22nd Meeting of the Austrian Society of Transplantation, Transfusion and Genetics. Zell am See, October 29–31, 2008. Guest Editor: Rainer Oberbauer, Linz, Austria

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22nd Meeting of the Austrian Society of Transplantation, Transfusion and Genetics Zell am See, October 29–31, 2008 Guest Editor: Rainer Oberbauer, Linz, Austria

01 The role of Lipocalin-2 as an inﬂammatory marker in acute allograft rejection? F. Aigner1, H. Maier1, P. Obrist2 , H. Schwelberger1, S. Sickinger1, J. Troppmair1, R. Margreiter1 1 Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria; 2 Department of Pathology, St. Vinzenz Hospital Zams, Tyrol, Austria

Background. Lipocalin-2 (Lcn-2) is associated with ischemia=reperfusion injury (IRI) in different organs. Data on Lcn-2 expression during allograft rejection have been missing so far. The main focus of this study was to analyze the possible implication of Lcn-2 during acute rejection following solid organ transplantation. Methods. We investigated the expression of Lcn-2 in two different settings: (1) Hearts from male inbred C3H mice were transplanted into C57BL=6 wildtype and Lcn-2= recipients. Hearts were recovered at different time points (0 min, 2, 4, 6 and 8 d) and Lcn-2 expression was analyzed by conventional histology, Western blot and immunohistochemistry. (2) Serum of 40 patients undergoing orthotopic liver transplantation was collected preoperatively and postoperatively from day 1 to 15. Lcn-2 was analyzed by ELISA and expression levels were correlated with parameters of allograft rejection. Results. (1) Lcn-2 expression already increased in early stages of acute rejection (2 d) and significantly correlated with the amount of infiltrating polymorphonuclear cells. Coincidental expression of migration molecules like MIP2 or MMP-9 with Lcn-2 upregulation was observed. (2) Serum levels of NGAL following liver transplantation were elevated 3 to 7-fold immediately after transplantation due to IRI and also increased prior to clinically Eur Surg Vol. 40 Supplement Nr. 226 2008

apparent acute rejection closely related to elevated laboratory parameters. Conclusions. Our data suggest a chemoattractant function of Lcn-2 in the allograft due to infiltrating immune competent cells. Lcn-2 is an inflammatory marker upregulated during acute graft rejection and its expression prior to clinical parameters of acute rejection might help to identify possible targets for therapeutic intervention.

02 Incidence and outcome of CMV infection in the newest cardiac transplant era A. Aliabadi1 , C. Bauer2, H. Kerschner2, M. Groemmer1, S. Mahr1, D. Zimpfer1, A. Zuckermann1 1 Division of Cardio-Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria; 2 Division of Clinical Virology, Medical University of Vienna, Vienna, Austria

Background. Cytomegalovirus (CMV) is a significant cause of morbidity and mortality in heart transplantation. However new diagnostic tools (CMV-PCR) as well as new treatment options (valganciclovir) have been adopted in clinical practice. The aim of this analysis was to evaluate the incidence of CMV infection and disease in the newest era between 2002 and 2008. Methods. We studied 201 heart transplant recipients who received quadruple-immunosuppressive therapy. The study population was categorized into 4 groups according to donor and recipient CMV serology at the time of transplantation (D=R, D=Rþ, Dþ=R, Dþ=Rþ) and was monitored by quantitative CMV-PCR blood tests. CMV infection was defined as increase of

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CMV-PCR results 1000 cps=ml. CMV disease was defined as CMV infection with clinical symptoms. All patients received CMVIG and CMV-high risk patients (Dþ=R) received prophylaxis with valganciclovir for three months. The incidence of CMV infection and CMV disease was analyzed among the four groups. Results. During the first year after transplantation, CMV infections and disease developed in 61 (30.3%) and 9 (4.5%) patients respectively. However, CMV disease rate was very low (9 cases, 4.5%). There was no death due to CMV infection. Comparison among the groups showed significantly different incidence of CMV infection (D= R: 2.3%, D=Rþ: 32.5%, Dþ=R: 23.5%, Dþ=Rþ: 47.5%, p < 0.05) and CMV disease (D=R: 0%, D=Rþ: 2.3%, Dþ=R: 11.7%, Dþ=Rþ: 5%, p< 0.05). Average time to CMV infection was longer in the Dþ=R: 22.4 14.8 weeks vs 6.7 12.2 weeks in the other groups. In the Dþ=R group, CMV infection occurred after the end of CMV prophylaxis. In all cases of CMV infection, PCR levels decreased after start of therapy. There were no ganciclovir resistant CMV infections. Conclusions. In the era of new CMV diagnostic and therapeutic options, CMV infection is diagnosed earlier and therapy can be started before CMV disease occurs. This approach can reduce morbidity and mortality due to CMV infection.

03 100 years of successful corneal transplantation. A review of the literature N. Ardjomand1 , F. FP Larkin2 1

Department of Ophthalmology, Medical University of Graz, Graz, Austria; 2 Moorﬁelds Eye Hospital, London, Great Britain

Background. The cornea was the first successful transplanted tissue more than 100 years ago. Our understanding about the immunological aspects of a corneal transplant has gained over the 100 years and surgical techniques and medical treatment of corneal grafts have changed within the last decades. The cornea is now the most successfully transplanted tissue worldwide. The success story of corneal transplantation began with the understanding of the immunological aspects and the introduction of topical and systemic treatment. Nowadays we have several surgical and medical options to get a prolonged graft survival, especially in eyes with a high risk for allograft rejection. Lamellar corneal grafts are standard operation techniques in the hands of experienced surgeons, which reduce the risk of graft rejection to almost zero, especially in eyes with anterior lamellar grafts. Conclusions. In this review, we summarize the history of corneal transplantation and possible medical and surgical options to increase the success of corneal grafts.

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04 First case of breakthrough pulmonary Aspergillus niveus infection in a patient following allogeneic hematopoietic stem cell transplantation J. Auberger1, C. Lass-Flörl1 , J. Clausen1 , R. Bellmann1 , W. Buzina2 , G. Gastl1 , D. Nachbaur1 1 2

Medical University of Innsbruck, Innsbruck, Austria; Medical University of Graz, Graz, Austria

Case Report. A 21-year-old woman underwent allogeneic HLA fully-matched-unrelated HSCT for secondary AML following reduced intensity conditioning with busulfan, fludarabine and thymoglobulin. The patient was considered being at high risk for a mold infection due to prolonged neutropenia (>6 months), steroid exposure over years and enduring hospital construction work. Thus, primary antifungal prophylaxis consisted of amphotericin B colloid dispersion (ABCD) (3 mg=kg=d). Routinely performed surveillance cultures during the transplant period repeatedly revealed colonization with MRSA. On day þ8, the patient developed fever up to 38.5 C, which was unresponsive to cefepime and linezolid. Concurrently, several nodules with halo-signs in the right upper lobe were detected in a CT scan, raising strong suspicion of invasive aspergillosis. Antifungal salvage combination therapy was initiated consisting of caspofungin (50 mg=d) and posaconazole (400 mg bid), the latter instead of the originally described voriconazole due to its broader spectrum including zygomycetes. On day þ13 after the transplant the patient, still being neutropenic, developed septic multiorgan failure necessitating catecholamines and mechanical ventilation. Blood cultures drawn at this time were positive for multiresistant Enterobacter cloacae, showing in vitro susceptibility only to polymyxin B and amikacin. On day þ21 successful neutrophil engraftment with an absolute neutrophil count >0.5 g=L was documented. CT scan performed on day þ26 showed significant progression of the pulmonary lesions in number and size. BAL revealed pulmonary IA caused by A. niveus being in vitro susceptible to all established mold-active azoles and echinocandins, but not to amphotericin B (AMB). Despite adequate antifungal treatment and antibiotherapy the patient succumbed on day þ31. At postmortem examination the diagnosis of pulmonary IA caused by A. niveus was confirmed. Species of Aspergillus other than A. fumigatus have been increasingly isolated in some institutions and the most important include A.flavus, A. niger and A. terreus. Accurate identification of species gains in importance to detect primary or secondary resistance to antifungal drugs.There is evidence to suggest, that pre-exposure of immunosuppressed patients to AMB or triazoles causes an emergence of AMB- resistant non-fumigatus Aspergillus species and of zygomycetes. The rise of non-A. fumigatus species is of serious concern since treatment of infection is challenged by frequent resistance to newer antifungals or Eur Surg Vol. 40 Supplement Nr. 226 2008

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to polyenes, as demonstrated in the present case.It is recognized that a prompt initiation of antifungal therapy is likely to improve patients outcome. Although standard definitions for optimal diagnostic criteria of IFI have been established in clinical practice it still remains difficult to obtain definite diagnosis of IFI. Diagnostic markers are of limited usefulness and applying invasive procedures such as BAL or CT-guided biopsies remains controversial. Conclusions. In conclusion, in times of increasing isolation of non- A. fumigatus species drug-resistant Aspergilli should be taken into account when pre-emptive antifungal treatment has been initiated.Antifungal treatment should be reassessed in case of persistent fever or progression during therapy. Species identification and in vitro susceptibility testing seem to be mandatory for probably life-saving therapeutic decision making.

05 Cryptosporidiosis increases tacrolimus levels in solid organ recipients L. F. Barroso1 , C. D. Sifri1 , R. G. Sawyer2, B. Wispelwey1, T. L. Pruett2 , C. N. Kotton3 , H. Bonatti2 1 Division of Infectious Diseases and International Health, Charlottesville, VA, USA; 2 Department of Surgery, University of Virginia Health System, Charlottesville, VA, USA; 3 Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA

Background. Diarrhea is a common condition after solid organ transplantation (SOT) and can be associated with an elevation of tacrolimus levels. Cryptosporidiosis is a rare pathogen after SOT in industrialized countries but common in some endemic areas. Methods. A retrospective chart review of all cases of Cryptosporidiosis in solid organ recipients at the University of Virginia (UVA) and Boston Mass General Hospital was performed. Cryptosporidiosis was defined by presence of diarrhea and detection of the pathogen using either microscopy or enzyme immuno assay. A literature search was performed with regard to reported cases of cryptosporidiosis in solid organ recipients. Results. Cryptosporidiosis was observed in five renal-, one lung- and one pancreas recipient All patients were male with a median age of 51 (range 36–57) years. Onset of cryptosporidial enteritis ranged was median 22.1 (range 3.4–53.1) months post transplant. Symptoms had existed for median 14 (10–42) days before diagnosis of cryptosporidiosis (microscopy: 3, EIA: 4) was made. Therapy consisted of azithromycin and=or nitazoxanide. TAC levels increased from baseline median 6 ng=ml to median 22 ng=ml causing a temporary rise in serum creatinine from median 1.2 mg=dl to median 2.4 mg=dl. All patients recovered completely from enteritis. Published articles on cryptosporidiosis after SOT revealed that in endemic Eur Surg Vol. 40 Supplement Nr. 226 2008

areas such as India, South America or the Middle East a high prevalence must be expected, whereas the vast majority of reports from Europe and the USA are sporadic case reports or case series frequently in intestinal recipients, pedsiatric SOT recipients and after travel to endemic areas. High TAC trough levels were reported in some cases, but were not interpretated as a direct sequaelae of the enteric infection. Conclusions. Cryptosporidiosis should be considered an important cause of diarrhea after SOT. Acquisition of the disease during travel and in immigrants may be present. A significant rise in TAC levels during cryptosporidial enteritis should be expected and early dose reduction is necessary.

06 Predictive factors of outcome in patients transplanted for hepatitis B S. Beckebaum1 , G. Sotiropoulos2 , C. Klein1 , A. Paul2 , G. Gerken3 , V. Cicinnati1 1

Interdisciplinary Liver Transplant Unit, University Hospital Essen, Germany; 2 Department of General, Visceral and Transplantation Surgery, University Hospital Essen, Essen, Germany; 3 Department of Gastroenterology and Hepatology, University Hospital Essen, Essen, Germany

In this study, we aimed 1) to investigate the impact of variables on graft and patient survival in hepatitis B virus (HBV)-infected liver transplant (LT) recipients and 2) to identify factors associated with a higher risk of graft cirrhosis at 5 years post-LT. A total of 104 chronically infected HBV patients (76% male, mean age of 49 10.8 years) transplanted between September 1992 and November 1997 were considered for this study. The overall 5 year patient and graft survival was 79.8% and 72.1%, respectively. All patients had cyclosporine A (CSA, n ¼ 58) or tacrolimus (TAC, n ¼ 46) based immunosuppressive regimens. Univariate and multivariate Cox s proportional hazards regression analysis indicated that recipient age and recipient BMI at LT and immunosuppression with CSA correlated with decreased patient survival. Older recipient age and higher recipient body mass index (BMI) at LT were associated with worse graft survival. In the log rank test, significant risk factors for graft cirrhosis within 5 years were qualitative and quantitative detection of HBV DNA and antiviral therapy at LT; significant posttransplant factors included biliary stenoses due to anastomotic strictures or ischemic type biliary lesions, hepatitis B recurrence, nucleos(t)id analogue monoprophylaxis (without hepatitis B immunoglobulin) and short term (<1 year) mycophenolate mofetil therapy. In conclusion, our study has shown that various recipient factors at LT as well as post-transplant virological status, antiviral prophylaxis, biliary complications and immunosuppressive regimen are related to the outcome of HBV recipients. Prospective studies are warranted to define the

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optimal immunosuppression in patients transplanted for hepatitis B.

07 Does the type of perfusion solution impact early endocrine function in clinical pancreas transplantation? – Results of a prospective randomized trial M. Biebl1 , S. Schneeberger1, W. Steurer1;2, U. J. Hesse3;4 , R. Toisi3 , J. M. Langrehr5, W. Scharek6 , M. Walter1, R. Margreiter1, A. Königsrainer1;2 1

Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria; 2 Department of General, Visceral and Transplant Surgery, University Hospital Tübingen, Tübingen, Germany; 3 Department of General, Hepato-Biliary and Transplantation Surgery, Ghent University Hospital, Gent, Belgium; 4 Department of Surgery, Hospital Bad Cannstatt, Stuttgart, Germany; 5 Department of General, Visceral and Transplantation Surgery, Charité Campus Virchow Klinikum, Humboldt Universität zu Berlin, Berlin, Germany; 6 Department of General, Vascular, Thoracic and Transplantation Surgery, University of Rostock, Rostock, Germany

Background. We aimed to evaluate early pancreas transplant graft function after Histidine-TryptophanKetoglutarate (HTK) versus University of Wisconsin (UW) perfusion. Methods. Prospective randomized multicenter study including 68 pancreas transplantations stratified according to preservation fluid used (27 HTK versus 41 UW). Primary endpoint was pancreas graft survival at 6 months. Serum -amylase, lipase, C-peptide, HbA1C and exogenous insulin requirement were compared at several timepoints. Results. Mean pancreas cold ischemia time was 10.8 3.7 (HTK) vs 11.8 3.4 h (UW) (p ¼ 0.247). Simultaneous pancreas-kidney transplantation was perfomed in 95.6%, pancreas transplantation alone in 2.9%, pancreas after kidney transplantation in 1.5%. Six months graft survival was 85.2% (HTK) vs 90.2% (UW) (p ¼ 0.703). Serum amylase and lipase values did not differ between both groups during the observation period. C-peptide levels were elevated in both groups without significant differences at each time point. Higher exogenous insulin requirement early after transplantation in the UW group

had resolved at three months. Six month patient survival was 96.3% (HTK) vs 100% (UW) (p ¼ 0.397). Conclusions. With a mean cold ischemia time of 10 h in this study, HTK and UW solutions appear equally suitable for perfusion and organ preservation in clinical pancreas transplantation.

08 Long term complications in HCV positive and HCV negative patients undergoing liver transplantation H. Bonatti1 , J. Aranda-Michel2 , M. Ghabril2 , D. M. Harnois2 , V. Machicao2 , J. Nguyen1 , R. C. Dickson2 1

Division of Transplant Surgery, Mayo Clinic, Jacksonville, USA; 2 Division of Hepatology and Gastroenterology, Mayo Clinic, Jacksonville, USA

Background. Up to 50% of liver transplants (LT) are performed for HCV associated liver disease in the US and some centers in Europe. These patients have a characteristic post LT course, which is primarily determined by interventions for recurrent hepatitis. Long term complications of LT include hypertension, metabolic disorders, infections and malignancies amongst many others and this may differ according to the underlying liver diseases. Aim. To describe long term complications after LT for patients with HCV compared to those without HCV. Methods. Longitudinal cohort study including 402 patients undergoing 467 LTs including 163 patients (193 transplants) with the diagnosis of HCV at Mayo Clinic Jacksonville between 1=98 and 12=0l. All transplants were performed using the piggyback technique. Immunosuppression consisted of tacrolimus, mycophenolate-mofetil and tapered steroids. Results. Patients with HCV differed in terms of baseline characteristic (Table 1) such as presence of hepatocellular cancer, gender, race and age, MELD score amongst others. In addition the incidences of some early complications differed (Table 2). Moreover, for 338 survivors >1 year, long term immunosuppression as well as incidences of some complications and some malignancies differed (Table 3). Incidence of obesity, metabolic changes such as DM and lipidemia and according complications such as hypertension and cardiovascular comorbidities (Table 4) differed.

Tab. 1 N N Median Median pat LTs age BMI

% % % % HBV MELD cauc male HCC core1 score

CMV Fast pos track

ECD preLT graft DM

non HCV

235

273

55(15–75)

26.6(15–51)

81%

57%

16%

18%

15(6–49)

72%

51%

59%

HCV

167

194

49(32–74)

27.5(18–51)

78%

72%

23%

41%

13.3(6–43)

68%

74%

59%

20%

0.061

0.17

0.367

0.002

0.05

<0.0001

0.294

0.278

<0.0001

0.537

0.499

p-value

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25%

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Tab. 2 Rejection rate

Denovo DM

IA bleed

Vasc compl

Biliary compl

Incisional hernia

Early renal failure

% Graft loss

% Died

non HCV

34%

13%

11%

10%

21%

7%

34%

16%

29%

HCV

31%

22%

6%

11%

19%

8%

29%

23%

26%

p-value

0.265

0.014

0.271

0.969

0.332

0.748

0.302

0.037

0.359

Tab. 3 N FU pat years

TAC Steroid Psych Non Smoker Alcohol PPI Any Skin Arthritis free compl H2Bl neoplasma cancer

non HCV 189

5.2(1–8.1) 74%

87%

50%

44%

6%

5%

61%

46%

13%

HCV

4.8(1–7.8) 83%

92%

69%

44%

17%

9%

67%

29%

8%

8%

<0.0001

0.212

0.001

0.53

0.002

0.087

0.162

0.001

0.078

0.059

p-value

149

0.04

14%

Tab. 4 Last BMI

Obesity

DM

Glucose intol

Lipi demia

Hypo thyroid

Osteo penia

HTN

Renal failure

Pulm

Cardio vas

non HCV

28(17–54)

56%

32%

30%

54%

21%

60%

70%

60%

23%

37%

HCV

27(18–51)

46%

30%

33%

46%

17%

47%

63%

43%

17%

27%

p-value

0.027

0.065

0.541

0.541

0.004

0.222

0.01

0.102

0.001

0.091

0.036

Survival of HCV patients was better for the first three years, however, thereafter became worse due to recurrent hepatitis C. This recurrence was associated with the utilization of extended criteria donor allografts. Such a worse outcome with ECD allografts was not observed in HCV negative individuals. In a multivariate analysis, HCV was not predictive for poor outcome, whereas a high MELD score and advanced donor age where indicative for poor survival. Conclusions. HCV patients differ in many regards to non-HCV patients. These differences need to be addressed in many regards including pre-LT management, perioperative management, organ allocation as well as long term management. An important finding was the fact that HCV patients had a lower risk for some malignancies.

09 Weird and worrysome: emerging pathogens in abdominal solid organ transplantation H. Bonatti1 , R. Sawyer1, A. J. Mathers2 , T. Pruett1 , C. Sifri2 1

Department of Surgery, University of Virginia, Virginia, USA; Division of Infectious Diseases, University of Virginia, Virginia, USA 2

Background. Changing demographics of organ donors and transplant recipients led to an increase in Eur Surg Vol. 40 Supplement Nr. 226 2008

patients who receive extended criteria donor organs with poor initial graft function and patients suffering from severe comorbidities. These changes and use of more powerful immunosuppression reduced rejection rates but also an increase in the rate of some infections. Methods. A review of infectious complications caused by rare or multi resistant pathogens in abdominal transplant recipients from 2007 to 2008 was performed. Results. Plesiomonas shigelloides grew from blood cultures from a donor who had a drowning accident and the liver was utilized; Moraxella osloensis was isolated from blood from a renal recipient; both patients were successfully treated with ciprofloxacin. One liver recipient developed Leuconostoc pseudomesenteroides cholangitis and sepsis after extended exposure to vancomycin and another had sepsis with Enterococcus casseliflavus. Both pathogens are resistent to vancomycin and patients were sucessfully treated with clindamycin and linezolid, respectively. Five liver recipients had pneumonia=peritonitis and=or sepsis with Carbapenemenase producing Gram negative rods (KPC þ GNR): Klebsiella pneumonia (n ¼ 2), Klebsiella oxytoca (n ¼ 1), E. coli (n ¼ 1), Citrobacter (n ¼ 1). All patients with KPC þ GNR were treated with colistin and all responded to therapy; however, one patient with hepatic artery thrombosis developed ischemic cholangiopathy and died shortly after retransplantation from septic shock and another patient died from multiorgan failure associated with graft failure and pulmonary hypertension. One patient had intraabdominal infection with multidrug resistant Acinetobacter baumanii together with vancomycin resistant Enterococcus faecium (VRE). She underwent

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surgical repair of a bile leak and was successfully treated with colistin=linezolid. In addition we observed one case of herpes simplex virus (HSV) and varicella zoster virus hepatitis each, one case of adenovirus nephritis and two cases of aseptic viral meningitis. Finally a dramatic increase in infections due to VRE, community-acquired methicillin resistant Staphylococcus aureus, Clostridium difficile colitis and opportunistic pathogens such as Nocardia and Cryptosporidium occurred. As a common risk factor, for the majority of patients, intensified immunosuppression using agents such as antithymocyte globulin or rituximab was identified and many of these patients become infected with multiple rare and=or multidrug resistent pathogens. Conclusions. Rare and multidrug resistant organisms are a challenge in modern transplant medicine and seem to be linked to the use of intensified immunosuppression and changing epidemiology of recipients and donors as well as environmental factors. Better infectious screening and prophylaxis protocols are urgently warranted.

10 Bariatric surgery in morbidly obese solid organ recipients H. Bonatti, B. Schirmer, T. Pruett, R. Sawyer, K. Brayman Department of Surgery, University of Virginia, Virginia, USA

Background. Obesity as part of the metabolic syndrome may cause significant complications in transplant recipients. Bariatric surgery may be a good option for obese transplant recipients. Methods. Fourteen patients (one pancreas=kidney, Two liver eleven renal recipients) were identified who had bariatric surgery pre (n ¼ 7) or following (n ¼ 7) their organ transplant. There were eight men and six women with a median age of 42.4 (range 30.1–59.3) years at transplantation and 44.7 (range 24.3–58.5) years at bariatric surgery. Procedures included vertical banded gastroplasty (n ¼ 1), open gastric bypass (n ¼ 5 including one who was converted after laparoscopic attempt), laparoscopic gastric bypass (n ¼ 1) and laparoscopic gastric banding (n ¼ 7). Results. Median follow up was 1251 (range 176– 5354) days, thirteen patients are currently alive, twelve with well functioning grafts. Body mass index pre transplant was 38.5 (range 25.0–44.8) kg=m2 , at bariatric surgery 46.7 (range 37.3–64.5) kg=m2 , and at last follow up 38.0 (range 23.5–47.5) kg=m2 . The patient with vertical banded gastroplasty had her renal transplant 1986 and lost the graft and died later from myocardial infarction. All three patients with gastric bypass had rejection associated with poor absorption of tacrolimus or cyclosporine. Four patients with gastric banding had good weight loss, two had moderate weight loss and

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the only pancreatic recipient required initially several changes in the filling volume due to diabetic gastroparesis before achieving acceptable weight loss. Both liver recipients had moderate weight loss but nevertheless developed NASH and underwent successful liver transplantation. Conclusions. Morbidly obese transplant recipients should undergo bariatric surgery and gastric banding seems to be the best option. Gastric bypass is associated with malabsorption of immunosuppressive drugs.

11 Bilirubin rinse of the graft suppresses MAPK activation and mediates protection in cardiac ischemia reperfusion injury F. Bösch1 , M. Thomas2 , P. Kogler1, D. Wiedemann3 , J. Troppmair1, R. Margreiter1, Öllinger1 1 Department of General and Transplant Surgery, D. Swarovski Research Laboratory, Medical University of Innsbruck, Innsbruck, Austria; 2 Department of Surgery, Ludwig Maximilians University Munich, Clinicum Munich, Germany; 3 Department of Cardiac Surgery, Medical University of Innsbruck, Innsbruck, Austria

Background. Induction of HO-1 ameliorates ischemia reperfusion injury (IRI). Bilirubin, a product of heme catabolism by HO-1 at least in parts accounts for the protective effects mediated by HO-1. Aim of this study was to determine the best strategy for administration of different forms of bilirubin to have beneficial effects on IRI by measuring mitogen activated protein kinase (MAPK) activation. To verify the results a specific MAPK inhibitor was used. Methods. Organ treatment prior to reperfusion (bilirubin and bilirubin ditaurate rinse at 125 mM), treatment of the donor or the recipient at 17.5 mg=kg and a combination were investigated. Isogenic C57Bl=6 hearts (n ¼ 4= group) were transplanted heterotopically after 18 hours of cold ischemia. Anastomosis time and reperfusion time were kept at 15 minutes. Western blot analysis was carried out for the total (T) and the phosphorylated (P) forms of Akt, ERK 1=2, JNK 1=2 and p38-MAPK. P=T ratio was quantified by ImageJ software and statistically analyzed using ANOVA. Graft function was assessed using an established score. Results. At 15 min after reperfusion phosphorylation of all MAPKs being investigated was dramatically increased, when compared to the non-reperfused isografts. At this time point, bilirubin rinse caused a significant inhibition of ERK and JNK (p < 0.001) as well as p38MAPK and Akt (p < 0.05) phosphorylation. Bilirubin ditaurate and systemic treatment had no effect on these MAPKs. Additionally, graft function of the bilirubin (score: 2 stdev) rinsed hearts was better when compared to the Eur Surg Vol. 40 Supplement Nr. 226 2008

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controls (score: 1 stdev), however data did not reach statistical significance. Conclusions. Bilirubin treatment inhibits MAPK activation what may account for its protective effects in a mouse model of ischemia reperfusion injury. Rinsing the graft before reperfusion might be a promising strategy to improve outcome after solid organ transplantation.

12 Salvage liver transplantation after resection for patients suffering from hepatocellular carcinoma (HCC) at the Vienna transplant center C. Burghuber1, C. Mayrhofer1, H. Pokorny2, M. Pones1 , M. Hofmann1 , F. Mühlbacher1, R. Steininger1 1

Division of Transplantation, Department of Surgery, Medical University of Vienna, Vienna, Austria; 2 Division of Surgery, Hospital Wiener Neustadt, Wiener Neustadt, Austria

Background. Liver transplantation (LT) is considered the best treatment for small HCC in most studies. But increasing waiting time and scarceness of organs threaten the outcome of these patients. The drop-out rate due to tumor progression is high, especially after introduction of the Milan-criteria. Various studies were published proposing primary surgical resection in appropriate patients and salvage transplantation if recurrence occurs. We tried to evaluate the safety of this concept in patients of our center. Methods. We conducted a retrospective study of prospectively collected data of all patients transplanted for HCC at our center between 1995 and 2005. Of 167 patients transplanted fifteen were primarily resected and subsequently transplanted for recurrence. They were separated as a group of secondary liver transplanted (SLT) and compared with the primarily transplanted patients (PLT). Baseline data such as age, sex, underlying liver disease, tumor characteristics and extent in histological examination were compared using t-test and chi-square-test. Overall-survival (OS) and recurrencefree-survival (RFS) were calculated using the Kaplan– Meier-method. Milan-criteria were introduced in 2000 at our center. Results. We found a significant difference between the two groups as far as the underlying liver disease was concerned. In the SLT group there were more patients without cirrhosis (26 vs. 4%) and less patients with nonviral cirrhosis (7 vs. 45%) compared to the PLT group (p ¼ 0.005), but the majority in both groups had a viral cirrhosis, 67 and 51% respectively. There was a difference concerning proportion of patients exceeding Milan-criteria in posthepatectomy-specimen (SLT 60 vs. PLT 37%), but it did not reach statistical significance. Eur Surg Vol. 40 Supplement Nr. 226 2008

Time from liver resection to SLT was median 32 months (IQR 22–39). 5-year-overall-survival was not statistically different in the two groups reaching 66 (SLT) vs. 53% (p ¼ 0.4). 5-year-disease-free-survival was 49 vs. 71% (p ¼ 0.1). Conclusions. Salvage transplantation seems to be a safe concept although there is a tendency to developing earlier tumor recurrence in the SLT group. This concept may help to avoid tumor progression during waiting time for transplantation in selected patients by treating them with tumor resection. Patients with recurrence thereafter can be successfully transplanted. A prospective randomized trial should be encouraged.

13 Predictive value of pre-transplant creatinine on renal impairment one year after liver transplantation C. Burghuber, G. Györi, I. Kristo, R. Steininger, F. Mühlbacher, T. Soliman, G. Berlakovich Division of Transplantation, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. Chronic renal failure after orthotopic liver transplantation (OLT) is common and is a cause for a reduced quality of life and survival. The course of renal function peri- and postoperatively is difficult to foresee. Means to predict the development of postoperative renal impairment (RI) would help to concentrate on early minimization of nephrotoxic medication in these patients. Methods. All adult patients who received a primary liver graft between 1999 and 2006 and were followed at our center for at least 1 year post-OLT were considered for analysis (n ¼ 269). Data were collected from our prospective electronic database. Postoperative RI was defined as a serum creatinine >1.5 at 1 year post-OLT. We conducted an analysis of the predictive value of creatinine 3, 2, and 1 months preoperatively and on the day of OLT. Sensitivity and specificity as well as positive and negative predictive values were calculated. Results. Renal impairment was diagnosed in 69 patients (26%) at 1 year after OLT. Pre-OLT serum creatinine values at 3-, 2-, 1-month and at transplant were compared with renal function 1 year post-OLT. The correlation coefficient was 0.43, 0.26, 0.44 and 0.33, respectively. Detection of RI post-OLT by pre-OLT serum creatinine at the 4 time points had a sensitivity of 25%, 27%, 33% and 7% and a specificity of 95%, 92%, 94% and 83%, respectively. The proportion of true positive test results among all the positive test results (positive predictive value) amounted to 44% and the negative predictive value to 75%.

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Conclusions. Pre-transplant serum creatinine had no predictive value for long-term renal function in patients listed for liver transplantation. Measuring serum creatinine alone does not permit to sort out patients with high risk for developing renal failure after OLT.

14 Combined mycophenolate mofetil and minimal dose calcineurin inhibitor therapy in stable liver transplant patients is nephro- and hepatoprotective V. Cicinnati1 , M. Lindemann2 , C. Klein1 , G. Sotiropoulos3 , G. Gerken4 , S. Beckebaum1 1

Interdisciplinary Liver Transplant Unit, University Hospital Essen, Essen, Germany; 2 Institute of Immunology, University Hospital Essen, Essen, Germany; 3 Department of General, Visceral and Transplantation Surgery, University Hospital Essen, Essen, Germany; 4 Department of Gastroenterology and Hepatology, University Hospital Essen, Essen, Germany

Background. Calcineurin inhibitor (CNI)-related nephrotoxicity significantly contributes to chronic renal failure after liver transplantation (LT). Methods. In this prospective study, LT patients with renal dysfunction were randomized to either receive mycophenolate mofetil (MMF) followed by stepwise reduction of CNI with defined minimal CNI-trough levels (MMF group) or to continue their maintenance CNI dose (control group). Immune monitoring was performed in a subgroup of the patients. Results. In the MMF group (n ¼ 50), renal function assessed by serum creatinine improved >10% in 62% of patients, was stable in 36% and deteriorated >10% in 2% after 12 months compared with baseline values. Mean serum creatinine levels (SD) significantly decreased from 1.90 0.44 mg=dL to 1.61 0.39 mg=dL and the corresponding calculated glomerular filtration rate significantly increased from 48.7 14.1 mL=min to 57.6 16.9 mL=min over a 12-month follow-up period. Blood presssure and levels of liver enzymes significantly decreased. In the control group (n ¼ 25), there were no significant changes with respect to the investigated parameters. The MMF group had significantly lower numbers of circulating cytotoxic T cells compared with the controls; whereas regulatory T cells significantly increased. Conclusions. Combined MMF and minimal dose CNI therapy after LT is nephro- and hepatoprotective, and may also promote allograft tolerance.

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15 Myocarditis or dilative cardiomyopathy? A 6-month follow-up of NT-proBNP and left ventricular diameters in children with left ventricular dysfunction F. Darbandi-Mesri, D. Luckner, E. Mlczoch, U. Salzer-Muhar Division of Pediatric Cardiology, Department of Pediatrics and Andolescent Medicine, Medical University of Vienna, Vienna, Austria

Background. Dilated cardiomyopathy (DCMP) is characterized by left ventricular dilation and dysfunction resulting in congestive heart failure. Most commonly DCMP is either idiopathic or caused by myocarditis and is one of the main indications for cardiac transplantation in children. Brain natriuretic peptide (BNP) is released by myocardial cells in the ventricles in response to increased wall stress. Plasma BNP levels correlate with elevated enddiastolic pressure and are considered to provide accurate profiling of heart failure. Methods. Retrospectively ten critically ill children with DCMP, either of idiopathic origin (n ¼ 4) or caused by myocarditis (n ¼ 6), were followed up for 6 months by review of medical=echocardiographic records and plasma NT-pro-BNP levels. Results. Plasma NT-pro-BNP levels were associated with clinical improvement (p < 0.0005) and decreased to normal in clinically recovered children. Echocardiographic findings (left ventricular enddiastolic diameter: p ¼ 0.06, left ventricular endsystolic diameter: p ¼ 0.003, fractional shortening: p ¼ 0.02) were associated with clinical improvement, but showed more delay than plasma NT-pro-BNP levels. Children with myocarditis presented with initially higher plasma NT-pro-BNP levels (mean plasma level: 29813.7 pg=ml [SD 3113.8 pg=ml]) and decreased to normal after clinical remission, whereas children with idiopathic DCMP initially presented with lower plasma levels (7813.8 pg=ml [SD 2192.3 pg=ml]) and did not normalize. This difference in plasma NT-pro-BNP levels was not significant (p ¼ 0.06) due to the small study population. Conclusions. Serial NT-pro-BNP measurements might contribute to the differential diagnosis between idiopathic DCMP and myocarditis and be of value in the pretransplant scenario. These findings have to be confirmed in an adequately powered prospective trial.

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16 The LB model: a new prognostic tool for prediction of short term survival in patients with end-stage liver disease D. Duller1, J. Haas2 , V. Stadlbauer3, H. Hetz4 , I. Kristo5 , F. Mühlbacher5, R. Stauber3 1

Division of Transplantation Surgery, Department of Surgery, Medical University of Graz, Graz, Austria; 2 Department of Gynaecology and Obstetrics, Medical University of Graz, Graz, Austria; 3 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; 4 Department of Anaesthesiology, Medical University of Vienna, Vienna, Austria; 5 Division of Transplantation Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. The model for end-stage liver disease (MELD) score, a prognostic tool comprising readily available laboratory parameters, is widely used for estimation of short term survival in cirrhotic patients. The aim of this study was to improve the diagnostic accuracy of MELD by creating a new prognostic tool based on laboratory parameters linked to liver failure as well as indocyanin green (ICG) clearance as a measure of hepatic excretory function. Methods. Between 2004 and 2006, routine laboratory parameters linked to liver failure as well as ICG clearance were determined in 70 consecutive patients with chronic liver failure. Plasma disappearance rate (PDR) of ICG was obtained by finger pulse densitometry following injection of ICG 0.25 mg=kg. Survival at day 90 was assessed. Logistic regression analysis was used to create a new prognostic model. The diagnostic accuracies of these prognostic models for prediction of 90-day survival were assessed by ROC analysis. Findings were validated in two separate cohorts, i.e. an internal validation set consistant of cirrhotics admitted to the Medical University Graz and an external validation cohort comprising transplant candidates from the Medical University of Vienna. Results. Univariate analysis revealed superior diagnostic accuracy for MELD as compared to ICG PDR. Multivariate analysis yielded a new prognostic model including white blood count and bilirubin as independent predictors. This new prognostic model, i.e. the leukocyte bilirubin (LB) model, was superior to MELD in predicting 90-day survival. In a subset of 90 patients admitted to the ICU, the LB model had superior diagnostic accuracy as the SOFA score. Conclusions. The LB model is a new prognostic model that shows superior diagnostic accuracy for prediction of short term survival as compared to MELD in patients with end stage liver disease.

Duodenal enteric drainage in pancreas transplantation combined with retroperitoneal placement: a more physiological method? D. Duller, D. Kniepeiss, S. Schaffellner, E. Jakoby, H. Müller, K. Tscheliessnigg, F. Iberer Division of Transplantation, Department of Surgery, Medical University of Graz, Graz, Austria

Background. Following the recommendations of the American Diabetes Association, pancreas transplantation is an acceptable treatment for type 1 diabetic patients. Until the mid-90ties 90% of pancreas transplantations were performed with enteral drainage into the bladder. Besides avoiding surgical complications this eased the monitoring of the transplanted graft through urinary amylases and lipase. The introduction of enteric drainage removed this opportunity. We report a new technique for pancreas implantation regarding implant site as well as enteric drainage into the duodenum. Case report. A 48-year-old patient was transplanted with a 17 year old pancreas with retroperitoneal placing. All anastomosis were sewed at the main vessels and enteric drainage was done through a duodenal-doudenal anastomosis. The patient was insulin independent the day after transplantation; both organs functioned well until now. No surgical complication was observed in a follow up period of 6 months. The transplant site of the donors duodenum allows easy graft monitoring through conventional gastroscopy. Conclusions. The routine method for enteric anastomosis into the recipients jejunum does not pose any opportunity for graft monitoring. The only possibility to monitor the transplanted organ is through CT guided biopsy. This new method allows a more physiological placement of the graft and enteric drainage on a physiological site with the new possibility to monitor the transplanted pancreas non invasively non risky for the recipient. In this single case and in both cases reported in the literature it did not pose any surgical problems nor require further intervention.

18 Serum Cystatin C as an easy to obtain biomarker for the onset of late renal impairment in heart transplantation D. Duller1, G. Wirnsberger2, P. Stiegler1, A. GrosejleStrehle3 , R. Roller-Wirnsberger2, A. Wasler1, D. Kniepeiss1 1

Division for Transplantation, Department for Surgery, Medical University of Graz, Graz, Austria; 2 Division for Nephrology, Medical University of Graz, Graz, Austria; 3 Department of Medical Statistics, Medical University of Graz, Graz, Austria Eur Surg Vol. 40 Supplement Nr. 226 2008

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Background. Improved survival of heart transplant recipients increased the incidence of chronic renal failure after transplantation. Highly sensitive, early and effective monitoring of post transplant renal function is still on research. This study aimed to evaluate the prognostic value of Cystatin C for patients being in various stages after heart transplantation. Methods. Seventy-three patients were included prospectively with a follow up of 4 years. Renal impairment was defined as Creatinine levels above 1.4 mg=dl. Serum Creatinine, renal glomerular filration rate calculated by the MDRD formula and serum Cystatin C levels were collected, risk factors for RF were assessed. Statistical analysis was performed for all patients regarding real function 12 months and 24 months and 48 months after study start. Results. Univariate analysis showed a prognostic impact of antihypertensive medication, onset of diabetes and pre-existing renal impairment (p < 0.001) on renal failure after transplantation. Serum Cystatin C levels strongly correlated with Serum creatinine levels (r ¼ 0.839). Multivariate analysis yielded Cystatin C measured at study start as superior prognostic parameter for all time points (AUROC 12 months: 0.879; 24 months: 0.934; 48 months: 0.930). Conclusions. Our results revealed an enormous potential of serum Cystatin C as an early prognostic and easy to obtain biomarker for renal dysfunction after heart transplantation.

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20 Sirolimus: a beneﬁt of use for liver transplant recipients with HCV recurrence?

Easy, safe and quick: a new parameter to monitor renal function after liver transplantation

D. Duller, D. Kniepeiss, S. Schaffellner, H. Müller, E. Jakoby, K. Tscheliessnigg, F. Iberer

D. Duller1, P. Stiegler1, A. Groselj-Strele2 , G. Wirnsberger3, K. Tscheliessnigg1 , F. Iberer1, D. Kniepeiss1

Background. Cirrhosis due HCV infection is one leading indication for liver transplantation. Triggered by immunosuppression the virus recurs nearly universally. A benefit of use and its effect on the viral load of SIR has never been examined. Aim. The aim of this study was to estimate a possible effect of Sirolimus as immunosuppressive agent on Hepatitis C recurrence in liver recipients. HCV positive patients transplanted in our centre were followed up prospectively regarding transaminases, immunosuppressive target levels, HCV RNA and survival. Results. Twenty-five patients were enrolled. Fourteen patients (gr. 1) were received a SIR including regimen (starting dose of 1–2 mg=day, target level treatment of 3–6 ng=ml), 11 patients (gr. 2) stayed on Calcineurin-inhibitor based IS. Median follow up periods were equal. Twenty-five patients showed stable transaminase levels. 1 rejection was treated with steroids in group 1 and 1 patients immunosuppressive regimen was adapted due to a massive viral recurrence in group 2. At time point 1 both group showed no significant difference in the viral

1 Division of Transplantation, Department of Surgery, Medical University of Graz, Graz, Austria; 2 Department of Internal Medicine, Medical University of Graz, Graz, Austria; 3 Ofﬁce for Biostatistic, Ofﬁce for Information Management, Center for Medical Research, Medical University of Graz, Graz, Austria

Background. Improved survival of liver transplant recipients increased the incidence of chronic renal failure after transplantation. Highly sensitive, early and effective monitoring of post transplant renal function is still on research. The aim of this study was to evaluate the prognostic value of Cystatin C for patients being in various stages after liver transplantation. Methods. Ninety patients were included prospectively with a follow up of 2 years. Patients were divided into two cohorts regarding study start: Recipients who received their graft prior to study start (GR1 ¼ 47) and recipients

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included with liver transplantation (GR2 ¼ 43). For GR1 all kidney functional parameters were collected prior to transplant retrospectively. Renal impairment was defined as serum creatinine levels above 1.4 mg=dl. Serum Creatinine, renal glomerular filration rate calculated by the Modification of Diet in Renal Disease formula and serum Cystatin C levels were collected for both groups, risk factors for renal failure were assessed. Statistical analysis was performed for all patients and for both subsets regarding real function 12 months and 24 months after study start. Results. Univariate analysis showed a prognostic impact of antihypertensive medication prior TX (p ¼ 0.0008) and serum Cystatin C one month after liver transplantation (p < 0.0001) on renal failure after transplantation at both time points. Multivariate analysis yielded Cystatin C as superior prognostic parameter in the whole patient group as well as in both subsets. (AUROC 0.84 for the whole set vs. AUROC 0.87 group 1 and 0.96 for group 2). Conclusions. Our results revealed an enormous potential of serum Cystatin C as an early prognostic easy to obtain biomarker for renal dysfunction after liver transplantation.

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

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load. At time point 2 group 2 showed significant decreases (p< 0.05) in the viral load as compared to group 1. Conclusions. Rare is known about a possible effect of Sirolimus on HCV infected recipients. This analysis suggests a potential benefit of SIR as immunosuppressant for HCV positive LT candidates to suppress viral recurrence.

22 Improved survival due to lower relapse risk after occurrence of GvHD after allogeneic retransplantation S. Eder, A. Schulenburg, W. Rabitsch, M. Mitterbauer, P. Kahls, C. Zielinski, H. Greinix

21 Ergebnisse nach PTCA, Stent und CABG bei Patienten nach Herztransplantation T. Dziodzio1 , A. Juraszek1 , S. Rödler1, M. Czerny1, D. Glogar2, M. Grimm1 , A. Zuckermann1 1

Klinische Abteilung für Herz-Thoraxchirurgie, Universitätsklinik für Chirurgie, Medizinische Universität Wien, Österreich; 2 Klinische Abteilung für Kardiologie, Universitätsklinik für Innere Medizin II, Medizinische Universität Wien, Österreich

Grundlagen. Ziel dieser Untersuchung war es, unsere Ergebnisse nach PTCA, Stentimplantation und Koronarbypassoperation zur Behandlung der Graftvaskulopathie nach Herztransplantation (HTX) zu evaluieren. Methodik. Im Zeitraum 1989 bis 2006 wurden 55 Patienten (11 % weiblich) aufgrund einer symptomatischen Graftvaskulopathie behandelt. Das Durchschnittsalter zum Zeitpunkt der HTX war 49 Jahre. Der Zeitraum zwischen HTX und der Revaskularisation war im Mittel 103 Monate. Es wurden insgesamt 298 La¨sionen behandelt. Dreiundachtzig La¨sionen sind prima¨r dilatiert worden, 124 La¨sionen sind prima¨r oder sekunda¨r mit einem Stent versorgt worden und 5 Patienten wurden prima¨r Bypass operiert. Die prima¨re Erfolgsrate, die Restenoserate sowie sekunda¨re kardiale Spa¨tkomplikationen wurden monitiert. Ergebnisse. Die prima¨re Erfolgsrate hat 99 % betragen. Der durchschnittliche Nachbeobachtungszeitraum nach der Revaskularisation war 72 Monate. Wa¨hrenddessen wurden 26 % La¨sionen nach prima¨rer PTCA und 15 % La¨sionen nach prima¨rem oder sekunda¨rem Stent nachinterveniert. In der Gruppe der Patienten nach Bypassoperation waren alle Bypa¨sse bei der jeweiligen Kontrolle einwandfrei offen. Zwei Patienten sind im Verlauf an einem Myokardinfarkt verstorben und zwei Patienten sind aufgrund der fortschreitenden ischa¨mischen Kardiomyopathie retransplantiert worden. Weitere zwei Patienten sind aus nicht kardialer Ursache verstorben. Schlussfolgerungen. Die Inzidenz der revaskuarisationspflichtigen Graftvaskulopathie nach HTX ist in dieser Serie niedrig. Der Großteil der La¨sionen ist mit einem individuellen Therapiekonzept gut behandelbar. Trotz einer geringen Inzidenz an spa¨ten Therapieversagern, bleibt die routinema¨ßige Kontrollangiographie ein unverzichtbarer Bestandteil der guten Nachsorge dieser Patienten. Eur Surg Vol. 40 Supplement Nr. 226 2008

Department of Medicine I, Medical University of Vienna, Vienna, Austria

Background. Relapse of malignant disease after high-dose therapy followed by hematopoietic stem cell transplantation (HCT) has a poor prognosis. Although HCT with reduced-intensity conditioning (RIC) is associated with lower transplant-related mortality (TRM), it is controversial whether RIC improves survival after retransplantation due to high rates of recurrent malignant disease. Methods. We retrospectively compared the outcome of 57 consecutive patients (31 male and 26 female) with a median age of 46 (range, 18–63) years who received either myeloablative (n ¼ 12) or reduced-intensity conditioning (RIC, n ¼ 45) for allogeneic retransplantation after first (n ¼ 31) or second (n ¼ 3) autologous or first allogeneic (n ¼ 23) HCT at our institution. Main diagnoses were non-Hodgkin’s lymphoma (NHL, n ¼ 12), acute myeloid leukemia (AML, n ¼ 12), and myeloma (MM, n ¼ 10). Time from first HCT until allogeneic retransplantation was a median of 24 (range, 2–144) months. Reasons for retransplantation were relapse of malignant disease (n ¼ 38), partial response to first HCT (n ¼ 16) and graft failure (n ¼ 3). Twelve patients received myeloablative conditioning for retransplantation, RIC for retransplantation consisted of either fludarabine=TBI of 2 Gy (n ¼ 23), FLAMSA (n ¼ 5) or other combination chemotherapy (n ¼ 17). Thirty-one patients had an HLA-identical related donor and 26 patients an unrelated one. In 7 patients bone marrow and in 50 peripheral blood stem cells were used as stem cell source. Results. Eight patients (7 after MA and 1 after RIC) died within 35 days of stem cell infusion and they are not available for response evaluation. Forty-four of 49 evaluable patients (90%, 39 after RIC, 5 after MA) achieved a complete remission after retransplantation whereas 4 (all after RIC) were refractory. Eleven patients experienced relapse after retransplantation (10 after RIC, 1 after MA). Kaplan–Meier probability of relapse at 5 years is 40% after RIC and 25% after MA conditioning for retransplantation (p ¼ 0.46). Acute and chronic graft-versus-host disease (GvHD) occurred in 25 patients, 6 patients suffered of acute GvHD, 12 patients of chronic GvHD and in 7 patients acute and chronic GvHD occurred. Incidence of relapse was 24% in patients experiencing GvHD compared to 53% in patients without acute or chronic GvHD (p ¼ 0.01). Kaplan–Meier probability of TRM is 67% after myeloablative and 23% after RIC HCT (p < 0.001) and not significantly different between patients with and without GvHD. With a median follow-up of 39 months after

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retransplantation 26 patients (46%; 24 after RIC, 2 after MA) are currently alive including 25 in continuous remission. Kaplan–Meier probability of five-year-survival is 46% after RIC and 25% after MA conditioning for retransplantation (p ¼ 0.01) and 25% in patients without GvHD compared to 58% in patients experiencing GvHD (p 0.01), respectively. Conclusions. Occurence of graft-versus-host disease after allogeneic retransplantation is associated with a significantly lower risk of relapse and significantly improved overall survival. Reduced-intensity conditioning for HCT did not increase relapse risk and thus, is of benefit for this poor risk patient population.

posCD3posCD4pos cells. However the functional properties of the respective fraction were not reduced. Allergen specific proliferation and cytokine production was not significantly altered compared to the control group. Th2 skewing experiments equally shut of Th-1 and Th-2 type response in vitro. Conclusions. In conclusion these results are in line with clinical data that demonstrated a persistence of symptoms. This insufficient suppression of Type I allergic reactions may be related to a lower frequency of regulatory cells.

24 23 Immunosuppression and allergy in organ transplant recipients: evidence for insufﬁcient control of allergen speciﬁc responses T. Eiwegger1, S. Gruber1, C. Geiger1, E. Dehlink1 , W. Klepetko2 , T. Frischer1, Z. Szépfalusi1 1

Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria; 2 Division of Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. The immunosuppressive therapy in solid organ transplantation targets mainly the T- and B-cell-mediated immune response. However, we have demonstrated that it does not suppress sensitization and clinical manifestation of type I allergies in organtransplanted patients. The present study addresses the question whether antigen-specific proliferative responses, cytokine profiles and regulatory markers differ between immunosuppressed and non-immunosuppressed (normal) individuals and whether this confers to an insufficient control of Th-2 type responses by a predominant effect on the suppression of Th-1 type cytokines. Methods. Peripheral blood mononuclear cells from 60 solid organ transplanted children (kidney, liver, lung) and 16 healthy, matched controls were assessed. Proliferative responses (thymidine incorporation assay) to a panel of allergens, intracellular cytokine production, the frequency of foxp3posCD3posCD4posCD25 high cells (flow cytometry), mRNA expression of IL-10, TGF-beta and foxp3 (RTPCR) was determined and in vitro Th-1 and Th-2 skewing experiments with naive CD4 T-cells (CD4RA-T-cells) (CD3CD28 beads, IL-2, IL-4; anti IL-12 treatment, CFSE labeling, intracellular cytokine staining) were performed. The inhibitory capacity of natural Tregs was evaluated by proliferation assays. Immunosuppression led to a significantly altered regulatory marker profile, expressed by enhanced TGF-beta mRNA production and a reduced frequency of foxp3-

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Pulmonale Komplikationen nach hämatopoetischer Stammzelltransplantation im Kindesalter: Inzidenz und Risikofaktoren T. Fazekas, A. Lawitschka, M. Seidel, A. Attarbaschi, C. Peters, S. Matthes St. Anna Kinderspital Wien, Wien, Österreich

Grundlagen. Ha¨matopoietische Stammzelltransplantation (HSZT) wird in der Behandlung von unterschiedlichen malignen und nicht-malignen Erkrankungen im Kindesalter eingesetzt. Die transplantationsassoziierte Mortalita¨t betra¨gt 10–30 % und ist ha¨ufig mit schweren pulmonalen Komplikationen assoziiert. Methodik. In einer retrospektiven Analyse wurde die Pra¨valenz pulmonaler Komplikationen bei 178 Patienten im Alter von 3,5 Monaten bis 28,1 Jahren erfasst, die zwischen 1997 und 2004 in unserem Zentrum wegen einer malignen oder nicht-malignen Grunderkrankung entweder allogenes Knochenmark oder allogene periphere Stammzellen aus dem Blut erhalten hatten. Ergebnisse. 50 Patienten (24 %) erlitten pulmonale Komplikationen, an denen 27 Kinder (12,4 %) innerhalb von zwei Jahren verstarben. Das mediane Zeitintervall zwischen HSZT und pulmonaler Komplikation betrug 6 Monate (12 Tage bis 32 Monate). Respiratorische Infektionen waren in 92 % der Fa¨lle die Todesursache, in erster Linie viraler (46 %) Genese, aber auch durch Pilzinfektionen (25 %) und Bakterien (13 %). Nur 8 % der Kinder verstarben an toxisch bedingten Komplikationen. Jene Kinder ¨ nohne pulmonale Beeintra¨chtigung waren signifikant ju ger (im Median 8,7 Jahre alt) als jene mit to¨dlicher pulmonaler Pathologie (13,1 Jahre). Die Mortalita¨t war bei Kindern mit einer schweren graft-versus-host Erkrankung signifikant ho¨her. Schlussfolgerungen. Wa¨hrend und nach HSZT sind infektiologische und immunologische Mechanismen fu¨r die Pathogenese von pulmonalen Komplikationen verantwortlich. Das Ausmaß an kumulativer Immunsuppression, sei es durch T-Zell Depletion oder immunsuppressive Therapie zur Behandlung einer GvHD, scheint die Mortalita¨t entscheidend zu beeinflussen. Mo¨gliche Risikofaktoren studieren wir derzeit durch serielle Messungen von Eur Surg Vol. 40 Supplement Nr. 226 2008

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Stickstoffmonoxyd in der Ausatemluft und in bronchoal¨ ssigkeit bei Kindern vor und nach veola¨rer Lavage Flu HSZT.

amyloid patients. For the first time in Austria a female patient with plasma cell dyscrasia and cardiotropic amyloidosis underwent total HTX. Subsequent treatment for amyloidosis is planned after a recovery period of 3 months.

25 Heart transplantation in a patient with cardiac amyloidosis B. Fellner1, D. Zimpfer2, G. Jakl1 , P. Kalhs3 , R. P. Linke4 , K. Huber1, A. Zuckermann2 1 Department of Medicine with Cardiology and Emergency Medicine, Wilhelminenhospital, Vienna, Austria; 2 Division of Cardio-Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria; 3 Division of Haematology and Haemostasis, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria; 4 amYmed, Reference Center of Amyloid Diseases, Martinsried, Germany

Background. Systemic amyloidosis complicated by heart failure is associated with high cardiovascular morbidity and mortality. In AL amyloidosis symptomatic cardiac involvement is associated with a particularly poor prognosis, typically 9 months. Patients suffering from severe cardiac amyloid infiltration are usually ineligible for the preferred treatment of chemotherapy with stem cell transplantation (SCT) rescue because of the high risk for treatment related mortality. Still, there are some rare cases reported about sequential heart and autologous SCT as a promising treatment option. Methods. A 47 years old woman with biopsy proven AL (primary) amyloidosis and signs of heart failure due to cardiac involvement was evaluated for heart transplantation (HTX) followed by high-dose chemotherapy and SCT. Results. The woman who had no former serious illness in her past medical history showed signs of heart failure with NYHA class III and NT-pro BNP levels about 9000 pg=ml. Cardiac amyloidosis was proven by endomyocardial biopsy. An initial attempt to treat the underlying plasma-cell dyscrasia only by chemotherapy had to be abandoned because of worsening of heart failure. So the patient was evaluated and finally accepted for HTX combined with SCT. Before listing for HTX CD34þ cells were collected for stem cell support. After two months on the waiting list the patient sucessfully underwent HTX. In order to remove all the diseased cardiac tissue a total HTX with resection as much of the atria as possible was performed. The postoperative care was not different as for standard HTX patients. The immunsupression consisted of a triple drug regimen (Cyclosporine, MMF and steroids) with ATG antibody induction. At the moment the patient is in NYHA I-II and scheduled 3 months for high dose chemotherapy and autologous SCT. Conclusions. Cardiac transplantation followed by SCT provides a novel therapeutic option for cardiac Eur Surg Vol. 40 Supplement Nr. 226 2008

26 Peripheral blood stem cell mobilisation with Pegﬁlgrastim versus Filgrastim in children and adolescents P. Fritsch, W. Schwinger, H. Lackner, P. Sovinz, M. Benesch, C. Urban Division of Pediatric Hemato-Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria

Background. Several studies have shown that Pegfilgrastim, the long acting agent of rh-GCSF, is as effective as Filgrastim in children undergoing cytotoxic chemotherapy by reducing the duration of neutropenia without increased adverse events as bone pain and headache. Recent studies in adults have shown that Pegfilgrastim is also effective to mobilize CD34þ stem cells. In contrast to Filgrastim studies showed a reduction of time to reach the peripheral blood CD34þ cell peak, resulting in earlier performance of leukapheresis. Although the use of Pegfilgrastim in children after conventional chemotherapy is established, only few data have been published yet describing the ability to mobilize CD34þ stem cells. The aim of the study was to compare the efficacy of Pegfilgrastim versus Filgrastim for CD34þ stem cell mobilization in children and adolescents. Methods. Patients admitted after 2006 were stimulated with Pegfilgrastim on day 4 following a chemotherapy course of the according treatment protocol followed by peripheral stem-cell-apheresis. Filgrastim was added when CD 34þ stem cells did not reach 15=ml between day 15 to 20. Patients were compared to historical controls (2002–2005) stimulated with conventional daily Filgrastim. Results. Three groups of patients were compared: Group 1 (historical group): six patients with Ewing’s Sarcoma undergoing chemotherapy according to the EURO-EWING protocol receiving Filgrastim 10–20 mg=kg (median 11 mg=kg) underwent a median of 3.8 peripheral stem cell collections (PSCC) between day 12–24 (median 15.4). A median of 16.9 CD34þ stem cells=kg BW were collected. Group 2: Three Patients with Ewing’s Sarcoma admitted after 2006 undergoing chemotherapy according to the EURO-EWING protocol received only Pegfilgrastim 100– 200 mg=kg (median 166 mg=kg). 3 PSCC were performed between day 11–13 (median 12) and a median of 24.3 CD34þ stem cells=kg BW were collected. Group 3: Five Patients suffering from recurrent neoplasm [B-NHL (n ¼ 1), medulloblastoma (n ¼ 1), ependy-

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moma (n ¼ 1) and germ cell tumor (n ¼ 2)]. Chemotherapy was performed according to relapse protocols followed by Pegfilgrastim 150-200 mg=kg (median 188 mg=kg), however 2 patients needed further cytokine stimulation with Filgrastim 10–15 mg=kg (median 12.5 mg=kg) combined with stem cell factor. A median of 3.2 PSCC was performed between day 8–30 (median 17). A median of 11.9 CD34þ stem cells=kg BW were collected. Conclusions. Our data show that stem cell mobilisation with Pegfilgrastim seems to produce earlier CD34þ peaks and better CD34þ yields than Filgrastim in children when performed during primary or without previous long lasting chemotherapy. However patients with previous long lasting chemotherapy might need additional mobilisation therapy with Filgrastim and stem cell factor.

reperfusion. This was associated with a significant raise in creatinine and urea (p < 0.05). Our data suggest that the use of small molecular weight p38-MAPK inhibitors (SB203580) have a beneficial effect on the ischemia reperfusion injury, showing a decrease in creatinine and urea as assessed the first days after reperfusion. Conclusions. Renal I=R injury is associated with a significant increase in MAPK activity. Isolated p38 MAPK inhibition during ischemia improves kidney function suggesting that p38 may provide a promising target for ameliorating reperfusion injury.

28 Characteristics of de novo malignomas after liver transplantation

27 p-38 MAPK inhibition attenuates kidney ischemia reperfusion injury P. Gehwolf1;2 , K. Fischler1;2, R. Oberhuber1;2, G. Brandacher1;2, S. Schneeberger1;2, R. Öllinger1;2 R. Margreiter1;2, J. Troppmair1;2, R. Sucher1;2 1 Center of Operative Medicine, Department of Visceral-, Transplant- and Thorax surgery, Medical University of Innsbruck, Innsbruck, Austria; 2 D. Swarovski Research Laboratory, Medical University of Innsbruck, Innsbruck, Austria

Background. Solid organ transplantation causes complex intracellular changes due to ischemia and subsequent reperfusion. Cell death, metabolic alterations and inflammation result in impairment of short- and long term function. Cells respond to extra cellular signals by transmitting intracellular instructions to coordinate appropriate responses. The group of mitogen activated protein kinases (MAPKs) was implicated through aberrant activation in many settings including I=R-associated organ damage. Although MAPKs are known as critical proteins in cell signaling their role during I=R is controversially discussed. Methods. To asses intracellular signaling and kidney function a rat ischemia reperfusion model was used. Ischemic renal injury was induced by clamping the left renal artery for 45 min in male Wistar-rats immediately after right-side nephrectomy. Animals received either SB203580 (2 mg=kg=BW) prior to reperfusion (Group I) or vector control (Group II). Sham-operated animals served as controls (Group III). Reperfusion was studied at different time points up to 7 days. Renal function was determined by plasma creatinine=urea and the alterations of MAPKs (ERK, JNK, p38) activation was analysed using phosphorylation specific antibodies. Results. In summary, reoxygenation was characterized by a dramatic increase in the activity of p-ERK (p < 0.05), p-JNK (p < 0.05) and p-p38 (p < 0.0001) during early

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I. Graziadei1 , A Finkenstedt1 , W. Oberaigner2, K. Nachbaur1, W. Mark3 , W. Vogel1 1

Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of Innsbruck, Innsbruck, Austria; 2 Department of Clinical Epidemiology, Tyrolean State Hospitals, Innsbruck, Austria; 3 Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria

Background. As improved patient survival has lengthened the observation period after liver transplantation (LT), development of de novo malignancies emerges as a serious long term complication and has become the second leading cause of death in this special cohort. Aim of this study was to characterize the types, specific mortality, risk factors and incidence of de novo malignancies after liver transplantation (LT) compared to the general population. Methods. We performed a retrospective analysis of all adult LT patients between 1982 and 2007 at our institution. Observed cancers in the study group were compared with expected cancers based on age-, sex- and calendar year-specific rates in the corresponding county excluding non melanoma skin cancer. Results. During a mean follow-up of 5.3 years, 96 out of 779 LT recipients (12.2%) developed 105 de novo malignancies. The cumulative risk for development of malignancy was 10.0%, 23.7%, 31.8% and 42.3% at 5, 10, 15 and 20 years after LT, respectively. Mean time from LT to diagnosis of the first de novo neoplasia was 61 (2–235) months and did not differ significantly among the different tumor types. There were 19 gastrointestinal tumors (6 colorectal, 5 gastric, 5 esophageal and 3 pancreatic carcinomas), 18 skin cancers, 17 lung cancers, 12 oropharyngeal cancers, 11 lymphomas, 11 prostatic cancers, 4 renal cell cancers, 4 breast cancers, 3 bladder cancers, 2 uterine cancers, 1 chondrosarcoma, 1 Kaposis sarcoma, 1 de novo HCC and 1 in situ carcinoma of the conjunctiva. The 1-, 5- and 10- year survival rates after the diagnosis of the first tumor were 76.0%, 49.6% and 46.3%, mean surEur Surg Vol. 40 Supplement Nr. 226 2008

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vival was 6.6 years. During the study period 37 of the tumor patients (39%) died due to their malignancy. Patients with gastrointestinal tumors, lung cancer, oropharyngeal tumors or lymphomas had a significant worse survival than patients with other tumors. Interestingly, the overall 1-, 5-, 10- and 15-year survival rates after LT were 99%, 80.8%, 59.4% and 34.8% in tumor patients and therefore not significant lower than in tumor free patients. Tumor patients were significantly older (p ¼ 0.004) and more often smokers (p ¼ 0.005). No correlation was seen regarding gender, etiology of liver disease, inflammatory bowel disease, malignancy or alcohol abuse before LT and use of monoclonal antibodies for immunosuppression. The overall standardized mortality ratio (SMR) for de novo malignomas was 1.9 (95% confidence interval 1.5– 2.4) and therefore significantly higher compared to the general population. The highest risk was found for lymphomas (SMR ¼ 8.1) and oropharyngeal cancer (SMR ¼ 5.5), while prostatic cancer did not occur more often than in the general population (SMR ¼ 0.9). In LT patients, gastrointestinal (60 vs. 72 years), oropharyngeal (55 vs. 60 years) and lung cancer (63 vs. 67 years) developed at a younger age than in the non transplant population. Conclusions. Our results confirm that de novo malignomas are an important complication after LT with a significantly increased incidence in this patient cohort. Careful long-term screening protocols are important to facilitate diagnosis at an early stage of disease and to provide better therapeutic options.

29 The Vienna experience with HU heart transplantation M. Grömmer, S. Mahr, A. Aliabadi, D. Zimpfer, A. Zuckermann Division of Cardio-Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. Mortality on the waiting list before cardiac transplantation is still a problem. In the Eurotransplant era, high urgency status is a method to increase the chance of transplantation in those patients who have the highest risk to die before transplant. This analysis evaluates outcome of high urgent patients in Vienna. Methods. Between 2000 and June 2008 28 (20% of total waiting list population) patients, waiting for heart transplantation, were listed as high urgent in Vienna. Causes of HU-listing were divided into 4 groups (inotropic dependent ICU patient (ICU), acute Retransplantation due to primary graft failure (Re-TX), pediatric patient in ICU (Ped) and VAD complications (VAD). Time on waiting list, wailting list survival and post-transplant outcome were calculated. Results. Twelve patients (43%) were ICU patients, 7 in the Ped group, 5 in the VAD group and 4 in the ReTX group. Patients were listed for heart transplantation Eur Surg Vol. 40 Supplement Nr. 226 2008

1.5 months before receiving HU status. As HU patients, waiting time was 13.2 days till transplantation. Between 2000–2004 only 6 patients received HU status, compared to 22 between 05 and 08. Mortality during this time period was 21.4%; 5 patients (17.8%) were removed due to different reasons (2 VAD implant, 3 stabilisation). 60.7% (n ¼ 17) patients were transplanted with HU status. 30 day and one year survival after HU transplant was 82.3% and 70.6%, respectively. 60% of patients died of infection. Conclusions. The selective use of HU status, is associated with acceptable outcome after HU transplant in this very sick patient group. Mortality on the waiting list is low and time to transplant has been decreased. The increase of HU requests lately has been associated with increase VAD complications due to longer VAD bridge times.

30 Does tacrolimus predispose for type 1 allergy after organ transplantation? S. Gruber1, K. Tiringer1, T. Eiwegger1, H. Konstantin2 , W. Hörl2 , A. Goll3 , Z. Szepfalusi1 1 Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria; 2 Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria; 3 Department of Medical Statistics, Medical University of Vienna, Vienna, Austria

Background. Solid organ transplantation requires lifelong immunosuppresive therapy. The immunosuppressive drugs applied target mainly the T cell-mediated immune response. As these cells also play an essential role in type 1 hypersensitivity, it should be expected that transplant recipients would not display allergic disease. However, type 1 allergies have repeatedly been reported after organ transplantation. A causal relationship to tacrolimus immunosuppression has been proposed by various authors. We recently performed a study in lung-transplanted adults, which showed a higher prevalence of type 1 allergy in tacrolimus-treated patients than in patients receiving other immunosuppressants. This difference was not significant in statistical analysis. The present study therefore aims to compare the occurrence of allergic disease under tacrolimus to that under cyclosporin A in a larger patient sample. Methods. The prevalences of IgE-mediated sensitization and allergy are assessed in 200 kidney-transplanted adults (100 patients receiving tacrolimus, 100 cyclosporin A). Mechanisms include a standardized questionnaire, skin prick test, and measurement of total and specific IgE against common nutritional and inhalant allergens. Results. To date 191 patients (100 tacrolimus, 91 cyclosporin A) have been evaluated. The groups are comparable regarding sex, age and underlying disease. Both sensitization and allergy rate are markedly higher in the

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tacrolimus-group than in cyclosporin A-receiving patients (33% and 20%, vs. 15.31% and 9.2%). Conclusions. Our results suggest that tacrolimus-immunosuppression is associated with an increased risk for the occurrence of IgE-mediated sensitization after organ transplantation. Further in vitro studies assessing the underlying mechanisms are required.

32 T-cell senescence and contraction of T-cell repertoire diversity in patients with chronic obstructive pulmonary ﬁsease S. Hacker1, C. Lambers2 , M. Posch3 , K. Hoetzenecker1, A. Pollreisz1 , W. Klepetko1 , H. J. Ankersmit1

31 Evaluation of pretransplant changes in creatinine and glomerular ﬁltration rate as indicator for long term renal function in liver recipients G. Györi, C. Burghuber, T. Soliman, R. Steininger, F. Mühlbacher, G. Berlakovich Division of Transplantation, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. Aim of this study was to analyse the predictive value of pretransplant changes in serum creatinine (Delta-SCr) and glomerular filtration rate (GFR) on post transplant renal function. Methods: Included were all adult patients from a single center receiving their 1st liver graft between 1999 and 2006 (n ¼ 269). All received standard immunosuppressive regimen with Thymoglobuline induction for 3 days, low dose calcineurininhibitors from day 4 and rapid steroid taper. GFR was calculated using the MDRD formula. Serum Creatinine (SCr) was measured 3 months (mo), 2 mo, 1 mo pretransplant and 1a after transplantation. Renal impairment (RI) was defined as SCr above 1.5 mg=dl or GFR lower 60 ml=min. Data were collected prospectively in our Transplant Surgery database and reviewed retrospectively. Sensitivity and specificity as well as positive and negative predictive values were calculated. Results. Sixty-nine patients with RI 1a post OLT were identified. In patients with a Delta-SCr > 0.5 correlation coefficient was 0.0933, sensitivity 13%, specificity 97%, positive predictive value 67%, negative predictive value 74%. In patients with a Delta-SCr > 0.25 correlation coefficient was 0.1553, sensitivity 20%, specificity 91%, positive predictive value 46%, negative predictive value 74%. GFR 1a post-OLT was

> 60 in 137 patients, < 60– >40 in 95 patients and < 40 in 37 patients

Correlation coefficient of GFR 3 mo, 2 mo, 1 mo pre OLT to 1a post OLT was 0.4190, 0.5025, 0.4953, 0.4385 respectively. Subgroup analysis of patients with renal impairment after 1a (n ¼ 132) showed a correlation coefficient of 0.4964, 0.4460, 0.2925, 0.1893 respectively. Conclusions. Neither Delta-SCr nor MDRD at 3 mo, 2 mo, 1 mo pre TX had predictive value for renal function post transplant.

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1 Department of Surgery, Medical University of Vienna, Vienna, Austria; 2 Department of Pulmonary Medicine, Medical University of Vienna, Vienna, Austria; 3 Department of Medical Statistics, Medical University of Vienna, Vienna, Austria

Background. Pathogenetic mechanisms leading to chronic obstructive pulmonary disease remain poorly understood. Clonogenic T-cells might be associated with worsening of chronic obstructive pulmonary disease (COPD). We sought to correlate systemic levels of CD4þ CD28null T-cells and their cytokine response with severity of chronic obstructive pulmonary disease. Methods. Sixty-four patients and controls were included. T-cell phenotype was analysed using flow cytometry. Enzyme-linked immunosorbent assays were utilized to determine cytokines. Statistical evaluations were performed using non-parametric group comparisons and correlations. A multivariate logistic regression model was used to determine predictive values of CD4þ CD28null in the diagnosis of chronic obstructive pulmonary disease. Results. Populations of CD4þ T-cells lacking surface co-stimulatory CD28 were significantly enlarged in evaluated patients when compared to controls. Natural-killer-like T-cell receptors (CD94, 158) and intracellular perforin, granzyme B were increased in CD4þ CD28null cells. Cytokine production after triggering of peripheral blood mononuclear cells was elevated in patients at early disease stages. Receiver operating characteristic curve plotting revealed that presence of CD4þ CD28null T-cells has a diagnostic value. Conclusions. For the first time elevation of CD4þ CD28null cells and their immunological functions are associated with chronic obstructive pulmonary disease.

33 Increased soluble serum markers ICE, ST2, caspase-cleaved Cytokeratin-18 and histones indicate apoptotic turnover and chronic immune response in COPD S. Hacker1, C. Lambers 2 , K. Hoetzenecker1, A. Pollreisz1 , A. Mangold1 , W. Klepetko1 , H. J. Ankersmit1 1

Department of Surgery, Medical University of Vienna, Vienna, Austria; 2 Department of Pulmonary Medicine, Medical University of Vienna, Vienna, Austria

Background. Chronic Obstructive Pulmonary Disease (COPD) is a worldwide burden and a major cause of Eur Surg Vol. 40 Supplement Nr. 226 2008

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death. The disease is accompanied by chronic inflammation and increased cell turnover due to apoptosis induction. We investigated the role of soluble serum proteins in patients with mild to severe COPD compared to healthy smokers and never-smokers. Methods. A total number of 64 patients were enrolled in the study. Groups (non-smoker, healthy smoker, COPD I and II, COPD III and IV) were age and sex matched. Enzyme-linked immunosorbent assays were used to evaluate concentrations of proteins in serum samples. Results. Levels of caspase-1=ICE correlated significantly with the number of pack years (p ¼ 0.007). Serum contents of apoptotic end-products caspase-cleaved cytokeratin-18 and histone-associated-DNA-fragments were increased in patients with COPD, whereas anti-inflammatory soluble Interleukin-1 Receptor 4=ST2 showed a peak in patients with COPD I and II (p ¼ 0.031) compared to healthy smokers. Conclusions. Our results indicate a systemic release of apoptosis-specific proteins as markers for increased cellular turnover accompanied with progression of COPD. Furthermore, soluble ST2 seems to play a critical role in the anti-inflammatory regulatory mechanism at early stages of the disease.

34 Elevated HSP27, HSP70 and HSP90a in chronic obstructive pulmonary disease: markers for immune activation and tissue destruction S. Hacker1, C. Lambers2 , K. Hoetzenecker1, M. Lichtenauer1, T. Niederpold1 , W. Klepetko1 , H. J. Ankersmit1 1 Department of Surgery, Medical University of Vienna, Vienna, Austria; 2 Department of Pulmonary Medicine, Medical University of Vienna, Vienna, Austria

Background. Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death. Although the underlying pathomechanism remains poorly understood, COPD is accompanied by increased cellular stress and inflammation. We investigated serum contents of heat shock proteins (HSP 27, 60, 70, 90alpha), 20S proteasomes and interleukin-6 (IL-6) in patients with mild or severe COPD and healthy smokers and non smokers. According to the literature, serum markers identifying patients with COPD have not been described. Methods. We enrolled 64 patients suffering from COPD, smoking and non-smoking controls. Groups were matched for age and sex. Severity of COPD was classified using spirometric analysis according to GOLD classification. Enzyme-linked immunosorbent assays (ELISA) were utilized to quantify serum levels of HSP27, HSP60, HSP70, HSP90, 20S Proteasome and IL-6. Results. HSP27, HSP70 and HSP90 were significantly altered in patients suffering from COPD as compared to Eur Surg Vol. 40 Supplement Nr. 226 2008

controls. A negative correlation was found between HSP27, 70 and lung function parameters. Receiver operating characteristic (ROC) curves were plotted to assess for sensitivity and specificity of HSPs to diagnose COPD. HSP27 and HSP70 may serve as novel serum markers for the diagnosis of COPD in the smoking population (area under the curve: HSP27: 0.763; HSP70: 0.885). Conclusions. This is the first study to demonstrate elevated serum levels of the described heat shock proteins in patients with COPD. These findings indicate a continuous activation of the immune system. We showed sensitivity and specificity of serum HSP27 and HSP70 as potential diagnostic markers for COPD.

35 Association of donor and recipient LCT-13910 genotypes with outcome after allogeneic hematopoetic stem cell transplantation in AML and MDS-patients H. Hauser, O. Zach, O. Krieger, J. Koenig, H. Kasparu, M. Girschikofsky, D. Lutz Department of Internal Medicine I, Hospital Elisabethinen, Linz, Austria

Background. The donor genotype of a single nucleotide polymorphism in a regulatory element of the lactase gene (LCT-13910) responsible for lactose tolerance is associated with overall survival after allogeneic hematopoetic stem cell transplantation (HSCT). Methods and results. To further evaluate the role of donor=recipient genotypes we retrospectively typed DNA from 71 consecutive patients with AML or MDS (60 AML, 11 MDS); median age 42 y (18–66); 32=39 female=male; 25=46 early=advanced stage of disease; 47 myeloablative and 27 reduced intensity conditioned; 65 peripheral blood stem cells) transplanted from 50 related and 21 unrelated donors. A lactase-non-persistent genotype (CC) was found in 11 patients (16%) and 22 donors (31%). Median overall survival was significantly shorter for patients whose donor had a lactase-persistent genotype, i.e. TC or TT (10.8 months) than for patients whose donor had a CC genotype (median overall survival not reached after 138 months, logrank P ¼ 0.0083). Multivariate analysis identified advanced stage of disease (HR 3.35, 95% CI 1.47–7.77, P ¼ 0.0041) and a donor LCT 13910 T allele (HR 5.64, 95% CI 1.69–18.81, P ¼ 0.0049) as the only two independent risk factors for death. In contrast, recipient genotypes were not significantly associated with OS (logrank P ¼ 0.2254) in this group of patients. However, patients with a CC genotype had a dramatic survival benefit when transplanted from donors with a CC genotype compared to donors with any T-allele (logrank P ¼ 0.0212). This results in a better survival for LCT–13910 CC=CC donor=recipient combinations (n ¼ 8) compared to donor=recipient pairs with any T allele (n ¼ 63; logrank

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22nd Meeting of the Austrian Society of Transplantation

P ¼ 0.0388). In this cohort containing many high risk patients the cumulative incidence of survival was 27.6% after 138 months (median survival time 11.1 months) for donor=recipient pairs with any T allele, while it was 85.7% when both donor and recipient had a homozygous C allele at the LCT–13910 locus. Conclusions. Donor but not recipient LCT–13910 genotypes were directly associated with survival. However, since the subgroup with a donor=recipient CC=CC combination had the best overall survival, there might be an association with recipient genotypes as well. Due to the small numbers in this cohort validation of these findings in an independent larger series is planned.

36 Human hand transplantation: characterization of the cellular inﬁltrate and adhesion markers in skin rejection T. Hautz1 , B. Zelger2, G. Brandacher1, H. G. Müller3, A. W. P. Lee4 , R. Margreiter1, S. Schneeberger1 1 Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria; 2 Department of Pathology, Medical University of Innsbruck, Innsbruck, Austria; 3 Department of Dermatology, Medical University of Innsbruck, Innsbruck, Austria; 4 Division of Plastic Surgery, University of Pittsburgh, Pittsburgh, US

Background. The postoperative course of human hand transplantation is mainly complicated due to problems regarding skin rejection. To contribute to the clarification of the molecular mechanisms concerned, we investigated eight key molecular markers involved in lymphocyte migration, cellular and antibody mediated rejection. Expression of FoxP3 and Indoleamine 2,3-dioxygenase (IDO), a key regulatory enzyme to induce T-lymphocyte unresponsiveness, were analyzed to better define the characterization of the cellular infiltrate. Methods. 104 skin biopsies taken from three bilateral hand transplant recipients over a period of 6 years were assessed by H&E histology and graded as per a previously published classification 0–4b. Further, biopsies were investgated by immunohistochemistry using antibodies for Lymphocyte Function-Associated Antigen (LFA)-1, Intercellular Adhesion Molecule (ICAM)-1, E-Selectin, P-Selectin, VE-Cadherin, Human Leukocyte Antigen (HLA)-II (DR), Psoriasin, IDO, FoxP3 and C4d. Levels of expression were assessed according to a 3-graded classification (0, 1, 2) and analysed with regard to severity of rejection and time after transplantation. Results. Rejection ranged between grade 0 and 4a with an average score of 0.94. In healthy skin, none of the markers was consistently up-regulated, except IDO was constitutively expressed in the vascular endothelium independent of rejection. Upon rejection, ICAM-1, Selectin-E and Selectin-P staining in endothelial cells was sig-

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nificantly increased. Expression of Selectin-E and Selectin-P correlated well with severity of rejection. VECadherin was detected on endothelial cells as well, but was not associated with rejection. The majority of infiltrating lymphocytes stained positive for LFA-1. Interestingly, also keratinocytes were positive for LFA-1 at the onset of rejection. Psoriasin staining was observed in keratinocytes as well in a basal and focal pattern and correlated well with rejection. For C4d, no consistent staining pattern was observed indicating that antibody mediated rejection did not play a role in these patients. IDO expression in the cellular infiltrate was significantly increased and correlated well with severity of rejection (grade 1, 0.91 0.85: grade 3, 2.30 0.82, p < 0.001). FoxP3 positive T-lymphocytes were mainly found in samples showing severe rejection (grade 1, 0.14 0.35: grade 3, 0.75 0.75, p ¼ 0.019). IDO expression was associated with FoxP3 expression, although the overall staining intensity for FoxP3 was lower at any time-point. Strong evidence towards higher expression of IDO and FoxP3 towards later time-points after transplantation was observed (year 1: FoxP3 0.07 0.26, IDO 0.75 0.92 [n ¼ 68], year 3: FoxP3 0.45 0.74, IDO 1.29 0.99 [n ¼ 24], year 5: Fox 0.25 0.45, IDO 1.4 0.97 [n ¼ 12]). Expression of IDO correlated closely with expression of E-Selectin, P-Selectin, ICAM-1 and LFA-1. Conclusions. Molecular markers involved in lymphocyte trafficking are up-regulated upon skin rejection after human hand transplantation. Therefore these molecules represent promising targets for prophylaxis and treatment of skin rejection after hand transplantation. Characteristics of the cellular infiltrate found in skin undergoing rejection indicate tolerogenic properties of a proportion of the cells. Thus, a tendency towards self-limitation of the alloimmune response towards the skin with both time after transplantation as well as severity of rejection can be speculated.

37 Unexpected malignancies in explanted lungs – incidence and outcome. The experience of the Vienna Lung Transplant Program M. A. Hoda, P. Jaksch, S. Taghavi, G. Lang, C. Aigner, A. Scheed, W. Klepetko Division of Cardio-Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. The detection of malignancy in a potential lung transplant recipient poses an absolute contraindication for transplantation. However undetected neoplasms in explanted lungs at transplantation are a rare finding and may effect survival of these patients. We describe our experience with the incidence and survival of undetected primary neoplasms in explanted lungs. Eur Surg Vol. 40 Supplement Nr. 226 2008

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Methods. Between 1989 and 2006 710 primary lung transplantations were performed at our institution. Among these patients we identified 7 patients with a pretransplant undetected malignancy in the explanted lungs. We collected and retrospectively reviewed the files of these patients. Results. Demographics: 3 women and 4 men with a mean age of 53.7 10.5 (range 32–65 years) received 1 single-lung and 6 bilateral lung transplants. The underlying diagnoses for transplantation were in 6 cases COPD and 1 case cystic fibrosis. Among the newly detected neoplasms in the explanted lungs the diagnosis was adenocarcinoma in 4 patients, squamous cell carcinoma in 2 patients and carcinoid in 1 patient. All patients underwent post-transplant staging and no incidence of lung or distal metastasis was detected. Restaging was performed on a yearly basis during follow up. Mean follow up was 1240.3 859.7 (range 576–2974 days). All 7 patients are still alive. No patient died of any other complication. All neoplasms were detected in an early stage (stage I, T1, No). No evidence for tumor recurrence was detected at any point of time during follow up. Conclusions. Malignancies in explanted lungs at transplantation are rare, with an incidence of 1% in all our patients. Adenocarcinoma was the most common cell type. The unexpected finding of a T1,N0 tumor in the explanted lungs does not to influence the further prognosis. For this reason no adjuvant chemotherapy is suggested.

38 Prognostische Wertigkeit des Brain Natriuretic Pepide A. Juraszek, T. Dziodzio, S. Rödler, M. Czerny, R. Gottardi, M. Grimm, A. Zuckermann Klinische Abteilung für Herz-Thoraxchirurgie, Universitätsklinik für Chirurgie, Medizinische Universität Wien, Wien, Österreich

Grundlagen. Ziel der Studie war die Evaluierung der Wertigkeit von Brain Natriuretic Peptide (BNP) bei Patienten nach Herztransplantationen (HTX) und dessen ¨ tzlichkeit bei der Ermittlung der Graftsklerose fu¨r den Nu klinischen Verlauf. Methodik. Eine Gruppe von 48 Patienten (post HTX) die sich aufgrund einer wirksamen Graftsklerose einer invasiven Revaskularisation unterziehen mussten, wurden mit einer Gruppe von Patienten nach HTX ohne wirksame Graftsklerose 1:1 gematcht. Die Gruppen unterschieden sich hinsichtlich Alter, Geschlecht, Patienten und Spendergeschlecht, Diabetes mellitus, Hyperlipida¨mie, Ventrikelfunktion (LVF), Hypercholesterina¨mie, Hypertonie, Cytomegalovirus Status und Grunderkrankung nicht. Patienten, die Abstoßungsreaktionen zeigten, wurden ausgeschlossen. Die BNP-Werte wurden nach einer mittleren Follow-up-Zeit von 72 Monaten nach der HTX analysiert. Es wurden zusa¨tzlich die mittleren Eur Surg Vol. 40 Supplement Nr. 226 2008

BNP-Werte von Patienten mit unstabilen (n ¼ 17) und stabilen (n ¼ 15) Graftklerose im Zeitraum von 6 Monaten verglichen. Ergebnisse. Die mittleren BNP-Werte von Patienten mit manifester Graftsklerose waren signifikant (3028 pg=ml vs. 493 pg=ml, p < 0,001) ho¨her als bei Patienten ohne Graftsklerose. Bei 89,58 % der Patienten mit Graftsklerose ist der mittlere BNP-Wert ho¨her als bei der Kontrollgruppe. Bei den Patienten mit unstabilen Graftsklerose liegt der mittlere BNP-Wert in sechsmonatigen Nachbeobachtungszeit bei 3977 3051 pg= ml und bei den Patienten mit stabilen Graftsklerose bei 1655 2040 pg=ml. Schlussfolgerungen. Das BNP ist ein wertvoller Mark¨r Patienten nach einer HTX in Hinblick auf die Erkener fu nung und die Nachsorge einer wirksamen Graftsklerose. Weiters stellte sich heraus, dass der BNP-Wert auch im Langzeit-Follow-up fu¨r Patienten nach Stentimplantation ¨ tzlicher Marker dient. und PTCA-Eingriffen als nu

39 Antithymocyte globulin use for treatment of biopsy conﬁrmed acute rejection is associated with prolonged renal allograft survival A. Kainz1;2 , A. Soleiman3 , B. Mayer4, R. Oberbauer1;2;5 1 Department of Nephrology, Krankenhaus der Elisabethinen, Linz, Austria; 2 Department of Nephrology, Medical University of Vienna, Vienna, Austria; 3 Department of Pathology, Medical University of Vienna, Vienna, Austria; 4 Emergentec Biodevelopment GmbH, Vienna, Austria; 5 Austrian Dialysis and Transplant Registry, Austria

Background. Antithymocyte globuline (ATG) and OKT3 have been used for the treatment of severe biopsy confirmed acute renal allograft rejection (BCAR). It remains elusive, however, which therapy is more efficient in terms of hard outcomes such as graft and patient survival. Methods. We identified all 399 subjects in the Austrian Dialysis and Transplant Registry who were diagnosed with BCAR between 1990 and 2005 and were treated with either ATG or OKT3. Multivariable analyses including Banff scores were performed by using three different strategies to account for confounding by indication, propensity scores, an automated approach and a clinical expertise model. Results. Half of the subjects in the OKT3 group exhibited a functioning graft at 6.3 years after the diagnosis of BCAR but 74% of the ATG patients grafts were still functioning at that time (logrank p ¼ 0.006). Median actual graft survival was only 4.6 years in the OKT3 subjects but 9.5 years for ATG treated patients (log rank p ¼ 0.004). Multivariable analysis revealed that the risk for functional graft loss was significantly elevated in the OKT3 vs. ATG

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patients (HR ¼ 1.79, 95%CI 1.06–3.02, p ¼ 0.029). The risk for actual graft, counting death as event, was also significantly elevated in the OKT3 patients (HR ¼ 1.73, 95%CI 1.09–2.74, p ¼ 0.019). The hazard of death was not different between groups (HR ¼ 1.55, 95%CI 0.87–2.77, p ¼ 0.137). Conclusions. These data suggest that rejecting renal allografts treated with ATG exhibit longer graft survival than OKT3 treated transplant kidneys. Causal inference, however, can not be drawn from this associational study.

40 Regulation of the RNA inhibition component Dicer in acute lung transplant rejection

Results. Immunohistochemical scoring of allograft biopsies revealed a high homogenous overall expression level of microRNA processing proteins in normal bronchiolar epithelia, pneumocytes and endothelial cells. Dicer expression was high within the parenchyma of the primary biopsies without rejection. A reduction of Dicer expression was observed in pneumocytes and bronchioles of acute rejection where loss of Dicer was restricted to areas of intense mononuclear inflammation. No alterations of Dicer expression were observed in blood vessels with or without rejection or in non-rejection related parenchymal damage. Conclusions. The key regulatory factor of microRNA biogenesis, Dicer, is regulated in pneumocytes and bronchioles within areas of mononuclear rejection indicating a local defect in microRNA biogenesis coinciding with the site of the cellular rejection process. Loss of Dicer indicates a rejection-driven impairment in RNA inhibition that might seriously affect the transplant response to immunological injury.

M. Keplinger1, P. Jaksch1 , B. Zweytizk1 , M. Saemann2 , W. Klepetko1 , A. Soleiman3 1 Division of Cardio-Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria; 2 Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria; 3 Department of Pathology, Medical University of Vienna, Vienna, Austria

Background. Pulmonary tissue is a major known site of physiological microRNA-based cell regulation involved in tissue morphogenesis and cell differentiation. MicroRNAs mediate post-transcriptional regulation of protein expression by mRNA silencing. However, RNAinhibition in the early post-transplant period of lung transplantation is a yet uncharted territory. An involvement of microRNAs in transplant rejection has been suggested in other organs namely through changes in the central regulator of microRNA biogenesis, the cytosolic RNase III, Dicer. Since Dicer down-regulation leads to a global repression of small RNA biogenesis the level of Dicer expression directly affects cell cycle and functional differentiation of parenchymal cells. Alterations of Dicer expression during lung transplant rejection in damaged cellular compartments have not been investigated to date. Methods. Diagnostic lung allograft biopsies from 24 lung transplant patients performed routinely without clinical dysfunction or histopathological rejection and consecutive biopsies of the same patients with allograft dysfunction with cellular rejection of A2 or higher (ISHLT95) were subjected to immunohistochemical analysis for Dicer protein. In parallel, serial biopsies from 15 patients without dysfunction and histological rejection were analyzed in the same way. All patients were devoid of clinical, serological or histological signs of concomitant infection to avoid interference with Dicer regulation in infection. Read out was made by semi-quantitative scoring of immunohistochemical signals in the different cellular compartments of the transplant organ. Dicer expression was analyzed for correlation with functional and histological parameters as well as patient outcome.

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41 The impact of Cyclosporine, Tacrolimus and Sirolimus on cellular senescence in renal tubule cells and ﬁbroblasts. A possible complicity in chronic GR G. Kern1 , P. Jennigs2 , M. Schaumeier1, W. Pfaller2, G. Mayer1, C. Koppelstaetter1 1 Division of Nephrology and Hypertensiology, Department of Internal Medicine IV, Medical University of Innsbruck, Innsbruck, Austria; 2 Division of Physiology, Department of Physiology and Medical Physics, Medical University of Innsbruck, Innsbruck, Austria

Background. The nephrotoxic potential of the calcineurin inhibitors Cyclosporine A (CsA) and Tacrolimus (FK506) is well recognized. Nevertheless, CN-inhibitors not only have been linked with toxicity, but also with cellular senescence and epithelial-to-mesenchymal transition (EMT), which both hinder parenchymal tissue regeneration and thus prime kidneys for subsequent insults. To minimize pathological effects on kidney grafts, alternative immunosuppressants like Sirolimus have been introduced more frequently. Methods. We compared the effects of CsA, FK506 and Sirolimus on human renal tubule cells and fibroblasts. Telomere length (by real time PCR), DNA synthesis (by BrdU incorporation), cell viability (by Resazurin conversion), gene expression (by RT-PCR), senescenceassociated (beta)-galactosidase (SA-beta-gal) activity and H2O2 production (by Amplex Red conversion) were evaluated. Results. H2O2 production was induced by all three immunosuppressants, although CsA lead to a more significant increase of H2O2 than FK506 and Sirolimus. Eur Surg Vol. 40 Supplement Nr. 226 2008

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Cyclosporine caused the most significant reduction both in telomere length (14%, p ¼ 0.063) and in BrdUincorporation (p 0.05) compared to FK506. SA-betaGal-staining revealed a time- and dose-dependent increase by CsA and at a higher level by FK506. Sirolimus on the contrary induced a decrease in SA-beta-gal activity. Senescence associated genes were in parts regulated in renal tubule cells but not in fibroblasts whereas no regulation in EMT-associated genes was observed in both cell types. Conclusions. In summary, renal tubule cells exposed to CsA and FK506 show clear signs of cellular senescence. No such effects could be observed in fibroblasts after the same treatment. Posttransplantative complications like chronic renal allograft disfunction may be partially induced by senescence of epithelial cells and a concomitant overgrowth of residential fibroblasts. The deceleration of cellular senescence by Sirolimus may contribute to the beneficial effects of a immunosuppression-switch.

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vival of 97.6% and a 5-year survival of 89.6%. Graft survival was 78.3% at 1 year and 60.2% at 5 years. Acute rejection was observed in 17 of the 42 patients (21 rejection episodes in total; 6 were C4d positive, 15 interstitial). Rejection lead to graft loss in 8 cases. In patients receiving a fourth renal transplant 1-year and 5-year patient survival was 77% and 68%, respectively. Two patients died of sepsis, 1 of acute liver failure and 1 of acute myocardial ischemia. Graft loss occurred in 7 of the 15 kidney transplant recipients. Conclusions. Third kidney transplantation yields satisfactory patient and graft survival rates. Renal fourth transplantation can be successful although the risk of allograft rejection and consecutive graft loss is high.

43 Serum catalase as marker of graft-versus-host disease in allogeneic stem cell transplantation B. Kircher, P. Schumacher, J. Clausen, D. Nachbaur

Single-center experience in third and fourth kidney transplantation

Division of Haematology and Oncology, Department of Internal Medicine V, Medical University of Innsbruck, Innsbruck, Austria

K. Kienzl, C. Bösmüller, G. Brandacher, R. Öllinger, M. Maglione, R. Margreiter, W. Mark

Background. Oxidative stress resulting from increased production of free radicals and reactive oxygen species may be involved in the pathogenesis of treatmentrelated morbidity and mortality after hematopoietic stem cell transplantation (HSCT). The reactive oxygen species content, however, is balanced by antioxidative systems which includes enzymes such as glutathione peroxidase, superoxide dismutase and catalase. Methods. In order to investigate these repair mechanisms we have analyzed the catalase levels of 22 patients with hematological malignancies undergoing allogeneic HSCT from HLA-matched donors. Catalase levels were measured in serum at various time points before and after HSCT and were expressed as median levels standard error. Results. Serum catalase levels of 16 patients determined on days -4=-5 before HSCT (146.9 46.4 mM) were significantly higher than those of eight healthy normal controls (46.7 2.1 mM; p< 0.005) indicating enhanced repair mechanisms present in leukemia patients already before chemotherapy start. During conditioning catalase levels decrease and rise again when leukocyte counts begin to increase. The catalase levels of patients developing acute graft-versus-host disease (GvHD) are, with two exceptions, at each time point higher than the catalase levels of patients without this complication. The median increase of catalase at onset of acute GvHD was 1.4 0.3, whereas no increase (1.0 0.3) was observed for patients without GvHD on days þ18 until þ21 (represent the median days of GvHD occurrence). Patients were further divided into a group with an increase of catalase levels higher or lower than 1.5. The cumulative incidence of

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

Background. Patients undergoing kidney retransplantation have a higher immunological risk than first renal allograft recipients. Especially in the population of third and fourth allograft recipients, data on patient and graft survival are rare. Methods. We retrospectively investigated 57 recipients of third and fourth kidney transplants in our center since 1997. For descriptive statistical analysis, mean values, standard deviations, absolute and relative frequencies were calculated. Patient and graft survival was evaluated according to Kaplan Meier survival statistics. Results. Among the 57 patients evaluated were 42 third and 15 fourth kidney transplantations. Mean age was 43.7 10.2 years. Recipients received kidneys from 5 living and 52 cadaveric donors with a mean donor age of 45.8 13.8 years. High immunological risk was defined by PRA > 5% and was detected in 39 patients with a mean of 40.4 29.6% and 50.7 23.0% preformed anti-HLA antibodies in third and fourth transplantations, respectively. Before 2001, immunosuppression was based on a triple regimen (CNI, MMF=Aza, steroids) IL2-R-antagonist or ATG induction therapy. Since then, initial immunosuppression consisted of alemtuzumab induction, FK506 and steroid treatment additional rituximab, MMF or plasmapheresis=immunadsorption. Patients with a third kidney graft displayed a 1-year patient surEur Surg Vol. 40 Supplement Nr. 226 2008

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developing acute GvHD was 83% (58–100%, 95% confidence interval [CI]) for patients with a 1.5 times higher catalase increase in comparison to 44% (25–76%, 95% CI) for patients with a lower catalase increase (p ¼ n.s.). Within this cohort we have lost only two patients due to treatment-related mortality and only two patients experienced a relapse so that these analyses could not be performed. The cumulative survival after 2.3 years was 56% (32–81%, 95% CI) for patients without a catalase increase and 17% (0–46%, 95% CI) for patients with an increase >1.5 (p ¼ 0.06). Six patients received dosis-reduced conditioning. Interestingly, catalase levels of this cohort were, with two exceptions, at each time point higher than the catalase levels of the 16 patients treated with a myeloablative regimen suggesting that the intensity of the conditioning regimen is not influencing catalase levels. In the myeloablative cohort catalase levels were, at most time points, higher in patients developing GvHD in comparison to patients without this complication. The cumulative incidence of acute GvHD was 46% (26–83%, 95% CI) for the cohort without a catalase increase whereas all three patients with an increase >1.5 developed acute GvHD. High catalase levels were again indicative for a reduced survival (33%, [0–87%, 95% CI] versus 62% [35–88%, 95% CI] in the low catalase increase cohort; p ¼ n.s.). Conclusions. In conclusion, this study revealed substantial catalase levels in sera of patients undergoing allogeneic HSCT which were enhanced during the occurrence of acute GvHD. However, larger number of patients and analysis of further antioxidative enzymes are needed to elucidate the chemistry of the antioxidative process during HSCT.

44 The distinct roles of the donor and recipient CD40 costimulation pathways in a mixed chimerism model C. Klaus, N. Pilat, E. Schwaiger, M. Gattringer, F. Muehlbacher, T. Wekerle Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. Bone marrow transplantation (BMT) together with costimulation blockade induces mixed chimerism and tolerance in different protocols with (antiCD154 [CD40L] mAb) being an essential component in all of them. Anti-CD154 mAbs are not available for clinical application, as their use was associated with severe thromboembolic side effects in early clinical studies. We therefore aimed to better define the role of the CD40CD40L pathway, and to examine if targeting=blocking donor CD40 alone would be a viable alternative to antiCD40L therapy. Methods. Varying strain combinations were employed: donors were wild type Balb=C or CD40KO Balb=C and recipients were wild type C57BL=6, CD40KO C57BL=6

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or CD154KO C57BL=6. Recipients received approximately 20 mio. fully mismatched Balb=C bone marrow (BM) cells (d0) and costimulation blockade consisting of CTLA4Ig (d þ 2) with or without MR1 (d0): 1) WT Balb=C to CD154 KO C57BL=6, 2) CD40KO Balb=C to CD40KO C57BL=6 and 3) CD40KO Balb=C to WT C57BL=6 (n ¼ 7– 8=group). Multi-lineage chimerism was followed by flow cytometry. Tolerance was assessed by grafting donor and 3rd party skin and verified through mixed lymphocyte reactions (MLRs). Results. Mixed chimerism and donor-specific skin graft tolerance (3rd party grafts were promptly rejected) developed without anti-CD154 mAb in BMT recipients deficient in CD154 (group 1). If both donor and recipients are deficient in CD40 (group 2) chimerism and tolerance also were established. In contrast, if only the donor lacks CD40 no chimerism and no skin graft tolerance developed without anti-CD154 (group 3) (p < 0.05). Besides, recipients of group 3 demonstrated anti-donor reactivity in MLRs at the time of take-down (p < 0.05 compared to self). Conclusions. These data reveal that interference with the CD40 costimulation pathway on donor APC alone would not be enough to allow engraftment of allogeneic BM. Blocking recipient CD40L (without triggering a signal in the target cell and without target cell destruction) is sufficient in this BMT model to establish chimerism and tolerance.

45 It is also a matter how to survive – A case of a septegarian liver transplant recipient D. Kniepeiss1 , D. Duller1, R. Roller2, H. Müller1, K. Tscheliessnigg1 , F. Iberer1 1

Department of Surgery and Transplantation, Medical University of Graz, Graz, Austria; 2 Department of Internal Medicine, Medical University of Graz, Graz, Austria

Background. Advanced age is considered a relative contraindication to solid organ transplantation because of increased morbidity and mortality especially from cardiac disease or infection. Case report. In December 1998, a 70-year-old woman with cirrhosis hepatitis (Child s C) due to hepatitis C was evaluated for liver transplantation (LTX) at our centre. As no contraindication and no relevant comorbidities were detected liver transplantation was performed. On day 2 after LTX liver parameters increased dramatically and angiography showed a hepatic artery thrombosis resulting in re- transplantation. Although during her 29 days stay at the intensive care unit liver function stabilized gradually, patients early recovery was disrupted by acute renal failure and bacterial pneumonia. The whole hospitalization lasted 52 days, followed by extensive therapeutic treatment for re-establishment of mental and physical performance. Eur Surg Vol. 40 Supplement Nr. 226 2008

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Patients ongoing history was marked by different infectious episodes under immunosuppressant maintenance mono- therapy with FK506 at a through level of 3–6 ng=mL. Organ transplant function was not affected critically throughout the whole time as monitored by laboratory values and ultra- sonography. As a consecutive co-morbidity non-insulin dependent diabetes mellitus developed 42 months following LTX. During her whole post-transplant life our patient did not receive supportive care for daily living. Being together with her spouse in their house, she was able to regain all parameters attributing to individual quality of life. From Geriatric assessment parameters of cognition, as measured by Mini Mental Status by Folstein (MMSE), functional abilities monitored by Instrumental activity of daily living (IADL), and mobility scoring by Tinetti showed quite persistent scorings over a period of 9 years following transplantation. The actual scorings during the last patients visit at our department were MMSE 30, IADL 16 and Tinetti 14, respectively. Conclusions. Understanding the additional life-years given to patients is necessary as a substantial investment, as the number of organ donors is deceased. Therefore introduction of quality of life measures and assessment procedures for evaluation of individual physical and psychosocial functionality during recruitment for LTX, especially in elderly patients, need to be established in transplant centres. Interdisciplinary collaboration has worked out with increasing post-transplant results in our hands and should also be considered elsewhere.

46 Through concentrations and dose requirement of immunosuppressive agents in relation to CYP3A5 genotypes after heart transplantation D. Kniepeiss1 , W. Renner2, A. Wasler1, G. Koshsorur2, D. Duller1, F. Iberer1, K. Tscheliessnigg1 1

Division of Transplantation, Department of Surgery, Medical University of Graz, Graz, Austria; 2 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria

Background. Tacrolimus and Everolimus are immunosuppressive drugs which are used worldwide in solid organ transplantation to prevent rejection. However, these drugs have a narrow therapeutic range and intensive drug monitoring is necessary to reach optimum target concentrations. Both Tacrolimus and Everolimus are metabolized by enzymes of the CYP3A subfamily. A common variant of the CYP3A5 gene, CYP3A5*3, results in strongly decreased CYP3A5 activity and has been shown to influence Tacrolimus blood concentrations in patients after renal transplantation, but its role for the pharmacogenetics of Everolimus remains unclear. Aim of the presEur Surg Vol. 40 Supplement Nr. 226 2008

ent study was to examine the role of CYP3A5*3 variant in Tacrolimus and Everolimus dose and drug levels in heart transplant recipients. Methods. The present study comprised 15 patients with Tacrolimus and 30 patients with Everolimus based maintenance therapy after heart transplantation. Individual levels and doses of Tacrolimus or Everolimus were documented at month 1, 3, 6 and 12 after medication start. CYP3A5 genotypes were determined by a 5’exonuclease (TaqMan) assay. Results. In the Tacrolimus group, 13 subjects were CYP3A5 non-expressors (*3=*3 genotype) and two subjects were heterozygous expressors (*1=*3). Tacrolimus dose was higher in subjects expressing CYP3A5 compared to non-expressors (month 1: p ¼ 0.076, month 3: p ¼ 0.019, month 6: p ¼ 0.019, month 12: p ¼ 0.030). Tacrolimus levels were not significantly different at any point of time. In the Everolimus group, 27 subjects were CYP3A5 non-expressors (*3=*3 genotype) and three subjects were heterozygous expressors (*1=*3). Neither Everolimus dose nor levels were significantly different between CYP3A5 expressors and non-expressors at any point of time. Conclusions. Our study demonstrated a strong correlation between CYP3A5 genotypes and Tacrolimus dose requirement, but showed no association concerning Everolimus. Since inadequate immunosuppression leads to graft rejection, evaluation of CYP3A5 polymorphism may be helpful in determining an appropriate starting dosage, rapidly achieving adequate immunosuppression and ultimately improving the outcome of heart transplantation in patients administered Tacrolimus. Pharmacogenetic analysis before transplantation can easily be done and may assist in guiding individual immunosuppressive therapy.

47 Non-HLA anti-endothelial cell antibodies do not lead to kidney injury N. Kozakowski1 , N. Huttary1, G. Böhmig2 , A. Auer1, T. Fahim3 , H. Regele1 1

Department of Pathology, Medical University of Vienna, Vienna, Austria; 2 Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria; 3 Department of Ear, Nose and Throat Diseases, HNO, Medical University of Vienna, Vienna, Austria

Background. Rejection of allografts is primarily mediated by (T-)cellular immune responses. Several lines of evidence suggest that alloantibodies are also potent mediators of tissue injury and their interaction with endothelial cells (EC) is of crucial importance in allograft rejection. MHC antigens are the most important targets of alloantibodies in blood group matched renal allografts. Several reports however also indicate the occurrence of clinically relevant non-HLA anti-endothelial alloantibodies. These antibodies might escape detection by conven-

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tional serological tests aimed at detection of anti-HLA antibodies. Methods. In order to directly detect non-HLA antiendothelial antibodies we isolated EC from kidney donors for flow-cytometric crossmatch testing (EC-FCXM) with serum samples from the respective donors. In parallel we also performed flow-cytometric T-cell crossmatches (T-cell-FCXM) and FlowPRA tests in order to detect anti-HLA antibodies. We analysed serum samples drawn before transplantation and at 1, 3, 6, 12 and 24 months after transplantation in a total of 75 kidney allograft recipients. In 49=75 patients reactive antibodies could be detected by at least one of the applied methods: Fortyone patients were positive in either T-cell-FCXM or FlowPRA and 26 patients were positive in EC-FCXM. In 8=75 patients we identified non-HLA antibodies reactive with EC (but not with T-cells or FlowPRA beads). Results. Transplant biopsies were available in only 2 of these 8 patients and did not show intra-graft complement activation (C4d negative). In contrast, 5=11 biopsies were C4d positive in the 18 patients with anti-HLA antibodies binding to EC (positive in T-cell-FCXM or FlowPRA and EC-FCXM). In 23 patients with detectable circulating anti-HLA antibodies, which however did not bind to the recipient’s EC in vitro, none of the 12 available biopsies was C4d positive. Patients with non-HLA anti-EC antibodies did not display a higher rate of graft loss or clinical signs of graft dysfunction at two years after transplantation. Conclusions. Based on these results we conclude that non-HLA anti-endothelial antibodies may be present in app. 10% of kidney allograft recipients. These antibodies however, in contrast to anti-HLA antibodies, do not lead to capillary complement activation and are not associated with inferior graft outcome.

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Non-heart-beating kidney donation: a single centre experience during 24 years

Radioimmunotherapy: hopeful treatment in refractory diseases

I. Kristo, M. Hofmann, M. Pones, C. K. Burghuber, G. P. Györi, R. Steininger, F. Mühlbacher

G. Kropshofer1, A. Schulz2 , W. Friedrich2 , A. K. Franke1 , B. Meister1

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

1 Devision of Pediatrics II, Department of Children and Adolescent Medicine, Medical University of Innsbruck, Innsbruck, Austria; 2 Department of Children and Adolescent Medicine, University of Ulm, Ulm, Germany

Background. The gap between performed kidney transplantations and active organ needs has resulted in alternative ways to increase the donor pool such as the procurement of non-heart-beating donor (NHBD) kidneys. In this study we present over two decades of experience with the NHBD program at our institution. Methods. All kidneys were allocated by Eurotransplant from 1984 to 2007. Demographic data of donors and recipients were collected in our transplant database prospectively and analysed retrospectively. The NHBDs were classified in accordance to Maastricht categories I–IV. Prognostic significance of cold ischemic time

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(CIT), warm ischemic time (WIT), delayed graft function (DGF), donor age, HLA-miss match and organ rejection were calculated according to graft survival. Results. A total of 81 kidneys from 48 NHBD were transplanted. One patient was lost to follow up, leaving a total of 80 patients for final analysis. The cohort consisted of 50 male (62.5%) and 30 female (37.5%) patients leading to a male female ratio of 1.7:1. The mean age of kidney recipients was 48.7 14.1 years (median 49.6 years, range 20.8–73.9 years). The mean age of NHBDs was 40.4 15.6 (median 42.0 years, range 9–66 years). The median follow up was 85.2 months (range: 2–286 months). NHBD kidneys were distributed according to Maastricht categories as followed: 4 kidneys (5%) category I, 32 kidneys (40%) category II, 23 kidneys (28.8%) category III and 21 kidneys (26.2%) category IV. Mean CIT was 17.4 6.8 h, mean WIT was 0.35 0.19 h, DGF was observed in 55 patients (68.8%) and primary non function in 10 patients (12.3%). The mean summation of HLA miss match was 2.6 1.17, acute rejections were observed in 32 patients (40%) postoperatively. Univariate analyses of prognostic factors in graft survival showed significance for WIT (p ¼ 0.01), DGF (p ¼ 0.001) and acute rejection (p ¼ 0.012), whereas none of these parameters showed significance in multivariate analyses. The estimated 5 year graft survival was 79.7%, 33 grafts at risk and the estimated 5 year patients survival was 84.1%, 44 patients at risk. Conclusions. Despite a high logistical effort the results prove that kidneys of NHBDs can be used successfully to increase the donor pool. However, this study shows that extended WIT, DGF and acute rejections have a negative impact on graft survival and therefore risk factors, such as prolonged WIT, have to be reduced by using standardised protocols.

Background. Conventional treatment including allogeneic stem cell transplantation (SCT) remains successful in most pediatric hematooncologic patients. Therefore there is a need of treatment in children with refractory disease and high comorbidity who remain not eligible for conventional transplant procedure. Methods. We report on two girls with refractory diseases. First on a 8 year old girl with cystic fibrosis and neuroblastoma stage IV. Response to chemotherapy was good and autologous SCT was performed after complete Eur Surg Vol. 40 Supplement Nr. 226 2008

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resection of the primary tumor and liver metastases. Five years later she developed a systemic relapse with extensive bone marrow infiltration, bone and liver metastases. Therefore we decided to perform an allogeneic SCT from her healthy HLA-identical sister. Considering her underlying illness and prior toxicity she received reduced intensity conditioning (RIC) with Radioimmunotherapy (RIT) (anti CD 66 antibody labelled with 90 Ytrium) followed by Fludarabin and Melphalan. RIT provided a dose of 25 Gy to the bone marrow without increasing organ toxicity. Full donor chimerism was obtained within four weeks. The second girl suffered from an Ewing sarcoma with pulmonal metastases. She received an autologous SCT and local radiation. Five years later she came down with a secondary AML. Additionally she acquired an invasive pulmonal mucormykosis and aspergillosis during induction chemotherapy. Allogeneic SCT from an unrelated matched donor was performed after reduced conditioning. Unfortunately she relapsed 4 months later, showing an increasing host chimerism. So we decided to perform a third transplant using RIT as part of conditioning (antiCD45 antibody labbeled with 90 Ytrium) also followed by Fludarabin and Melphalan. There were no complications post transplant and no signs of GVHD. She is in good clinical condition and in third remission with full donor chimerism more than a year. Conclusions. RIT offers a novel therapy with intensification of the conditioning regimen and concomitant reduction of extramedullary organ toxicity especially in heavily pretreated patients.

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intraop and rituximab (375 mg=m2 ) on day 1 post renal transplantation followed by maintenance therapy with tacrolimus, steroids and MMF (if lymphocytes reached 5% of WBC). Results. After a median follow up of 453 275 days, patient and graft survival was 87% and 78%, respectively, with excellent renal function (median creatinine 1.51 0.64 mg=dl, median urea 46.5 35.7 mg=dl). Median cold ischemia time was 15.2 6.2 h and median anastomosis time was 27 8 min. Delayed graft function as defined by required posttransplant dialysis was 39.1% Induction therapy was well tolerated, however in two patients positive for HCV antigen severe infectious complications were observed. Acute cellular rejection episodes could be controlled by bolused steroids (n ¼ 1; 4.3%). In three (13%) cases humoral rejection was suspected or diagnosed by positive C4d staining upon histology and treated, successfully in one patient, by plasmapheresis. Conclusions. Highly immunized renal transplant recipients should be considered for combined alemtuzumab=rituximab induction, which may be considered a valuable approach for successful transplantation in view of this difficult immunological setting.

51 Effect of body mass index on survival and development of cardiac allograft vasculopathy of cardiac transplant receipients: the Vienna experience

Combined Alemtuzumab and Rituximab induction therapy in highly sensitized kidney transplant recipients

S. L. Mahr, M. Grömmer, A. Aliabadi, D. Zimpfer, U. Hamscha, D. Dunkler, A. Zuckermann

M. Maglione, G. Brandacher, C. Boesmueller, R. Oellinger, P. Hengster, R. Margreiter, W. Mark

Background. The relationship between post transplant obesity or cachexia with mortality and morbidity in cardiac transplant receipients has poorly been reported yet. Methods. We retrospectively reviewed 783 consecutive adult patients (645 male, 138 female, mean age 50.8) who received primary heart transplants between January 1985 and December 2007. Of these 67 cachectic (body mass index BMI< 20 kg=m2 ), 282 overweight (BMI ¼ 25–30 kg=m2 ) and 44 morbidly obese (BMI > 30 kg=m2 ) patients were compared with 390 normal weight receipients (BMI ¼ 20–25 kg=m2 ). We evaluated the influence of pretransplant BMI as well as BMI 1 year after transplant on morbidity and mortality after cardiac transplantation. Kaplan Meier survival distribution model and Logrank test was used for statistical analysis. Results. There was a significant difference between male and female in BMI (p< 0.0001), in the cachectic group 59% male and 40% female whereas in all other groups male represented over 80% and female less than

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

Background. The increasing pool of multiple kidney graft recipients requires refined immunological strategies for HLA-sensitized patients. In this context, high levels of pretransplant panel reactive antibodies (PRA) have been associated with poor outcome due to an increased risk for acute as well as chronic rejection. Therefore, we have investigated a novel treatment protocol containing combined induction with alemtuzumab and rituximab in sensitized renal allograft recipients. Methods. In this retrospective study twenty-three patients (13 f=10 m; median age: 43 12.2 years) with median historical PRA of 78 32.5% (0–100%) and actual PRA of 41 36.1% were transplanted from 1=2006 to 12=2007 and were included in this study. Patients received induction therapy with alemtuzumab (30 mg) Eur Surg Vol. 40 Supplement Nr. 226 2008

Division of Cardio-Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria

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10% of patients. The BMI at transplant showed no significant difference on the survival at 1 month after transplant; p ¼ 0.25) and at 5, 10 and 20 years after transplantation (p ¼ 0.6) with a mean and median BMI of 24.4 and 24.3 respectively. Cardiac allograft vasculopathy (CAV) develloped significantly higher in the pre transplant obese patients (p ¼ 0.03). BMI 1 year after transplant had no impact on the long term survival (p ¼ 0.1). On the other hand a 3 unit BMI gain or loss resulted in a significant worse survival 53% at 10 years (BMI loss), 46% (BMI gain) against 70% (no BMI difference); p ¼ 0.03. BMI at 1 year after transplant showed to be a significant risk factor for development of CAV (p ¼ 0.005) whereas the BMI gain or loss of over 3 units did not affect CAV. Conclusions. A high selection before transplantation shows to be of great benefit. The low number of obese patients shows a selection bias in the BMI > 30 group. Patients who manage to maintain there weight after transplantation have a better survival, though a high BMI one year after transplant shows to be a great risk for CAV development.

52 The role of Matrix Metalloproteinase 9 and Lipocalin-2 complex in ischemia and reperfusion of the transplanted heart H. Maier1, A. Muigg2 , E. Wallnöfer2, H. Schwelberger1, J. Troppmair2, R. Margreiter1, F. Aigner1 1

Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria; 2 Daniel-Swarovski-Research Laboratory, Medical University of Innsbruck, Innsbruck, Austria

Background. Matrix Metalloproteinase 9 (MMP-9) is known to be involved in cell migration, tissue repair and remodelling. Lipocalin-2 (NGAL=Lcn-2) forms a complex with MMP-9 preventing it from degradation. Retrieved from previous data about the role of NGAL= Lcn-2 during ischemia and reperfusion (IR) following murine heart transplantation, this study focuses on the interaction between MMP-9 and NGAL=Lcn-2 in analogous settings. Methods. Male inbred C57BL=6 and the Lcn-2= mouse were used for heterotopic heart transplantations. RT–PCR, Western blot, gelatine zymography and immunohistochemistry were performed to visualize MMP-9 and NGAL=Lcn-2 expression in the graft at different time points (0, 2, 12, 24 and 48 h). Polymorphonuclear cells (PMN) isolated from wildtype and Lcn-2= mice were used for in vitro experiments. Results. MMP-9 expression was upregulated 8 fold and peaked at 48h compared to NGAL=Lcn-2 expression which peaked at 24h. Similar expression patterns were observed in the different experimental settings (Lcn-2= donor Lcn-2= recipient, wildtype donor

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and recipient and mixed wildtype=Lcn-2= transplantations). However, MMP-9 protein levels were significantly lower in Lcn-2= grafts corresponding to a reduced infiltration of PMN in Lcn-2= recipients. In vitro PMN retrieved from Lcn-2= mice showed lower levels of MMP-9 than from wildtype mice. Stimulation of PMN with recombinant NGAL=Lcn-2 did not increase MMP-9 expression. Conclusions. Our data demonstrate an interaction between MMP-9 and NGAL=Lcn-2 expression during IR in heart transplantation. Reduced MMP-9 expression in Lcn-2= grafts is directly related to the simultaneously decreased infiltration of PMN described previously, thus suggesting a chemotactic function of the MMP-9=Lcn-2 complex during IR of the transplanted heart.

53 Survival of pancreas allografts despite partial thrombosis of the Y-graft used for arterial reconstruction C. Margreiter1, W. Mark1 , M. Freund2 , D. Wiedemann1 , R. Sucher1, R. Margreiter1, F. Aigner1 1

Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria; 2 Department of Radiology, Medical University of Innsbruck, Innsbruck, Austria

Background. A complex system of arterial anastomoses between the gastroduodenal, splenic (SA) and superior mesenteric artery (SMA) exists. During pancreas transplantation arterial reconstruction between the SA and SMA a donor iliac Y-graft is used. Graft thrombosis is still responsible for up to 10% of graft losses. The aim of this study was to investigate arterial vascularization of the pancreas with special regard to the anastomoses within the pancreatic head. Methods. Ten pancreas grafts considered not suitable for transplantation were removed and a canule was inserted into the splenic artery and the SMA ligated. Angiography was performed with 5, 10 and 15 ml of contrast medium. In addition we retrospectively analyzed 15 patients with radiologically proven thrombosis of the SMA with stable graft function. Results. Angiography showed excellent perfusion of the pancreaticoduodenal arcade via anastomoses in all cases after ex-vivo injection of 5 ml of contrast medium into the splenic artery only. All 15 patients analyzed had normal graft function and sufficient perfusion of the pancreatic head. Conclusions. This study demonstrates that the whole pancreas is sufficiently perfused by the splenic artery only through a complex system of intraparenchymal anastomoses. This finding can be important not only for pancreas transplantation but also for all kinds of pancreatic surgery. Eur Surg Vol. 40 Supplement Nr. 226 2008

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54 Prevention of endothelial dysfunction as a novel approach to attenuate ischemia reperfusion injury following murine pancreas transplantation R. Oberhuber1, G. Brandacher1, M. Hermann2 , W. Mark1 , E. R. Werner3, R. Margreiter1, M. Maglione1 1

Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria; 2 Center for Islet-Cell Transplantation, Medical University of Innsbruck, Innsbruck, Austria; 3 Institute of Medical Chemistry and Biochemistry, Medical University of Innsbruck, Innsbruck, Austria

Background. Tetrahydrobiopterin (H4B) is both an essential cofactor of nitric oxide synthases (NOS) and thus a critical determinant of nitric oxide (NO) production and a strong antioxidant. When H4B is partially oxidized to dihydrobiopterin by oxidative stress NOS is known to produce oxygen radicals rather than NO by uncoupling, a phenomenon thought to contribute to endothelial dysfunction in a variety of vascular diseases. In previous work we observed dramatically reduced ischemia=reperfusion injury (IRI) following murine pancreas transplantation after donor pre-treatment with H4B. In order to differentiate unspecific reductant effect from uncoupling prevention we compared in this study H4B pre-treatment with supplementation of 4-amino-tetrahydrobiopterin (A-H4B) and tetrahydroneopterin (H4N), two pteridin analogues with similar chemical redox behaviour but no cofactor activity. Methods. Male syngeneic C57BL6 (H-2b) mice, 10– 12 weeks old, were used as size-matched donor and recipient pairs. Murine cervical heterotopic vascularized pancreas transplantation was performed with a modified no-touch technique. Pancreatic grafts were subjected to 16 h prolonged cold ischemia time (CIT) as well as 45 min warm ischemia time (WIT) and different treatment regiments: untreated animals (I), H4B 50 mg=kg i.m. prior to organ retrieval (II), A-H4B 50 mg=kg i.m. prior to organ retrieval (III) and H4N i.m. prior to organ retrieval (IV). Non transplanted animals served as controls (V). After 2 h of reperfusion intravital fluorescence microscopy was used for analysis of graft microcirculation by means of functional capillary density (FCD) and capillary diameters (CD). Quantitative assessment of parenchymal damage was analyzed by histology (H&E) and by performing nitrotyrosine-immunostaining to determine peroxynitrite formation. Intragraft pteridin levels were measured by HPLC. Results. Following prolonged CIT and 45 min of WIT only pancreatic grafts treated with H4B prior to organ explantation (II) displayed NOS stabilization and hence markedly higher values of FCD and CD compared to non treated animals (I) (p < 0.01 and p < 0.05 respectively). In contrast, pre-treatment of donor animals with A-H4B (III) as well as with the pteridin anaEur Surg Vol. 40 Supplement Nr. 226 2008

logue H4N (IV) did not improve FCD or CD levels. However, compared to non treated animals application of both pteridin analogues A-H4B and H4N significantly attenuated parenchymal damage as well as peroxynitrite formation (p < 0.05). Nevertheless, early parenchymal damage after murine pancreas transplantation was significantly reduced only by H4B pre-treatment compared to the two pteridin analogues (p < 0.05). Finally, partially oxidized H4B was significantly decreased in the H4B treatment group. Conclusions. The protective effect of H4B supplementation prior to organ procurement on microcirculation relies on its NOS cofactor activity rather than on its antioxidative capacity. H4B pre-treatment of the donor prior to organ retrieval might therefore be a novel, promising agent to attenuate IRI in clinical pancreas transplantation preventing endothelial dysfunction early after graft reperfusion.

55 Virtual histology intravascular ultrasound in late cardiac allograft vasculopathy T. Oberndorfer1, M. Frick1 , J. Doerler1, C. Mussner1, D. Hoefer2, H. Antretter2, G. Poelzl1 1

Division of Cardiology, Department of Internal Medicine, Medical University of Innsbruck, Innsbruck, Austria; 2 Department of Cardiac Surgery, Medical University of Innsbruck, Innsbruck, Austria

Background. Allograft vasculopathy, the leading cause of late mortality after cardiac transplantation, manifests as a diffuse obliterative process characterized by concentric intimal proliferation. Investigation of coronary plaque composition in vivo has become possible with the introduction of virtual histology (VH) intravascular ultrasound (IVUS). In this study we evaluated the feasibility of in vivo plaque characterization in late allograft vasculopathy using VH-IVUS. Methods. Thirty-nine patients (median age 64 years; 32–73 years) 8 years (2–16 years) after heart transplantation were included into this study. In all patients at least one coronary artery was investigated using VH-IVUS. VHIVUS data were obtained using a continuous pullback (0.5 mm=s) and a commercially available mechanical sector scanner (Eagle Eye GoldTM, Volcano Therapeutics). A region of interest (10–15 mm) was selected and analyses were done offline with pcVH-review software (Volcano Therapeutics). Four histological plaque components (fibrous, fibrolipidic, necrotic and calcified) were correlated with a specific spectrum of the radiofrequency signal. Results. Median plaque length was 13.2 mm (10– 15 mm), vessel segment volume 201 mm3 (88–374 mm3 ), and plaque-plus-media-volume 94 mm3 (35–180 mm3 ), resulting in a volume stenosis of 45% (26–62%). The remodeling index was 1.03 (0.80–1.49). VH demonstrated that 67% (38–80%) of plaque volumen was composed of

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fibrous, 12% (2–27%) of fibrolipidic, 13% (3–41%) of necrotic and 5% (1–24%) of calcified tissue. Conclusions. In vivo analysis of plaque composition in cardiac allograft vasculopathy is feasible using VHIVUS. Future studies will examine whether the addition of VH to standard IVUS may be helpful for the differentiation between early allograft vasculopathy from donortransmitted atherosclerosis, and for investigating the effects of new therapy on disease progression.

56 Bilirubin and biliverdin di-taurate for the treatment of renal ischemia reperfusion injury R. Öllinger1, M. Thomas2 , F. Bösch1 , C. Koidl3 , P. Kogler1, R. Sucher1, R. Margreiter1 1 Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria; 2 Department of Surgery, Klinikum Großhadern, Munich, Germany; 3 Department of General, Visceral and Transplant Surgery, University of Tuebingen, Tuebingen, Germany

Background. Heme Oxygenase-1 (HO-1) is playing a crucial role in ischemia reperfusion injury (IRI). HO-1 degrades heme into carbon monoxide, free iron and biliverdin, which subsequently is converted to bilirubin by the enzyme biliverdin reductase. Experimentally both, biliverdin and bilirubin, at least in part, account for the protective effects mediated by HO-1. In order to develop strategies how to potentially clinically use the bile pigments for the treatment=prevention of IRI, we tested conjugated, thus water soluble, forms of bilirubin and biliverdin at various doses and time points in a model of warm renal IRI and a model of kidney transplantation in rats. Methods. Renal arteries of contralaterally nephrectomized Wistar rats were clamped for 45 min prior to reperfusion. Further, Wistar rat kidneys were transplanted after 18 h of cold ischemia into isogenic recipients. Animals were treated with saline (as a control) or various doses of bilirubin or biliverdin di-taurate, applied as three i.v. injections covering the perioperative period or a single injection given either before or after reperfusion. Serum total bilirubin was assessed five minutes and serum urea and creatinine was assessed 24 h after reperfusion (n ¼ 4 in each group). Activation of mitogen activated protein kinases (MAPK) has been assessed by western blot and measurement of optical densities. Results. Perioperative and preoperative treatment with bilirubin di-taurate at 3, 10 and 30 but not 1 mg=kg resulted in a significant increase in serum total bilirubin levels after reperfusion (p < 0.0001 vs. control). Similarly, treatment with biliverdin di-taurate prior to reperfusion increased serum total bilirubin (p < 0.0001 vs. control). The increase in serum total bilirubin was associated with

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significantly lower serum creatinine and urea levels at 24 h after reperfusion as well as substantial changes in MAPK activation in the clamp model when compared to the control (p < 0.05) with no significant difference within the various doses. However, when the bile pigments were applied post reperfusion, no effect on kidney function was being observed. In the transplant model, application of biliverdin di-taurate at 30 mg=kg but not at 3 or 10 mg=kg significantly improved kidney function when compared to the control (p < 0.05). Conclusions. Intravenous application of water soluble bilirubin and biliverdin di-taurate is feasible and effective in improving kidney function after renal IRI. Bilirubin serum levels should be increased prior to reperfusion in order to achieve sufficient renoprotection. Bilirubin and biliverdin di-taurate may become a valuable tool to improve outcome after kidney transplantation.

57 Intensivierte ernährungsmedizinische Intervention bei Nierentransplantierten (DALMART-Studie) R. Otto1 , A. Sylle1 , M. Leopold2 , H. Holzer1, G. Wirnsberger1 1 Klinische Abteilung für Nephrologie und Hämodialyse, Universitätsklinik für Innere Medizin, Medizinische Universität Graz, Graz, Österreich; 2 Diätbüro, LandeskrankenhausUniversitätsklinikum Graz, Graz, Österreich

¨ r nierentransplantierte Grundlagen. Bislang fehlen fu Patienten Daten u¨ber die Langzeitauswirkung einer intensivierten erna¨hrungsmedizinischen Intervention auf die Transplantatfunktion. Da mit der Einfu¨hrung von Mycophenolat-Mofetil (MMF) als Basisimmunsuppressivum eine erho¨hte gastrointestinale Nebenwirkungsrate in Kauf genommen werden musste, stellte sich in dieser Studie die Frage, ob eine Besserung dieser Symptome durch eine gezielte Dia¨tintervention mo¨glich ist. Methodik. 193 nierentransplantierte Patienten wurden von Oktober 2006 bis Ma¨rz 2008 im Rahmen einer prospektiven Single-Center-Interventionsstudie einer intensivierten erna¨hrungsmedizinischen Beratung=Schulung zugefu¨hrt. In weiterer Folge wurden die Patienten u¨ber ein Jahr in regelma¨ßigen klinischen Visiten kontrolliert und relevante Laborparameter fu¨r die Nierenfunktion und dem Metabolismus erhoben. Alle Patienten nahmen MMF als Mono- oder Kombinationstherapie ein. Um Nebenwir¨ r MMF ein AUC-Bereich kungen zu minimieren, wurde fu von 30–60 mg=L.h angestrebt. Gleichzeitig wurde bei den Patienten zu Studienbeginn der Gastrointestinal-Qualityof-Life-Index evaluiert und die subjektive Befindlichkeit ¨berpru ¨ft. bei den einzelnen klinischen Visiten u Ergebnisse. Im Verlauf der Untersuchung verbesserte sich das Cystatin C als muskelunabha¨ngiger Marker fu¨r die Nierenfunktion um 0,12 mg=dl signifikant im Vergleich zu den Ausgangsdaten. 9 Patienten wurden wa¨hrend des Untersuchungszeitraums dialysepflichtig. Es fanden Eur Surg Vol. 40 Supplement Nr. 226 2008

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sich keine akuten Abstoßungsreaktionen. Der bei 180 Patienten auswertbare Gastrointestinal-Quality-of-LifeIndex zeigte einen Durchschnittswert von 106,5 Punkten von max. 144 erreichbaren. Nur ein Patient musste wegen gastrointestinaler Nebenwirkungen auf eine MMF freie Immunsuppression umgestellt werden. Schlussfolgerungen. In dieser Studie konnte gezeigt werden, dass eine intensivierte Dia¨tschulung von nierentransplantierten Patienten einen positiven Einfluss auf die Transplantatfunktion als auch auf die gastrointestinale Nebenwirkungsrate von MMF hat.

58 An irradiation-free protocol for mixed chimerism and tolerance through Treg therapy N. Pilat1 , U. Baranyi1 , C. Klaus1 , E. Jaeckel2 , E. Schwaiger1, F. Mühlbacher1, T. Wekerle1 1 Department of Surgery, Medical University of Vienna, Vienna, Austria; 2 Hannover Medical School, Hannover, Germany

Background. Routine clinical application of bonemarrow (BM) transplantation for induction of tolerance by the concept of mixed chimerism is inhibited by the requirement of cytotoxic host treatment (irradiation, cytotoxic drugs or cytotoxic mAbs). Tregs have potent effects in autoimmune and allo-transplantation models, but do not induce skin graft tolerance across MHC barriers on their own. Antigen-specific TCR-transgenic T cells retrovirally transduced to express FoxP3 exert potent suppressor function in certain autoimmune disease models, but little is known about the ability of polyclonal FoxP3-transduced T cells to suppress alloreactivity in wild-type transplantation models. We explored whether treatment with polyclonal FoxP3-transduced Tregs leads to engraftment of clinically realistic doses of allogeneic BM and donor-specific tolerance without cytotoxic host conditioning. Methods. MACS-separated C57BL=6 CD4þ cells were retrovirally transduced to express FoxP3 and GFP under the control of an internal ribosomal entry site. Regulatory function and phenotype were examined in vitro in proliferation assays and by flow cytometry. Non-irradiated C57BL=6 recipient mice received 20 106 fully mismatched Balb=c bone marrow cells (d0) under the cover of costimulation blockade (anti-CD154 mAb, MR1 d0 and CTLA4Ig d þ 2) and short-course rapamycin (d-1=0=þ 2). A separate group of BMT recipients was treated in addition with 2–4 106 FoxP3-transduced CD4 cells (d0). Multi-lineage chimerism and deletion of donor-reactive T cells were followed by flow cytometry. Tolerance was assessed by grafting donor and 3rd party skin. Results. Mice receiving additional Treg treatment developed long-term multilineage chimerism, whereas BMT recipients without Tregs uniformly failed to do so (6=7 vs 0=7 at wk 23, p < 0.01; 1.61% mean B-cell chimerism, 2.68% mean chimerism in myeloid lineage among chiEur Surg Vol. 40 Supplement Nr. 226 2008

meras). Treg-treated recipients showed profound and specific deletion of donor-reactive CD4 T cells in comparison to control group (1.87% vs 3.75% for Vbeta11 þ P < 0.05, 0.86 vs 2.46 for Vbeta5 þ P < 0.01; 13 weeks after BMT) and naı¨ve B6 mice (1.87% vs 4.96% for Vbeta11 þ P < 0.05, 0.86% vs 1.72% for Vbeta5 þ P < 0.05). Notably, additional treatment with FoxP3-transduced Tregs led to tolerance induction with this otherwise unsuccessful regimen (long-term donor skin graft acceptance> 120 days in 7=7 mice in Treg-group vs 1=6 control group, p < 0.005). Conclusions. Robust non-cytotoxic BMT regimens with clinically obtainable BM doses have remained elusive so far. The results shown here demonstrate that polyclonal FoxP3-Tregs lead to engraftment of achievable allogeneic BM doses without cytotoxic recipient treatment. Lasting mixed chimerism, deletion of donor-reactive T cells and donor-specific skin graft tolerance were achieved in an irradiation-free BMT protocol.

59 Inﬂuence of donor BMI on organ procurement M. Pones, I. Kristo, M. Hofmann, T. Soliman, G. Berlakovich, R. Steininger, F. Mühlbacher Division of Transplantation, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. As the number of patients on waiting lists is increasing and donor organs are scarce our interest in this study was to evaluate the impact of body-massindex (BMI) on organ procurement. Due to the increase of BMI among the Austrian population in the last decade we evaluated its influence on all procured donors in the Eastern Austrian Region. Methods. We identified all procured donors and the number of organs procured from each donor in the Eastern Austrian Region between 01.01.1998 and 31.12.2007. The BMI and number of organs procured were collected in our transplant donor database prospectively and retrospectively analysed. Statistical analyses were performed with SPSS 15.0 using the Fischer exact test and T-test wherever appropriate. Results. A total of 681 donors were procured. The mean number of donors procured per year was 68.1 12.56 (range [46–87]). Fourteen out of these were identified as tissue donors only, leaving 667 organ donors for further analyses. From these a total of 2212 organs were procured, subdivided into 1286 kidneys, 433 livers, 58 pancreata, 245 hearts and 190 lungs. The mean number of organs procured per donor was 3.32 1.24. The mean number of hearts procured per donor was 0.37 0.48. The mean number of lungs procured per donor was 0.28 0.45. The mean number of livers was 0.65 0.48. The mean number of kidneys per donor was 1.93 0.33. The mean BMI of all procured donors was 25.13 3.68. The impact of BMI on whether an organ from a

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donor was transplanted or not was significant for heart (p ¼ 0.01), for lung (p ¼ 0.002), for liver (p ¼ 0.008) and not significant for pancreas (p ¼ 0.056) and kidney (p ¼ 0.745). The analyses whether or not organs from donors with a BMI 30 compared to donors with a BMI 30 were procured showed a significance for heart (p ¼ 0.01), for lung (p ¼ 0.00), for pancreas (p ¼ 0.03) and liver (p ¼ 0.00) and no significance for kidney (p ¼ 0.66). Conclusions. This study shows a negative impact of BMI on organ procurement for all organs except kidney and pancreas. If the donor is obese with a BMI 30 all organs except the kidney are more likely to be not accepted for transplantation. The increasing number of people suffering from obesity in Austria may lead to a significant decrease in Cadaveric donor organs in the future. This study is limited by the fact that no other co-factors which influence the outcome of donor organ procurement were included into statistical analyses.

60 Ten year analysis of reported donors outcome in the Eastern Austrian Region M. Pones, G. Györi, I. Kristo, C. Burghuber, M. Hofmann, R. Steininger, F. Mühlbacher Division of Transplantation, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. As the number of patients on waiting lists is increasing and donor organs are scarce our interest in this study was to evaluate all reported donors in the Eastern Austrian Region for the last decade and identify reasons for failure of donor procurement. Methods. We identified all donors reported in the eastern Austrian region between 01.01.1998 and 31.12.2007. The eastern region is formed by three counties, Vienna, Lower Austria and Burgenland with a total of 68 hospitals. Demographic data such as age, sex, height, weight, BMI and standard clinical variables such as date of admission, diagnosis on admission, date of donor report to transplant center, date of explantation, reasons for failure of organ procurement were collected in our transplant donor database prospectively and retrospectively analysed. Results. A total of 1049 potential donors were reported to our transplant center between 1998 and 2007. Mean number of donors reported per year is 104.9 13.51 (median ¼ 106 range[r] ¼ 85–125). Mean age is 47.9 16.57 (median ¼ 48 r ¼ 0.75–90). 41.65% (n ¼ 437) of reported donors were female and 58.35% (n ¼ 612) male with a female-male-ratio of 1:1.4. Mean height is 1.73 m 0.99 (median ¼ 1.75 r ¼ 0.8–1.95). Mean weight is 76.26 kg 15.3 (median ¼ 75 r ¼ 10 ¼ 182). Mean BMI is 25.45 4.34 (median ¼ 24.7 r ¼ 11.57–69.3). Diagnosis on admission was intracerebral bleeding (n ¼ 320), subarachnoidal bleeding (n ¼ 229), craniacerebral trauma

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(n ¼ 329), hypoxia (n ¼ 94), stroke (n ¼ 57), braintumor (n ¼ 17), encephalitis, brain abscess and meningitis (all n ¼ 1) whereas the last three were only diagnosed in not procured donors. A total of 681 donors were procured. Mean number of donors procured per year is 68.1 12.56 (median ¼ 69 r ¼ 46-87). Mean time from reporting to explantatian in days is 1.89 2.13 (median ¼ 1 r ¼ 0–19). A total of 368 donors could not be procured. Mean number of donors not procured per year is 36.8 10.37 (median ¼ 33.5 r ¼ 25–55). Mean age between procured donors (47.04 15.78 [median ¼ 48 r ¼ 2–86]) and those not procured (49.62 17.92 [median ¼ 50 r ¼ 0.75–90]) compared by t-test is significant (p ¼ 0.02). Mean BMI between procured donors (25.16 3.74 [median ¼ 24.69 r ¼ 11.57–47.75]) and those not procured (26.1 5.38 [median ¼ 25.1 r ¼ 13.2–69.3]) compared by t-test is significant (p ¼ 0). Mean time from admission to report in days between procured donors (3.04 4.14 [median ¼ 1 r ¼ 0–30]) and those not procured (2.99 4.2 [median ¼ 1 r ¼ 0–21]) compared by t-test is not significant (p ¼ 0.579). Reasons for not procuring a donor where defined as death before confirmed braindeath (n ¼ 127, 12.11%) of all reported donors, family opposition (n ¼ 56, 5.34%), no consent (n ¼ 17, 1.62%), organ quality (n ¼ 75, 7.15%) and not braindead (n ¼ 83, 7.91%). Mean conversion rate over ten years, defined as percent of donors procured from all reported donors, is 64.9% 8.64 (median ¼ 66.69 range ¼ 49.54–73.91). Causes for not procuring a donor where defined as death before confirmed braindeath (n ¼ 127, mean ¼ 0.36 0.48) in 12.11% of all reported donors, family opposition (n ¼ 56, mean ¼ 0.16 0.36) in 5.34%, no consent (n ¼ 17, mean ¼ 0.47 0.21) in 1.62%, organ quality (n ¼ 75, mean 0.21 0.41) in 7.15% and not braindead (n ¼ 83, mean 0.23 0.42) in 7.91%. Conclusions. In years with low conversion rate reasons for failure of procurement were mainly organ quality due to advanced age and high BMI of the donors and not because of time at ICU.

61 Establishment of a mixed chimerism model employing memory cell-enriched recipients H. Ramsey, E. Schwaiger, C. Klaus, N. Pilat, U. Barayani, F. Muehlbacher, T. Wekerle Division of Transplantation, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. Current murine models of transplantation tolerance use specific pathogen free mice, which have a largely naı¨ve T cell repertoire. Memory T cells, present in high frequency in large animals and in humans, potently resist tolerization which is thought to be one of the main reasons why many rodent protocols fail in non-human primate models. Therefore, we started Eur Surg Vol. 40 Supplement Nr. 226 2008

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to establish a murine mixed chimerism model in which the recipient is enriched in memory cells. Methods. Allospecific primed T cells were generated by grafting C57BL=6(H2-b) mice with BALB=c(H2-d) skin. Sensitization was confirmed by a flow cytometry cross match assay (FCXM), measuring anti-donor IgG levels in sera. C57BL=6 recipient splenocytes were harvested and primed T cells were then purified using a MACS Pan T cell kit yielding> 96% purity. The purified spleen cells were then adoptively transferred into secondary B6 recipients, followed by an established treatment employing nonmyeoablative TBI of 2 Gy, transplantation of 15 106 Balb=c bone marrow cells and costimulation blockade (CTLA4Ig, anti-CD154). 10 106 sensitized T cells (Group B), 10 105 (Group C), and 10 104 (Group D) were transferred, with a control group receiving the same treatment without splenocytes transfer (Group A). Results. Sensitized B6 recipients showed production of high levels of donor-specific IgG shown by flow cytometry. It was shown that the number of sensitized splenic T cells administered in these experiments did not abrogate chimerism. Mixed chimerism developed in comparable levels across all groups, appearing in CD4þ (2.3–7.4%), B-cell (15.3–22.4%) and myeloid lineages (27.0–42.0%, p ¼ n.s.). Currently, all groups are grafted with BALB=c and third party C3H tail skin to assess tolerance (follow-up ongoing). Conclusions. These results suggest that moderate numbers of primed T cells seem not to prevent chimerism and tolerance induction. Based on these findings we have increased the number of transferred primed T cells. Once our model has been established, it will be of great interest for further use in mixed chimerism and tolerance studies.

62 Umstellung von Calzineurinihibitoren auf Sirolimus bei chronischer Transplantat-Dysfunktion nach pädiatrischer Nierentransplantation A. Schneider1, J. Falger1, M. Böhm2 , C. Aufricht2 , K. Arbeiter2, A. Soleiman3 , T. Müller2 1 Abteilung für Pädiatrie, Landeskrankenhaus Weinviertel Mistelbach, Mistelbach, Österreich; 2 Pädiatrische Nephrologie, Universitätsklinik für Kinderund Jugendheilkunde, Medizinische Universität Wien, Wien, Österreich; 3 Klinisches Institut für Pathologie, Medizinische Universität Wien, Wien, Österreich

Grundlagen. Die chronische Transplantatdysfunktion ¨r den spa¨ten Organist eine der ha¨ufigsten Ursachen fu verlust nach pa¨diatrischer Nierentransplantation (NTX). Neben vielen anderen Ursachen scheint auch eine nephrotoxische Immunsuppression mit Calzineurininhibitoren (CNI) einen Einfluss auf die Progression der Transplantatdysfunktion zu haben. Ziel der ersten Studie Eur Surg Vol. 40 Supplement Nr. 226 2008

war es, das Fortschreiten der chronischen Transplantatdysfunktion durch Elimination der CNI und Umstellung auf Sirolimus zu stabilisieren oder zu verbessern. In dieser Studie pra¨sentieren wir 7-Jahres-Langzeitdaten. Methodik. Im Zeitraum von 7=2001 bis 7=2008 wurden 14 Patienten mit chronischem Transplantatversagen mit Cyclosporin-A (n ¼ 4) oder Tacrolimus (n ¼ 10) nach Transplantatbiopsie auf Sirolimus umgestellt. Die 14 Patienten (6 weibl.) waren bei NTX 11,75 (4,1–17,5) Jahre und zum Zeitpunkt der Umstellung seit 22,9 (3–72) Monaten transplantiert. Ergebnisse. Die Umstellung auf Sirolimus erfolgte mit einer mittleren Dosis von 0,21 mg=kg KG=d, wobei ein Talspiegel von 15–20 ng=ml angestrebt wurde. Die Nierenfunktion wurde als Vera¨nderung der GFR nach der Schwartz Formel (-GFR) in den letzten 3 Monaten vor bzw. 3 Monate nach Umstellung sowie 12, 24, 36, 48, 60 und 72 Monate nach Umstellung berechnet. Der GFR-Verlust in den 3 Monaten vor Umstellung betrug im Mittel 2,4 (25,7) ml=min=1,73m2 . 3 Monate nach Umstellung wurde ein GFR-Gewinn von 2,2 ( 26,9) ml erreicht, nach 12 Monaten ein Gewinn von 4,5 ( 14,2) ml und nach 24 Monaten von 11,3 ( 15,9) ml. Nach 3 Jahren betrug die Zunahme der GFR þ 11,6 ( 9,1) ml, nach 48 Monaten konnte ein GFR-Gewinn von þ 13,4 ( 3,7) ml erreicht werden. Nach 60 Monaten war ein Plus von 13,1 ( 10,7) ml, und nach 72 Monaten nach TX ein Plus von 12,8 ml verzeichnet worden. Zum Zeitpunkt 24 Monate zeigten 5 Patienten einen deutlicher GFR-Gewinn, zwei Patienten einen GFR-Verlust. Sirolimus wurde klinisch gut vertragen. Weiters kam es zu keinen signifikanten Vera¨nderungen des Serumcholesterins, der Thrombozyten oder des Blutdruckes. Schlussfolgerungen. Diese Daten an 14 pa¨diatrischen Patienten mit chronischer TX-Dysfunktion zeigen, dass durch Elimination einer CNI basierten Immunsuppression und Umstellung auf Sirolimus eine Stabilisierung und sogar eine Verbesserung der Transplantatfunktion erreicht werden kann.

63 DPPIV-Inhibition as a strategy to enhance stem cell engraftment in a murine mode of mixed chimerism E. Schwaiger1, C. Klaus1 , U. Baranyi1 , N. Pilat1 , I. De. Meester2, F. Mühlbacher1, T. Wekerle1 1 Division of Transplantation, Department of Surgery, Medical University of Vienna, Vienna, Austria; 2 Department of Pharmaceutical Sciences, Laboratory of Medical Biochemistry, University of Antwerp, Belgium

Background. Enhancing stem cell engraftment would be important for the development of minimally toxic bone marrow transplantation (BMT) protocols. CXCL12=SDF1-alpha has an important role in homing of hematopoietic stem cells to bone marrow (BM).

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SDF1-alpha is cleaved by the ectopeptidase dipeptidylpeptidaseIV (DPPIV=CD26) which thus inhibits homing. Consequently, inhibition of DPPIV enzymatic activity would be an appealing strategy for promoting BM engraftment. The DPPIV activity inhibitor DiprotinA has been reported to promote engraftment of purified syngeneic stem cells in lethally irradiated recipients, but little is known about the effects of DPPIV inhibition in non-myeloablative BMT protocols for the induction of mixed chimerism and transplantation tolerance. We therefore explored the inhibition of DPPIV enzymatic activity with DiprotinA or the clinically available inhibitor Sitagliptin for the purpose of enhancing engraftment of unseparated BM after non-myeloablative recipient conditioning. Methods. In a congenic model B6 CD45.1 mice were conditioned with 1 Gy total body irradiation (TBI, d 1) and received 15 mio (group A and B) or 10 mio (group C, D and E) congenic bone marrow cells (BMCs, CD45.2). Donor BM DPPIV activity of group B was inhibited in vitro by incubation of BM with DiprotinA (Ile-Pro-Ile, 5 mM). In group E mice were additionally treated with DiprotinA in vivo (4mM iv d0 and 5mM sc d0-2) and in group D, recipients DPPIV serum enzymatic activity was inhibited with Sitagliptin (200 mg=kg twice daily, oral gavage, 3 days). In an allogeneic model B6 mice received 1 Gy TBI, 15 mio fully MHC-mismatched BALB=c BMCs, costimulation blockade (MR1 1 mg d0 þ CTLA4Ig 0.5 mg d2) and 2 200 mg=kg Sitagliptin for 2 days. Multi-lineage macrochimerism was followed by flow cytometry. Blood samples were collected 2 and 24 h and DPPIV serum enzymatic activity was measured by a kinetic fluorimetric enzyme assay with glycyl-L-roline-4-methoxy-2-naphthylamide as substrate. Results. Similar rates and levels of multi-lineage chimerism were observed with or without DPPIV enzymatic inhibition. One hundred and fifty three days after BMT mean chimerism levels of group A (control) and B (DiprotinA treated) were comparable [approx. 40% vs 36% in the CD4 T-cell and 32% vs 23% in the CD8 T-cell lineage (p ¼ ns)]. In the allogeneic model in vivo use of Sitagliptin during the engraftment period caused a complete inhibition of DPPIV enzymatic activity (þ2 h 100%; p < 0.001, þ12 h 50%; p < 0.09) but no increase in chimerism levels was observed (2.3% vs 2%in CD4 Tcell and 2.7% vs 1.6%in CD8 T-cell lineage, d58; p ¼ ns). Preliminary results of the congeneic model using 10 mio BMCs indicate that neither DPPIV inhibition in vitro and in vivo by using DiprotinA (group E) nor in vivo by using Sitagliptin (group C) was able to enhance engraftment (d27). Conclusions. With the clinically approved DPPIV inhibitor Sitagliptin we identified a dosing regimen that completely blocks recipients DPPIV serum enzymatic activity during BMT. No evidence was found that inhibition of recipient DPPIV enzymatic activity with Sitagliptin or of donors BM DPPIV enzymatic activity with DiprotinA was able to enhance engraftment of unseparated BMCs in a CD45-congenic model or in an allogenic model using non-myeloablative conditioning.

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64 First documented case of Paracoccus yeei infection in a transplanted heart M. Schweiger1, P. Stiegler1, A. Wasler1, G. Prenner1, M. Scarpatetti2 , K. Tscheliessnigg1 1

Divison for Transplantation, Department of Surgery, Medical University of Graz, Graz, Austria; 2 Department of Pathology, Medical University of Graz, Graz, Austria

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 transplantation. 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 administration of ciprofloxacin clinical conditions ameliorated and further biopsy showed a regression of infiltrates in the cardiac specimens. The patient finally was dismissed in a good state of health. Conclusions. Resumptive Paracoccus yeei, a gramnegative bacterial eugenic oxidizers should be included in diagnostically thoughts in remarkably cases of myocarditis. Treatment with quinolones resulted in clinical and histological improvement.

65 Results of lung transplantation for COPD=emphysema S. Semsroth1 , C. Geltner2, C. Hörmann3 , E. Ruttmann-Ulmer1, H. Hangler1, G. Laufer1, L. Müller1 1 Department of Cardiac Surgery, Medical University of Innsbruck, Innsbruck, Austria; 2 Department of Pulmology, Hospital Natters, Natters, Austria; 3 Department of Anaesthesia and Intensive Care, Medical University of Innsbruck, Innsbruck, Austria

Background. Lung transplantation (LUTX) for COPD and emphysema is still under discussion, since survival advantages compared to conventional treatment or other forms of surgical therapy (LVRS) or interventional treatment are difficult to assess. Furthermore, due to organ shortage the recommended transplant type (uni- vs. bilateral) is not decided although bilateral transplantation (BLTX) gives superior survival compared to unilateral lung transplantation (SLTX). Eur Surg Vol. 40 Supplement Nr. 226 2008

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Methods. A continuous series of 137 primary lung transplants performed between 11=1993 and 03=2008 was analysed. Ninety nine patients (72.3%) were transplanted for COPD or alpha-1 antitrypsin deficiency emphysema; of these (71.7%) received BLTX and 28 SLTX. Immunosuppression included induction therapy, calcineurin inhibitors, cortisone and antimetabolites and TOR inhibitors in late post transplant phase. Results. Total one, three and five year survival was 85.2, 76.5 and 67.9%. For recipients< 60 years respective survival rates were 86.0, 79.7 and 73.3%. BLTX was associated with 87.2, 80.4 and 76.2% survival, BLTX in patients< 60 years with 89.9, 85.2 and 82.4% survival after one, three and five years. Conclusions. In a patient population transplanted predominantly for COPD excellent survival results can be achieved by BLTX. Center volume does not necessarily match with results, if patient treatment is confined to a small dedicated team of surgeons, pulmonologists and anaesthesiologists.

Results. Sixty min after brain death induction neurological examination and EEG examination confirmed brain death. Intracranial pressure increased continuously and stayed stable after occurrence of brain death. All 16 animals showed typical signs of brain death such as diabetes insipidus, hypertensive and hypotensive periods and tachycardia. All symptoms could be treated using standard medication. After 24 h of brain death successful multiorgan donation was performed. After organ retrieval, abdominal and thoracic organs could be analysed for tissue damage and organ quality. Conclusions. It is feasible to induce brain death in a pig model by inserting a catheter after trepanation of the skull. According to standard guidelines brain death diagnostic was performed, 0 line EEG occurred in all animals 60 min after brain death induction. Using this method, a suitable brain death donor model could be establish that will enable us not only to investigate in detail effects and pathophysiology after occurrence of brain death but also to evaluate new strategies to ameliorate organ quality and even to enlarge the donor pool for multiorgan donation.

66 Establishing a brain death donor model in pigs M. Sereinigg1 , P. Stiegler1, M. Schweiger1, T. Marko3 , T. Seifert2 , F. Iberer1, K.-H. Tscheliessnig1 1 Division for Transplantation Surgery, Department of Surgery, Medical University of Graz, Graz, Austria; 2 Department of Anaesthesiology and Intensive Care, Medical University of Graz, Graz, Austria; 3 Department of Neurology, Medical University of Graz, Graz, Austria

67 Transplantation of bone marrow stem cells, immobilized in biodegradable 3-D matrix as bioartiﬁcial neo-tissue for functional supporting damaged organ M. Shagidulin1 , M. Krasheninnikov2, I. Iljinsky3, N. Mogeiko3 , E. Nemets4 , V. Sevastjanov4, N. Onishchenko2 1

Background. Several factors influencing organ quality and recipient survival after multiorgan donation and transplantation are still unknown and difficult to investigate in humans. Therefore the need for an animal model that imitates human conditions might be useful not only to be able to monitor pathomechanisms of brain death and biochemical cascades in the organisms after brain death but also to be able to investigate novel strategies to ameliorate organ quality and functionality after multiorgan donation. Therefore the aim of this study was to establish a brain death donor model in pigs. Methods. Sixteen pigs were used for these experiments in accordance with the Austrian animal law. Brain death was induced by inserting a catheter into the intracranial space after trepanation of the skull and augmenting intracranial pressure until brain stem herniation occurred. Intracranial pressure was monitored continuously and after 60 min brain death diagnostics was performed by a neurologist including EEG examination and clinical examination. Donor care was performed according to standard guidelines and after 24 h of brain death and intensive care multiorgan donation was performed. Eur Surg Vol. 40 Supplement Nr. 226 2008

National Research Institute of Transplantology and Artiﬁcial Organs, Moscow, Russia; 2 Stem Cells Laboratorium, Moscow, Russia; 3 Department of Pathomorphology, Moscow, Russia; 4 Center of Research of Biomaterials, Moscow, Russia

Background. Cell therapy is a new perspective method treatment of different diseases. Safe and prolonged cell cultivation in vivo for regulatory peptides production and functional support of damaged organs forms the base of this method. This investigation was undertaken in order to make bioengineering construction for prolonged bone marrow (BM) stem cells cultivation after the transplantation of this construction into an organism. Methods. Adult dogs (28–30 kg) were used as donors of BM stem cells (multipotent mesenchymal stromal cells – MMSC). The survival of MMSC, immobilized on biodegradable matrixes ElastoPOB+ 3-D was investigated. Matrixes being produced by the Center on Research of Biomaterials at the National Scientific Research Institute of Transplantology and Artificial Organs. Matrixes were prepared on the basis of hydroxybutyrate and oksyvalerate copolymer, which was synthesized at Scientific Research Institute of Biophysics of the SBRAS, Krasnoyarsk. Now this biopolymer is resolved to medical

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application. BM cells were received by femur washing with Hanks solution, which contained 200 mkg=ml of gentamycin and 250 un=ml of a heparin. Suspension of BM cells was centrifuged and cell sedimentation was resuspended in lysing solution (114 mM NH4CI; 7.5 mM KHC03; 100 mkM EDTA). BM cells were sown in Petri dishes and cultivated during 10–14 days with medium changing every 4 days at constant microscope control of cell viability (MMSC). In 10–14 days MMSC were sown on matrixes and incubated in vitro within 3–4 days for immobilization. After that matrixes with autological MMSC were implanted into small bowel mesentery of earlier operated animals. The dynamics of cell proliferation in matrix pores and cell vitality in matrixes were investigated in 30, 60 and 90 days. Histology. Morphological structures of MMSC and surrounding tissue were studied using hematoxylin and eosin stain. Vessels were studied using PAS staining. Results. It was determined that MMSC viability immediately after discharge from BM was 92–96%. Results of our researches have show viable and proliferative activity of MMSC for a long time on the biodegradable matrixes ElastoPOB+ 3-D. We have detected neogenic plethoric vessels, growing through matrixes and viable cells, which proliferated on them even 90 days after implantation of matrixes with MMSC into mesentery of experimental animals. Conclusions. Our preliminary studies have shown a long surviving and viability of isolated autologous MMSC, attached on the three-dimensional biodegradable matrix ElastoPOB+ 3-D in vivo. We suppose that matrixes ElastoPOB+ 3-D can be applied as a tissue engineering method for building intracorporated bioartificial neo-tissue and functional support of damaged organs.

68 Lipocalin-2: a new regulator of the inﬂammatory response during ischemia=reperfusion S. Sickinger1, H. Maier1, H. Schwelberger1, B. Redl2 , R. Margreiter1, J. Troppmair1, F. Aigner1 1 Daniel-Swarovski-Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck, Austria; 2 Division of Molecular Biology, Medical University of Innsbruck, Innsbruck, Austria

Background. Lcn-2 is upregulated during ischemia=reperfusion in the course of murine heart translantation and correlates with granulocyte infiltration into the transplanted hearts. To get insight into the regulatory role of Lcn-2 we sought to i) elucidate the molecular mechanism underlying increased Lcn-2 expression, and ii) analyze differences in the expression of chemokines between Lcn-2 þ=þ and Lcn-2= hearts and granulocytes. Methods. The murine heterotopic heart transplant model and additional procedures have been described

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(see above). COS-7 cells were transiently transfected using lipofectamine (Invitrogen); hypoxic treatment was done in a hypoxia chamber; reporter gene assays were performed using the Dual Luciferase Assay (Promega). Results. While neither hypoxia itself or following overexpression of HIF-1 or induced Lcn-2 promoter activity, transfection of HIF-1, but not HIF-1, incresased reporter gene activity (2.29-fold 0.68, n ¼ 3). The expression of the chemokine MIP-2, but not of KC, MCP-1, or LIX, was significantly downregulated 48 h after reperfusion only in wildtype grafts transplanted into Lcn-2= recipients, but not in the reverse setting. Furthermore, LIX expression was upregulated in primary granulocytes from Lcn-2= mice that was enhanced by hypoxia. Conclusions. The Lcn-2 promoter is regulated by overexpression of HIF-1=ARNT but not by hypoxia itself. Downregulation of MIP-2 in the Lcn-2= recipient point to a possible chemotactic role of Lcn-2. Lcn-2 might have an effect on the expression of LIX mRNA in granulocytes. Understanding these regulatory mechanisms will be crucial to establish treatment strategies for ischemia= reperfusion injury during solid organ transplantation.

69 Allo-reactive non-HLA speciﬁc antibodies in haemodialysis patients before renal transplantation are mainly targeted against distinct cytoskeletal proteins S. Skeledzic1 , M. Veitinger1, D. Cejka2 , C. Reichel3 , G. Boehmig2 , F. Mühlbacher1, R. Oehler1 1

Department of Surgery, Medical University of Vienna, Vienna, Austria; 2 Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria; 3 Department Life Sciences Proteomics Austrian Research Centers GmbH ARC, Seibersdorf, Austria

Background. The presence of allo-reactive antibodies in renal transplant recipients is one of the main determinants of organ rejection. Antibodies against donors HLAproteins lead to severe rejection events. However, good HLA-Matching does not guarantee rejection-free longterm graft survival. Recent data suggest that also nonHLA antigens might be targets for antibody-mediated rejection. Objective of the study. This proteomics study aims at investigating the frequency and specificity of alloreactive non-HLA-specific antibodies in haemodialysis patients. Methods. Serum-samples were taken before grafttransplantation from 29 haemodialysis patients. Lymphocytes from 10 patients as well as from 20 healthy volunteers were used a as source of antigen. Allo-reactive anti-lymphocytic antibodies were investigated by one & two dimensional gel electrophoresis and Western Eur Surg Vol. 40 Supplement Nr. 226 2008

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blotting. Antigenic proteins were identified by mass spectrometry. Results and Conclusions. Presence of antibodies against a variety of lymphocytic antigens was observed in high and low PRA haemodialysis patients. This includes always also antibodies against proteins with a molecular weight other than HLA. The specificity of these non-HLA targeting antibodies differs between patients. In addition, antibodies from each single patient diversify between lymphocytes from different healthy volunteers. The antigenic proteins were identified as the cytoskeletal proteins Vimentin, alpha and beta Tubulin, and Lamin B1 and the cytoplasmatic protein Rho GDP-dissociation inhibitor 2. Since the presence of allo-reactive non-HLA specific antibodies have been already associated with impaired long term graft survival, the specificity of these antibodies – as identified here – could be used to improve donor-recipient matching in future.

70 Intracellular signaling pathways as targets for the prevention of ischemia=reperfusioninduced damage during solid organ transplantation J. Smigelskaite1 , A. Kuznetsov1, M. Hermann2 , R. Sucher1, P. Gehwolf1 , C. Doblander1, M. Maglione1 1 Daniel Swarovski Research Laboratory, Department of Visceral-, Transplant- and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria; 2 KMT Laboratory, Department of Visceral-, Transplantand Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria

Background. Ischemia (I) and reperfusion (R) trigger a series of events, which culminate in severe injury to the transplanted organ. The formation of mitochondrial reactive oxygen species (ROS), linked to the perturbation of mitochondrial Ca2þ homeostasis is most critical for the development of IR-associated tissue damage. Our work aims at devising strategies to interfere with the early steps of mitochondrial perturbation, before damage amplification occurs. Methods. Growth factor abrogation form factor-dependent cells results in mitochondrial changes in ROS and Ca2þ comparable to alterations observed under IR. Using this model and the overexpression or ablation (siRNA) of central intracellular signaling molecules we tested for the effects on survival and mitochondrial Ca2þ=ROS homeostasis. Alteration in mitochondrial ROS and Ca2þ levels were monitored by confocal imaging following loading of cells with MitoSOXTM Red or Rhod-2, respectively. Results. We provide evidence for the first time that key signaling molecules involved in cell survival regulation (RAF, AKT, Bcl-2) prevent apoptosis by maintaining perEur Surg Vol. 40 Supplement Nr. 226 2008

missive levels of mitochondrial ROS and Ca2þ. Cell death is preceded by a ROS-dependent increase in mitochondrial Ca2þ, which was absent in cells protected by these proteins. Our findings also demonstrate a role for intracellular signaling in controlling mitochondrial homeostasis in cardiomyocytes under hypoxia and reoxygenation. Conclusions. These data show that mitochondrial changes, which normally proceed the onset of cell death, are subject to extrinsic control. This raises the possibility to develop novel approaches for the prevention of IR-induced organ damage using the targeted modulation of intracellular signaling.

71 Regulation of RNA inhibition components in renal allograft dysfunction – local loss of Dicer in tubulo-interstitial rejection A. Soleiman1 , L. Strebinger1, A. Prvulovic1 , M. Saemann2 , R. Oberbauer3 1 Department of Pathology, Medical University of Vienna, Vienna, Austria; 2 Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria; 3 Department of Internal Medicine III, Hospital Elisabethinen Linz, Linz, Austria

Background. RNAinhibition in post-transplant renal allograft dysfunction is a yet uncharted territory. The complex reno-cellular processes underlying rejection and other causes of early graft dysfunction have not been elucidated in detail. Regulation of protein expression by microRNAs is a major step in tissue development and maintainance of differentiated function. MicroRNAs mediate post-transcriptional regulation by mRNA silencing. MicroRNA biogenesis and function depends on a multistep pathway of RNA processing molecules that is centrally driven by the cytosolic RNase III Dicer. Methods. Diagnostic renal allograft biopsies from 75 renal transplant patients (first transplantation) performed for severe allograft dysfunction within the first two posttransplant months (serumcreatinine > 4mg=dl) due to exclusive interstitial rejection (BANFF1, n ¼ 25), vascular rejection (BANFF2 n ¼ 25) or non-rejection caused acute tubular damage (BANFF0, n ¼ 25) were subjected to immunohistochemical analysis for microRNA processing proteins Drosha, Dicer, Argonaute 1 and TARBP. Read out was made by semi-quantitative scoring of immunohistochemical signals in the different cellular compartments of the transplant organ. Results. Immunohistochemical scoring of allograft biopsies revealed a high overall expression level of microRNA processing proteins in normal renal tubuli and endothelial cells. A strong reduction of Dicer expression was observed in proximal tubuli of BANFF1 rejection where loss of Dicer was restricted to areas of tubuloaggressive inflammation (tubulitis) while a slight reduction of the other monitored proteins was observed in the same

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cells. No alterations of microRNA processing proteins were observed in glomeruli, blood vessels with or without vascular rejection or in non-rejection related acute tubular damage. Namely, proximal tubular cells with acute dystrophy unrelated to rejection=tubulitis displayed stably high Dicer expression unaltered even by morphologically severe damage. Conclusions. The key regulatory factor of microRNA biogenesis, Dicer, is massively down-regulated in proximal tubular cells with T-cellular tubulitis thus revealing local impairment of microRNA regulation under immunological attack in acute T-cell driven renal allograft rejection. Dicer downregulation at the site of cellular rejection was found specific for rejection associated tubular damage suggesting importance of the microRNA pathway in immunologically targeted cells.

Functionality of transplanted porcine islet cells was detected by insulin measurement and detection of C-Peptide. After scarification, histological and immunohistochemical evaluation showed no signs of fibrosis or inflammation in the surrounding tissue. Viability of microencapsulated porcine islet cells after explantation was proven by immunohistochemical viability stains. Conclusions. It is feasible to reverse diabetes in rats by transplanting porcine islet cells microencapsulated in NaCS. Rats stayed normoglycaemic until the end of the study period. No signs of fibrosis could be detected in the surrounding tissue. NaCS seems to be a promising material for microencapsulation of porcine islet cells in order to treat diabetes. Further studies have to be carried out to show long term survival of transplanted porcine islet cells microencapsulated in NaCS in diabetic rats.

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72 Xenotransplantation of microencapsulated porcine islet cells in diabetic rats P. Stiegler1, V. Stadlbauer1, F. Hackl1 , S. Schaffellner1, C. Lackner2, F. Iberer1, K. Tscheliessnigg1 1

Division of Transplantation Surgery, Department of Surgery, Medical University of Graz, Graz, Austria; 2 Institute of Pathology, Medical University of Graz, Graz, Austria

Background. Xenotransplantation of microencapsulated porcine islet cells might be a possibility to overcome the shortage of human donor organs for pancreas transplantation. Several materials for microencapsulation of cells are described in literature which all show severe disadvantages. NaCS is easy to produce, does not show any cytotoxicity and cell lines survive for a nearly unlimited time-spam after microencapsulation. However, this material has not been tested for microencapsulation and xenotransplantation of porcine islet cells. Methods. Porcine islet cell isolation and purification was performed according to a newly modified Ricordi method. Porcine islet cells were microencapsulated with NaCS. Diabetes was induced in Sprague Dawley rats by intraperitoneal injection of STZ. Only rats that showed polydipsia, polyuria and blood sugar levels higher than 400 mg=dl over a time period of 14 days were used for the experiments. Microencapsulated porcine islet cells were transplanted under the kidney capsule of the animals. Blood sugar levels were monitored on a weekly basis, porcine C-Peptide levels and insulin levels were measured using ELISA. Intravenous glucose tolerance testing was performed once a months. After 4 months, the animals were sacrificed, the kidney containing the microencapsulated porcine islet cells was retrieved and processed for histological and immunohistochemical examination. Results. After xenotransplantation of microencapsulated porcine islet cells diabetes was reversed in rats. Animals stayed normoglycaemic up to four months.

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Prevention of oxidative stress induced organ damage in a porcine brain dead donor model P. Stiegler1, M. Schweiger1, J. Greilberger2, S. Hallström2 , C. Lackner3, F. Iberer1, K. Tscheliessnigg1 1

Division of Transplantation Surgery, Department of Surgery, Medical University of Graz, Graz, Austria; 2 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria; 3 Institute of Pathology, Medical University of Graz, Graz, Austria

Background. The autonomic storm initiated after brain death is known to induce a cascade of chemokine and cytokine release which induces oxidative stress and consecutively causes cell damage and diminished organ quality. In order to ameliorate donor organ quality new strategies to prevent the detrimental effects of the autonomic storm induced cascades have to be investigated. Recently published data on donor pre-treatment showed limited success. Therefore, this study aimed to characterize in detail the pathophysiological mechanisms after brain death and to evaluate the impact of an antioxidative treatment after brain death induction on organ quality and oxidative stress induced cell damage in a pig model. Methods. Brain death was induced in 16 pigs by trepanation of the skull and increasing intracranial pressure until brain stem herniation occurred. Brain death was confirmed by EEG exanimation, clinical neurological and CCT exanimation. 5 non-brain dead animals served as controls. Ten hours after brain death diagnosis, the pigs were randomized in two groups (n ¼ 8). Group 1 was infused 500 ml of a solution containing alpha-ketoglutaric acid and 5-MMF over 4 h whereas group 2 received 500 ml NaCl. Blood samples were taken at defined time points and carbonyl proteins (CP) and malondialdehyde (MDA) were measured in order to monitor Eur Surg Vol. 40 Supplement Nr. 226 2008

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oxidative stress induced cell damage. Monocyte chemoattractant protein 1, macrophage inflammatory protein, soluble platelet vascular cell adhesion molecule and soluble platelet selectin, sCD40L and the interleukins IL-6 and IL8 are determined using FlowCytomic, a multiple analytic detection method on the flow cytometer. Twenty-four hours after brain death multiorgan donation was performed and tissue samples were taken immediately after organ retrieval and after 4 h of cold ischemia time (CIT) for the heart and 6 h of CIT for the abdominal organs in order to analyse ATP levels. Histology and immunohistochemistry were performed to quantify occurrence of apoptosis and of oxidative stress induced cell damage. Results. Analysis of the blood samples allowed us to describe exactly the chemokine and cytokine cascades initiated during the autonomic storm in this pig brain dead donor model. Markers of oxidative stress as well as the concentration of the interleukins analysed were highest in all animals 8 h after brain death induction. It was feasible to lower CP and MDA levels as well as chemokine and cytokine concentrations significantly after application of the solution containing alpha-ketoglutaric acid and 5MMF. Histology and immunohistochemistry revealed significantly lower apoptotic cells as well as lower anti-nitrotyrosine positive cells in group 1 when compared to group 2 immediately after explanation and after CIT. ATP levels were highest in the control group, but significantly higher in group 1 when compared to group 2 at any time point. Conclusions. We could diminish oxidative stress induced cell damage and prevent the detrimental effects of the autonomic storm by applying a solution containing alpha-ketoglutaric acid and therefore achieved better organ quality after multiorgan donation in a pig brain death model. Further investigations have to be carried out to investigate these effects in detail. However, this method seems to be a promising tool to ameliorate organ quality in brain dead donors.

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critical determinant of NO production. BH4 depletion during cold ischemia leads to uncoupling of NOS and contributes to reperfusion injury (IRI) due to increased superoxide formation. The role of BH4 during warm ischemia, however, is still largely unknown. Methods. Ischemic renal injury was induced by clamping the left renal artery for 45 min in male Lewis rats immediately after right-side nephrectomy. Reperfusion was studied at R0 (no reperfusion), 15 min (R1), 2 hours (R2) and 7 days (R3). Animals received either BH4 (20 mg=kg=BW) prior to reperfusion (Group I) or saline (Group II). Sham operated animals served as controls (Group III). Renal function was determined by plasma creatinine=urea. BH4 tissue levels were assessed by HPLC. Morphologic changes were quantified by H&E histology. Peroxynitrite formation was assessed by nitrotyrosineimmunostaining and kidney microcirculation was analyzed by means of functional capillary density and capillary diameters using intravital microscopy. Results. BH4 tissue levels significantly decreased after 45 min of warm ischemia (p < 0.05) up to two days (R1, R2) when compared to non-ischemic controls. Additional BH4 treatment prior to ischemia significantly improved renal function at all time points studied following reperfusion (all p < 0.001). Furthermore, BH4 reduced ischemia induced histologic damage (increased inflammation, interstitial edema, hemorrhage, tubular atrophy and focal areas of necrosis) and diminished peroxynitrite formation and hence nitrotyrosine staining (R1–R3). Subsequently, microcirculatory changes correlated with kidney peroxynitrite generation, and improved considerably through BH4 treatment. Conclusions. BH4 treatment significantly improves post-ischemic renal function as well as histologic and microcirculatory damage and might be a promising novel therapeutic strategy in attenuating IRI via maintenance of NO homeostasis.

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Tetrahydrobiopterin abrogates microvascular kidney ischemia reperfusion injury via maintenance of NO homeostasis

Haploidentical stem cell transplantation with subsequent donor lymphocyte infusions following reduced intensity conditioning in three children=adolescent with refractory=relapsed malignancies

R. Sucher1;2, P. Gehwolf1;2 , N. Fischler1;2, R. Oberhuber1;2, C. Margreiter1;2, M. Maglione1;2 , S. Schneeberger1;2, R. Öllinger1;2, E. R. Werner3, P. Obrist3 , R. Margreiter1;2, G. Brandacher1;2

C. Urban, W. Schwinger, P. Sovinz, H. Lackner, M. Benesch, A. Moser

1

Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria; 2 D. Swarovski Research Laboratory, Innsbruck, Austria; 3 Division of Medical Biochemistry, Medical University of Innsbruck, Innsbruck, Austria

Background. Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthases (NOS) and thus a Eur Surg Vol. 40 Supplement Nr. 226 2008

Division of Paediatric Haematology and Oncology, Department of Paediatrics and Adolescent Medicine, Medical University of Graz, Graz, Austria

Background. The outcome of patients with refractory malignant diseases is poor. Since these patients are generally not amenable to further treatment intensification, alternative immunotherapeutic approaches would probably allow to induce an immune-mediated destruction of tumor cells.

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Methods. Three extensively pre-treated patients (male, n ¼ 1; female, n ¼ 2) received haploidentical (haplo-) stem cell transplantation (SCT) for refractory (neuroblastoma IV) or recurrent (Hodgkin disease [second relapse following autologous SCT]; Ewing sarcoma [relapse following autologous SCT]) following reduced intensity conditioning (RIC) with OKT-3 (MuromonabCD3, 0.1 mg=kg) from day 8 to þ17=þ18, fludarabine (40 mg=m2 =day) on days 8 to 5, thiotepa (2 5 mg=kg) on day 4, and melphalan (70 mg=m2 =day) on days 3 and 2. Stem cells were manipulated by CD34 positive selection and CD3=CD19 depletion. Additional graft versus host disease (GVHD) prophylaxis included administration of mycophenolat mofetil (MMF, 1.2 g=m2 =day) for 12, 30, 50 days. The number of transplanted CD34þ and CD3þ cells was 21.6, 19.3, 14.9 106=kg, and 1 105 =kg, 5.3 104 =kg, 6 104 =kg recipients body weight. Additional donor lymphocyte infusions (DLI) were given starting on day þ99, þ72, þ40 after haplo-SCT. Results. All patients showed rapid trilineage engraftment. One patient developed GVHD of the skin grade II prior to the initiation of DLI therapy. In the absence of active GVHD patients received a total of 7, 4 and 50 DLIs containing 2.5 104 =kg-3.5 106=kg donor lymphocytes. All patients developed DLI-induced GVHD (skin grade, skin gut grade II, skin grade II–III), but responded quickly to intensified immunosuppression. Follow-up is now þ594, 370, þ 351 days. All patients are alive in excellent clinical condition with signs of limited chronic GVHD and achieved either complete (n ¼ 1) or partial remission (n ¼ 2). Conclusions. Although haplo-SCT with additional DLIs after RIC carries a high risk for GVHD, this approach may induce and=or maintain remissions in heavily pretreated patients with recurrent malignant diseases by an immunologic graft versus tumor effect. DLIs have to be stopped and immunosuppressive therapy initiated in patients who develop DLI-induced GVHD.

76 Clinical relevance of preformed C4d-ﬁxing and non-C4d-ﬁxing HLA single antigen reactivity in renal allograft recipients M. Wahrmann1 , G. Bartel1 , M. Exner2, H. Regele3 , G. F. Körmöczi4 , G. F. Fischer4, G. A. Böhmig1

C4d-fixing ability a particular pathogenetic impact was speculated. Methods. Pre-transplant sera obtained from 338 kidney transplant recipients were pre-screened by FlowPRA. To evaluate the clinical effects of C4d- or non-C4d-fixing (IgG) HLA sensitization with or without detectable DSA, 71 [IgG]FlowPRA positive recipients were retrospectively analysed by Luminex single antigen testing applying a novel fluorescent-labeled anti-C4d reagent for detection of antibody-triggered C4d deposition in addition to IgG binding. Results. Fifty-five recipients were positive by [IgG], 46 by [C4d]Luminex. Patients with [IgG]DSA (N ¼ 39) showed a substantially higher rate of C4d positive rejection (33%) than 16 patients with [IgG]nonDSA (0%) or 283 [IgG] Luminex or FlowPRA negative patients (4%, multivariate analysis excluding retransplantation because of high co-linearity: P < 0.0001), and adversely affected 5-year death-censored graft survival (74% versus 81% and 90%, respectively, multivariate model: P < 0.05). Assessment of the C4d-fixing ability of DSA failed to improve test performance. Without any apparent difference, both [C4d]DSA (N ¼ 21) and [C4d]nonDSA (N ¼ 25) recipients showed increased rates of C4d positive rejections (24% and 28% versus 4% in recipients without C4d-fixing reactivity; multivariate analysis: 0.002) and a trend towards adverse 5-year graft survival (76% and 76% vs. 90% 0.2). Conclusions. This study reinforces that Luminexbased identification of IgG DSA may be an attractive strategy for pre-transplant risk stratification. However, even though C4d-fixing HLA reactivity in general was found to be associated with adverse graft outcomes, identification of C4d-fixing DSA failed to distinguish a patient subgroup at particular immunological risk.

77 Long-term safety of Belatacept: 6 year results of phase II study T. Wekerle1 , F. Mühlbacher1, B. Charpentier2, C. Larsen3 , M. Agarwal4 , F. Vincenti5 1 Department of Surgery, Medical University of Vienna, Vienna, Austria; 2 Department of Nephrology, Hospital Le Kremlin Bicetre, Le Kremlin Bicetre, France; 3 Department of Transplant Surgery, Emory University, Atlanta, GA, USA; 4 Bristol-Myers Squibb Company, Princeton, NJ, USA; 5 Transplant Svc, University of California San Francisco, San Francisco, CA, USA

1

Department of Medicine III, Medical University of Vienna, Vienna, Austria; 2 Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria; 3 Department of Pathology, Medical University of Vienna, Vienna, Austria; 4 Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria

Background. Donor-specific alloantibodies (DSA) uncovered by sensitive solid phase techniques may pose allograft recipients at risk for rejection. For DSA with

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Background. Belatacept is a first-in-class T-cell costimulation blocker which inhibits CD28 mediated T-cell activation and provides selective immunosuppression. Belatacept is being evaluated in Phase 3 trials as CNI-free immunosuppression in renal transplant. An interim report of an open-label long-term extension (LTE) of the phase 2 trial is presented. Methods and Results. The Phase 2 trial design and results have been described previously. For the LTE, Belatacept arms were dosed as either q4week or q8week Eur Surg Vol. 40 Supplement Nr. 226 2008

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maintenance infusions of 5 mg=kg; control patients received Cyclosporine (CsA), dosed to target C0 levels (150–300 ng=ml). All patients continued to receive MMF and steroids per protocol. Results for outcomes and serious adverse events (SAE) are presented as incidence rates=100 patient-years of drug exposure. No formal comparisons or statistical testing were applied. 128 of 218 original patients elected to participate in the LTE and remained on their assigned treatment; 102 received Belatacept, 26 received CsA. Among LTE patients enrolled, 92 (72%) patients remain (76=102, 75% Belatacept; 16=26, 62% CsA). Median follow-up from original randomization was 60 months (range 15–78 m). Results for SAE categories, acute rejection (AR), and death=graft loss are shown. Conclusions. Through six years, belatacept continues to be safe with continued low rates of acute rejection, death and graft loss as part of a long term- CNI-free immunosuppression regimen in renal transplant. Phase III studies are ongoing.

78 The ﬁbrin derived peptid B-beta15–42 protects hearts and kidneys from ischemia-reperfusion injury in rat models D. Wiedemann1 , S. Schneeberger2, S. Scheidl2 , P. Petzelbauer3, R. Margreiter2, G. Laufer1, S. Semsroth1 1 Department of Cardiac Surgery, Medical University of Innsbruck, Innsbruck, Austria; 2 Department of General and Tranplant Surgery, Medical University of Innsbruck, Innsbruck, Austria; 3 Department of General Dermatology, Medical University of Vienna, Vienna, Austria

Background. Ischemia-reperfusion injury still represents one of the major problems in solid organ-transplantation. Reperfusion of ischemic organs is associated with an inflammatory response caused by leukocytes migrating from the bloodstream into allograft tissue. The fibrin derived peptide B-beta15–42 has shown to reduce Ischemia-reperfusion injury in local reperfusion models after myocardial infarction (LAD ligation and reperfusion) by inhibiting leukocyte migration into the tissue. The purpose of our study was to investigate the protective effects of B-beta15–42 on ischemia-reperfusion injury after organ-transplantation. First of all we tested B-beta15–42 in rat-heart-transplant model. In a second study we tried to show if B-Beta15–42 is also protective for the rat kidney after ischemia and reperfusion. Methods. Hearts of male Lewis rats(250–300 g) were flushed with chilled (0–1 C) custodiol preservation solution and either transplanted immediately or stored in the same solution for 4 or 8 h and then transplanted into syngenic recipients. 1.2 mg of B-beta15–42 were given i.v. immediately after implantation of the heart or added to the preservation solution prior to harvest. At 24 h and 10d hearts were retrieved for histological Eur Surg Vol. 40 Supplement Nr. 226 2008

evaluation and MPO (myeloperoxidase) immunohistochemistry. At time of harvest, serum samples were collected for troponinT level analysis. Pumpfunction of hearts subjected to 8 h of cold ischemia followed by 24 h of reperfusion was evaluated in an isolated working heart model (pressure-volume). To show if B-beta15–42 has also protective effects on other organs, we tested the peptide in a renal ischemiareperfusion model: After removal the right kidney we clamped the left renal artery for 60 min followed by reperfusion periods of 24 h or 7 days. 1.2 mg of B-Beta15–42 were given i.v. 1 min before reperfusion. To determine renal function we took serum-samples at several timepoints and measured Creatinine and BUN levels. Results. Hearts transplanted immediately or after 4 h of cold ischemia did not show any morphological damage. In contrast 8 h of cold ischemia showed severe myocardial damage associated with an inflammatory response at 24 h. Lesions further progressed after 10d. Administration of B-beta15–42 resulted in a significant amelioration of myocardial necrosis together with a reduced leukocyte infiltration. The protective effect of the peptide was further underlined by reduced troponin T levels in the treatment groups. The working heart preparation showed poor myocardial pump function in the control group. Administration of B-beta15–42 resulted in a significant amelioration of graft function. Renal ischemia-and reperfusion resulted in elevated levels of Creatinine and BUN in untreated animals with a peak at day 2. From day 5 on renal function recovers and finally reaches normal levels on the 7 again. Animals receiving B-beta15–42 showed significantly lower levels of Creatinine and BUN on day 2 and day 5. Conclusions. B-beta15–42 is a promising novel therapeutic agent against ischemia-reperfusion injury. Our data shows that it is not only usefull for treatment of reperfusion injury after myocardial infarction but also for cardiac tranplantation. Our recent findings show that B-beta15–42 is also protectiv for the kidney. So that we can sumarize that B-beta15–42 seems to be a potential candidate drug for the clinical treatment of ischemia and reperfusion injury.

79 Molecular predictors for kidney post-transplant anaemia J. Wilﬂingseder1, A. Kainz1;2 , P. Perco2;3 , B. Mayer3, R. Oberbauer1;2;4 1

Department of Nephrology Hospital Elisabethinen, Linz, Austria; 2 Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria; 3 Emergentec Biodevelopment GmbH, Vienna, Austria; 4 Austrian Dialysis and Transplant Registry, Austria

Anemia of chronic kidney disease is a well studied comorbidity, but the molecular predictors of post-transplant anemia remain elusive.

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In this case control study, 25 subjects with post transplant anemia defined as ESA requirement within the first post-transplant year were matched to 25 control recipients with comparable demographics but no anemia using the Austrian Dialysis and Transplant Registry. Genome wide gene expression analyses of deceased donor kidney biopsies obtained immediately before engraftment were performed using custom cDNA microarrays. Significant molecular features were included together with clinical variables in a multivariable logistic regression analysis and further analyzed with respect to their molecular functions, biological processes, cellular locations using gene ontology terms and protein–protein interactions. Within the expression data sets we found a number of significantly differentially regulated features potentially associated with the need for ESA. In particular proteins involved in immunity response were upregulated in the ESA patient group, suggesting the involvement of the inflammation cascade, as seen with deceased donor organs, as predictor of ESA need within one year after engraftment. From the list of differentially expressed genes we identified the best expression features allowing a prediction of ESA need utilizing a stepwise gene selection algorithm. Following this procedure the gene expression of SPRR2C (OR ¼ 0.24, 95%CI 0.07–0.85, p ¼ 0.027) and GSTT1 (OR ¼ 2.40, 95%CI 1.21–4.77, p ¼ 0.013) remained significant for prediction of ESA after adjusting for donor age, eGFR, BCAR and CRP. On the basis of the identified molecular markers a correct prediction of the need for ESA within the first year after transplantation was possible in 93% of the cases. Causal inference of these proteins with anaemia however needs to be further tested.

80 Biocompatibility of peritoneal dialysis solutions determined by genomics of human leukocytes: a cross-over study J. Wilﬂingseder1, P. Perco2;3 , A. Kainz1;2 , B. Mayer3, R. Oberbauer1;2;4 1 Department of Nephrology Hospital Elisabethinen, Linz, Austria; 2 Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria; 3 emergentec biodevelopment GmbH, Vienna, Austria; 4 Austrian Dialysis and Transplant Registry, Austria

Optimal biocompatibility of solutions for peritoneal dialysis (PD) is vital for allowing continuous use with minimized side effects. Understanding the molecular profiles induced by peritoneal fluids used may provide important insight for further optimizing peritoneal fluids. This present study evaluated the differential gene expression profiles of peripheral blood mononu-

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clear cells (PBMCs) from patients treated with two different PD solutions. In a random cross over design five patients received a glucose-based fluid (GBF), and an icodextrin-based (IBF) fluid, harvesting a comparatively analyzing the PBMCs after each peritoneal fluid use for each patient. Unsupervised hierarchical clustering of the gene expression profiles suggested a distinct molecular signature for IBF samples. Differentially regulated genes were found to be associated with activation of inflammation, and 34 genes upregulated as a consequence of IBF treatment were associated with processes categorized as immunity and defence. Genes upregulated in the context of GBF treatment on the other hand were found to be mainly involved in signal transduction processes. These data suggest reduced inflammation and consequently an improved biocompatibility of glucose-based peritoneal dialysis fluids.

81 Biocompatibility of hemodialysis membranes determined by genomics of human leukocytes: a randomized cross-over study J. Wilﬂingseder1, P. Perco2;3 , A. Kainz1;2 , B. Mayer3, R. Oberbauer1;2;4 1

Department of Nephrology Hospital Elisabethinen, Linz, Austria; 2 Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria; 3 emergentec biodevelopment GmbH, Vienna, Austria; 4 Austrian Dialysis and Transplant Registry, Austria

Biocompatibility of hemodialysis (HD) membranes is the most important quality criteria to ensure longterm dialysis without major harmful effects. This study sought to evaluate the genomic signatures of PBMCs in HD-patients treated with two different dialyzer membranes. In this random cross over study we investigated the genome wide molecular signature of PBMCs with cDNA arrays in four stable HD patients before and after dialysis comparing semi-synthetic and full-synthetic dialysis membranes. The aims of the study were to find the more biocompatible membrane and to elucidate the underlying molecular mechanism. Differentially expressed genes were analyzed on a functional level using the PANTHER classification system. Pathways and molecular function groups holding a significant number of genes were additionally determined. Selection of twofold differentially regulated genes yielded 172 up-regulated sequences by the semi-synthetic membrane that can be categorized according to PANTHER ontologies into several immunity and defense, signal transduction processes and apoptosis. Dialysis with Eur Surg Vol. 40 Supplement Nr. 226 2008

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full-synthetic membrane caused an activation of only 72 genes which are mainly involved in cell cycle and cell cycle control. The over-representation of up-regulated immunity= defence, signal transductions molecules and proteins which are involved in apoptosis suggests that full-synthetic membranes are more biocompatible than semisynthetic membranes. Nevertheless, the molecular pathways causally involved in this processes need to be established in further experimental studies.

82 Stem cell collection and autologous transplantation after TD or VTD (Velcade, thalidomide, dexamethasone) induction regimens results in high complete N. Worel1 , M. Mitterbauer2, J. Ackermann3 , H. Greinix2 , C. Zielinski3 , P. Kalhs2 , J. Drach3 1 Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria; 2 BMT-Unit, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria; 3 Division of Oncology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria

Background. During the last years front-line therapy for multiple myeloma has been rapidly evolving with the development of new highly active regimens. Thalidomide and bortezomib (velcade) based therapeutic strategies are demonstrating substantial efficacy, particularly in patients with poor prognostic features and=or high tumor burden. Given as induction therapy prior to hematopoietic stem cell transplantation (HCT) TD and VTD offer rapid and durable responses with high rates of complete response (CR), a surrogate end point for improved overall survival. Methods. We report our experience in 10 patients (7 male, 3 female; median age 49 years, range, 35–65 years) where TD or VTD were used as induction treatment prior to autologous stem cell collection and transplantation. Results. Patients had a high tumour burden seen in marrow infiltration of 30 to up to 95%(n ¼ 6), serum free-kappa light-chains of 4000 mg=l (n ¼ 1), serum IgG> 8300 mg=dl (n ¼ 1), serum IgA of 3990 mg=dl (n ¼ 1) and bulky plasmacytomas in various bones (n ¼ 2) and=or high-risk cytogenetic abnormalities. TD or VTD resulted in a rapid response after a median of 5 (range: 3–7) cycles, 6 patients (60%) achieved a CR, and 4 a partial response (PR, including two very good PR, VGPR). After G-CSF priming, a median number of 5.02 (range: 2.23–8.2) 10E6 CD34þ cells=kg b.w. was collected. Following high-dose melphalan (200 mg=m2) patients received a median of 2.94 (range: 2.23–5.15) 10E6 CD34þ cells=kg b.w. and showed absolute neutrophil counts (ANC) above> 0.5 G=L and platelet counts above 20 G=L after a median of 11 (range: 9–26) and 12 (range: 10–30) days, Eur Surg Vol. 40 Supplement Nr. 226 2008

respectively. After a median follow-up of 9.4 (range: 2.3– 28.6) months, 8 (80%) patients are in continuous CR and 2 (20%) patients in VGPR. Conclusions. Induction treatment with TD or VTD results in rapid tumour mass reduction and a high remission rate in MM patients even with poor prognostic features. These preliminary results suggest that TD and VTD are effective and safe first-line treatments allowing autologous stem cell collection for consolidating high-dose therapy and autologous HCT in MM.

83 Improved mobilization of peripheral blood CD34þ cells by AMD3100 plus granulocyte-colony-stimulating factor (G-CSF) in hard to mobilize patients N. Worel1 , B. Pribitzer1, K. Gerhartl1 , H. Gisslinger2, M. Mitterbauer3, G. Leitner1 1 Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria; 2 Division of Hematology and Hemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Austria; 3 BMT-Unit, Department of Medicine I, Medical University of Vienna, Vienna, Austria

Background. Since the 1990s, almost exclusively peripheral blood stem cells (PBSC) are used for autologous stem cell transplantation (ASCT) instead of bone marrow due to the more rapid recovery of hematopoiesis. However, some patients do not adequately mobilize PBSC. Risk factors for difficult mobilization are: older age, bone marrow involvement, prior radiotherapy to marrow sites and extensive pretreatment. Various strategies such as escalation of G-CSF dosage or combination with GM-CSF, IL-3 or SCF to improve mobilization in these patients have been described. Another exciting option for these patients is the new cytokine, AMD3100. This agent is an inhibitor of SDF1 binding to CXCR4 and appears to promote mobilization of CD34þ cells into the circulation. The use of AMD3100 in combination with G-CSF in patients unable to collect adequate CD34þ cells with G-CSF alone was recently reported in 280 patients with lymphoma and multiple myeloma (MM). There, success was defined as a collection of 2 10E6 CD34þ cells=kg with this regimen. In contrast, clinical studies showed that AML, CLL and PCL cells may also be mobilized by AMD3100 via CXCR4 inhibition. Due to these concerns, AML, CLL and PCL patients are excluded from AMD3100 trials. Methods and results. We here report 2 patients with MM III A who failed stem cell mobilization after cyclophosphamide and G-CSF administration. Pretreatment consisted of 3 cycles vincristine, adriamycin, dexamethasone (VAD), thalidomide, deaxamethason and lenalidomid in 1 patient and 5 cycles of dexamethasone and

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22nd Meeting of the Austrian Society of Transplantation

lenalidomide in the other one, respectively. Patients received 5 mg=kg G-CSF bid. for 4 days followed by 240 mg=kg of AMD3100 given subcutaneously 10–11 h before collection. Our aim was to assess the effect of AMD3100 on the mobilization of CD34þ cells. Administration of G-CSF and AMD3100 were continued daily until> 2.5 106 CD34þ cells=kg were collected. Conclusions. Adequate collection of CD34þ cells (3.58 and 5.54 10E6 CD34þcells=kg) were achieved in boths patients within 2 days. In conclusion, AMD3100 in combination with G-CSF was generally safe and offers a new treatment to collect CD34þcells for autologous transplant from poor mobilizers. Due to the reported mobilization of leukemic cells, AMD3100 should be restricted to patients with lymphomas and MM.

84 Catastrophic graft-versus-host disease after lung transplantation proven by PCR-based chimerism analysis N. Worel1 , A. Bojic2 , M. Binder3, G. Mitterbauer-Hohendanner4, B. Streubel5 , T. Staudinger2, G. Locker2 1

Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria; 2 Intensive Care Unit, Department of Medicine I, Medical University of Vienna, Vienna, Austria; 3 Department of Dermatology, Medical University of Vienna, Vienna, Austria; 4 Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria; 5 Department of Pathology, Medical University Vienna, Vienna, Austria

Acute graft-versus-host disease (GvHD) is a rare complication after solid organ transplantation. We describe a 52-year-old female developing neutropenia and fever 48 days after single lung transplantation for chronic obstructive pulmonary disease. Bone marrow biopsy suggested drug-induced marrow failure, so immunosuppression was reduced. Five days later a maculopapular skin rash was observed, progressing to a generalized erythema with desquamation. Skin biopsy was suspective of GvHD, so immunosuppression was re-initiated. PCR-based chimerism analysis of bone marrow revealed 78% donor cells. Intensified immunosuppression resulted in temporary improvement, but bone marrow aplasia recurred and the patient experienced severe GvHD of gut and liver. Despite extensive immunosuppression the patient died from multi-organ-failure 99 days after transplantation. This report describes the occurrence of neutropenia as an early presenting sign of acute GvHD after lung transplantation. We therefore recommend to integrate GvHD in the differential diagnosis of neutropenia after solid organ transplantation necessitating early chimerism analyses.

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85 Safety and efﬁcacy of statin therapy in patients switched from Cyclosporine A (Csa) to Sirolimus (Srl) after cardiac transplantation A. Zuckermann, A. Aliabadi, D. Dunkler, S. Mahr, M. Grömmer, D. Zimpfer, M. Grimm Division of Cardio-Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria

Background. Statins are an established therapy against hyperlipidemia after cardiac transplantation. However, their interactions with CsA bear the danger of rhabdomyolysis. Srl is a novel immunosuppressive drug that has been used successfully in cardiac transplant patients. Yet it s interactions with statins are similar to CsA. Furthermore, Srl increases lipid blood levels in transplant patients. The aim of the study was to evaluate the safety and efficacy of statin therapy after switch from Csa- to Srl-based immunosuppression. Methods. Ninety-eight long-term patients were switched from Csa to Srl. All patients received also MMF steroid therapy. Reasons for switch were renal dysfunction (88%), graftvasculopathy (7%) and skin cancer (5%). Average patient age at switch was 59.9 9.8 years and 92% were male. Patients were switched 7.8 4.7 years after transplant. Total examinational period was 12 months before and 12 months after switch (total of 1088 patient-months). Safety Evaluation consisted of regular measurements of CK-levels as well as evaluation of incidence of CK elevation ( > 200) myalgia, myositis (CK> 500), and rhabdomyolysis (CK> 1000). Moreover, hepatoxicity (AST > 100, ALT> 140) was closely monitored. Efficacy analysis was performed by serial blood lipid assessments (LDL, HDL, total cholesterol, triglyzerides). Results. 43 (44%) of patients received atorvastatin, 28 (29%) pravastatin and 28 (27%) received other drugs, were converted from one statin to another or therapy was temporarily suspended. There were a total of 29 CK elevation cases (0.03 events=person-months [EPM]). CK elevations were seen in 19 cases (0.02 EPM), myositis in 9 cases (0.008 EPM) and rhabdomyolysis in 1 case. Incidence of EPM was similar between atorvastatin (0.03) and pravastatin (0.02). In case of CK elevation statins were reduced or paused. Only four cases of hepatoxicity (0.003 EPM) were detected during follow-up. Most lipid blood levels increased significantly after switch (cholesterol: pre: 192.9 38.6 vs. post: 219.9 42.5, p < 0.01; LDL: pre: 107.9 35.6 vs. post: 122.5 37.6, p < 0.01; Triglycerides: pre: 178.3 88.2 vs. post: 221.7 138.7, p < 0.01). Only HDL cholsterol remained unchanged (pre: 55.2 15.4 vs. post: 56.3 13.3, n.s.). Blood lipid levels increased significantly in patients no matter if they were treated with either atorvastatin or pravastatin. Only in patients in whom statin therapy was increased after switch (pre: 13.8 5 mg vs. post 31.3 12.6 mg; p < 0.01), blood lipid levels remained similar to pre-switch levels (cholesterol: pre: 187.5 31.8 Eur Surg Vol. 40 Supplement Nr. 226 2008

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vs. post: 221.6 59.8, p ¼ 0.07; LDL: pre: 110.5 46.9 vs. post: 128.6 48.2, n.s.; Triglycerides: pre: 175.4 82.2 vs. post: 180.2 80.6, n.s.). Conclusions. Statin therapy after switch from Csa to Srl in long-term cardiac transplant patients is safe. However, blood lipids increase significantly after the switch unless statin therapy is increased. There were no differences in safety and efficacy between atorvastatin and pravastatin.

86 Hohe Prävalenz von Osteoporose und Wirbelkörperfrakturen nach Lungentransplantation bei Patienten mit Cystischer Fibrose B. Zweytick1 , S. Holzer1, P. Jaksch1 , M. Koeller2, F. Kainberger3, W. Klepetko1 1 Klinische Abteilung für Herz-Thoraxchirurgie, Universitätsklinik für Chirurgie, Medizinische Universität Wien, Wien, Österreich; 2 Klinische Abteilung für Rheumatologie, Universitätsklinik für Innere Medizin III, Medizinische Universität Wien, Wien, Österreich; 3 Klinische Abteilung für Neuroradiologie und muskuloskeletale Radiologie, Universitätsklinik für Radiodiagnostik, Medizinische Universität Wien, Wien, Österreich

Grundlagen. Patienten mit Cystischer Fibrose (CF) haben im Vergleich zu Gesunden bedingt durch Malnutrition, verminderte Sexualhormone, Inaktivita¨t und chronische Infekte eine geringere Knochenmasse. Mit steigender Lebenserwartung dieser Patienten gewinnt die Osteoporose immer mehr an Bedeutung. In der Lit-

Eur Surg Vol. 40 Supplement Nr. 226 2008

eratur ist die Pra¨valenz an Osteoporose mit 30% und an Wirbelkompressionfrakturen mit 7–35% in erwachsenen CF sehr hoch. Keine osteologischen Daten existieren nach Lungentransplantation (LUTX), wenn CF einer hohen Immunsuppressions und Glucocorticoidtherapie ausgesetzt sind. Methodik und Ergebnisse. Wir haben retrospektiv Skelettro¨ntgen und Densitometrie (DXA) von 21CF (9 m=12 w; Alter: 28 9 a). Wir analysierten Daten von allen CF, die zwischen 01=99 und 08=04 lungentrans¨berlebten. plantiert wurden und zumindest 2,5 Jahre u Das mittlere Follow up war 2 Jahre (0,1–8,5 a). Nur 1CF (4,7 %) hatte normale T-Werte (Mittlere Knochenmineralisationsdichte von jungen Erwachsenen), 3 CF (14,3 %) hatten eine Osteopenie, 1110 CF (52,4 %) hatten eine Osteoporose and 6 CF (28,6 %) hatten Wirbelfrakturen, wovon 50 % innerhalb eines Jahres nach Lungentransplantation diagnostiziert wurden. Die Knochenmineralisationsdichte war deutlich reduziert (LWS: Mittel: 0,97 g=cm2 ; T-Score: 2,9. Femur Mittel: 0,73 g=cm2 , TScore: 2,64) und pra¨diktiv fu¨r Kompressionsfrakturen. Das Parathormon (PTH) war erho¨ht in 82 %, Osteocalcin in 45 % and 25-Hydroxyvitamin D war vermindert in 36 %. Die Kreatininclearance betrug im Mittel 56,4 ml=min. Alle CF hatten eine ada¨quate Osteoporosetherapie nach LUTX. Schlussfolgerungen. Wegen der hohen Pra¨valenz an ¨ hen Auftreten Osteoporose in CF pra¨operativ und dem fru von Kompressionsfrakturen postoperativ sollten alle CF ¨ he pra¨operativ auf Osteoporose gescreent werden. Um fru Kompressionsfrakturen zu verhindern, sollten alle erwachsenen CF mit Osteopenie oder Osteoporose eine Biphosponat=Kalzium=Vit D Therapie vor LUTX und zumindest ein Jahr nach LuTX erhalten.Nur eine optimale perioperative medikamento¨se Therapie in Kombination mit Muskelaufbautraining kann die Inzidenz an Wirbelko¨rperfrakturen innerhalb des ersten Jahres nach LUTX reduzieren.

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Nach Redaktionsschluss eingelangt 87 Fatal disseminated toxoplasmosis via kidney allograft in a 15-month-old infant M. Gnigler1, A. Krezcy2, L. B. Zimmerhackl1 , R. Margreiter3, M. Frühwirth1 1 Department of Pediatrics, Medical University of Innsbruck, Innsbruck, Austria; 2 Institute of Pathology, Medical University of Innsbruck, Innsbruck, Austria; 3 Department of Operative Medicine, Medical University of Innsbruck, Innsbruck, Austria

Background. Toxoplasmosis is a known complication of transplantations in adults, mostly described in bone marrow and heart transplant recipients. The infection is rare following renal transplantation in children. Methods. We report the unusual case of an infant who underwent renal transplantation at the age of 11 months because of end stage renal failure due to hypoplastic-dysplastic kidney disease. The patient developed toxoplasmosis, most likely transmitted by the allograft. The child received the kidney of a 3.5-year-old donor who had IgG but no IgM antibodies against toxoplasmosis. The initial immunosuppression consisted of Tacrolimus, Azathioprin and Aprednisolon. Six days after transplantation the boy developed fever and the renal function deteriorated. Acute cellular rejection was suspected and treated with Methylprednisolon. Since the fever persisted antibiotic treatment was started even though the CRP was within the normal range. One month later the patient’s state of health worsened, so a biopsy was performed which excluded acute cellular rejection. Furthermore all serologic investigations including CMV and EBV were negative. On day 121 after transplantation the patient showed signs of sepsis. A fundoscopic examination showed retinochoroiditis, and consequently an infection with Toxoplasma gondii was suspected. Serologic investigations were positive for Toxoplasma IgG and IgM, and a PCR of the cerebro-spinal-fluid confirmed the diagnosis. Therapy was immediately changed to Sulfadiazine and Pyrimethamin and immunosuppression was changed to Prednisolon only. The patient developed a multi-organ failure and died on day 137 after his renal transplantation. Autopsy showed cysts of Toxoplasma gondii in heart and lungs but not in the transplanted kidney. Results. In pediatric renal transplant recipients the risk of toxoplasmosis is low. In literature there are reports about 31 cases of toxoplasmosis after kidney transplantations in adults and only four cases of children described. We add the case of a 15-month-old infant. Diagnosis of toxoplasmosis is difficult, since symptoms like fever are unspecific and seroconversion can be delayed because of immunosuppression. In all four patients the transplanted

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organ is suspected to be the source of infection. Also in our case transmission over the allograft seems the most likely explanation, since we assume the child was seronegative and therefore in the high-risk group because he received a seropositive organ. Antirejection treatment may have contributed to the development of severe immunosuppression and the critical clinical course in our patient. Conclusions. In summary toxoplasmosis is a rare complication after renal transplantations in children, which is potentially fatal, but can be treated if detected early enough. We recommend a screening of donor and recipient as it is routinely done for CMV prior to transplantation, which makes it possible to detect high-riskconstellations of seronegative recipients of seropositive organs. If the donor is toxoplasmosis positive a continuous monitoring should be done.

88 Targeting adhesion molecules for prophylaxis and treatment of skin rejection in composite tissue allotransplantation T. Hautz1 . B. Pulikkottil2 , B. Zelger3, G. Brandacher1, A. WP Lee2 , R. Margreiter1, S. Schneeberger1 1 Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria; 2 Division of Plastic Surgery, University of Pittsburgh, PA, USA; 3 Medical Center, Department of Pathology, Pittsburgh, PA, USA

Background. The skin as the primary target of rejection in a composite tissue allograft needs to be specifically addressed to prevent and treat skin rejection episodes and therefore to allow for wide spread application of Composite Tissue Allotransplantation. Investigation of the expression of molecular markers of leukocyte and lymphocyte trafficking in skin biopsies after human hand transplantation showed VE-Cadherin as well as E-Selectin up-regulated in association with skin rejection. Methods. In a Brown-Norway to Lewis rat hind-limb allotransplant model Efomycine-M, a blocker of the adhesion molecule E-Selectin and 15-42, a fibrin derivate that binds VE-Cadherin were used for topical treatment of the skin. Animals received either Efomycine-M (5 mg=kg= week subcutaneously) alone, in combination with systemic immunosuppression consisting of Tacrolimus for 50 days (30 mg=kg=day i.p. to POD 30, 25 mg=kg i.p. to POD 50) or Tacrolimus alone for 50 days (30 mg=kg=day i.p. to POD 30, 25 mg=kg i.p. to POD 50). Another group received 15-42 (5 mg=kg=week subcutaneously) and subtherapeutic doses of Tacrolimus (0.1 mg=kg=day i.p.). Untreated animals served as controls. Results. Rats that were treated with Tacrolimus for 50 days showed high-grade rejection of the skin on postoperative day 66 1. Additional treatment with topical Efomycine-M prolonged limb graft survival up to 130 days until present. Treatment with 15-42 together Eur Surg Vol. 40 Supplement Nr. 226 2008

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with subtherapeutic doses of Tacrolimus significantly prolonged limb allograft survival from 7 0 days (controls) to 17.25 2.872 days (p ¼ 0.0056). However, results of Efomycine-M treatment as well as 15-42 treatment alone without any systemic immunosuppression was similar to untreated controls. Conclusions. Local administration of adhesion molecule blockers for E-Selectin or VE-Cadherin together with short-term or low-dose systemic immunosuppression significantly prolong allograft survival in a rat hind-limb transplantation model. These findings indicate a valid strategy to minimize the need for systemic immunosuppression in Composite Tissue Allotransplantation.

89 Histogenomics: Association of gene expression patterns with histological parameters in kidney biopsies P. Perco1;2;3 , A. Kainz1;2 , J. Wilﬂingseder1;2, A. Soleiman4 , B. Mayer3, R. Oberbauer1;2;5 1 Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria; 2 Department of Nephrology, Krankenhaus der Elisabethinen, Linz, Austria; 3 emergentec biodevelopment GmbH, Vienna, Austria; 4 Department of Pathology, Medical University of Vienna, Vienna, Austria; 5 Austrian Dialysis and Transplant Registry

Background. Several studies investigated the association of histological scores of donor kidney biopsies obtained before engraftment with post transplant outcomes. Discrimination and goodness of fit of these scores however is low. Methods. Thus we sought to identify and elucidate the performance of molecular rather than histological markers for this purpose using whole genome gene expression microarray experiments. Results. We identified 80 unique differentially regulated genes in 82 samples showing no histological damage versus those with histological damage based on the Chronic Allograft Damage Index (CADI) and acute tubular injury. Main biological categories enriched with upregulated genes in damaged tissue were ‘‘immunity and defense’’, ‘‘cell communication’’, or ‘‘apoptosis’’. Interestingly genes involved in cell structure, cell adhesion and protein trafficking were specific for tubular atrophy. Histology (CADI score) explained only 14% of the variability of one year creatinine (adjusted R2 for panel reactive antibodies, biopsy confirmed acute rejection, and sum of HLA mismatches) whereas a combination of three biomarkers without clinical covariables explained 28%. The three molecular markers are the NLR family, pyrin domain containing 2 (NLRP2), immunoglobulin J polypeptide (IGJ), and the regulator of G-protein signaling 5 (RGS5). Conclusions. In summary, we identified biomarkers in transplant kidney biopsies, which are predictive for medium term allograft function.

¨ sterreich. – Datenkonvertierung und Umbruch: Medieninhaber und Herausgeber: Springer-Verlag GmbH, Sachsenplatz 4–6, 1201 Wien, O ¨ sterreich. – Verlagsort: Thomson Press (India) Ltd., Chennai; Druck: Druckerei Ferdinand Berger & So¨hne Gesellschaft m. b. H., 3580 Horn, O Wien. – Herstellungsort: Horn.

Printed in Austria P. b. b.==Erscheinungsort: Wien==Verlagspostamt 1201 Wien Eur Surg Vol. 40 Supplement Nr. 226 2008

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22nd Meeting of the Austrian Society of Transplantation, Transfusion and Genetics. Zell am See, October 29–31, 2008. Guest Editor: Rainer Oberbauer, Linz, Austria

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