved for clinical use, has high potential for clinical

translation.

51Role of Lipocalin-2 in chemotaxis duringischemia and reperfusion injury

F. Aigner, St. Sickinger, H. Maier, H. Schwelberger,N. Vallant, M. Kofler, St. König, R. Öllinger,St. Schneeberger, J. Troppmair, R. Margreiter


Background. Neutrophil Gelatinase-associated Lipocalin

(NGAL/Lcn-2) expression is associated with ischemia/reperfu-

sion injury (IRI) following transplantation and correlates with

polymorphonuclear cell infiltration. To get insight into the regu-

latory role of Lcn-2 we aimed to analyze differences in the ex-

pression of chemokines in wildtype (wt) and Lcn-2–/– hearts and

granulocytes.

Methods. The murine heterotopic heart transplant model

also implying the Lcn-2–/– mouse with C57BL/6 background

was used for the in vivo experiments. Primary granulocytes

from Lcn-2–/– and wt mice underwent hypoxic treatment in

a hypoxia chamber. The mRNA expression of the chemokines

MIP-2, LIX, KC and MCP-1 was analyzed by qPCR in the Lcn-

2–/– and wt setting, respectively. Recombinant Lcn-2 was la-

belled with FITC and uptake into target cells (COS-7, HL-1,

HUVEC, MDCK) was analyzed by confocal fluorescence mi-

croscopy.

Results. Significant lower granulocyte infiltration during IRI

was observed in the Lcn-2–/– setting. Lcn-2 expression peaked at

24h after reperfusion. In the Lcn-2–/– setting MIP-2 and KC were

strongly up-regulated in the early phase after reperfusion (2h),

but down-regulated at later time points (48h). LIX expression

was dramatically upregulated (600-fold) at 12h of re-perfusion.

MCP-1 was only weakly up-regulated (max. 6-fold) peaking at

24h and 48h of reperfusion. In the wt setting MIP-2 mRNA was

up-regulated more than 50-fold at 2h of reperfusion. Up-regula-

tion of LIX, MCP-1 and KC was delayed and induced at 24h after

reperfusion. In primary granulocytes KC, MIP-2 and MCP-1 are

abundantly expressed. LIX mRNA is only weakly expressed in wt

but 5-fold up-regulated in Lcn-2–/– granulocytes, significantly

enhanced by hypoxia.

Conclusions. Our data point to a possible chemotactic role

of Lcn-2 which may also affect the expression of particular

chemokines in infiltrating granulocytes. We suggest that Lcn-

2 has an inhibitory effect on LIX expression in granulocytes.

The magnitude and kinetics of IRI influence Lcn-2 expression

and susceptibility of various cell types in the reperfused myo-

cardium (cardiomyocytes, neutrophils, endothelial cells). Un-

derstanding these regulatory mechanisms will be crucial to

establish treatment strategies for IRI during solid organ trans-

plantation.

52Stable vascular regeneration throughco-application of adult blood-derivedendothelial colony – forming cells (ECFCs)and mesenchymal stromal cells (MSCs)

A. Reinisch1, N. A. Hofmann, A. Obenauf,K. Kashofer, E. Rohde, K. Schallmoser,D. Thaler, M. Fruehwirth, K. Flicker,W. Linkesch, M. Speicher, D. Strunk1Stem Cell Research Unit, Department of Hematology,Medical University of Graz, Graz, Austria; 2Institute of HumanGenetics, Medical University of Graz, Graz, Austria; 3Instituteof Pathology and BloodGroup Serology and TransfusionMedicine,Medical University of Graz, Graz, Austria

ECFCs have recently been described as vascular progeni-

tor cells with robust proliferative potential and vessel-forming

capacity. Vascular integrity depends on endothelial and

pericyte functions. This study was performed to establish con-

ditions for the generation of stable vessels by ECFC/MSC-co-

transplantation.

MSC were propagated as previously described. ECFCs

were isolated with a novel recovery strategy and propagated

under animal protein-free culture conditions with pooled

human platelet lysate (pHPL) replacing fetal bovine serum

(FBS). ECFC long-term proliferation potential was monitored

and phenotype was analyzed by flow-cytometry and

immune- cytochemistry. Functionality was studied during

vascular network assembly in vitro and in human vessel for-

mation in immune-deficient mice in vivo. Genomic stability

was assayed with chromosome G-banding and array-

comparative genomic hybridization (array-CGH). Additionally

we compared telomere-length and telomerase-activity of

ECFCs at different time points of culture with flow-

fluorescence in situ hybridization (Flow-FISH) and telomere

repeat amplification protocol-assay (TRAP).

A mean of four ECFC colonies/mL of peripheral blood

could be recovered. The progeny of these cultures could be

expanded to mean 1.5 � 0.5�108 ECFCs within 11–25 days.

Consecutive analysis confirmed ECFC purity, immune

phenotype and sustained proliferation potential for >30

population doublings with preserved progenitor hierarchy.

Genomic stability was confirmed by karyotyping and array-

CGH. Large-scale expanded ECFCs functioned even after

cryopreservation to form complex vascular networks in vitro

and assembled stable CD31þ/Vimentinþ/von Willebrand

factorþhuman vessels when transplanted with MSC in vivo.

These human vessels were connected to the mouse circula-

tion as indicated by a rich content of Ter119þ murine

erythrocytes.

This demonstrates that functional and stable human vessel

can be generated in vivo as result of ECFC/MSC-cotransplanta-

tion. This procedure represents a promising tool to develop in-

novative experimental, diagnostic and therapeutic strategies. It

should help to set a new standard to study therapeutic applica-

bility and risk profile of vessel-forming ECFC-based investiga-

tional new drugs.


Supplement 231

53Bronchialstenosen nachLungentransplantation sind einsporadisches Problem mit derfortlaufenden Nahttechnik. Erfahrungenbei über 1000 Anastomosen

A. Scheed, L. Hatos-Agyi, M. A. Hoda, B. Moser,C. Aigner, G. Lang, S. Taghavi, B. Zweytick,P. Jaksch, W. Klepetko


Grundlagen. Bronchiale Heilungsprobleme sind eine wich-

tige Komplikation nach Lungentransplantation. In der Literatur

wird eine Inzidenz von bis zu 20% beschrieben. Das Ziel

dieser Studie war, die Inzidenz von Bronchialstenosen bei Ver-

wendung einer fortlaufenden Nahttechnik, an unserem Zentrum

zu analysieren.

Methodik. Alle Lungentransplantationen an unserem

Zentrum zwischen Januar 1999 und Dezember 2009 wurden

retrospektiv analysiert. Re-Transplantationen, kombinierte Herz-

Lungentransplantationen und Patienten, welche die ersten 7

postoperativen Tage nicht uberlebt haben, wurden exkludiert.

Inkludiert wurden: 454 bilaterale und 122 einseitige Lungentrans-

plantationen, respektive 1030 Bronchialanastomosen. Fur alle

Anastomosen wurde eine fortlaufende Nahttechnik benutzt.

Die Kontrolle der Anastomosen erfolgte im Rahmen der

postoperativen Routinebronchoskopien oder aufgrund klinischer

Notwendigkeit.

Ergebnisse. Keine Deshiszenz oder Malazie wurde be-

obachtet. Nach 193 � 167 (mean � SD) Tagen entwickelten 27

Patienten (2,6%) eine interventionsbedurftige Bronchialste-

nose. 9 Patienten wurden mittels endoskopischer Stentim-

plantation, 15 mittels Ballondilatation behandelt und bei 3

Patienten war eine chirurgische Intervention notig. Die mittlere

Nachbeobachtungszeit betrug 1328,8� 829,9 Tage. Es ergaben

sich keine signifikanten Unterschiede hinsichtlich Alter,

Geschlecht, Uberlebenszeit, Ischamiezeit der Spenderlunge, Alter

des Spenders oder in der Entwicklung eines Brochiolitis

Obliterans Syndroms (BOS) zwischen Patienten mit oder ohne

Bronchialstenose.

Schlussfolgerungen. Mit einer fortlaufenden Nahttechnik

konnen exzellente Ergebnisse, sowohl in der Fruh- als auch in

der Spatphase der Bronchialheilung erzielt werden. Die Technik

kann deshalb uneingeschrankt empfohlen werden.

54Elevated urine NGAL (Lipocalin-2) levelsare predictive for ischemia and reperfusioninjury after kidney transplantation

H. Maier, H. Schwelberger, J. Troppmair, R. Öllinger,S. Schneeberger, R. Margreiter, F. Aigner

Daniel-Swarovski-Research Laboratory, Department of Visceral,Transplant and Thoracic Surgery, Center of Operative Medicine,Medical University of Innsbruck, Innsbruck, Austria

Background. Neutrophil Gelatinase-associated Lipocalin

(NGAL/Lcn-2) expression is associated with ischemia/reperfu-

sion injury (IRI) following transplantation and correlates with

polymorphonuclear cell infiltration. It has been found to play a

role in kidney development and tubular regeneration after IRI. In

clinical studies, urine NGAL has been found to be an early pre-

dictor for acute kidney injury. This study focuses on the urine

NGAL expression of patients following kidney transplantation

with regard to short-term clinical outcome.

Methods. Urine of 11 patients following kidney transplanta-

tion was analyzed for NGAL expression at day 0 (preoperative),

and days 1, 2, 3, 4, 5, 10 and 15 after transplantation by ELISA

technology and correlated with standard clinical parameters. For

renal NGAL/Lcn-2 uptake studies the Lcn-2 knockout mouse

was used. Recombinant murine Lcn-2 (200mg/100mL PBS) was

administered intraperitoneally and the kidneys were harvested at

2h after application for immunohistochemical and Western

blot analysis. Urine was analyzed for NGAL expression by

Western Blot.

Results. Urine NGAL expression following kidney transplan-

tation decreased in all 11 patients from mean 141.3 (day 0) to

99.3 (d1), 58.8 (d2), 27.6 (d3), 34.7 (d4), 31.7 (d5), 13.7 (d10) to

11ng/ml at day 15 after transplantation. Three patients with

delayed graft function due to ischemic tubulopathy requiring

dialysis showed an increase in NGAL levels compared to mean

urine levels (2–3 fold). None of the patients developed acute

allograft rejection. Accumulation of Lcn-2 protein in murine

proximal tubule cells in the knockout setting was found at 2h

following intraperitoneal administration of exogenous recombi-

nant Lcn-2 but was absent in the urine.

Conclusions. NGAL is not only a marker for chronic renal

failure. Renal clearance of NGAL might be a useful tool to moni-

tor graft function since urine NGAL levels are increased during

IRI. NGAL/Lcn-2 accumulates in the proximal tubule cells after

exogenous application.

55Size reduced lung transplantation – 9 yearsof experience by a single centre

B. Ghanim, M. Winter, A. Alimohammadi, M. A. Hoda,P. Jaksch, C. Aigner, S. Taghavi, G. Lang, W. Klepetko


Background. Reducing waiting time mortality and expand-

ing the donor pool for patients on the waiting list is still a great

challenge in lung transplant surgery. Size reduced lung trans-

plantation is an opportunity to offer optimal size matching to

especially young patients or adults with small chests. Howev-

er, size reduced lung transplantation is still no standard

procedure.

Methods. We retrospectively reviewed all 162 patients

(m¼ 71, f¼ 91, mean age: 40þ 17a) who underwent size

reduced lung transplantation from January 2000 to January

2009 at our institute. All patients (pts) were analyzed with

regard to postoperative outcome and were compared to

all other pts who underwent standard lung transplantation

(n¼ 434) during the observation period.

Results. During the observation period 162 (27.2%) of all 596

lung recipients underwent size reduced lung transplantation.

Downsizing was achieved by lingula resection n¼ 13 (8%), lobar



resection n¼ 136 (84%) and split lung transplantation n¼ 13

(8%). Within the observation period 49 pts (36%) died (infections:

n¼ 26, graft failure: n¼ 5, bronchiolitits: n¼ 4, others: n¼ 14) in

the size reduced group and 133 pts (31%) in the standard group.

There was no statistically significant survival difference

(Log Rank p¼ 0.259) between the size reduced group (mean

survival 1962 dþ 117 d) and the standard transplantation group

(mean survival 2175þ 72 d).

Conclusions. Size reduced lung transplantation, including

split lung transplantation, lobar transplantation and wedge

resection, is a reliable procedure providing equal survival results

compared to standard lung transplantation.

56Effect of hemodialysis before transplantsurgery on renal allograft function – a pairof randomized controlled trials

�Z. Kikić1, M. Lorenz2, G. Sunder-Plassmann1,M. Schillinger3, H. Regele4, G. Györi5, F. Mühlbacher5,W. C. Winkelmayer6, G. A. Böhmig1

1Department of Medicine III, Medical University of Vienna, Vienna,Austria; 2Department of Medicine, Hospital Barmherzige Brüder,Vienna, Austria; 3Department of Medicine II, Medical Universityof Vienna, Vienna, Austria; 4Institute of Clinical Pathology, MedicalUniversity of Vienna, Vienna, Austria; 5Department of Surgery,Medical University of Vienna, Vienna, Austria; 6Divisionof Pharmacoepidemiology and Pharmacoeconomics and RenalDivision, Brigham andWomen's Hospital, Harvard Medical School,Boston, MA, USA

Background.Hemodialysis immediately before kidney trans-

plant surgery has been suggested to adversely affect early

graft function. On the other hand, considering its profound an-

ti-inflammatory effects, a beneficial impact of regional citrate

anticoagulation on the evolution of graft function can be specu-

lated. We sought to assess the clinical impact of preoperative

hemodialysis and dialysis anticoagulation in two related random-

ized trials.

Methods. Eligible kidney transplant candidates with a

serum potassium �5.0mEq/L were randomized to receive

dialysis or no dialysis prior to deceased donor transplantation.

Patients with a potassium >5.0mEq/L were randomized to

receive dialysis with heparin or citrate anticoagulation. The

primary endpoint was the estimated glomerular filtration rate

(eGFR) at post-transplant day 5.

Results. The first comparison (56 versus 54 patients) revealed

no effect of dialysis on eGFR at day 5 [primary endpoint; 12

(interquartile range: 5–36) versus 13 (5–37) mL/min/1.73m2,

P¼ 0.98], rates of delayed graft function (DGF, 22% versus 27%,

P¼ 0.66), cellular rejection (20% versus 24%, P¼ 0.65) and C4d-

positive dysfunction (2% versus 9%, P¼ 0.11), or 1-year death-

censored graft survival (89% versus 91%, P¼ 0.51). Comparing

citrate with heparin anticoagulation (44 versus 66 patients), no

differences in eGFR at day 5 [17 (8–31) versus 14 (6–38) mL/min/

1.73m2, P¼ 0.57], DGF (21% versus 30%, P¼ 0.28), cellular

rejection (23% versus 33%, P¼ 0.29) and graft survival (90%

versus 88%, P¼ 0.44) were found. For citrate anticoagulation, less

C4d-positive rejection episodes (P¼ 0.08) and higher 1-year eGFR

levels (P¼ 0.03) were observed.

Conclusions. Pre-transplant hemodialysis and anticoagula-

tion may not affect early graft function in a meaningful way.

57Vitamin D deficiency, hyperparathyroidismand bone turnover in patients listed for livertransplantation (LTX): preliminary resultsof a cross-sectional study

D. Wagner1, H. Dobnig2, D. Kniepeiss1, F. Iberer1,K. H. Tscheliessnigg1, T. R. Pieber2,B. M. Obermayer-Pietsch2, M. Trauner3,A. Fahrleitner-Pammer2

1Department of Transplantation Surgery, Medical Universityof Graz, Graz, Austria; 2Endocrinology and Nuclear Medicine,Medical University of Graz, Graz, Austria; 3Department ofGastroenterology and Hepatology, Medical University of Graz,Graz, Austria

Background. Although transplantation bonedisease is a com-

mon complication following liver transplantation (LTX), screening

and preventive measures during the pre-transplant period are not

part of routine patient care. Aim of the current analysis was to

evaluate bone metabolism of liver transplant candidates.

Methods. Currently 60 patients (mean age 57 � 7) with

end stage liver disease (mean MELD 13 � 4) are in evaluation

for LTX and included into the present study. Blood sampling

was performed in the morning following an overnight fast.

Aside of routine parameters 25-hydroxyvitamin D [vitD],

parathyroid hormone [iPTH], bone specific alkaline phospha-

tase [bALP], osteocalcin [OC], tartrate resistant alkaline phos-

phatase 5b [TRAP5b] and serum crosslaps [sCTX] levels were

analyzed.

Results. Mean VitD serum level of the patient cohort was

17.8 � 11ng/ml. Only 14% of the patients had levels within the

normal range (30–65). Nearly two thirds (64.3%) of the patients

had levels below 20ng/ml, indicative of a high prevalence of

vitamin D deficiency. Serum VitD levels were negatively correlat-

ed to iPTH values [r¼ –0.58; p¼ 0.001], and 54% of the patients

had secondary hyperparathyroidism (sHPT). Patients with sHPT

had comparable kidney function and bone formation markers

(OC, bALP) when compared to those without sHPT, however,

had significantly higher sCTX (p¼ 0.01) and TRAP5b (p¼ 0.005)

levels, indicating a negative balance of bone turnover.

Conclusions. Vitamin D deficiency and sHPT are common in

patients with end stage liver disease who would very likely benefit

from vitamin D supplementation. Evaluation of bonemetabolism

and eventually DXA measurements should routinely be per-

formed in patients evaluated for LTX.

58Ungeplante Revisionen nach NTX alsIndikator der Qualitätskontrolle

A. Krause, O. Gangl, N. Roth, J. Huber, U. Fröschl,R. Függer

Chirurgische Abteilung, Krankenhaus der Elisabethinen,Linz, Österreich

Grundlagen. Die ungeplante Revision gilt als Qualitatsindi-

kator in der Chirurgie. Daten fur die NTX liegen nicht vor.


Supplement 231

Methodik. Retrospektive Analyse von 223 NTX zwischen

9/2002 und 5/2009. Zielkriterium: Anzahl der ungeplanten (oper-

ative, endoskopisch oder perkutane) Revisionen, Alter >60,

Mehrfachtransplantationen, Seite und Art (lebend – Leiche) der

Spenderniere wurden als mogliche Risikofaktoren evaluiert.

Ergebnisse. Die Analyse unseres Patientenkollektives bei 223

Nierentransplantationen uber 81 Monaten stellte eine 18,80% ige

Revisionsrate dar, dies entspricht bei 42 Patienten 58 Revisionen.

An 32 Patienten entsprechend 14,30% war eine operative

Revision notwendig. Anzahlsmaßig im Vordergrund standen

dabei Blutungen (1,8%), Hamatomausraumungen (4,9%) und

Nephrektomien (2,7%) wegen Thrombosen. Bei einer Subanalyse

dieser Komplikationen konnte hinsichtlich Patientenalter,

Implantationsseite bzw. Ein- oder Mehrfach-Transplantation

keine erhohte Risikoratifizierung dargestellt werden. Diese opera-

tiven Revisionen traten lediglich bei Leichennieren auf, bei Ver-

wandtenspenden im angefuhrten Zeitraum war keine

Reoperation notwendig. Ebenso war dies auch bei den Nierenve-

nenthrombosen der Fall. Hier konnte auch hinsichtlich einer 2.

oder 3. Transplantation kein erhohtes Risiko gesehen werden. Bei

den endoskopisch interventionellen Revisionen (3,1%) standen

Hydronephrosen bzw. Paravasate, welche im Großteil der Falle

durch retrograde Splintlegungen bzw. perkutanen Nephrosto-

mien therapiert wurden, im Vordergrund. Eine operative Revision

wegen Ureternekrosen war in 2 Fallen notwendig.

Schlussfolgerungen. Die Aufarbeitung der ungeplanten

Revisionen stellt unseres Erachtens einen relativen einfach

erhebbaren und guten Indikator fur das perioperative Qualitats-

management dar. Unsere Daten hinsichtlich der Nierentrans-

plantationen sind dabei mit Ergebnissen anderer chirurgischer

Bereiche durchaus vergleichbar.

59VITA-D: Cholecalciferol substitutionin vitamin D deficient kidney transplantrecipients: a randomized, placebo-controlled study to evaluate thepost-transplant outcome

U. Thiem1, G. Heinze2, R. Segel3, Th. Perkmann4,F. Kainberger5, F. Mühlbacher6, W. Hörl1, K. Borchhardt1

1Division of Nephrology and Dialysis, Department of InternalMedicine III, Medical University of Vienna, Vienna, Austria; 2Core Unitfor Medical Statistics and Informatics, Section of Clinical Biometrics,Medical University of Vienna, Vienna, Austria; 3Hospital Pharmacyof the Vienna General Hospital, Vienna, Austria; 4Departmentof Laboratory Medicine, Medical University of Vienna, Vienna,Austria; 5Department of Diagnostic Radiology, Medical Universityof Vienna, Vienna, Austria; 6Division of Transplantation, Departmentof Surgery, Medical University of Vienna, Vienna, Austria

Background. Vitamin D does not only regulate calcium ho-

meostasis but also plays an important role as an immune mod-

ulator. It influences the immune system through the induction of

immune shifts and regulatory cells resulting in immunologic

tolerance. As such, vitamin D is thought to exert beneficial effects

within the transplant setting, especially in kidney transplant reci-

pients, considering the high prevalence of vitamin D deficiency

in kidney transplant recipients.

Methods. The VITA-D study, a randomized, placebo-con-

trolled, double-blind study with two parallel groups including

a total of 200 kidney transplant recipients, is designed to in-

vestigate the immunomodulatory and renoprotective effects of

cholecalciferol (vitamin D3) within the transplant setting. Kid-

ney transplant recipients found to have vitamin D deficiency

defined as 25-hydroxyvitamin D3 <50 nmol per liter will be

randomly assigned to receive either oral cholecalciferol thera-

py or placebo and will be followed for one year. Cholecalciferol

will be administered at a dose of 6800 International Units daily

over a time period of one year. The objective is to evaluate the

influence of vitamin D3 substitution in vitamin D deficient

kidney transplant recipients on the post-transplant outcome.

As a primary endpoint glomerular filtration rate calculated

with the MDRD formula (modification of diet in renal disease)

one year after kidney transplantation will be evaluated. Inci-

dence of acute rejection episodes, and the number and severi-

ty of infections (analyzed by means of C-reactive protein)

within the first year after transplantation will be monitored

as well. As a secondary endpoint the influence of vitamin D3

on bone mineral density within the first year post-transplant

will be assessed. Three DXA analyses will be performed, one

within the first four weeks post-transplant, one five months

and one twelve months after kidney transplantation.

60Toxocara-associated eosinophilicascites in a pancreas-kidney transplantedpatient; first documented case of zoonosisin Graz

M. Sereinigg1, S. Schaffellner1, P. Stiegler1,T. Valentin2, F. Iberer1, K. H. Tscheliessnigg1

1Division of Transplantation Surgery, Department of Surgery,Medical University of Graz, Graz, Austria; 2Infectiology,Department of Internal Medicine, Medical University of Graz,Graz, Austria

Background. In the 1990’s patients after solid organ trans-

plantation were strictly advised not to keep pets because of the

risk to contract zoonosis. No transmission of an animal disease

occurred in the last 20 years at our centre. Besides some of our

patients are farmers, so we got more liberal.

Case report. A 47 year old patient suffering from Type I

diabetes underwent combined pancreas-kidney transplantation

in 2001. Organ functions were stable (Creatinin 1.72mg/dl, GFR:

45.71ml/min, C-Peptide: 3.7 ng/ml) under immunosuppression

with Tacrolimus, Sirolimus and MMF. In 2008 the patient pre-

sented with massive ascites. Ascites and blood samples showed

eosinophilia. Initial infectiological analysis showed no signs of

parasitic infection. Clinical symptoms were nausea and vomiting.

Gastroscopy, CT-scans and MRI of the abdomen did not show

relevant pathology. The sudden onset ascites could not be

explained. Screening for malignancy, liver and kidney dysfunc-

tion were negative. The ascites proceeded to be therapy resistant.

More than 15 punctures had to be performed during 18 months.

Then serology for toxocara became positive. The result was con-

firmed by a second analysis. Serologic screening for other para-

sites was negative. Our patient was treated with two five-day-

cycles of albendazole 400mg twice a day. After that, the ascites

stabilized and the eosinophilia vanished.

Conclusions. It’s apparent that pet ownership provides an

enormous psychological benefit to people with chronic diseases.

Toxocara are ubiquitary distributed parasites and may infect



humans with a variety of symptoms. Guidelines to avoid zoonotic

diseases should be respected in general. Larvae may survive years

in humans and indication for and duration of treatment remains

unclear in immunosuppressed patients.

61Successful treatment of wound healingdisorder after surgically treated anklefracture in a pancreas kidney transplantedpatient with dual cell stimulation system(Vivostat® system)

E. Jakoby, S. Schaffellner, D. Wagner, D. Kniepeiss,F. Iberer, K. H. Tscheliessnigg


Background. The treatment of slow healing wounds after

transplantation is often crucial due to infections. Stimulation of

the vagal nerve through electrical point stimulation (P-STIM) and

usage of autologous fibrin in the VIVOSTAT-PRF+ system

(Vivolution A/S, Birkeroed, Denmark) have shown promising

results in patient cohorts with wound healing disorders. We pres-

ent the usage of the combined P-STIM+ and VIVOSTAT-PRF+

treatment after an unsuccessful VAC (vacuum assisted closure)

in wound healing disorder in a patient after pancreas kidney

transplantation.

Case report. A 51 year old female Caucasian patient, who

was grafted with a pancreas and kidney 2006, suffered a traumat-

ic tri-malleolar fracture. 21 days after surgical fixation the patient

developed a wound healing disorder located on the right lateral

ankle that was intentionally treated with VAC closure. After 3

months an administered VAC treatment was changed to

IVOSTAT-PRF+ in combination with P-STIM+. Complete wound

closure was observed within 2 month after the treatment change.

The patient did not suffer from any further wound infections or

complications.

Conclusions. The development of chronic wounds is com-

mon especially for transplanted patients. We present a highly

interesting case of a patient under immunosuppression who de-

veloped wound healing disorders due to pre-existent severe

peripheral artery disease. Applied VAC therapy was able to clean

but not to close the wound. To avoid further surgical procedures

an autologous fibrin based closing system combined with vagal

stimulation were used and were able to assist wound closure

within a short period of time.

62De novo bronchiogenic malignanciesfollowing kidney transplantation –

a single center report

M. Maglione, F. Augustin, C. Boesmueller, T. Schmid,R. Margreiter


Background. Chronic immunosuppressive therapy (IS)

implicates a higher risk for cancer development. In Europe, bron-

chial cancer (BC) is associated with the highest mortality rate

compared to other malignancies. Aim of this study was to evalu-

ate incidence and outcome of patients developing BC following

kidney or simultaneous pancreas kidney (SPK) transplantation.

Methods. Patients, who have undergone kidney or SPK trans-

plantation at our institution between 1996 and 2008 and later on

had developed BC, were analized retrospectively.

Results. Among 1641 patients, 10 (0.61%) developed BC. Two

patients had a SPK and 8 a kidney transplant; 2 patients were

female. Median age at transplantation was 60 (range 39–72)

years. Five patients suffered from diabetic nephropathy, 3 from

glomerulonephritis, 2 had nephrosclerosis. Prophylactic IS con-

sisted of calcineurin-based (5 cyclosporine, 5 tacrolimus) triple

drug therapy. Pretransplant chest X-rays were without abnormal

findings, even retrospectively reviewed. Nine patients had a

smoking history. Median interval from transplantation to tumor

diagnosis was 36 (range 7–149) months. In 9 patients (90%),

carcinomas were diagnosed at UICC stage IV (1 Small Cell Lung

Cancer, 8 Non Small Cell Lung Cancers). Six patients were treated

with palliative radio-chemotherapy; 3 patients received symp-

tomatic therapy. The only carcinoma diagnosed at UICC stage

IB was curatively resected; the patient is still alive without recur-

rent disease after a follow-up of 390 days. A second patient with

recent diagnosis is alive with stable disease under polyche-

motherapy (Carboplatin/Taxotere/Cetuximab). All other patients

suffered from progressive disease with a median survival of 7

(range 1–13) months.

Conclusions. The cumulative incidence (0.61%) of BC is

higher when compared to a non immunosuppressed population.

Nearly all tumors were alreadymetastatic at diagnosis. Due to the

poor prognosis, special screening strategies and intensive educa-

tional training of transplanted patients with a smoking history

should be considered.

63De novo malignancies in a cohort of 402liver transplant recipients

H. Bonatti, J. B. Albright, J. Aranda Michel,R. C. Dickson, W. W. Nields, J. Stauffer, R. Hinder,D. Harnois, J. Nguyen

Department of Surgery, Mayo Clinic Jacksonville, Universityof Virginia Health Services, Charlottesville, VA, USA

Background. Advances in liver transplantation (LT) have led

to excellent long-term survival. De novo malignancy is one of the

leading late causes of death in LT recipients. The most common

cancers experienced following LT are skin and post-transplant

lymphoproliferative disease (PTLD). Little is known about the

significance of solid tumors when using newer immunosuppres-

sive regimes.

Methods. From January 1998 to December 2001, 402 patients

underwent 467 LT at our facility. Of these, 85.1% (342/402) sur-

vived longer than one year following transplant.

Results. Of the 342 patients surviving greater than one year,

22.5% developed de novo malignancies during a median follow-

up of 5.9 (range 1.0–8.5) years. A total of 51 patients developed

163 skin cancer lesions with 24 having multiple skin cancers. Six

patients had melanomas (all single lesions), 29 had basal cell

carcinomas (57 lesions), and 34 had squamous cell cancers


Supplement 231

(100 lesions). HCV patients had a lower incidence of skin cancer

than other patients. PTLD developed in seven patients and other

types solid tumors in 26 patients (non-skin squamous cell carci-

noma (SCC) 9 patients, adenocarcinoma (ADC) 16 patients) of

which seven had multiple malignancies. These malignancies in-

cluded lung SCC (3), oral SCC (1), hypopharyngeal SCC (1),

esophageal ADC (1), gastric ADC (1), colorectal ADC (3), anal

SCC (2), pancreatic ADC (2), renal cell carcinoma (1), and bladder

transitional cell carcinoma (1). Of the 127 women, three devel-

oped breast cancer, two cervical, two uterine and two vaginal

cancers; of the 215 men, four had prostate cancer. The mean

time from LT to diagnosis of non-PTLD non skin cancer malig-

nancies was 1380.2� 619.0 days and at diagnosis, distant meta-

static disease was noted in 26.9% (7/26) of patients and nodal

metastases in an additional 7.7% (2/26) of patients. The mortality

rate for these 26 patients was 23.1% (6/26) at a median time of

60.5 days (range 6–1126) following diagnosis of cancer and 1575

(range 563–2364) days post LT. An additional 7.7% (2/26) had

non-fatal progression of disease at last follow up. Patients with

non skin cancer malignancies were significantly more likely to

have alcoholic liver disease as the etiology of liver failure than

those without malignancy (38.5% vs. 18.9%, p¼ .016). Gender,

other liver failure etiology, and age at transplant were not

significant risk factors. Of patients with solid organ malignancies,

42.3% (11/26) had significant smoking histories.

Conclusions. Cancer is one of the most common long term

complications post LT with skin cancer having a very high

incidence and prevalence. Patients with alcoholic liver disease

had an increased risk to develop cancer and smoking must be

considered an important risk factor.

64Liver transplantation for nodularregenerative hyperplasia associatedprimary portal hypertension in a patientwith common variable immunodeficiencysyndrome

A. L. Roman1, C. D. Sifri2, T. Flohr1, K. D. Hagspiel3,T. L. Pruett1, J. C. Iezzoni4, R. G. Sawyer1,H. J. R. Bonatti1

1Department of Surgery, Mayo Clinic Jacksonville,University of Virginia Health Services, Charlottesville, VA, USA;2Division of Infectious Diseases, Department of Internal Medicine,Mayo Clinic Jacksonville, University of Virginia Health Services,Charlottesville, VA, USA; 3Department of Radiology, Mayo ClinicJacksonville, University of Virginia Health Services, Charlottesville,VA, USA; 4Department of Pathology, Mayo Clinic Jacksonville,University of Virginia Health Services, Charlottesville, VA, USA

Background. Common variable immunodeficiency (CVID)

can be associated with various hepatic conditions, the most

common being nodular regenerative hyperplasia (NRH).

These patients are at high risk for a variety of opportunistic

infections.

Methods. We report on the first patient with CVID who

underwent liver transplantation (LT) for non cirrhotic NRH asso-

ciated portal hypertension. A review of the literature with regard

to LT in CVID patients was performed.

Results. Receiving tacrolimus/steroid immunosuppres-

sion, our patient remained rejection free, but developed

CMV disease, disseminated nocardiosis, Pseudomonas pneu-

monia and Clostridium difficile associated colitis. All infec-

tions were all successfully managed and the patient is alive

with a well functioning graft after 18 months, however, liver

biopsy suggests the possibility of recurrent NRH and the pa-

tient was started on a steroid taper. In retrospective review, we

concluded that a more intense infectious prophylaxis and less

intense immunosuppression may have prevented the severe

infectious complications. Thus far ten additional cases of LT in

individuals with CVID have been reported and nine had cir-

rhosis due to HCV infection, which was acquired through

contaminated immune globulin preparations. Rejection and

infection rate was high in this cohort and HCV recurrence

was a common cause of death.

Conclusions. Portal hypertension associated with NRH may

be more commonly observed as an indication for LT in patients

with CVID. Optimal immunosuppression and infection prophy-

laxis need to be developed for this specific patient population.

Long term follow up is necessary to determine if NRH may

relapse post LT.

65Successful liver transplantation usinga liver from a donor with plesiomonasshigelloides sepsis following fresh waterdrowning: case report and reviewof the literature on gram negativeaspiration during drowning and utilizationof organs from bacteraemic donors

H. Bonatti1, C. Sifri2, T. Pruett1, R. Sawyer1

1Division of Transplantation, Department of Surgery, Mayo ClinicJacksonville, University of Virginia Health Services, Charlottesville,VA, USA; 2Division of Infectious Diseases and International Health,Mayo Clinic Jacksonville, University of Virginia Health Services,Charlottesville, VA, USA

Background. Plesiomonas shigelloides is a freshwater, non-

fermentative Gram negative rod, which is associated with spo-

radic and epidemic diarrheal disease and rarely with invasive

infection in humans. Many reported cases have occurred in

immunosuppressed individuals.

Case report. We report on a patient who underwent suc-

cessful liver transplantation (LT) using a graft from a 14 year

old boy who had drowned in a freshwater lake. Perioperative

prophylaxis consisted of piperacillin/tazobactam. One day

post transplant Gram negative bacteria were isolated from

blood cultures in the donor and reported to our center. As

part of our antibiotic rotational protocol, the liver recipient

was started on cefepime, however, on day 2 post LT, we were

informed that the organism was identified as Plesiomonas

shigelloides, which is known to be resistant to third and

fourth generation cephalosporins. The patient was started

on ciprofloxacin for seven days and continuous surveillance

cultures remained negative. After an uneventful course the

patient was discharged on day 10 post LT without any signs

of infection. A review of the literature revealed a single case

of a drowing victim in whom tissue procurement was

attempte. Due to isolation of Plesiomonas shigelloides from

heart valves and blood, the organs were discarded. In fresh-



water drowning victims universally bacteraemia is found on

post mortem and isolated pathogens correspond to speci-

mens from the drowning site. Aeromonas spp. are amongst

the most common pathogens in this scenario, however, many

other bacteria and fungi were isolated in such cases. In terms

of donors suffering from sepsis, in most cases good outcome

without development of graft transmitted infections have

been reported if the donor has been appropriately treated

and adequate perioperative prophylaxis was administered.

Conclusions. This is the first case of a LT using a graft from

a donor who suffered from Plesiomonas shigelloides sepsis

infection. Drowning victims should be considered potentially

infected with rare pathogens and therefore represent extended

criteria donors.

66Posterior reversible encephalopathysyndrome (PRES) after lungtransplantation

L. Hatos-Agyi, A. Scheed, M. Keplinger, P. Jaksch,W. Klepetko


Background. PRES is a clinicoradiological syndrome associ-

ated with immunosuppression, infection and pregnancy. The

clinical presentation is characterised by headache, visual, motor-

ic and mental disturbances, often progressing to convulsion. The

neuroimaging presents a vasogenic edema predominantly in the

white matter of the occipitoparietal region.

Methods. From January 2007 until July 2009 10 cases of PRES

after lung transplantation were diagnosed at our centre. Medical

data (medication, infection parameters at the time of PRES and

outcome) were collected retrospectively.

Results. 10 patients (2 male, 8 female, mean age 21.1� 13.1

years) developed PRES within 64.3� 124 (range 5–395)

days after lung transplantation. The underlying disease

for transplantation was cystic fibrosis (5), bronchiolitis obliterans

(2) and others (3). The overall incidence was 4.38% of all

performed lung transplantations in the same period. Main clini-

cal manifestations were seizures (9), tetraplegia (3), poor vision

(2), disturbances of mental health and consciousness (1) and

intracranial hemorrhage (1). The radiological findings were

distributed in the occipital (8), parietal (3) and frontal (1) regions.

Primary immunosuppression was maintained by combination

of tacrolimus, mycophenolate mofetil and steroids (mean

concentration of tacrolimus at the day of PRES 12.2� 5.2ng/ml).

Bacterial (4) and CMV (1) infection, acute renal insufficiency (4),

arterial hypertonia (3) and acute allograft rejection (2) were

associated with the occurrence of PRES. Eight patients were

switched to cyclosporine A and 7 patients received antiepileptic

treatment. Six patients were free of neurological residue in follow

up (mean 363.5 � 250.3 days). Four patients died during

hospitalisation (of sepsis, GvHD, intracranial hemorrhage

and acute rejection, respectively). Mean survival for them was

96.7 � 78 days.

Conclusions. PRES is a complication after lung transplanta-

tion not to be underestimated. Rapid diagnosis and treatment

(immunosuppression switch, blood pressure regulation and in-

troducing antiepileptics) have important impact on the revers-

ibility and outcome.

67Hypernatriämie und Herztransplantation –

ein Fallbericht

M. Schweiger1, P. Stiegler1, A. Zuckermann2,G. Prenner1, A. Wasler1, M. Sereinigg1,K. H. Tscheliessnigg1

1KlinischeAbteilungfürTransplantationschirurgie,Universitätsklinikfür Chirurgie, Medizinische Universität Graz, Graz, Österreich;2Klinische Abteilung für Herz-Thorax-Chirurgie, Universitätsklinikfür Chirurgie, Medizinische Universität Wien, Wien, Österreich

Grundlagen. Natriumspiegel von uber 155mmol/ml bei

Organspendern fuhren zu schlechter Graftfunktion mit erhohter

Mortalitat. Studien belegen fur Leber-transplantierte Patienten

das bei aggressiver Korrektur der Hypernatriamie unter

155mmol/ml bis zum Entnahmezeitpunkt gute Langzeitergeb-

nisse zu erzielen sind. In Zeiten marginaler Donatoren stellt sich

bei der HTX, bei welcher die initiale Graftfunktion entscheidend

ist, die Frage ob diese Hypothese ebenfalls zutrifft.

Fallbericht. Vier Tage nach einem Verkehrsunfall wird ein

22-jahriger, mannlich Patient mit Subarachnoidalblutung und

offener Gesichtsfraktur nach Versorgung in einem peripheren

Krankenhaus mit Zeichen des Hirntodsyndroms an unser Kli-

nikum geflogen. Nach der Hirntoddiagnostik ergaben USKG

und EKG außer einer Sinustachycardie keine Pathologien.

Die Kreislaufunterstutzung mit 0,11 mg/kg/min Noradrenalin

war ausreichend, weder CPR noch hypotone Phasen wurden

vermerkt. Die Lungen wurden auf Grund von Aspiration und

schlechten 100% Astrup als nicht transplantabel eingestuft.

Eine Contusio cordis wurde vom erstversorgenden Kranken-

haus diagnostiziert, ohne dass an unserer Klinik diese Diag-

nose verifiziert werden konnte. Auffallend war ein

Natriumspiegel von 166mmol/l, welcher trotz adaquater Thera-

pie bis zum nachsten Tag auf 178mmol/l anstieg. Auf Grund

dieses Befundes wurde weitere 24h zugewartet und das Na auf

150mmol/l gesenkt. Dennoch wurde das Herz an unserem Kli-

nikum auf Grund dieser Hypernatriamie und der nicht sicher

ausgeschlossenen Contusio cordis abgelehnt. Es wurde fur einen

HU-Empfanger, der ansonsten wohl keine weitere Alternative

gehabt hatte, akzeptiert. Die Abdominalorgane sowie das Herz

wurden an anderen Zentren erfolgreich transplantiert. Postoper-

ativ war der HTX-Verlauf komplikationslos und der Patient

konnte nach 3 Monate in hausliche Pflege entlassen werden.

Schlussfolgerungen. Trotz passagerer hoher Natriumspiegel,

welche bei Organspendern durchaus haufig zu beobachten sind,

war in diesem Fall eine erfolgreiche HTX komplikationslos

durchfuhrbar und das Zuwarten von 24h zur Therapieoptimie-

rung gerechtfertigt.

68Rauchen vor und/oder nachHerztransplantation

S. Mahr, F. Eskandary, M. Grömmer, A. Aliabadi,D. Dunkler, B. Bunzel, R. Herics, D. Zimpfer,G. Laufer, A. Zuckermann



Supplement 231

Grundlagen. Obwohl Rauchen eine Kontraindikation zur

Herztransplantation darstellt und eine Nikotinkarenz von 6

Monaten Voraussetzung fur eine Listung zur Herztransplan-

tation ist, ist der Ruckfall nach erfolgter Transplantation ein

nicht seltenes Ereignis. Graftvaskulopathie als auch Malig-

nome stellen die zwei haufigsten Todesursachen nach Herz-

transplantation dar und stehen mit Nikotinabusus in

gewissem Zusammenhang. Dies stellt die Notwendigkeit

einer verlasslichen Uberprufung des Raucherverhaltens mit-

tels laborchemischer Untersuchung.

Methodik. Wir haben eine Kohorte von 143 Patienten

mittels Testung von Kotinin im Harn analysiert, die seit

1985 an unserem Zentrum transplantiert wurden. Ein Wert

von uber 500 ng/ml Kotinin im Harn wurde als regelmaßiger

Tabakkonsum definiert. Aus der Pratransplantanamnese

wurde das Raucherverhalten vor der Transplantation

festgestellt.

Ergebnisse. Pratransplant waren 69 Patienten (48,2%)

Nichtraucher, 62 Raucher (43,3%), 12 Patienten (0,08%)

haben einen unbekannten Raucherstatus. 30 Patienten

(20,9%) wurden uber 500 ng/ml getestet, davon waren 22

(73,3%) pratransplant Raucher. 105 Patienten (73,4%) waren

unter 100 ng/ml, davon waren 61 Patienten (58%) pratrans-

plant Nichtraucher. 8 Patienten (0,05%) wiesen Werte

zwischen 100 und 500 auf, davon waren 6 Patienten pratrans-

plant Raucher.

Schlussfolgerungen. Die Ruckfallrate von Patienten mit

Nikotinabusus nach erfolgter Herztransplantation, aber auch

de-novo Raucher zeigen, welch große Bedeutung die Verhinde-

rung dieses Phanomens hat. Insgesamt rauchen nach Transplan-

tation weniger Patienten, jedoch ist diese Schlussfolgerung

aufgrund von potentiellen Fehlangaben der Patienten vorsichtig

anzusehen.

69Imitation einer arrhythmogenenrechtsventrikulären Dysplasie durch einekardiale Sarkoidose

C. M. Steger1, T. Hager2, H. Antretter1, J. Altenberger3,G. Pölzl4, L. Müller1, D. Höfer1

1Universitätsklinik für Herzchirurgie, Department OperativeMedizin, Medizinische Universität Innsbruck, Innsbruck,Österreich; 2Institut für Pathologie, Medizinische UniversitätInnsbruck, Innsbruck, Österreich; 3Universitätsklinik für InnereMedizin II, Landeskrankenhaus Salzburg, Österreich;4Universitätsklinik für Innere Medizin III (Kardiologie), MedizinischeUniversität Innsbruck, Innsbruck, Österreich

Grundlagen. Eine isolierte kardiale Sarkoidose ist außerst

selten und kann sich durch verschiedenste Symptome manifes-

tieren, so auch durch ventrikulare Rhythmusstorungen. Dadurch

kann eine arrhythmogene rechtsventrikulare Dysplasie (ARVD/C)

vorgetauscht werden.

Methodik. Anhand eigener Erfahrungen sowie der vorhan-

denen Literatur wurde die Problematik der Differenzierung

zwischen kardialer Sarkoidose und ARVD/C behandelt.

Ergebnisse. Bisher wurden insgesamt 14 Falle publiziert,

welche das Bild einer ARVD/C boten. Die endgultige Diagnose

einer Sarkoidose wurde bei allen Patienten histologisch gestellt.

2 Patienten wurden erfolgreich transplantiert, bei den anderen

Uberlebenden erfolgte eine antiarrhythmische und immunsup-

pressive Therapie.

Schlussfolgerungen. Eine klinische Differenzierung

zwischen isolierter kardialer Sarkoidose und ARVD/C ist

außerst schwierig. Die Endomyokardbiopsie ist ein wertvolles

Hilfsmittel fur die Unterscheidung, wenn auch aufgrund der

fleckformigen Auspragung der kardialen Sarkoidose falsch

negative Befunde erhoben werden konnen. Wahrend die

gesicherte kardiale Sarkoidose durch Kortikosteroidtherapie

in ihrer Progredienz deutlich verzogert werden kann,

fuhrt die ARVD/C letztlich zur transplantationspflichtigen

Herzinsuffizienz.

70Implantation des CardioWest-Kunstherzens und konsekutiveHerztransplantation beim irreversiblenkardiogenen Schock nach Drug-eluting-stent-Implantation und konsekutiveroperativer Myokardrevaskularisation

N. Reiss, L. Arusoglu, M. Morshuis, J. Gummert

Klinik für Thorax- und Kardiovaskularchirurgie, HerzzentrumNordrhein-Westfalen, Ruhr-Universität Bochum,Bad Oeynhausen, Deutschland

Ein 74-jahriger Patient erhielt bei hochgradiger Stenose der

RCA einen Drug-eluting-stent. Drei Wochen spater wurde er

erneut mit Angina pectoris-Symptomatik aufgenommen. Die

erneut durchgefuhrte Koronarangiographie zeigte eine akute Dis-

sektion der LAD unter Einschluss eines großeren Diagonalastes.

Die rechte Kranzarterie zeigte ein optimales Ergebnis nach Stent-

Implantation.

Der Patient wurde in unsere Klinik transferiert und notfall-

maßig mit einem Sequentialbypass zum LAD und Diagonalast

unter Verwendung der linken Arteria mammaria versorgt. Der

postoperative Verlauf war zunachst vollkommen komplikations-

los. Postoperativ wurde eine Therapie mit Marcumar und zusatz-

lich Clopidogrel eingeleitet.

Am neunten Tag nach dem Eingriff entwickelte der Patient

dann ein Low-output-Syndrom. Die sofort durchgefuhrte Koro-

narangiographie zeigte einen Verschluss der rechten Kranzar-

terie. Eine Wiedereroffnung des okkludierten Gefaßes gelang

nicht. Bei weiterhin trotz maximaler medikamentoser Therapie

bestehendem kardiogenen Schock wurde zunachst eine IABP,

dann im weiteren Verlauf ein femoro-femoraler Bypass implan-

tiert. Es wurde die Indikation zur Implantation eines Ventrikel-

unterstutzungssystems gestellt. Bei der Operation zeigte sich in

weiten Teilen des Myokards eine ausgedehnte Schadigung, so

dass letztendlich ein CardioWest Total Artificial Heart implantiert

wurde. An diesem System erholte sich der Patient von seinem

Multiorganversagen und wurde nach einer Unterstutzungszeit

von 150 Tagen erfolgreich transplantiert.

Der vorliegende Fall verdeutlicht eindringlich die Wich-

tigkeit der Antikoagulation und Antiaggregation bei

Patienten, deren Koronargefaße teilweise durch Stent-Im-

plantation, teilweise operativ versorgt sind. Die Kasuistik

belegt zusatzlich, dass auch Patienten >70 Jahre von der hier

beschriebenen maximalen Therapie mit Implantation eines

Total Artificial Heart und nachfolgender Herztransplantation

profitieren.



71First documented case of Paracoccusyeei infection in a transplanted heart

M. Schweiger, P. Stiegler, M. Scarpatetti, G. Prenner,A. Wasler, K. H. Tscheliessnigg


Background. Cardiac transplantation remains the only

curative therapy apart of supportive mechanical support for

end-stage heart disease due to inflammatory cardiomyopathy.

Detection of pathogenic viruses and bacteria is crucial in order

to avoid reinfection and may be challenging as described in this

case report.

Case report. A 36 year old male patient with inflammatory

cardiomyopathy underwent successful cardiac transplanta-

tion. First eight consecutive endomyocardial biopsies showed

severe infiltrates comparable with bacterial myocarditis

resulting clinically in dyspnea and NYHA stage II-III. PCR

analysis of native myocardial samples revealed infection with

Paracoccus yeei sp. nov and Parvovirus B-19. After adminis-

tration of ciprofloxacin clinical conditions ameliorated and

further biopsy showed a regression of infiltrates in the cardi-

ac specimens. The patient finally was dismissed in a good

state of health.

Conclusions. Resumptive Paracoccus yeei, a gram-

negative bacterial eugenic oxidizers should be included in

diagnostically thoughts in remarkably cases of myocarditis.

Treatment with quinolones resulted in clinical and histologi-

cal improvement.

72Use of posaconazole for prophylaxisof mycosis in lung transplant recipientswith augmented immunosuppression –

single centre experience

Ch. Geltner1, B. Bucher1, C. Lass-Flörl2,L. Ch. Mueller3, H. Bonatti4

1Department of Pulmonology, Hospital Natters, Natters, Austria;2Division of Hygiene and Medical Microbiology,Department of Hygiene, Medical Microbiology and SocialMedicine, Medical University of Innsbruck, Innsbruck, Austria;3Department of Heart Surgery, Center of Operative Medicine,Medical University of Innsbruck, Innsbruck, Austria; 4Departmentof Surgery, Virginia Medical University, Charlottesville, VA, USA

Background. We want to demonstrate the efficacy of posa-

conazole in avoidance of invasive pulmonary mycosis in lung

transplant recipient receiving monoclonal antibody treatment

with alemtuzumab or rituximab.

Methods. Retrospective analyses of all patients receiving

posaconazole as prophylaxis for invasive mycosis. 10 patients

out of 150 consecutive lung transplant were included. The

observation period was 6 months. Patients had standard im-

munosuppression with CyA or Tac, MMF and steroids and

routinely antimicrobial prophylaxis with valganciclovir, Cotri-

moxazol, inhaled amphotericin and cephalosporin 3rd/4th

generation.

Results. 10 patients were included. Seven patients were

treated with alemtuzumab (MabCampath+) for recurrent

steroidresistent acute rejection. Four had treatment either

with alemtuzumab or rituximab (MabThera+) for post trans-

plant lymphoproliferative disease. Rituximab was given in

dose of 375mg/m2 q 21d for PTLD in combination with

CHOP. Antimicrobial prophylaxis was done with posacona-

zole (400mg q 12 h, 8–12 weeks), valganciclovir (450mg q

12 h, 12 weeks), piperacillin/tazobactam (4.5 g q 8–12 h, 5 to

10 days). Immunosuppression was reduced concerning the

therapeutic protocol. Three patients developed colonization

or infection with filamentous fungi. 3 colonization with As-

pergillus fumigatus and coinfection with zygomyces occurred.

3 out of 10 patients developed invasive mycosis during treat-

ment. One case with azole resistant zygomycosis (absidia

corymbifera) and one case with penicillium crysogenum.

Both newly described facultativ pathogenic species. 5 of the

10 patients had no signs of colonization or occurrence of

invasive pulmonary mycosis. Two patients died within the

observation period.

Conclusions. Posaconazole is a potent prophylaxis against

aspergillus infection in states of augmented immunosuppression

in patients after lung transplantation that are treated with mono-

clonal antibodies. Non-aspergillus species as zygomyces and

penicillium are emerging.

73Intracellular ROS production undercellular stress: a comparison of differentdetection methods

A. V. Kuznetsov1, J. Smigelskaite1, M. Hermann2,P. Gehwolf1, C. Doblander1, A. V. Kozlov3, R. Margreiter1,J. Troppmair1

1Daniel Swarovski Research Laboratory, Innsbruck, Austria;2KMT Laboratory, Department of Visceral, Transplant and ThoracicSurgery, Center of Operative Medicine, Medical Universityof Innsbruck, Innsbruck, Austria; 3Ludwig Boltzmann Institutefor Experimental and Clinical Traumatology, AUVA ResearchCenter, Vienna, Austria

Intracellular overproduction of reactive oxygen species

(ROS) and oxidative stress have been proposed as fundamen-

tal mechanisms in ischemia-reperfusion injury (IRI), hypoxia-

reoxygenation (HR) and cancer. Moreover, a moderate ROS

formation was shown to be crucial for important cellular

responses. However, the presence of different cellular sources

for ROS, as well as distinct chemical properties of individual

ROS may produce different outcomes and for this an optimal

strategy for the precise ROS determination in living cells

under various pathological or stressful conditions is required.

The aim of the study therefore was to compare different

methods for ROS detection.

Using various cultured cell models and stressful conditions

(promyeloid IL-3-dependent 32D cells, NIH 3T3 fibroblasts, or

melanoma A375 cells under starvation, and HL-1 cardiomyocytes

during 45–60min hypoxia at 0.5% O2 and after reoxygenation),

we evaluated different fluorescent probes, such as 2,7-dichloro-

dihydro-fluorescein diacetate (DCF-DA), MitoSOXTM and re-

duced MitoTracker Red CM-H2XRos, and different methods


Supplement 231

(spectrofluorophotometry of cell suspensions and confocal im-

aging of confluent cells) for intracellular ROS detection. In addi-

tion, the fluorescent approaches were compared with EPR/ESR

method using various spin-trap probes: PPH (4-phosphono-oxy-

2,2,6,6-tetramethyl-piperidine-N-hydroxyl) or CPH (1-hydroxy-3-

carboxy-pyrrolidine).

Using all methods and probes, we detected markedly

higher ROS production under cell starvation (IL-3 or serum

depletion) or hypoxia, with significant further ROS increase

after reoxygenation. Quantitative spectrofluorometrical anal-

ysis of cells loaded with DCF-DA or MitoSOXTM correlated

well with the results of fluorescent confocal imaging, where

mitochondrial origin of ROS was confirmed by colocalization

with established mitochondria-specific probe MitoTracker+Green. The increase in ROS levels was �3-fold in IL-3 deplet-

ed 32D cells, �3.5-fold in serum deprived NIH cells, and

�2.5-fold in hypoxic HL-1 cells, respectively. Notably, confo-

cal acquisition of DCF-DA green fluorescence can be easily

combined with ROS detection by MitoSOXTM or by reduced

MitoTracker Red. In all cases, antioxidants, such as N-acet-

ylcysteine (NAC) or trolox diminished ROS production to its

initial levels, whereas addition of pro-oxidant, tert-butyl hy-

droperoxide (t-BHP) or hydrogen peroxide (H2O2) rapidly in-

duced high ROS generation. The best probe for mitochondrial

ROS detection was reduced MitoTracker Red CM-H2XRos,

whereas MitoSOXTM was less sensitive and produced less

stable results. DCF-DA was found to be a suitable probe for

both mitochondrial and non-mitochondrial ROS, having,

however, a disadvantage of laser irradiation-induced photo-

oxidation during confocal imaging, in particular under con-

ditions of long acquisition time and high laser power.

74Oxygen sensing as a key factorregulating somatic endothelialprogenitor cell function

N. A. Hofmann1, A. Reinisch1;2, K. Schallmoser1;3,E. Rohde1;3, S. Chatterjee1, R. Birner-Gruenberger4,D. Strunk1;2

1Stem Cell Research Unit, Medical University of Graz, Graz,Austria; 2Department of Hematology, Medical University of Graz,Graz, Austria; 3Clinic of Blood Group Serology and TransfusionMedicine, Medical University of Graz, Graz, Austria;4Centre for Medical Research, Medical University of Graz,Graz, Austria

The integrity of the vasculature is maintained by a tightly

regulated proliferative function of somatic endothelial progenitor

cells (EPCs). Endothelial colony forming cells (ECFCs) have re-

cently been described as prototype blood- or vessel wall-derived

EPCs. Both resident and circulating cells of the vascular system

are exposed to different physiological oxygen levels in the arteries

or veins defined as EUOXIA. We hypothesized that various oxy-

gen levels ranging from HYPEROXIA to EUOXIA and HYPOXIA

differentially regulate the functionality of adult human blood-

derived ECFCs.

Adult human ECFCs were isolated from whole blood and

propagated with pooled human platelet lysate replacing fetal

bovine serum during cell culture. Progenitor cell hierarchy,

long-term proliferation, wound repair, migratory and vasculo-

genic functions were monitored under defined oxygen levels.

Molecular regulation of ECFC responses to different oxygen

levels was assessed by flow-cytometry, immune cytochemistry

and proteomic profiling.

Results revealed a decrease in EPC colony size under HYP-

OXIC (27.4� 7.3mmHg) compared to EUOXIC (41.5� 3.4mmHg)

oxygenation. Cell number reduction was paralleled by a loss of

high proliferative potential (HPP) ECFCs under HYPOXIA.

HYPEROXIC conditioning (139.8 � 2.9mmHg) resulted in a

significant increase in HPP-colonies (60 � 18% of total colonies)

as compared to HYPOXIC (0%) and EUOXIC (9� 6%) oxygen

levels. The absolute colony number per seeded ECFCs remained

independent of oxygenation. Increasing oxygen concentration

resulted in a rise in ECFC proliferation in primary and long term

cultures. With escalating oxygen supply vascular wound repair

was improved in scratch assays in vitro and vascular-like network

formation in Matrigel+ was promoted. Proteomics revealed

several heat shock and antioxidant proteins involved in the

oxygen dependency of ECFCs.

These data demonstrate that varying oxygen levels differ-

entially regulate functions of EPCs. Oxygen sensing mediated

by stress-induced proteins may be a key factor for vascular

homeostasis and repair in situ making it relevant for regener-

ative medicine.

75Treatment of chronic liver failureby hepatocyte transplantation(morphological investigation)

M. Shagidulin, M. Krasheninnikov, I. Iljinsky,N. Mogeiko, V. Sevastjanov, N. Petrova,N. Onishchenko, S. Gaultier

National Research Institute of Transplantology and ArtificialOrgans, Moscow, Russia

Background. Because of the deficiency of donor livers the

working out of an effective way for prolong support liver function

during the pretransplant period at chronic hepatic failure (HF) is

an actual problem.

Methods. Adult Wistar rats were used as donors of hepato-

cytes (H). Isolated H were obtained by a standard procedure with

0.12% collagenase solution. HF was modeled with rat-recipients

by using 0.2–0.5ml of 60% CCl4/100g per weight according to

the scheme. The suspension of H was seeded on matrixes

Sphero+ Gel as 1–2,5�106cells/cm3. H-seeded matrixes (HSM)

were implantated into rat’s livers. Morphology of implanted H

and liver tissue were investigated 90 days later after implantation

of HSM.

Results. At modeling HF liver morphology was character-

ized as: fatty dystrophia and focal necrosis of liver H, dilata-

tion and swelling of sinusoids and central veins, thin

lymphoid-cellular infiltration and porto-portal sclerosis. 90

days after implantation HSM there were found the preserved

structures of liver; the fatty dystrophia was only at single H;

there were inexpressed dilatation of Disse’s spaces and thin

paravasal mononuclear infiltration. Islets of implanted H

were also discovered. Near the central veins H-islets

expressed mononuclear infiltration preferentially on the pe-

riphery of the islets.

Conclusions.Our preliminary studies asserted the survival

of H on matrixes after their transplantation into damaged

livers, the possibility of treatment of chronic HF by HSM



transplantation into livers and also the necessity to continue

these investigations.

We consider that the present data are an important

step toward clinical application of HSM in future as a bridge

to OLT.

76Eine runde Sache: standardisierterTransport von Inselzellen unterRotationsbedingungen

M. Hermann1, M. Wurm2, C. Mittermair1,A. Deutschmann1, A. Draxl1, R. Margreiter1;2,P. Hengster1;2

1KMT-Labor, Department Operative Medizin, Universitätsklinikfür Visceral-, Transplantations- und Thoraxchirurgie, MedizinischeUniversität Innsbruck, Österreich; 2IBAL-Projekt, DepartmentOperative Medizin, Universitätsklinik für Visceral-,Transplantations- und Thoraxchirurgie, Medizinische UniversitätInnsbruck, Österreich

Der Aufbau einer GMP-konformen Inselzellisolationseinheit

ist ein Kosten- und Zeit-intensiver Prozess. Konsequenterweise

haben sich zahlreiche klinische Zentren entschlossen, die Insel-

zellen in einem bereits etablierten Inselzellisolationszentrum iso-

lieren zu lassen. Anschließend werden die frisch isolierten

Inselzellen zu den entsprechenden Zentren transportiert. Ein

solcher Transport kann mehrere Stunden dauern und bedingt

daher einen zusatzlichen Stress fur die Inselzellen. Es stellt sich

daher die Frage nach den bestmoglichen Transportbedingungen,

um so diese Form des Stresses so gering wie moglich zu halten.

Hier stellen wir eine solche Losung vor.

Humane Inselzellen wurden nach der Isolation in einem, im

Rahmen des ,,IBAL-Projektes‘‘ entwickelten rotierenden Zellkul-

tur-Perfusions-Reaktor, fur 24 Stunden simulierten Transportbe-

dingungen ausgesetzt. Als Vergleich dienten Inselzellen, die unter

wie bisher ublichen Transportbedingungen inkubiert wurden

(statisch, in einem 50ml Behalter bei Raumtemperatur). Die

Qualitatskontrolle erfolgte mittels ,,real time live‘‘ konfokaler Ana-

lyse. Als Farbstoffe wurden Tetramethylrhodamin Methyl Ester,

Acetoxymethylester, Propidiumiodid, Annexin-FITC und FITC-

markiertes Weizenkeimlektin verwendet, um mitochondriale

Potentiale, Kalzium, Zelltod, Apoptose und Zellmorphologie dar-

zustellen.

Die verwendeten Zellviabilitatsfarbungen lieferten nach

24 Stunden eindeutige Resultate: Alle untersuchten Zellviabi-

litatsparameter zeigten signifikant bessere Werte bei den

unter Rotationsbedingungen ,,simuliert transportierten‘‘

Inselzellen an. Wahrend die unter statischen ,,standard‘‘-

Bedingungen inkubierten humanen Inselzellen deutliche

Anzeichen von Zell-Stress bzw. Zelltod aufwiesen, war die

Zellviabilitat der unter Rotationsbedingungen inkubierten

Zellen vergleichbar mit den Ausgangswerten vor dem simu-

lierten Transport.

Die hier vorgestellte Transportmethode fur Inselzellen

unter Rotatonsbedingungen zeigt eindeutige Vorteile gegen-

uber den bisher ublichen Transportmodalitaten auf. Abgese-

hen von der wesentlich besseren Zellviabilitat erlaubt auch

die aktive Kontrolle vitaler Parameter wie etwa Sauerstoff

oder Temperatur eine Standardisierung des Inselzelltrans-

portes, was bei den bisher ublichen Transportmethoden nicht

der Fall ist.

77Intracellular signaling pathways astargets for the prevention of ischemia/reperfusion-induced damage during solidorgan transplantation

M. I. Ashraf1, R. Sucher1, J. Smigelskaite1, M. Haller1,M. Hermann2, Ph. Gehwolf1, M. Maglione1,A. Kuznetsov1, R. Oberhuber1, Th. Ratschiller1,A. Kozlov3, G. Brandacher1, St. Schneeberger1,R. Margreiter1

1Daniel Swarovski Research Laboratory, Innsbruck, Austria; 2KMTLaboratory, DepartmentofVisceral, Transplant andThoracicSurgery,Center of Operative Medicine, Medical University of Innsbruck,Innsbruck, Austria; 3Ludwig Boltzmann Institute for Experimentaland Clinical Traumatology, AUVA Research Center, Vienna, Austria

The formation ofmitochondrial reactive oxygen species (ROS)

during early periods of reperfusion, linked to perturbation of mi-

tochondrial Ca2þ homeostasis is essential for the development of

ischemia (I)/reperfusion (R)-associated tissue injury (IRI). Our

work aims at devising strategies to interfere with these mitochon-

drial alterations before damage amplification or cell death occur in

order to decrease or prevent IRI. We have demonstrated recently

the activation of several canonical intracellular signalling path-

ways very early during reperfusion. We are particularly interested

to establish a link between these signalling activities and mito-

chondrial changes. Experimental setups studied included animal

models for heart and kidney transplantation as well as several in

vitro systems. Expression of transgenes, small molecular weight

inhibitors and conditional knockdown using siRNA were used to

modulate intracellular signalling pathways. Alteration in mito-

chondrial ROS and Ca2þ levels were monitored by confocal imag-

ing following loading of cells with MitoSOXTM Red or Rhod-2,

respectively. In vivo ROS measurements were carried by using

electron paramagnetic resonance (EPR) and the spin trap CP-H.

IR as well as hypoxia/reoxygenation (HR) resulted in pertur-

bation of mitochondrial ROS and Ca2þ homeostasis, which was,

where tested, essential for cell death. Key regulators of cell sur-

vival prevented apoptosis by maintaining permissive levels of

mitochondrial ROS and Ca2þ while the activation of signalling

proteins preferentially involved in cell death induction (e.g. p38),

resulted in a further deterioration. Cell death is preceded by a

ROS-dependent increase in mitochondrial Ca2þ, which was ab-

sent in protected cells. Taken together our data suggest that

modulation of signalling activities occurring during IR may pro-

vide a feasible approach to control the extent of IRI.

78Heightened extracellular levels of calciumand magnesium induce secretionof chemokines and anti-inflammatorycytokines

S. Nickl, K. Hoetzenecker, A. Mangold, B. Moser,M. Zimmermann, S. Hacker, T. Niederpold,A. Mitterbauer, H. J. Ankersmit, M. Lichtenauer


Supplement 231

Department of Cardiac and Thoracic Surgery, Medical University ofVienna, Vienna, Austria

Background. Heightened levels of extracellular ions, mainly

calcium, have been associated with the initiation of an inflam-

matory cascade and cellular chemotaxis. In the present study we

evaluated pro- and anti-inflammatory cytokine and chemokine

secretion of peripheral blood mononuclear cells (PBMC) incu-

bated in medium containing different concentrations of extracel-

lular calcium and magnesium. Furthermore, solutions of

phosphate buffered saline containing different concentrations

of calcium and magnesium are commonly used as an additive

for cell culture and in cell preparation protocols and might influ-

ence the cellular microenvironment and secretion of cytokines.

Methods. Human PBMC were incubated in culture medium

with different concentrations of calcium and magnesium. Cells in

medium alone or in suspensions supplemented with ETDA plas-

ma served as controls. Supernatants were analyzed, amongst

others, for Interleukin-1beta, Interleukin-1 receptor antagonist,

Interleukin-4, Interleukin-10, Transforming growth factor beta,

Interleukin-8, Epithelial cell-derived neutrophil-activating pro-

tein-78, Growth related oncogene-alpha, Monocyte chemoattrac-

tant protein-1 and Regulated upon Activation, Normal T-cell

Expressed, and Secreted (RANTES).

Results. A dose dependent increase of Interleukin-1 receptor

antagonist, Interleukin-8 and Monocyte chemoattractant pro-

tein-1 was found when PBMC were cultured in medium supple-

mented with increasing concentrations of calcium and

magnesium. Cells incubated in cultures supplemented with

EDTA exhibited a total abrogation of cytokine and chemokine

secretion (p<0.01).

Conclusions. The release of the anti-inflammatory cytokine

Interleukin-1 receptor antagonist in addition to unchanged levels

of pro-inflammatory mediators suggests an important modulato-

ry mechanism of heightened extracellular calcium levels. Addi-

tionally, these data suggest that mononuclear cells respond to

altered extracellular ion levels by releasing chemokines subse-

quently leading to the local accumulation of immune cells.

79Resveratrol und Ginseng-Extraktverbessern die Organfunktion ineinem Rattennieren-Ischämie-Reperfusionsmodell

P. Gehwolf, F. Bösch, A. Kostron, K. Stromberger,F. M. Struller, F. Aigner, S. Sickinger, R. Sucher,R. Margreiter, R. Öllinger

Universitätsklinik für Visceral-, Transplantations- und Thoraxchir-urgie, D. Swarovski Forschungslabor, Department OperativeMedizin, Medizinische Universität Innsbruck, Innsbruck, Österreich

Grundlagen. Die induzierbare Hamoxygenase-1 (HO-1)

katalysiert die Oxidation von Ham zu Biliverdin, CO und freiem

Eisen. Eine systemische Induktion der HO-1 wirkt antiinflamm-

atorisch sowohl auf das spezifische als auch das unspezifische

Immunsystem. Eine klinische Anwendung ist derzeit noch nicht

moglich, da die bis dato im Tiermodell verwendeten HO-1-

Induktoren aufgrund ihrer Hepatotoxizitat am Menschen nicht

angewendet werden durfen. Resveratrol und Ginseng-Extrakt

sind naturlich vorkommende Substanzen und Bestandteil von

Nahrungsmitteln. In vitro fuhrt deren Applikation zu einer Akti-

vierung der HO-1. Hypothese dieser Studie ist, dass Resveratrol

und Ginseng-Extrakt in vivo mittels HO-1-Induktion Ischamie-

Reperfusionsschaden mildern.

Methodik. Lewis-Ratten wurden unilateral nephrektomiert,

die kontralaterale Nierenarterie wurde fur 45min geklemmt und

das Organ anschließend reperfundiert. H2O (Kontrolle), Resverat-

rol oder Ginseng-Extrakt (jeweils 100mg/kg) wurden 24h vor

Ischamie und unmittelbar nach Reperfusion oral appliziert

(n¼ 6). Kreatinin und Harnstoff der Tiere wurden 0, 24, 48 und

72h nach Reperfusion mittels Enzym-Assays bestimmt. Die HO-

1-Expression in peripheren Monozyten und in den Nieren wurde

mittels quantitativer PCR und Western Blot bestimmt.

Ergebnisse. Sowohl Resveratrol als auch Ginseng-Extrakt

fuhrten zu einer vermehrten Expression von HO-1 in peripheren

Monozyten, nicht jedoch in den Nieren selbst. In der H2O-Kon-

trollgruppe kam es zu einem Anstieg von Kreatinin und Harnstoff

mit einem Maximum am 2. Tag nach Reperfusion (Kreatinin

3, 1 � 1,0mg/dl, Harnstoff 360,7 � 94,2mg/dl). Resveratrol ver-

besserte die Nierenfunktion signifikant (Kreatinin 1,46� 0,7mg/dl;

Harnstoff 199,6 � 75,5mg/dl, p<0,01 vs. Kontrolle). Noch deut-

licher wurde die Organfunktion bei Behandlung der Ratten mit

Ginseng-Extrakt verbessert (Kreatinin 0,83� 0,1mg/dl; Harnstoff

135,5 � 31,6mg/dl, p<0,001 vs. control).

Schlussfolgerungen. Ginseng-Extrakt und Resveratrol sind

vielversprechende naturliche Substanzen, die zur Verminderung

des Ischamie-Reperfusionsschadens Anwendung finden konnten.

Es ist wahrscheinlich, das Resveratrol und Ginseng-Extrakt nicht

am Organ selbst, sondern durch HO-1-Induktion im unspezi-

fischen Immunsystem ihren Effekt erzielen.

80Genexpression von A20 und HO-1in humanen Nierenbiopsien währendder kalten Ischämiezeit als Prognosefaktorfür die initiale Transplantatfunktion

L. Lohkamp1, G. Kern2, C. Koppelstätter2, G. Mayer2,M. Rudnicky2, M. Haller1, F. Bösch1, P. Kogler1, W.Mark1,J. Troppmair1, R. Margreiter1, R. Öllinger1

1Universitätsklinik für Visceral-, Transplantations- und Thoraxchir-urgie, Department Operative Medizin, Medizinische UniversitätInnsbruck, Innsbruck, Österreich; 2Universitätsklinik für InnereMedizin IV (Nephrologie und Hypertensiologie), Department InnereMedizin, Medizinische Universität Innsbruck, Innsbruck, Österreich

Grundlagen. Die verzogerte Transplantatfunktion ist eine

haufige Folge des Ischamie-Reperfusionsschadens wahrend der

Transplantation und verbunden mit einem verminderten Lang-

zeituberleben. A20 ist ein durch TNF-� induzierbares Gen, das

als Teil einer negativen Ruckkopplungsschleife die Aktivierung

des Transkriptionsfaktors NF-kB hemmt. HO-1 katalysiert den

geschwindigkeitsbestimmenden Schritt im Abbau von Ham zu

Biliverdin, Kohlenmonoxid und freiem Eisen. Beide werden

basierend auf experimentellen Studien als ,,protektive‘‘ Gene

betrachtet, deren Expression infolge eines Organschadens erfolgt

und deren Induktion das Organ schutzt. Ziel der Studie war es,

den Zusammenhang zwischen der Expression von A20 und HO-1

in humanen Spendernieren vor Reperfusion/wahrend der kalten

Ischamiezeit und einer verzogerten Transplantatfunktion zu

untersuchen.



Methodik. Es wurden 57 Nullbiopsien von humanen Spen-

dernieren am Ende der kalten Ischamiezeit entnommen und

darin mittels RT-PCR die mRNA-Konzentrationen von A20 und

HO-1 quantifiziert. Die Dauer der kalten Ischamiezeit in Stunden

und die Anzahl der notwendigen postoperativen Dialysen seitens

des Empfangers als Referenz fur die initiale Organfunktion wur-

den mit der Genexpression korreliert.

Ergebnisse. Beide Gene konnten in den Nieren nachgewie-

sen werden. Die HO-1-Expression korrelierte nicht mit der KIZ

(r-Wert 0,0173). Im Gegensatz dazu stieg die m-RNA-Konzentra-

tion von A20 linear in Relation zur Dauer der KIZ (r-Wert 0,2276).

In Korrelation zu den nach Transplantation notwendigen

Hamodialysen zeigte die HO-1-Expression keine Signifikanz

(r-Wert 0,017). Bei Patienten, die eine oder mehrere Hamodialy-

sen nach Transplantation benotigten, fanden sich deutlich

erhohte mRNA-Mengen von A20 (r-Wert 0,51).

Schlussfolgerungen. A20, nicht jedoch HO-1-Expression

korreliert mit der Dauer der kalten Ischamiezeit und der initialen

Organfunktion in humanen Nierentransplantaten. A20 ist somit

ein Pradiktor der Organfunktion nach Nierentransplantation, die

Modulation der A20 Expression konnte zur Verbesserung des

Ischamie-Reperfusionsschadens beitragen.

81Tacrolimus-induzierte pulmonaleHypertonie nach Nierentransplantation

F. Knoll, C. Mayr, E. Zitt, K. Lhotta

Abteilung für Nephrologie und Dialyse, LandeskrankenhausFeldkirch, Österreich

Wir berichten uber einen 54-jahrigen Patienten, der zwei

Wochen nach Nierentransplantation einen akuten Anstieg des

pulmonal-arteriellen Drucks mit Rechtsherzdekompensation

mit massiven peripheren Odemen entwickelte. Nach Absetzen

von Tacrolimus und Beginn mit Rapamycin nahm die pulmonale

Hypertension schrittweise ab, und die Odeme bildeten sich zu-

ruck. Drei Monate spater waren keine Zeichen fur eine pulmonale

Hypertonie mehr nachweisbar. Allerdings stieg der arterielle Blut-

druck deutlich an, und der Patient benotigte wieder eine antihy-

pertensive Mehrfachtherapie wie vor Transplantation. Wir

vermuten, dass Tacrolimus eine Vasokonstriktion der pulmona-

len Arteriolen induzierte. Ein gleichzeitig bestehendes Lungen-

emphysem konnte einen Vulnerabilitatsfaktor darstellen, der zu

der bisher nicht beschriebenen Nebenwirkung von Tacrolimus

auf die pulmonalen Gefaße beitrug.

82Immunologisches Immunesuppressiva-Monitoring auf dem ARCHITECT-Analyzermittels halb- und vollautomatischerProbenvorbereitung

St. Schmid, B. Gruber, R. W. Schmid

Department für Medizinische und Chemische Labordiagnostik,Medizinische Universität Wien, Wien, Österreich

Grundlagen. Die neuen immunologischen Chemoluminis-

zenz-Assays zur Bestimmung von Tacrolimus, Sirolimus und

Ciclosporin in Vollblut auf der ARCHITECT-Plattform zeigen eine

Reihe von Vorteilen zu bisherigen Immunoassays. Sie bringen

aber auch gleichzeitig den Nachteil mit sich, dass zur Vorberei-

tung der Vollblutproben manuelle Arbeitsschritte erforderlich

sind, die nicht nur den Arbeitsaufwand erhohen, sondern auch

die Gefahr von Probenverwechslung mit sich bringen.

Methodik. Es wurde ein neues Probenvorbereitungs-Rack-

system entwickelt, mit dem die Primar-Vollblutproben logisch

mit den Sekundargefaßen verknupft sind. Dieses Racksystem

erlaubt nicht nur das Schutteln der Proben gemeinsam, sondern

auch die Zentrifugation, ohne dass Proben einzeln gehandhabt

werden mussen. Im Falle des Sirolimus-Assays konnen Proben in

dem Racksystem gemeinsam thermisch inkubiert werden. Paral-

ell dazu wurden alle diese manuellen Arbeitsschritte auf einer

automatischen Pipettierstation integriert: Kernpunkt dabei ist das

automatisierte Schutteln der Vollblutproben und auch der gefall-

ten Proben durch eine spezielle Rotations-Einheit oder uber eine

automatisierte Schuttelstation. Dadurch wird das Sedimentieren

der Vollblutproben vermieden, und es erfolgt eine sofortige

Durchmischung der gefallten Proben.

Ergebnisse und Schlussfolgerungen. Mit dem neu konzi-

pierten Racksystem kann gezeigt werden, dass eine Probenver-

wechslung sehr unwahrscheinlich wird, auch bei sehr großen

Probenzahlen und bei paralleler Abarbeitung mehrerer Assays,

wobei die einzelnen Sekundarrohrchen nicht mehr einzeln be-

schriftet werden mussen. Die arbeitsintensiven Probenvorberei-

tungs-Schritte wie das Vortexen, Inkubieren und Zentrifugieren

sind wesentlich einfacher und schneller durchzufuhren. Durch

Integration dieses Racksystems in eine automatisierte Pipettier-

station wird der manuelle Arbeitsaufwand weiters reduziert bei

gleichzeitiger Erhohung der Prazision der Immunoassays der drei

Immunesuppressiva.

83Falsch positive Tacrolimus-Werteim Blut bei einem Patienten nachNierentransplantation bei Messungenmit dem ACMIA-Immunoassay

A. Winter1, R. Schmid1, G. Sunder-Plassmann2

1Department für Medizinische und Chemische Labordiagnostik,Medizinische Universität Wien, Österreich; 2Universitätsklinikfür Innere Medizin III, Medizinische Universität Wien, Wien,Österreich

Das routinemaßige Monitoring von Immunsuppressiva-

Medikamenten ist eine Voraussetzung fur die optimale klinische

Therapie von organtransplantierten Patienten geworden. Wir

berichten von einem Fall von systematisch falsch hohen Tacro-

limus-Werten im Vollblut eines Patienten, die mittels ACMIA-

Immunoassay routinemaßig erhoben wurden.

Bei dem Patienten mit HIV-Co-Therapie wurden regelmaßig

unveranderte Tacrolimus-Werte im Bereich von 10ng/ml erho-

ben, obwohl die Tacrolimus-Dosis bereits bis auf nur mehr

2�wochentlich reduziert worden war. Vergleichsmessung mit

dem Referenzmessverfahren LC-MS ergaben tatsachliche Blut-

werte von <2ng/ml. Messungen von der Pharmakokinetik uber

einen Dosiszeitraum von 12h zeigten einen nur geringgradigen

Anstieg der Peak-Werte auf 2,8ng/ml. Vergleichsmessungen mit

einem zweiten immunologischen Verfahren der Fa. Abbott, bei


Supplement 231

dem ein Probenvorbereitungsschritt vorgeschaltet ist, ergaben

idente Tacrolimus-Konzentrationenwiemittels LC-MS.AufGrund

dieser Tatsachen wurde im Weiteren bei diesem Patienten die

Tacrolimus-Dosis nur mehr auf Basis von LC-MS-Messungen

uberpruft.

Im vergangenen Jahr wurde in der Literatur wiederholt von

falsch hohen Tacrolimus-Spiegel bei Transplant-Patienten be-

richtet, die mittels des homogenen ACMIA-Assays auf der DIMEN-

SION-Analyzer-Plattform erhoben worden waren. Der Grund fur

diese falschen Messwerte konnte bisher nicht festgestellt werden,

denn wie bei diesem berichteten Fall wurden bei keinem dieser

Patienten signifikant erhohte Parameter im Blut festgestellt.

Schlussfolgerungen. Klinisch unplausibel hohe Tacrolimus-

Werte nach ACMIA-Tacrolimus-Messungen sollten immer mit

einem Referenzmessverfahren uberpruft werden.

84Alemtuzumab as first line treatmentof T-cell posttransplantlymphoproliferative disease

Ch. Geltner1, B. Bucher1, A. Gschwendtner2,H. Bonatti3, L. Ch. Mueller4, M. Freund5

1Department of Pulmonology, Hospital Natters, Natters, Austria;2Department of Pathology, Klinikum Coburg, Coburg, Germany;3Department of Surgery, University of Virginia, Charlottesville,VA, USA; 4Department of Heart Surgery, Center of OperativeMedicine, Medical University of Innsbruck, Innsbruck, Austria;5Department of Diagnostic Radiology, Medical Universityof Innsbruck, Innsbruck, Austria

Background. Efficacy of first line treatment with alemtuzu-

mab in monoklonal T-cell PTLD.

Methods. Case presentation.

Results. Patient’s history: The patient (male, 5 years) was

transplanted for emphysema and alpha-1-antitrypsin deficiency

(ZZ) in January 2008 (left single lung transplant). The patient had

primary graft failure (PGF 3) and pulmonary venous stenosis with

necessity of surgical redo of the pulmonary venous/atrial anasto-

mosis. His initial graft function was and remained reduced to

60% pred. FEV1 at time of dismission from hospital. 3 months

later he had pulmonary embolism and bronchial aspergillosis

that was successfully treated with voriconazole. No episode of

rejection or CMV reactivation/infection was detected (CMV sta-

tuts D–/Rþ). He received standard immunsuppression with

daclizumab, cyclosporine A, MMF, steroids and antibiotic pro-

phylactic regimen. Epicrisis: 6 months after transplant he pre-

sented with a progressive pulmonary nodule. Histology was

obtained through perthoracic CT-guided needle biopsy and

revealed polyclonal T-cell PTLD. Exact immunohistochemistry

staining showed monoclonal gamma-delta T-cell receptor differ-

entiation, CD20, CD52 and EBV markers were negative. There

were no B-cell markers and clonal B-cell-proliferation found.

Previous studies and reports showed alemtuzumab being used

in treatment of T-cell lymphatic leucemia and refractory allograft

rejection. We decided to treat the patient with four courses of

alemtuzumab (d1 3mg, d2 10mg, d3 30mg; q 3 wks). Valganci-

clovir (450mg q 12h, 3 months), posaconazole (400mg q 12h, 3

months) and tazobactam (4.5g q 8h, 14 days) was given as

prophylaxis. Immunosuppression was reduced to cyclosporin

(C0 80–100ng/ml) and prednison 5mg. The tumor size de-

creased within six weeks and stood in remission without signs

of recurrence. The patient’s course was complicated with inva-

sive mycosis and bacterial pneumonia without recurrence of

lymphoma.

Conclusions. Alemtuzumab is a new tool in treatment of

post transplant lymphoproliferative disease (PTLD) with mono-

clonal T-cell proliferation.

85A low or high body-mass index is notpredictive for outcome following allogeneichematopoietic stemm cell transplantation

J. Auberger, J. Clausen, B. Kircher, D. Nachbaur

Department of Internal Medicine V, Hematology and Oncology,Center of Internal Medicine, Medical University Innsbruck,Innsbruck, Austria

Background. Recently it was hypothesized that a low (<20)

body-mass index (BMI) is significantly correlated with an in-

creased transplant-related mortality, decreased survival and

relapse-free survival after allogeneic SCT (K Le Blanc,

Haematologica 2003;88:1044).

Methods. 208 patients receiving a first allogeneic transplant

were studied. Underlying diagnoses were acute myeloid leukemia

(AML) (n¼ 71), acute lymphoblastic leukemia (ALL) (n¼ 41),

lymphoma (n¼ 11), and other diseases (n¼ 75). Median patient

age at time of transplant was 45 (range, 18–76) years. 108 patients

were grafted from an HLA-identical sibling donor and 110

patients received grafts from volunteer unrelated donors. Condi-

tioning was myeloablative in 60 and of reduced intensity in the

remaining 148 patients.

Results. Overall survival for the entire cohort was 34%

(24–45%, 95% Confidence Interval, CI). There was a trend for a

poorer outcome in patients with <25% and >75% percentile BMI

(i.e. BMI � 21 and�27) (OS 48% vs. 34%, p¼ 0.1 log rank test)

due to a higher non-relapse mortality in this patient cohort (37%

vs. 30%). These differenceswere observed inboth, themyeloablative

as well as reduced intensity transplant cohorts. The BMI had no

influence on relapse incidence in either patient cohort.

Conclusions. By dividing patients into percentiles BMI had

no significant impact on outcome and non relapse mortality

neither following myeloablative nor following reduced intensity

allogeneic stem cell transplantation.

86Spontaneous pneumomediastinum,pneumothorax and subcutaneousemphysema complicating bronchiolitisobliterans syndrome after bone marrowtransplantation (BMT) in a pediatric patientwith AML

G. Kropshofer, R. Crazzolara, A. Klein-Franke,B. Meister

Department of Children and Adolescent Medicine,Medical University of Innsbruck, Innsbruck, Austria



Background. Bronchiolitis obliterans syndrome (BOS) is a

form of obstructive airway disease and is considered a manifes-

tation of chronic graft versus host disease (cGvHD). Here we

describe a boy who developed pneumomediastinum, subcutane-

ous emphysema and pneumothorax secondary to BOS. Simulta-

neously he suffered from systemic nocardiosis ensured by blood

culture.

Methods and results. A meanwhile 17-year old boy under-

went allogeneic BMT from his HLA-identical mother for re-

lapsed acute myeloblastic leukemia (AML). The intensified

conditioning regimen consisted of b usulfan, cyclophospha-

mid and melphalan. GvHD prophylaxis started with cyclo-

sporine monotherapy. His post transplant course was

complicated by a severe septicemia resulting in acute respi-

ratory distress syndrome and lung bleeding. Simultaneously

he developed skin and liver GvHD and two weeks later he

also developed gastrointestinal GvHD. Initial response to

steroids was good. Six months after transplantation he

showed up with increasing dyspnea and productive cough.

CT scan showed mucous plugging and infiltrates resembling

bronchiolitis organizing pneumonia (BOOP). Pulmonary

function test was consistent with a mixed restrictive/obstruc-

tive airway dysfunction. He was treated with increasing doses

of steroids, antibiotics and CyA, and then switched to myco-

phenolat because of incompliance with CyA intake. Showing

signs of recovery he was dismissed in good clinical condition.

Two weeks later physical examination showed a distinctive

area of crepitus involving the anterior chest and neck due to

subcutaneous emphysema and moderate dyspnoe. The CT

scan demonstrated a right ventral pneumothorax, a pneumo-

mediastinum, a dense pulmonal infiltration and milk glass

like changes. The patient received high-flow oxygen and an-

tibiotic therapy for systemic nocardiosis, diagnosed by blood

culture and radiographic findings. Within ten days the sub-

cutaneous emphysema resolved also did his nocardiosis un-

der long term antibiotic treatment.

Conclusions. Pulmonary air-leak syndromes represent a rare

complication in patients with cGvHD related BOS. Infectious

complications like pulmonary mycosis, nocardiosis and cytome-

galy should be considered in the differential diagnosis.

87Cidofovir treatment of cmv diseaserefractory to ganciclovir treatmentin solid organ recipients

H. Bonatti1;3, C. Larcher2, St. Schneeberger3,W. Mark3, C. D. Sifri4, R. Margreiter3, Ch. Geltner5

1Department of Surgery, Mayo Clinic Jacksonville,University of Virginia Health Services, Charlottesville, VA, USA;2Department of Hygiene, Microbiology and Social Medicine,Medical University of Innsbruck, Innsbruck, Austria; 3Departmentof Visceral, Transplant and Thoracic Surgery, Medical UniversityInnsbruck, Innsbruck, Austria; 4Division of Infectious Diseasesand International Health, Department of Medicine, Mayo ClinicJacksonville, University of Virginia Health Services, Charlottesville,VA, USA; 5Department of Pulmonology, Hospital Natters, Austria

Background. Solid organ transplantation (SOT) is frequently

complicated by viral infections. Cidofovir (CDV) is active against

most herpesviruses including ganciclovir (GCV) resistant

mutants.

Methods. Seven men and two women (median age of 50.1

years) including 2kidney/pancreas, 4 lung, 1 small bowel and

2 hand recipients received CDV for CMV disease.

Results. All patients received CMV positive grafts (three CMV

mismatch transplants). Five patients received antithymocyte-

globulin, four daclizumab induction, seven experienced rejection

(5 multiple episodes); one suffered from common variable

immunodeficiency. Six presented with other infections (five in-

vasive fungal, four bacterial infections). Eight patients received

prophylactic GCV, eight were pretreated for CMV infection/dis-

ease (GCV: 8, CMV-hyperimmune-globulin: 3, foscarnet: 3). Indi-

cations for CDV included UL97 mutation (n¼ 2), GCV-induced

neutropenia/ongoing CMV disease (n¼ 4), clinical resistance to

GCV (n¼ 3). Seven patients cleared CMV, two had a partial re-

sponse, four experienced relapsed CMV requiring GCV (n¼ 2),

repeat CDV (n¼ 1), CMV-hyperimmune-globulin (n¼ 1). Four

patients had mild nephrotoxicity, three developed renal failure

(all in association with other factors). No patient died from CMV

disease, but one from invasive aspergillosis and another from

nocardiosis.

Conclusions. CDV was useful as a second line treatment of

CMV disease after SOT that did not respond to GCV.

88Invasive pulmonary mycosis dueto Penicillium crysogenum – a newinvasive pathogen

Ch. Geltner1, B. Bucher1, A. Gschwendtner2,C. Lass-Flörl3, H. Bonatti4, L. Ch. Mueller5

1Department of Pulmonology, Hospital Natters, Natters, Austria;2Department of Pathology, Klinikum Coburg, Coburg, Germany;3Division of Hygiene and Medical Microbiology, Departmentof Hygiene, Medical Microbiology and Social Medicine, MedicalUniversity of Innsbruck, Innsbruck, Austria; 4Departmentof Surgery, University of Virginia, Charlottesville, VA, USA;5Department of Heart Surgery, Center of Operative Medicine,Medical University of Innsbruck, Innsbruck, Austria

Background. We present a case of invasive pulmonary my-

cosis (Penicillium crysogenum) in a lung transplant recipient.

Methods. Case presentation.

Results. Previous history: The patient (male, 56 years) had a

left single lung transplant for alpha-1-antitrypsin deficiency (phe-

notype ZZ) with severe lung emphysema in January 2008. His

early postoperative period was complicated with a pulmonary

venous stenosis and primary graft failure. Later on he developed

pulmonary embolism and 6 months later a pulmonary nodule in

his left upper lobe. Histologic specimen revealed posttransplant

lymphoproliterative disease (polymorphic T-cell lymphoma,

CD20 neg, CD52 neg, EBV neg). Further treatment was alentuzu-

mab, valganciclovir, posaconazole as antimycotic prophylaxis

and reduction of immunosuppression. Epicrisis after 2 months

the patient developed pulmonary infiltrates and nodules mainly

in left lower lobe. The patient was still on posaconazole. Trans-

bronchial biopsy and BAL revealed mycosis and a treatment with

voriconazole and caspofungin was started. After further evalua-

tion of the pathogen (suspected zygomycosis) liposomal ampho-

tericin was added. Infiltrates and all over condition of the patient

deteriorated. Microbiological analyses showed Penicillium cryso-

genum as main pathogen with invitro resistency (according to

EUCAST methodology): amphotericin B 16g/ml; voriconazole


Supplement 231

0.025 g/ml; caspofungin 0.019 g/ml; posaconazole 0.025g/ml.

Further treatment with posaconazole and caspofungin without

clinical benefit. The patient deteriorated and died with signs of

invasive mycosis and multiorgan failure.

Conclusions. Penicillium crysogenum is a new pathogen

with potency of invasive pulmonary mycosis in immunocompro-

mized host and unclear therapeutic possibilities.

89Das transplantierte okkulte Melanom –

ein Fallbericht

M. Hofmann1, J. Marschalek1, G. Györi1, M. Pones1,G. Jomrich1, J. Kleinert2, G. Berlakovich1,F. Mühlbacher1, R. Steininger1

1Klinische Abteilung für Transplantation, Universitätsklinikfür Chirurgie, Medizinische Universität Wien, Wien, Österreich;2Klinische Abteilung für Nephrologie und Dialyse, Universitätsklinikfür Innere Medizin, Medizinische Universität Wien,Wien, Österreich

Grundlagen. Etwa 0,2% aller Tumore bei Organempfangern

sind allogen- das metastasierte allogene Melanom macht etwa

30% dieser Tumore aus.

Fallbericht. Eine 56-jahrige Europaerin war die Organspen-

derin (HLA-A: 1, 10, 26, HLA-B: 5, 18, 51, HLA-Bw: 4, 6, HLA-DR:

1, 10). Sie verstarb an einer intrazerebralen Blutung. Die Anam-

nese zeigte keine Auffalligkeiten, die Untersuchungen waren

unauffallig, sodass beide Lungen, die Leber, die Nieren, die Cor-

neae und die Herzklappen zur Transplantation entnommen und

uber Eurotransplant alloziert wurden, auf eine Autopsie wurde

verzichtet. Der Empfanger beider Lungen verstarb kurz nach der

Transplantation an Herz-Kreislaufversagen.

Die Leber wurde gesplittet. Der erste Empfanger verstarb

kurz nach der Transplantation an chirurgischen Komplikatio-

nen. Der Empfanger der zweiten Split-Leber wurde 6 Monate

nach der Transplantation im reduzierten Allgemeinzustand wie-

der stationar aufgenommen – im Zuge der Abklarung fanden

sich multiple Leberherde, die biopsiert wurden. Die histolo-

gische Aufarbeitung zeigte das Vorliegen eines metastasierten

malignen Melanoms. Der Patient verstarb kurz nach der Auf-

nahme im Multiorganversagen.

Der erste Nierenempfanger wurde zeitgleich mit generalisier-

ter Lymphadenopathie stationar aufgenommen – die Histologie

zeigte ebenso das Vorliegen eines metastasierten Melanoms. Der

Patient verstarb kurz danach an den Folgen der generalisierten

Metastasierung.

Der zweite Nierenempfanger wurde bei volliger Beschwer-

defreiheit zur Kontrolle einberufen – in den Untersuchungen

fanden sich multiple kleine Herde in der transplantierten Niere

sowie hepatal als auch intrazerebral. Die Immunsuppression

wurde sofort beendet und die zerebrale Lasion mittels Gam-

ma-Messer entfernt, eine weiterfuhrende Therapie wird derzeit

noch evaluiert.

Schlussfolgerungen. Trotz genauer Evaluation der Spender

lasst sich das Vorliegen einer malignen Erkrankung nicht voll-

standig ausschließen – obwohl die apparative und laborche-

mische Diagnostik in den letzten Jahrzehnten immer genauer

geworden ist. Es wurde jedoch die pathologische Diagnostik

immer mehr in den Hintergrund gedrangt. Da intrazerebrale

Melanome oftmals zu Blutungen fuhren konnen, scheint die For-

derung einer Autopsie nach der Organspende mit entsprechen-

der histologischer Aufarbeitung unumganglich.

90Konsequenz einer Midazolamtherapiefür die Diagnose des Hirntodes beimpotentiellen Organspender – eineretrospektive Single-Center-Analyse

J. Marschalek1, G. Jomrich1, A. Schober2,M. Hofmann1, M. Pones1, I. Kristo1, R. Schmid3,S. Rasoul-Rockenschaub1, G. Berlakovich1,F. Mühlbacher1, R. Steininger1

1Klinische Abteilung für Transplantation, Universitätsklinikfür Chirurgie, Medizinische Universität Wien, Wien, Österreich;2Universitätsklinik für Notfallmedizin, Medizinische UniversitätWien, Wien, Österreich; 3Klinisches Institut für Medizinischeund Chemische Labordiagnostik, Medizinische Universität Wien,Wien, Österreich

Grundlagen. Da sowohl die klinisch-neurologische Untersu-

chung als auch das EEG durch eine Midazolam- bzw. Barbitu-

rattherapie beeintrachtigt werden konnen, sehen die

Empfehlungen des Obersten Sanitatsrates zur Durchfuhrung

der Hirntoddiagnostik (HTD) beim potentiellen Organspender

eine Spiegelbestimmung dieser Substanzen aus dem Serum,

Plasma oder Vollblut vor. Ziel dieser Studie war es, eine mogliche

Konsequenz der Midazolamtherapie bei einem potentiellen

Organspender hinsichtlich der Dauer bis zur Diagnose des Hirn-

todes zu identifizieren.

Methodik. 69 Patienten, bei denen in den Jahren 2006 bis

2009 im Allgemeinen Krankenhaus Wien der Hirntod diagnosti-

ziert wurde, wurden retrospektiv einer von zwei Gruppen

zugeordnet: der Kontroll-Gruppe (CON: keine oder einmalige

Midazolamgabe als Bolus) und der Midazolam-Gruppe (MID:

mehrmaliger Bolus oder kontinuierliche Midazolamapplikation).

Analysiert wurde unter anderem die Zeitspanne zwischen dem

vollstandigen Absetzen der Sedierung und der Diagnose des

Hirntodes. Spiegelbestimmungen wurden bei anamnestischem

Hinweis oder Verdacht auf Midazolamapplikation mittels High-

Performance-Liquid-Chromatography durchgefuhrt.

Ergebnisse. Von 69 Patienten erhielten 34,8% eine

kontinuierliche Infusion oder mehr als einen Bolus Midazolam

(n¼ 24). 65,2% konnten der Kontroll-Gruppe (n¼ 45) zugeordnet

werden. Das durchschnittliche Alter in der MID-Gruppe betrug

40,4 Jahre (SD¼ 13,63) versus 48,6 Jahren (SD¼ 18,65) in der

CON-Gruppe (p¼ 0,042). Hinsichtlich des Body-Mass-Index

(BMI), der Kreatinin-Clearance, der PTZ sowie der Cholinesterase

unterschieden sich die Gruppen nicht. BMI: MID 24,9kg/m2

(SD¼ 3,92), CON 25,2kg/m2 (SD¼ 4,48); durchschnittliche

errechnete Kreatinin-Clearance: MID 114,5ml/min (SD¼ 43,10),

CON 100,4ml/min (SD¼ 50,61); PTZ: MID 92,7% (SD¼ 26,76),

CON 80,7% (SD¼ 30,45); CHE: MID 4,6 kU/l (SD¼ 1,80), CON

5,3 kU/l (SD¼ 2,32). Die mittlere Dauer vom Zeitpunkt des

Absetzens der Sedierung bis zur Diagnose des Hirntodes betrug

bei der Midazolam-Gruppe 3,02 (SD¼ 2,28) und bei der Kontroll-

Gruppe 1,37 Tage (SD¼ 0,69) (p¼ 0,002).

Schlussfolgerungen. Es konnte gezeigt werden, dass nach

wiederholter oder kontinuierlicher Midazolamtherapie beim

potentiellen Organspender die Diagnose des Hirntodes im

Durchschnitt um 1,65 Tage verzogert wird.



Documents

23rd Meeting of the Austrian Society of Transplantation