key: cord-0034122-n3o4rtz4
authors: nan
title: 34th Seminar of the Austrian Society for Surgical Research. Wagrain, November 11–13, 2010. Guest Editor: H. Cerwenka, Graz, Austria
date: 2010
journal: Eur Surg
DOI: 10.1007/s10353-010-0565-7
sha: 832810181971b5c777d2aabb2e20c8734b9515d0
doc_id: 34122
cord_uid: n3o4rtz4

nan

Clinical and basic researcha symbiosis?

H. Cerwenka

In biology, the term ''symbiosis'' (from the Greek: syn ''with'' and biosis ''living'') is used to describe the coexistence of different species with close and often long-term interactions to the mutual benefit of the organisms involved. Many feel that symbiosis between clinical and basic research should be encouraged through more extensive contact and cooperation.

The path from ''bench to bedside'' (and to the operating room) may be hard and stony. In his article entitled ''The long road from basic science to clinical medicine,'' Niederau [1] wrote that -in the area of free radicals -the process of translating the exploding knowledge from basic science to clinical medicine may take longer than many scientists and physicians had hoped. This might also apply to many other domains. One aspect at least is very different now: It used to be that new findings were often published only after a long delay due to the time-consuming review and printing processes. Today, Internet, e-mail and electronic publishing have considerably changed the scene. Direct worldwide contact is possible and space limitations (as in printed journals) are no longer an important issue.

For those in basic medical research, it is important to keep up with front-line clinical medicine, and for clinicians, it is essential to be open to new findings -including, of course, critical perception and evaluation. For a long time, surgery was dominated by outstanding personalities and their doctrines. More recently, not only has surgical technique been revolutionized by many innovations, but also the role of the surgeon has changed, with much more involvement in a ''symbiosis'' with internal medicine, interventional radiology and many other specialties -liver surgery is a good example with recent developments in the management of hepatic metastases [2] or liver abscesses [3] . Interdisciplinary meetings and tumour boards should enhance rapid integration of research findings from all fields into clinical decision-making. In a recent study on the techniques of hernia surgery in different hospitals, however, we found that the strate-gies used not only varied greatly but also were often ''eminence-based'' rather than ''evidence-based'' [4] .

Taking a closer look at the matter, we have to accept that only a few of our surgical policies in routine use are really ''evidence-based'' and many of them would not be suitable for prospective randomized studies as, for instance, ''operative versus non-operative treatment in patients with diffuse peritonitis due to visceral perforation'' -this has been compared with a demand that the effect of a parachute be proven in a randomized controlled trial [5] . Another important issue is that every patient requires tailored individual treatment and statistical results cannot be automatically applied to single persons and their specific situations (-globalize the evidence, but localize the decision).

The seminar of the Austrian Society for Surgical Research (some highlights of which are published in this supplement) is an excellent opportunity to intensify the symbiosis between clinical and basic research. I would like to express my sincere thanks to everyone contributing to its success.

The innate immune response against cancer A. Cerwenka

Innate Immunity Group, German Cancer Research Center=D080, Heidelberg, Germany

The immune system represents a complex network of molecules, cells, tissues and organs that have evolved to protect the organism from pathogens. It consists of an innate and an adaptive arm that are distinguished based on the type of antigens they recognize, their recognition strategies and kinetics of their responses. A major feature of the immune system is its ability to recognize ''non-self'' as well as ''altered-self'' molecules. ''Altered-self'' recognition enables the detection of transformed cells and potentially the prevention of the development of malignancies.

Sequential recruitment to tumor tissues and efficient activation of specialized effector cell populations determine successful anti-tumor immune responses. The innate immune response serves as a first line of immune defense against cancer. The main population of innate immune cells infiltrating tumors is cells of myeloid origin such as subsets of macrophages and dendritic cells. In most cases, myeloid subpopulations support an immunosuppressive, tumor-promoting tumor microenvironment. Thus, high numbers of myeloid cells in the tumor tissue often correlate with a bad prognosis for tumor patients. Other types of innate immune cells such as Natural Killer (NK) cells that infiltrate tumors can directly lead to tumor cell destruction and are crucial for the efficient activation of the adaptive immune response exerted by T lymphocytes. Therefore, high numbers of NK cells in tumors often correlate with a good prognosis for tumor patients [1, 2] .

The activation of NK cells is determined by a delicate balance between signals delivered by inhibitory receptors, most of which are specific for self-MHC class I molecules and activating receptors. Ligands for activating receptors are often expressed on tumor cells but not on healthy cells. Many tumors in cancer patients lose expression of MHC class I molecules to escape from direct recognition by CD8 þ T cells, and thus become more susceptible to attack by NK cells. In patients suffering from acute myeloid leukaemia who undergo bone marrow transplantation, adoptive transfer of alloreactive NK cells significantly improves patients' survival [3] . However, eradication of large solid tumors by NK cells is usually not efficient despite low expression of MHC class I molecules on many tumor cells. Indeed, our study revealed that in cervical carcinoma expression of MHC class I was low in many biopsies. In addition, ligands for activating NK cell receptors were quite frequently expressed suggesting that cervical carcinoma cells should be efficiently attacked by NK cells [4] . However, numbers of NK cells in these tumors were low and tumor infiltrating NK cells were of a non-cytolytic phenotype. Thus, the focus of our studies is to harness NK cells against tumors. We are following multiple strategies including (i) Enhancing NK cell function and numbers in tumorbearing hosts and (ii) Increasing the susceptibility of tumor cells to NK cell mediated killing.

In the first step, we characterized the in vivo NK cell response in a mouse model of subcutaneous injection of the MHC class I-deficient lymphoma RMA-S. We observed that after subcutaneous injection of RMA-S tumor cells, NK cells infiltrating the tumor tissue show a less mature phenotype as compared to blood NK cells. In addition, gene expression profiling using whole genome microarrays revealed a strikingly different transcription profile of tumor infiltrating NK cells. When compared to blood NK cells, NK cells from the tumor tissue displayed a signature of genes involved in cell inhibition. Therefore, therapies that involve the blockade of NK cell inhibitory molecules should be beneficial to reactivate NK cells against tumors. Furthermore, we observed that NK cells in tumors have downregulated the chemokine receptor CXCR3 suggesting that this receptor might have been used for migration to the tumor tissue [5] . Indeed, CXCR3-deficient NK cells were defective in their ability to accumulate in the tumors. Furthermore, CXCL10, the ligand of CXCR3, recruited high numbers of NK cells to the tumor correlating with improved survival of recipient mice.

In addition, we are developing strategies to upregulate ligands for activating receptors on tumor cells in order to increase the visibility of tumor cells for NK cells. One of the activating receptors, NKG2D, interacts with several virus-or stress-inducible ligands, including ULBP1 and -2 [6] . Recently, it has been shown that induction of the DNA damage and=or cellular senescence pathway in tumor cells resulted in activation of NK cells through up-regulation of certain NKG2Dligands. Both pathways activate the transcription factor and tumor suppressor p53 that is mutated in 50% of cancers. Our study reveals that induction of wildtype (wt) p53, but not mutated (mut) p53, led to a pronounced up-regulation of ULBP1 and -2 expression in tumor cells resulting in enhanced NKG2D-dependent degranulation and IFN-production by NK cells. Accordingly, treatment of certain wtp53-expressing tumor cell lines with the p53-reactivating small molecular compound RITA resulted in ULBP2 mRNA and cell surface up-regulation. Thus, a combination of p53reactivating drugs, such as RITA, with adoptive transfer of NK cells might constitute an efficient novel NK cell-based immunotherapy against wtp53 expressing cancers.

In conclusion, our studies suggest that increasing the numbers of tumor infiltrating activated NK cells combined with enhancing expression of activating ligands on tumor cells might lead to successful strate-gies for NK cell-based immune therapies against solid tumors. Background. Previous experiments with low porosity grafts revealed fast host cell immigration into nanostructured, electrospun grafts. However, vascular specific cells did not populate the inner sections of the graft wall and showed a continuous decrease in the long-term. This study investigates the effects of varied graft porosity on the healing characteristics of electrospun, small diameter conduits.

Methods. Vascular grafts (length 15 mm, inner diameter 1.5 mm) were fabricated by electrospinning using polyether-urethane. Fine mesh grafts (void fraction 70%) or coarse mesh grafts (void fraction 80%) were interposed into the abdominal aorta of 56 rats for either 7 days or 1, 3 or 6 months. Specimens were analyzed after retrieval by histology.

Results. Coarse mesh grafts showed significant increased host cell immigration in all areas of the conduit wall and improved cell survival after long-term implantation. Grafts with greater pores showed also increased formation of neointima and synthesis of extracellular matrix compounds.

Conclusions. Host cell ingrowth and host cell survival benefit from high graft porosity. The effect of the increased extracellular matrix synthesis on biomechanical properties has to be evaluated in further studies.

The impact of homocystein on neointimal hyperplasia in experimental vein grafts Methods. C57BL6 mice (WT) and Cystathione betasynthase heterozygous knockout mice (CBSAE) as a model for hyperhomocysteinemia underwent interposition of the vena cava of the donor mice into the carotid artery. Three experimental groups were used: group 1: WT donor and WT recipient, group 2: CBS AE donor and CBS AE recipient and group 3 WT donor and CBS knockout recipient.

Four weeks after surgery veins were harvested, paraffin embeded and underwent histopathological investigation. Hematoxylin=Eosin, Elastika, Alcian and Masson trichrom staining of the samples was performed.

Results. At 4 weeks postoperatively the neointimal thickness in controls (group 1) was 53.8 (16-59) mm, whereas CBS knock out animals (group 2) showed significantly increased neointimal thickness of 157.4 (121.2-183) (p ¼ 0.03). Animals of group 3 (WT donor and CBS knockout recipient) showed neointimal thickness levels of 114.7 (58.3-130.9) this was significantly more than in control animals (p ¼ 0.005) but significantly less than in group 2 (p ¼ 0.032).

Elastica staining revealed that animals of group 2 and group 3 had significantly more elastic fibres than control animals (p ¼ 0.008). Alcian staining showed significantly less acid mucoplysaccharids than in control animals (p ¼ 0.008). There were no significant differences in the Masson Trichrom staining (p ¼ n.s.)

Conclusions. Hyperhomocysteinemic cystathione beta-synthase knockout mice exhibit increased neointimal formation in arterialized vein grafts. Background. Negative pressure therapy is an established therapy since many years for open abdominal therapy. A new creation of foil for abdominal use (Suprasorb CNP, Lohmann & Rauscher) was thought to optimize fluid flow out of the abdomen. In order to get information for a safe human application, the influence of the foil on the different abdominal organs with continuous negative pressure application was tested on an animal model.

Methods. On seven domestic pigs of approx. 30 kg, laparotomy was performed. On four locations, a cholecystectomy-bed, exposed pancreas surface, small bowel and colon anastomosis and suture, suction pads were positioned and the abdomen was covered with Suprasorb CNP foil, before closing the abdominal wall. Suction pads consisted of gauze with a drainage core covered with a double layer of the foil. Suction pads were positioned in the way treated and untreated areas were covered. Then, continuous negative pressure, 40-60 mmHg, was applied for 8 h under general anaesthesia. The abdomen was reopened and the foil was observed for adhesion and influence of the organ surfaces. Organ parts were removed for histological studies.

Results. For the amount of continuous flow of secretion, a mean of 564 ml was found, ranging from 250 to 800 ml. The organs and tissues so treated showed no signs of macroscopic or microscopic damage and no signs of impaired microcirculation. Bowel suture and anastomotic sites did not develop fistulas. Histological studies did not indicate signs of developing impairments.

Conclusions. The results of seven animal studies suggest a safe use of Suprasorb CNP in the abdominal use in humans. Furthermore the results suggest the direct contact use of the suction device for different organ treatments. Background. The development of microsurgical techniques has facilitated the establishment of vascularized composite tissue transplant models in small mammals. Since the mouse would be the ideal model to study various CTA-related problems, we designed two new surgical techniques for orthotopic (ORT) and heterotopic (HET) hind limb transplantation.

Methods. BALB=c hind limbs were transplanted to BALB=c or C57BL6 recipients using a non-suture cuff technique. ORT: Donor femoral vessels were anastomosed to recipient femoral vessels, the sciatic nerve approximated end-to-end and osteosynthesis performed using an intramedullary rod. HET=cervical: Donor femoral vessels of a reduced size osteomyocutaneous hind limb CTA were anastomosed to recipient common carotid artery and external jugular vein without nerve approximation.

Results. Both procedures could be performed with a high success rate (ORT: 62%; HET 90%). Donor operation lasted 100 AE 12 min and recipient operation 114 AE 27 min (ORT); 54 AE 16 min (HET). Complication rates in terms of bleeding, and thrombosis at the cuff side was slightly higher in the ORT group. All syngeneic grafts survived long-term (>100 days). FK506 (2 mg=kg) significantly prolonged graft survival (87 AE 22 days) when compared to untreated controls (6 AE 1 day). Functional evaluation of ORT grafts by means of video gait kinematics and Cat-Walk analysis revealed specific differences of gait parameters when compared to not-transplanted controls (p < 0.05).

Conclusions. The ORT hind limb transplant model seems to be best suited to study functional outcome and nerve regeneration in CTA. The technically less demanding HET=cervical model may be used to investigate basic immunology and ischemia reperfusion injury in reconstructive transplantation. Background. Ischemia-reperfusion injury (IRI) following pancreas transplantation represents a major cause for graft pancreatitis, affecting short-and long-term graft survival. The essential cofactor of the nitric oxide synthase (NOS) and potent antioxidant tetrahydrobiopterin (H4B) was shown to dramatically reduce IRI-related organ damage in a murine pancreas transplantation model. Since the underlying mechanism is still under debate, aim of this study was to gain further insights using eNOS-=mice.

Methods. In the murine heterotopic pancreas transplantation model, male syngeneic C57Bl6 (h-2b) mice were used as recipients. C57Bl=6-based eNOS-=-mice, as well as eNOS wild types served as donors and where either pre-treated with 50 mg=kg b.w. H4B i.m. or untreated. Following pancreas retrieval, grafts were subjected to 16 h cold ischemia time (CIT) and 45 min warm ischemia time (WIT) to induce pancreatitis. Non-transplanted animals of both genotypes served as controls. Following 2 h reperfusion, microcirculation was analyzed by confocal intravital fluorescence microscopy and quantified by means of functional capillary density (FCD). Histopathological evaluation occurred by application of the Schmidt pancreatitis score. Formation of intragraft peroxynitrite was assessed by nitrotyrosine-immunohistochemistry. Intragraft H4B levels were determined by HPLC. Finally, since graft pancreatitis in this model was found to be lethal, different groups were tested for recipient survival.

Results. Compared to non-transplanted controls, prolonged CIT resulted in a pronounced microcirculatory breakdown. Independently from genotype, H4B-treated grafts reperfused for 2 h displayed markedly enhanced microcirculation compared to untreated animals (p ¼ 0.09). IRI-induced parenchymal edema, acinar necroses, hemorrhage and fat necroses were decreased following H4B pre-treatment, reaching statistical significance in wild type grafts (p < 0.05). Likewise, intragraft nitrotyrosine formation was decreased in both treated groups, reaching statistically significant differences only in wild type organs (p < 0.01). H4B donor pre-treatment significantly enhanced intragraft H4B levels (p < 0.01). Independently of the graft genotype, tetrahydrobiopterin pre-treatment significantly prolonged recipient survival (p < 0.001). Microarray analysis and RTqPCR detected among the complete mouse genome two differently regulated genes (>5-fold) in treated grafts already 15 min following reperfusion.

Conclusions. Comparable early intraparenchymal damage of transplanted grafts and similar recipient survival in both genotypes if treated with H4B, suggests, that in this model eNOS is not the major target of H4B. Future analysis of the two identified genes may provide further mechanistic insights in IRI following pancreas transplantation and in the therapeutic action of H4B.

Myeloperoxidase and carbonyl proteins are promising markers for non-invasive monitoring after heart transplantation concerning graft rejection Background. After heart transplantation (HTX) endomyocardial biopsy (EMB) is presently the standard method to diagnose acute graft rejection. As an alternative a non-invasive marker of rejection would be desirable to avoid or to permit more selective use of the costly and wearing EMB.

Methods. In our preliminary retrospective study, outcomes of routinely taken EMBs were used to select 28 patients after HTX EMB grade 0 R (8 patients), 1 R (9 patients) or 2R (11 patients). In these patients myeloperoxidase (MPO) and carbonyl proteins (CP) in serum were measured using ELISA.

Results. In this study, MPO and CP levels in patients after HTX with rejection grade 2R were significantly (MPO: p < 0.01 and CP: p < 0.001) elevated at the time of rejection in comparison with levels one month before. MPO and CP levels predicted rejection grade 2R and the best cut-off point was 237.5 mg=l for MPO and 222.5 pmol=mg for CP, respectively. Clinically most important was the obvious increase (doubling of basic values within one month) of MPO and CP levels in case of rejection grade 2R in patients after HTX.

Conclusions. MPO and CP seem to be appropriate parameters to monitor rejection events non-invasively and to reduce the application of EMBs after HTX. For that reason we start a prospective study according to our preliminary results. Background. As skin rejection still remains the major obstacle in reconstructive transplantation, attention is being directed towards this process, which has a T-lymphocytic infiltrate into the graft as its quintessential action. We herein investigated the effects of Correolide C, known to block a Kv1.3 potassium channel, and therefore to prevent activation and proliferation of a subtype of T cells, in an experimental rat limb transplantation model.

Methods. After orthotopic rat hind limb allotransplantation (BN-LEW) animals received Correolide C either i.p. (5 mg=kg=day) or as intra-graft treatment (3 mg=kg twi-ce=week s.c. into the limb) in combination with tacrolimus, given i.p. for 30 days (0.3 mg=kg=day) or 50 days (0.3 mg=kg=day 0-30 and 0.1 mg=kg=day 31-50) and ALS i.p. (0.5 mg=kg on day 0 and 3). Untreated animals, placebo (vehicle) treated animals and animals receiving a tacrolimus monotherapy or tracrolimus þ ALS served as controls. Rejection was assessed by daily inspection and H&E-histology of skin biopsies. Grade III rejection was defined as end-point. Tacrolimus 24 h-trough blood levels were measured regularly after pod 30.

Results. Untreated and placebo treated controls rejected at day 8.83 AE 0.98 and 9.00 AE 2.83 (p ¼ 0.894).

When given i.p., correolide C monotherapy resulted in slight but significant prolongation of allograft survival (10.50 AE 1.38, p ¼ 0.037). Histology showed only a mild lymphocytic infiltrate and single vacuolized keratinocytes in the epidermis on pod 10 in 4=6 correolide C treated animals. After weaning tacrolimus on pod 30, limbs were rejected by pod 40.00 AE 1.00 (grade III), and histology revealed necrosis of the epidermis. Additional treatment with local correolide C resulted in an insignificant prolongation of graft survival (pod 43.00 AE 3.74; p ¼ 0.24). 2=5 animals showed intact skin with a mild dermal infiltrate until day 45. Animals receiving additional ALS therapy rejected on pod 41.80 AE 2.05. Weaning tacrolimus on pod 50 resulted in rejection of the limb by day 55.00 AE 0.00 regardless of correolide therapy. Tacrolimus mean blood levels were 2.97 AE 0.98 ng=ml when tacrolimus was given at 0.3 mg=kg=day and undetectable (<0.6 ng=ml) 5 days after weaning.

Conclusions. Systemic administration of a Kv1.3 blocker results in slight prolongation of graft survival after rat hind-limb allotransplantation while local administration into the skin has no effect under low dose tacrolimus therapy. Background. With the progress in the field of immunosuppressive therapy, transplanted patients now face a different a kind of problem: chronic complications. As the incidence of acute rejection decreases, the frequency of chronical problems induced by long term immunosuppression rises.

Long term problems such as nephrotoxicity and cardiotoxicity are being investigated, especially in the context of calcineurin inhibitors (CNI) treatments, involving Cyclosporine A (CsA) or Tacrolimus. CsA is the most frequently used immunosuppressant in organ transplantation, but while it has been proven to limit acute complications, its long term effect on other organs such as the heart or the kidney remain controversial. This study aims to (1) evaluate the effects of chronic Cyclosporine treatment on the kidney and heart function in rats and (2) test the impact of the antioxidant solution Sanopal on the countering Cyclosporine effects.

Methods. Twenty-four male Wistar (250-300 g) rats were divided into 4 experimental groups as following: CsA Control Group: 6 rats were subjected to a daily subcutaneous olive oil (vehicle for CsA) and intraperitoneal NaCl injection for 45 days. All rats received a low-sodium diet, water and food intake were ad libitum. Sanopal Control Group: six rats were subjected to a daily subcutaneous olive oil injection and intraperitoneal Sanopal injection for 45 days.

CsA Group: six rats were subjected to a daily subcutaneous Cyclosporine A injection in a dose of 15 mg=kg for 45 days as reported by B.A. Young to induce chronic kidney failure. In addition they received NaCl intraperitoneally.

Sanopal Group: six rats were subjected to a daily Cyclosporine A injection as described above and an intraperitoneal injection of Sanopal.

On days 0, 7, 14, 21, 28, 35, and 42 of treatment, blood was taken from every animal to measure the following parameters: (1) kidney parameters (creatinine, potassium) (2) cardiac parameters (pro BNP, TnT) for surveillance of kidney and heart function. The kidneys were fixated for histological investigation and heart function was evaluated on the Langendorff apparatus (ASI, ECG, coronary flow, systolic and diastolic tension).

Results. Results will be presented at the congress, as the study is still ongoing.

Conclusions. Oxidative stress plays an important role in Cyclosporine toxicity. We hope that the concomitant Sanopal administration will proof to reduce the organ dysfunctions.

Surgical Oncology and Immunology I One of the strategies of cancer gene therapy is a delivery of different cytokine genes into the tumor cells using virus based vectors resulting in a local recruitment of inflammatory cells that inhibit tumor growth. The establishment of reverse-genetics systems has allowed to use influenza virus as a gene-delivery vehicle. Since influenza virus can stand insertion of foreign proteins we engineered a replication-defective influenza A virus expressing human IL-2. In order to increase genetic stability of this virus and intensify an expression of foreign gene viral NS1 gene was truncated to the N-terminal 21 amino acids and additional reading frame was inserted by using stop-start pentamer (UAAUG). C57BL=6 mice bearing established B16.F1 melanoma tumors were treated with intratumoral injection of viruses with or without IL-2 insertion. We observed a statistically significant prolongation of treated animals' lifespan and in case of cytokine therapy the rate of tumor growth was decidedly lower compared to that in mice in control group. Background. Due to the fact that biliary complications are very common in the course of liver transplantation, organ preservation and especially preservation of the bile duct plays an important role for graft function. To figure out best preservation conditions for the bile duct, different preservation solutions were tested in cell culture as well as large animal experiments.

Methods. The human biliary tract carcinoma cell line SK-ChA-1 was incubated in the preservation solutions UW, HTK, Celsior (CS), 0.9% NaCl, Ringer solution and growth medium for 6, 10 and 12 h at 6-8 C. Additionally we incubated the cells in UW, HTK, Celsior and NaCl containing different amounts of bile. Metabolic activity was tested immediately after exposure to the different solutions. Livers of pigs were preserved according to standard procedures using the different preservation solutions with or without flushing the bile duct. Histology and immunohistochemsitry for detection of apoptosis as well as oxidative stress induced cell death were applied and PCR was performed for Bcl2, Bax, GSH, OXSR, NFkappeB as well as HPRT.

Results. Immediate measurement of metabolic activity showed that CS seems to be the best solution in cell culture. Incubation of SK-ChA-1 with HTK, CS and NaCl showed a statistically significant increase of the metabolic activity with decreasing percentage of bile in the solutions. Preserving UW showed a consitant activity about 100% and no impact of bile on the cells. These findings are supported by the data gained from the large animal experiments.

Conclusions. UW seems to be superior in cell culture experiments when preserving Sk-ChA-1 cells. First results in large animals showed less apoptosis and more integrity of the bile duct epithelium flushing the bile duct with UW compared to the non-flushed bile ducts. Influenza A virus induced-ARDS can lead to a life threatening disease. Yet the mechanisms are still not elucidated. An exacerbated immune system might play a role in pathogenesis. In order to investigate the impact of atypical expression of cytokines in virus-induced lung pathology, we expressed different cytokines (IL-2, IL-15, IL-24 and CCL20) in the context of the replication defective influenza A virus delNS1. We then evaluated the pathogenicity and immunogenicity of those viruses in syngeneic Black=6 mice following intranasal administration at high concentration of 10E6 plaque forming units. In contrast to wild type virus which leads to lethal pneumonia at 1000-fold lower concentration, the empty vector (delNS1 virus) and the virus expressing IL-24 did not show any pathology. In contrast, IL-2-and CCL20-expressing viruses showed severe pneumonia-like lung pathology during the first four days, including animal death. IL15 expression showed less severe lung pathology. Serum IgG1, IgG2a and nasal wash IgA was determined 21 days after initial immunisation. Only the mice immunized with the control and with NS1-IL-24 virus developed substantial IgG2a titers. No trend in the polarisation towards the Th1 type (IgG2a) of immune response could be determined. The nasal wash virus specific IgA titers were undetectable. Lowering the concentration abolished toxicity of replication defective cytokine expressing viruses. In a second trail mice were immunized intraperitoneally with the same viruses. No illness was observed. There was no difference in serum levels of Th1=Th2 cytokines in the different groups detectable. The data indicate that cytokines might play a role in virus-induced ARDS. Furthermore, replication defective viruses expressing cytokines induce a cytokine specific immune-pathology in selected tissues. Background. Donor T cells have pleiotropic effects in allogeneic bone marrow transplantation (BMT), which include the enhancement of BM engraftment, the induction of graft-versus-leukemia effects and improved immunity, but also the induction of graft-versus-host disease (GVHD). We recently found that, surprisingly, transplantation of high doses of donor T cells together with donor BM causes rejection of donor BM despite costimulationblockade. This phenomenon is NK-independent but otherwise remains poorly understood. In the present study we investigate whether the release of distinct cytokines plays a role in triggering the rejection of donor bone BM and whether neutralization of selected cytokines overcomes the detrimental effect of donor T cells.

Methods. Recipients (C57BL=6) were treated with 3 Gy TBI (dÀ1) and received approximately 20 Â 10 6 fully mismatched Balb=c BM cells (d0) and costimulationblockers anti-CD154 mAb (1 mg; d0) and CTLA4Ig (0.5 mg; d þ 2). Groups received additional 25 Â 10 6 Balb=c T cells (isolated from Balb=c spleen and lymph nodes by MACS separation), with or without anti-IL-6 mAb (1 mg; dÀ1, þ 1, þ 3 and weekly thereafter). Multilineage chimerism was followed by flow cytometry. Splenocytes isolated from BMT recipients (d5 post BMT) were stimulated with irradiated donor splenocytes and a panel of cytokines was analyzed with a CCD camera and by ELISA.

Results. Co-transplantation of 25 Â 10 6 T cells triggered BM rejection under costimulation blockade within two weeks in an otherwise successful protocol (1=4, 4=4 chimeras; B-cell chimerism: 25 Â 10 6 T cells ¼ 3.57%; control group ¼ 24.28%, p ¼ 0.02 at 5 days post BMT). The level of IL-6, IFN-and IL-17A was found to be significantly higher in mice treated with additional donor T cells (p < 0.05) and TGF-was also increased (although not significantly). These cytokines might be candidates causing the detrimental effect of donor T cell involved in BM rejection. Neutralization experiments are currently ongoing.

Conclusions. The abrogation of the BM engraftment through co-transplantation of donor T cells seems to be T cell-mediated. Thus, donor T cells appear to activate recipient T cells despite costimulation-blockade, revealing a potentially important mechanism of costimulation blockade-resistant rejection. The activation might be due to the release of distinct cytokines from the donor T cells (IL-6, IFN-, IL-17A and TGF-) which offers potential targets for therapeutic intervention. Background. Chronic rejection characterized by allograft vasculopathy is still a major obstacle to long term graft survival. Among different triggers a prominent nonimmunologic factor associated with chronic allograft vasculopathy (CAV) is ischemia reperfusion injury. Recently, the essential cofactor for nitric oxide synthases tetrahydrobiopterin (H4B) was shown to significantly reduce ischemia reperfusion injury (IRI). Herein we analyzed whether H4B supplementation may also attenuate CAV by prevention of IRI.

Methods. A fully MHC mismatched (Balbc to C57bl6) mouse heterotopic cervical aortic transplantation model was used. Transplanted grafts were subjected to 24 h prolonged cold ischemia time (CIT) and to 45 min warm ischemia time. Donor animals received either H4B (50 mg=kg=BW) prior to organ retrieval or saline. Grafts without CIT served as controls. Aortic grafts were retrieved for further analysis 10 h and 4 weeks following reperfusion, respectively. Glutathion (GSH) tissue level measurements as well as HSP-70 western blot were performed to assess IRI-related tissue damage (10 h following reperfusion). Quantification of intimal hyperplasia as CAV-indicator was done by histology (hematoxylin and eosin) 4 weeks following reperfusion.

Results. Prolonged CIT resulted in a significant reduction of GSH tissue levels when compared to control animals (p < 0.05), in parallel HSP-70 expression was elevated (p < 0.05). Furthermore, supplementation of H4B resulted in significant restoration of GSH tissue levels, as well as in a significant reduction of HSP-70 expression when compared to untreated animals (p < 0.05). Histopathology at 4 weeks after transplantation revealed significant CAV characterized by prominent intimal hyperplasia in the untreated group. In contrast, pretreatment with H4B leads to a significant reduction of intimal hyperplasia (p < 0.001).

Conclusions. These data suggest that the degree of IRI strongly correlates with CAV development and that donor pre-treatment with H4B might therefore represent a novel agent in transplantation to attenuate CAV.

Tetrahydrobiopterin protects from ischemia-reperfusion injury: first results using a porcine kidney autotransplantation model Background. Ischemia-reperfusion injury (IRI) associated delayed graft function is known to affect graft survival. In various transplantation models administration of exogenous tetrahydrobiopterin (H4B), an essential cofactor of nitric oxide synthase (NOS) and potent antioxidant, resulted in substantial protection form IRI. Herein we present first results using a kidney autotransplantation model in pigs.

Methods. Male common house pigs (50-60 kg b.w.) were used. For pharmacokinetic studies H4B distribution in renal tissue was analysed (n ¼ 3). To avoid any confounding effects of an alloimmune response the well established kidney autotransplantation technique in pigs was chosen. Left kidney was explanted and stored on ice for 24 h. Before implantation of the autograft, contralateral nephrectomy was performed, so that renal function of the animal relied purely on the autotransplanted kidney. Before autotransplantation pigs were either pre-treated with H4B 20 mg=kg b.w. (n ¼ 3) or were untreated (n ¼ 3). Parenchymal damage was evaluated by H&E histology and nitrotyrosine western blot. H4B renal tissue levels were determined by HPLC.

Results. Maximal H4B tissue levels were observed 15 min post application (22.840 nmol=mg). Before reperfusion of the autograft H4B treated pigs displayed numerically higher creatinine (1.8 AE 0.6 vs. 4.3 AE 2.4; p ¼ 0.15) and urea levels compared to non treated animals (32.3 AE 2.7 vs. 74.0 AE 32.1; p ¼ 0.09) indicating a possible nephrotoxic effect. However, in contrast to non treated pigs H4B treated animals showed urine production already immediately following kidney reperfusion. Two out of three treated animals survived the entire 7-day observation period. One animal died two days after transplantation because of a gastrointestinal bleeding. Peak creatinine (18.5 mg=dl and 19.7 mg=dl) and urea (284.8 mg=dl and 338.6 mg=dl) levels were reached at day 3 and 5, respectively. Only one non treated animal survived for 7 days with creatinine and urea levels still rising at day seven (24.5 mg=dl and 246.7 mg=dl, respectively). Two animals died anuric two days post transplantation. Histopathological and nitrotyrosine western blots revealed no differences two hours post transplantation.

Conclusions. To our knowledge this is the first study observing nephrotoxic effects of H4B. Despite this finding, immediate urine production and better survival confirm the protective effect of this pterin in IRI; however, further dose finding studies have to be performed. Background. The global imbalance between organ supply and demand requires new approaches to minimize tissue damage and preserve function within the complex sequences of solid organ transplantation. Events of warm and cold ischemia followed by reperfusion contribute to acute and chronic graft failure due to the generation of reactive oxygen and nitrogen species.

The present study evaluated for the first time the impacts of the combination of alpha ketoglutaric acid (AKG) and 5 hydroxymethylfurfural (5HMF) (Karal + solution) on cardiac function of isolated rat hearts subjected either to warm or cold ischemia.

Methods. Two different experimental protocols were carried out. All hearts were attached to a Langendorff perfusion system via an aortic stump, retrogradely perfused with 37 AE 1 C warm Krebs Ringer solution at a constant perfusion pressure of 70 mmHg and continuously oxygenated with 5% CO 2 þ 95% O 2 .

After stabilization period and basal time (each 20 min), warm ischemia was induced by a ligation of the left descending coronary artery for 15 min, followed by 30 min of reperfusion.

Cold ischemia was induced by Celsior + cardioplegia. Hearts were stored at 4 C for 6 h then reperfused for 60 min.

Results. In control hearts, ventricular tachycardia and=or fibrillation during reperfusion were observed in 100%. The occurrence of irreversible arrhythmias was abolished in the hearts perfused with KRS containing Karal + 20%.

Furthermore this concentration induced a significant increase in the coronary flow as well as in the diastolic function of the isolated hearts, in both, warm and cold ischemia.

Conclusions. In this work we introduce a new antioxidant solution increasing cardiac function during reperfusion and explain the underlying biochemical mechanisms.

Effects of bilirubin on renal ischemia reperfusion injury and novel strategies for its application Background. Ischemia-reperfusion injury (IRI) represents an unresolved issue in renal transplantation. Currently, no satisfying strategies for the prevention of IRI are available. Recent findings indicate that the bile pigment bilirubin can exert beneficial effects. However, detailed protocols for its application and final proof for its effectiveness remain elusive. The aim of this study was to investigate the effects of bilirubin on transplant organ function and the determination of application strategies in an experimental setting of renal IRI.

Methods. In two Wistar rat models of renal IRI, kidneys were either transplanted to isogenic recipients after 18 h of cold ischemia, or, in a second model, renal artery clamping of contralaterally nephrectomised rats was performed for 45 min. Various doses (ranging from 1 to 30 mg=kg) of bilirubin, or its precursor bile pigment biliverdin, were administered by intravenous injection pre-, peri-, or postoperatively. Serum bilirubin levels were quantified shortly after reperfusion to assess bilirubin bioavailability. Kidney malfunction relating from IRI was assessed by serum urea and creatinine analysis after 24 and 48 h.

Results. Pre-and perioperative administration of bilirubin resulted in a significant increase of serum total bilirubin levels after reperfusion (ED 3-30 mg=kg). Similarly, treatment with biliverdin ditaurate could elevate serum bilirubin. The increase in serum bilirubin caused a decline in both, creatinine and urea levels, already after 24 h in the clamp model. Interestingly, no significant difference resulted from dose elevation over 3 mg=kg. However, postoperative bile pigment administration did not cause any beneficial effects. Similar findings were observed in the transplantation model, although higher dosages of biliverdin were required (>30 mg=kg).

Conclusions. Intravenous application of bilirubin or its precursor biliverdin are effective in preventing renal transplant malfunction from IRI. Bilirubin serum levels must be increased prior to reperfusion. The application of bile pigments in renal transplantation might serve as a novel strategy in the prevention of IRI. In previous work we constructed oncolytic influenza A viruses, which selectively replicate on malignant transformed cells, but spare healthy cells. Similar to other oncolytic viruses, intratumoral application of the oncolytic influenza A viruses resulted in reduced tumor growth of murine cancer but did not abolish the tumors. It now becomes evident, that the main mechanism by which oncolytic viruses reduce tumor growth is the local induction of tumor ablative immune response. In order to improve the therapeutic effect we want to construct a modified virus which promotes a better outcome for the standard therapy in cancer. The aim of this work is the expression of an additional immune enhancing cytokine, such as interleukin 24. Melanoma differentiation-associated gene 7 (mda7)=interleukin-24 (IL-24) is a unique member of the IL-10 gene family, which displays a broad range of antitumor properties, including induction of cancer specific apoptosis. We therefore expressed IL-24 as a chimeric gene in the background of a replication defective influenza A virus delNS1 by a new cloning strategy, using specific splice-site mutations. To show the effect of IL-24 expressing virus (delNS1-IL24) on apoptosis we performed Annexin V measurements. The tumor cells CaCo 2 and SKMel28 cells infected with delNS1-IL-24 virus showed enhanced apoptotic features as compared to empty viral vector. Enhancement of apoptosis was associated with decreased viral growth, a phenomenon, which was also observed for adenoviral vectors expressing IL-24. We conclude, that influenza A viruses expressing MDA7=IL-24 expression might be applicable as a safe oncolytic therapy for cancer and warrant further preclinical assessment in animal trials. Background. Cancer is the second leading cause of death worldwide and according to the World Cancer Report 2009 it will become number one by 2010. Development of new strategies for cancer prevention or therapy is of particular importance. Previously we could show that bile pigments (BV=BR) do have distinct effects on tumor growth, leading to cell cycle arrest and apoptosis. Biliverdin reductase (BVR), a kinase with dual pH=cofactor reductase activity is known to potentially influence cell proliferation and apoptosis.

Methods. SureSilencing TM shRNA was used for permanent knock down of BVR. Transfected cells were cultured in the presence of BV=BR and cell lysates were probed for MAPK's, AKT, Caspases, PARP, p53 and p27 by Western blot. Expression of BVR was investigated using immunohistochemistry. Statistical analysis was performed using SPSS-15.

Results. BVR plays an important role in bile pigment mediated tumor inhibition as knock down diminishes apoptosis and tumor growth reduction. Furthermore BVR influences cellular signaling, reversing the changes on the MAPK pathway mediated by BV=BR. Statistical analysis showed its intense expression in tumors with poor outcome (disease free=overall survival).

Conclusions. Not solely BV=BR but BVR itself may play an important role in defending cancer by regulating cell proliferation=apoptosis via alterations on cellular signaling.

The effect of reverse transcriptase inhibitors on colon cancer cells One mechanism for cancer cell survival is maintenance of telomers, which are repetitive sequences found on the ends of the chromosomes. Reverse Transcriptase Inhibitors (RTI) as used in HIV therapy are known to interfere with telomer maintance and are therefore discussed as anti-cancer agents. However, clinical use is hampered by the high concentrations of RTI needed to inhibit telomer elongation. Here we demonstrate that the combinatorial therapy using RTI inhibits telomer maintance at much lower concentrations of the drugs in colorectal cancer cell lines. Inhibition of telomer maintenance is associated with reduced proliferation, cell cycle arrest, increased induction apoptosis and reduced tumor growth in mice. Thus we conclude that RTI should be considered as therapy for solid cancer other than virus-associated T-cell lymphoma, where RTI are part of the current standard. Cancer is associated with immunosuppression. This is thought to diminish efficiency of conventional chemotherapy and possibly also of monoclonal antibody-based therapy. Animal data indicate, that cancer-associated immunosuppression is based on prolonged or repeated stimulation of Toll-like receptor (TLR) 4. This leads to hyporesponsiveness of monocyte-derived macrophages, the latter appears to be a hallmark of immunosuppression related to human cancer. Two negative regulators of TLR-4 signalling are IL-1 receptor-associated kinase M and B-cell leukemia 3. Here, we demonstrate that the expression of both proteins is inhibited when the TLR-7=TLR-8 agonist CL097 is added to monocyte cultures despite costimulation with the TLR-4 agonist LPS or hyaluronic acid. Reduction of IL-1 receptor-associated kinase M and B-cell leukemia 3 was paralleled by a significantly increased cytokine induction of TNF-, IL-10, and IL-12 observed after intracellular and extracellular TLR stimulation. In ex vivo stimulated whole blood of patients who have prolonged metastatic cancer, TLR-7=TLR-8 agonists retained their ability of increased stimulation of TNF-. Preliminary experiments further indicate that the CL-079-stimulation might have an enhancing effect on ADCC. These data further support the development of TLR-agonists as adjuvant to immuno-and=or chemotherapy in man.

Bringing evidence into daily practice Background. Evidence-based medicine (EBM) aims to apply the best available external evidence gained from scientific research to clinical decision making. The highest level of available evidence originates from randomized controlled trials (RCT) and systematic reviews of medical literature including meta-analysis of RCTs. Compared to other medical disciplines, EBM in surgery, so-called evidence based surgery (EBS), is mainly based on retrospective clinical studies, whereas RCTs in surgery are commonly infrequent.

Methods. We conducted a survey in the field of hernia surgery to determine if surgeons follow EBM criteria in their daily routine. Surgical departments in Styria received a short 10-item questionnaire concerning hernia surgery. Furthermore, we searched the literature for examples of following EBS in daily surgical practice (other than hernia surgery).

Results. We analyzed 15 completed surveys. A variety of different procedures were performed. The frequency and indication of use of each technique varied considerably among the hospitals.

An overview of EBS literature including EBS guidelines and realisation in daily practice is given.

Conclusions. Our survey showed that, based on the great variance among the evaluated departments as to surgical methods and indications, hernia surgery does not currently comply with EBS. Rather, ''eminence-based surgery'', based on expert's opinion, seems to be followed in daily routine. On the one hand, more high-quality surgical literature seems to be necessary for decision making at an EBS level. On the other hand, training courses are needed to show how to use available EBS in daily practice for a better quality in surgical care.

Untersuchung von Ischämie= =Reperfusions-Schäden bei ,,Composite Tissue"-Transplantationen Grundlagen. Die Auswirkungen der kalten Ischämiezeit und von Konservierungslösungen bei ,,Composite Tissue"-Allotransplantationen (CTA) sind noch unzureichend beschrieben. Ziel unserer Forschung ist die Aus-wirkung der kalten Lagerung sowie der Konservierung mit HTK-oder UW-Lösung anhand eines Ratten-Modells zu untersuchen.

Methodik. Die hinteren Extremitäten von Lewis Ratten wurden gespült und für 0, 2, 10, 30 und 40 h in HTKoder UW-Reperfusionslösung bei 4 C aufbewahrt. Nach jeder dieser Zeitspannen wurden Haut-, Muskel-, Knochen-und Nerven-Biopsien zur HE-Histopathologischen-Untersuchung entnommen.

Transplantierte Beine wurde am ersten und am zehnten Tag nach Transplantation mit 2, 10 oder 30 h kalter Ischämiezeit histopathologisch und konfokal-mikroskopisch hinsichtlich Veränderungen untersucht. Bei der histologischen Beurteilung ist folgendes standardisiertes Bewertungssystem verwendet worden: 0 ¼ keine Veränderungen, 1 ¼ leichte Veränderungen, 2 ¼ schwere Veränderungen, 3 ¼ Nekrose.

Ergebnisse Background. HO-1 is an antioxidant enzyme acting beneficially in transplant related ischemia reperfusion injury. Induction thereof is successful experimentally, however, the compounds being used so far cannot be administered to humans because of hepatotoxicity. Herein, we tested various nutraceuticals for their potential to induce HO-1 and prevent renal ischemia reperfusion injury.

Methods. Ten nutraceuticals known to induce HO-1 in vitro have been tested for their potential in vivo. HO-1 induction=expression has been assessed by PCR. Further, renal arteries of contralaterally nephrectomized Lewis rats have been clamped for 60 min 24 h after oral administration of one of the nutraceuticals, in presence or absence of the HO-1 inhibitor SnPP at 10 mg=kg. Serum creatinine and urea have been measured at defined time points after reperfusion.

Results. Two of the nutraceuticals tested showed significant upregulation of HO-1 (N18519 11-fold, N791419 17-fold, p < 0.0001 for both). Forty-eight hours after reperfusion mean serum creatinine was 3.06 mg=dl (SD 0.86) in controls. Application of both, N18519 (0.54 mg=dl 0.23) or N791419 (0.53 mg=dl AE 0.06) dramatically reduced renal damage. When HO-1 activity was blocked by SnPP, the beneficial effect was reversed.

Conclusions. Nutraceuticals with the potential to induce HO-1 substantially should be considered for the prevention=treatment of transplant related ischemia reperfusion injury. Background. Human serum albumin (HSA) binds and detoxifies endotoxin in healthy people. Oxidative stress leads to protein oxidation and thus to impaired binding properties of HSA. This, in combination with increased gut permeability leads to endotoxemia in the systemic circulation and further to impaired organ function. Based on this, we hypothesised that these processes occur in serum of brain-dead organ donors.

Methods. Endotoxin was determined with an adapted limulus amoebocyte lysate (LAL) assay. Albumin fractions and albumin binding capacity were determined by HPLC. FlowCytomix TM was used for determination of cytokine levels by means of flow cytometry. RT-PCR was used for analysis of tight junction protein (TJP) mRNA expression. Brain-dead organ donors were categorized by the length of intensive care unit (ICU) stay based on the mean ICU stay of all patients.

Results. Eighty-four consecutive brain-dead organ donors were studied. Albumin binding capacity for dansylsarcosine was reduced in brain-dead patients compared to controls. Endotoxin positivity, which was found in 16.7% of the serum samples, but not length of ICU stay was associated with a further decrease of binding capacity. In organ donors albumin was higher oxidized than in controls. An ICU stay of more than 6 days increased albumin oxidation. In addition, IL-6, IL-8, IL-10 and IL-1 levels were elevated in patients whereas IFN-levels were within the normal range. Preliminary data of TJP expression in duodenum samples showed a trend towards lower expression in the endotoxin positive sample.

Conclusions. We conclude that oxidative stress, albumin dysfunction as well as systemic endotoxemia are present in brain-dead organ donors. High endotoxin levels might be due to increased gut permeability and decreased binding capacity of albumin due to higher albumin oxidation. Background. Obesity is a worldwide epidemic. The number of overweight renal transplant recipients and cadaveric donors is increasing. We investigated whether donor and=or recipient body mass index correlate with the occurrence of delayed graft function after kidney transplantation.

Methods. Retrospective analysis of 708 consecutive cadaveric kidney transplants between January 2003 and December 2009. Delayed graft function was defined as requirement for more than one dialysis post-transplant dialysis. Impact of body mass index, gender, age, re-transplant, cold ischemia and anastomosis time on the occurrence of delayed graft function were analyzed using uni-and multivariate analyses.

Results. DGF rate was 25.2%, 29.8%, 40.9% and 52.6% in recipients with a body mass index <20, 20-25, 25-30 and >30 kg=m 2 respectively (p ¼ 0.0002). Donor body mass index <20, 20-25, 25-30 and >30 kg=m 2 resulted in a DGF rate of 22.5%, 31.0%, 37.3% and 51.2% (p < 0.0001). Multivariate analysis revealed overweight in the recipient as well as in the donor as an independent risk factor for delayed graft function.

Conclusions. Donor as well as recipient body mass index correlate with the incidence of delayed graft function. is still an area of much debate. Low pressure pulsatile lavage (LPPL) is suitable for relatively clean wounds less than three hours from time of injury, while at 6 h high pressure pulsatile lavage (HPPL) may result in greater removal of bacteria and debris.

Main principle of infection surgery has always been radical debridement. ''Ubi pus, ibi evacua'', often cited by surgeons, is not enough. All necrotic and infected tissue should be resected in case of limb or life threatening infections. Surgical and sharp debridement has become the gold standard, especially in complicated skin and soft tissue infections, whereas in chronic wounds alternative techniques (mechanical, enzymatic, autolytic and biological debridement, honey) may be more appropriate.

Wound irrigation can be helpful, but has no effect on remaining pathogens. HPPL may be deleterious by causing bacterial penetration into depth of tissues, while instillation of antiseptic fluids into a wound has an ongoing effect, when it is used for a longer time, and when the antiseptic agent remains in the wound for at least 20 min.

Methods. For more than seventeen years the V.A.C. + Therapy System has been used successfully in treating wound infections. Preparing wounds for closure means to clean wounds, to achieve wound retraction and minimize following surgical procedures.

When infection is under control, the wound should be closed at the earliest time possible. Sometimes a simple split-thickness skin graft may be suitable for wound closure. In case of exposed bone or orthopaedic hardware the use of local, pedicled and free flaps including microsurgical procedures is essential not only for wound closure but also for infection control. Well perfused tissue of the flap serves as carrier of systemic antimicrobial agents, which are absolutely necessary to kill remaining pathogens at the site of infection.

Infection control can be difficult or even impossible in case of insufficient debridement, persistence of bacteria, and insufficiency of antimicrobial agents in case of multidrug-resistant bacteria like MRSA and Acinetobacter Baumannii.

Instillation technology is able to combine and optimize the effects of permanent elimination of bacteria by NPWT and to kill and wash out pathogens by cyclic instillation of antiseptic fluids.

The V.A.C.-instill + System enables a 3-stage working cycle: Vacuum therapy=instillation of antiseptic fluids= hold time.

Results. Our experiences of treatment of infected wounds include chronic wounds with local infection, acute infections originating from chronic wounds as well as limb-and life-threatening infections caused by multidrug-resistant (MDR) bacteria. In some patients NPWT and Instillation Technology were used as a salvage procedure following failure of multiple plastic surgical procedures. One thing was equal in all patients. Debridement and subsequent plastic surgical procedure seemed to be insufficient for infection control, or time to closure seemed to be too early. We have always been using Polyhexanid (Lavasorb + ) for instillation. Instillation time directly depended on wound size. Time of therapy lasted at least 4 days, at most 18 days. Dressing changes were per-formed every two to four days. Surgical closure was performed by direct secondary suture, skin grafting or flap surgery. In all cases infection control and complete healing were achieved, despite of incomplete debridement and leaving Prolene-Mesh + in situ in two cases.

Conclusions. We believe the V.A.C.-instill + Wound Therapy System to be a useful tool for infection control in difficult anatomical regions (e.g. open wrist and talocrural joint), in case of impossibility of totally debridement and multidrug resistant bacteria.

Deckung von Bauchwanddefekten bei Neugeborenen=Säuglingen mit Permacol TM , einem azellulären xenogenen Meshimplantat -eine Single-Center-Studie Grundlagen. Die Wahl des operativen Vorgehens zum Verschluss kongenitaler Bauchwanddefekte (Omphalozele bzw. Gastroschisis) ist abhängig von der Defektgröße, die außerordentlich variieren kann, vom Ausmaß des Eingeweideprolaps, das sehr unterschiedlich sein kann, und von der Größe der Bauchhöhle, die ihrerseits vom Ausmaß des Eingeweidevorfalls abhängig ist. Kleinere Defekte mit geringgradigem Eingeweidevorfall und adäquat großer Bauchhöhle sind gewöhnlich durch eine lineare Naht zu verschließen. Wenn, unabhängig von der Defektgröße, das Gros der Eingeweide vorgefallen ist (bei einer Omphalozele typischerweise auch die Leber) und die Bauchhöhle demzufolge sehr klein ist, ist, von wenigen Ausnahmen, bei denen ein linearer Nahtverschluss oder eine primäre Patchdeckung des Defekts möglich ist, abgesehen, die Anlage einer passageren künstlichen Bauchhöhle mit einem Silastik-Silo unumgänglich. Unter langsamem Hineindrücken der Eingeweide und gleichzeitiger Reduktion der Silastic-Sheets vergrößert sich die Bauchhöhle sukzessive. Wenn das Silomaterial Bauchdeckenniveau erreicht, wird es ausgebaut. Der verbliebene Bauchwanddefekt kann im Einzelfall linear verschlossen werden, meist ist aber eine Defektdeckung mit einem Patch notwendig. Das Einheilen des Patch wird nicht selten durch Infektionen bzw. durch Durchblutungsstörungen der Bauchhaut beeinträchtigt. In einem solchen Fall muss der Patch ausgebaut und ein -passagerer -häutiger Bauchwandverschluss angestrebt werden. Etwa drei bis sechs Monate später sollte ein neuerlicher Patchverschluss des Bauchwanddefekts Methods. We report our experience with endoscopic stenting as a palliative procedure. We performed a retrospective analysis of 59 patients treated at our institution in the recent years, who were suffering from symptoms of malignant gastric outlet obstruction.

Results. Obstructive symptoms improved in 55 of the 59 patients (93.2%) after endoscopic stenting. Five patients (8.4%) had procedure-related complications (aspiration, stent dislocation and perforation) after stent implantation and one of them died (1.7%). The patients tolerated oral nutrition after one day on average and the median hospital stay was 4 days after stent implantation.

Conclusions. Endoscopic stent implantation as palliative treatment for malignant obstruction in the upper GI-tract distal of the esophagus is an effective and safe method, which leads to a rapid improvement of symptoms. It should be the first choice in the palliative treatment of malignant obstruction in this part of the GI-tract. Background. Bacterial infections of diabetic feet may lead to loss of the whole leg. Minor traumas or neuropathic plantar ulcers can cause underlying infections involving bones or joints. Often resection of infected bone and systematic antibiotic therapy is required to provide healing or at least to avoid lower limb loss. Infection control and healing is only possible, if bone resection is sufficient and concentration of antimicrobial agents reaches adequate levels.

Methods. Prospective study of measuring Fosfomycin in infected bone compared to collateral healthy subcutaneous soft tissue and to serum levels using in vivo microdialysis. Including criteria were diabetes in combination with severe diabetic foot infection and underlying osteomyelitis and the surgical indication for metatarsal bone resection. Nine patients (3 women and 6 men) were included. After the surgical procedure and shortly before wound closure microdialysis probes were inserted percutaneously through venflons into a drilled hole in the adjacent bone after resection and sequestrectomy, as well in contralateral subcutaneous tissue in order to compare levels including serum concentration during 6 following hours after intravenous injection of Fosfomycin Sandoz 100 mg=kg body weight.

Results. The concentrations of Fosfomycin found in serum and subcutaneous tissue were similar to previously published levels in healthy patients. In the first 3-4 h the bone levels were clearly lower than the levels found in serum and subcutaneous tissue but all levels equilibrated at the latest after 4 h. The minimal inhibitory concentration for staphylococcus aureus and MRSA was achieved.

Conclusions. To our knowledge in-vivo microdialysis in bone of diabetic foot patients was successfully performed worldwide for the first time finding sufficient concentrations of parenterally given Fosfomycin in bony tissue in order to treat infections. 

NK cells, viruses and cancer

Natural killer cell-directed therapies: moving from unexpected results to successful strategies

Natural killer cell allorecognition of missing self in allogeneic hematopoietic transplantation: a tool for immunotherapy of leukemia

Activating NK cell receptor ligands are differentially expressed during progression to cervical cancer

NK cell accumulation in tumors is dependent on IFN-and CXCR3 ligands

New twist on the regulation of NKG2D ligand expression

durchgeführt werden, um dessen Risiken (rasche Größenzunahme durch das Wachstum, mechanische Gefährdung des Darmes) zu begegnen. Zu diesem Zweck stehen vor allem alloplastische und seit kurzen auch wieder xenogene Patchmaterialien zur Verfügung. Die ersten Erfahrungen mit einem -xenogenen, d.h. porcinen -Patchmaterial (Permacol TM ) werden im Folgenden vorgestellt und diskutiert.Kasuistik. Während der letzten 15 Monate implantierten wir bei 4 Kindern (2 Neugeborenen und zwei Säuglingen (Alter 11 bzw. 19 Monate) einen Permacol TM -Patch zur Deckung relativ großer, d.h. im Längsdurchmesser zwischen 6 und 8 cm und im Querdurchmesser zwischen 4 und 6,5 cm haltender Bauchwanddefekte. Von den 2 Neugeborenen hatte ein Kind eine sog. Giant Omphalocele und das andere eine Gastroschisis. Von den beiden Säuglingen hatte ein Kind, das mit einer Gastroschisis geboren worden war, einen Zustand nach häutiger Defektdeckung wegen einer protrahierten Wundheilungsstörung nach Implantation eines alloplastischen Patch (Neuro-Patch + ); das zweite Kind, bei dem der durch eine Omphalozele bedingte Bauchwanddefekt linear verschlossen worden war, hatte eine halbgrapefruitgroße Narbenhernie. Bei den beiden Säuglingen wurde initial jeweils eine Schusterplastik angelegt und nach deren Ausbau der Bauchwanddefekt wegen der kritischen Lokalverhältnisse (ausgedehnte Defekte, Hautverschluss nur unter Spannung möglich) mit einem Permacol TM -Patch gedeckt. Bei den beiden Säuglingen waren ebenfalls die problematischen Hautverhältnisse, beide hatten eine deutlich überdehnte Haut mit einer minimalen subkutanen Fettschicht, der Grund für die Verwendung eines Permacol TM -Patch zum Defektverschluss.Ergebnisse. Bei den beiden Neugeborenen ging die Wundheilung auf Grund umschriebener Hautnekrosen sehr protrahiert vor sich, letztlich heilten die Patches aber sehr gut ein. Bei den beiden Säuglingen ergab sich trotz der schlechten Hautverhältnisse keine Wundheilungsstörung. Der bisherige postoperative Verlauf (Beobachtungszeitraum (Ende Juli 2010) zwischen 5 und 13 Monaten) gestaltete sich im Wesentlichen zufriedenstellend, wenngleich die vom Hersteller angegebene Durch-bzw. Abbaubarkeit des Patchmaterial bei keinem Kind bislang wirklich nachweisbar war. Im Gegenteil, bei den beiden Säuglingen sind die Patches unter der Bauchhaut, da leicht prominent, gut erkennbar und lassen sich als derbe ,,Platten" tasten; bei einem dieser Kind imponiert der untere Patchrand sogar wie aufgeworfen. Sonographisch nimmt sich die Gefäßeinsprossung bei allen Kindern eher bescheiden aus.Diskussion. Kongenitale Bauchwanddefekte werden bei Neugeborenen bzw. Säuglingen seit ca. 35-40 Jahren u.a. mit Hilfe unterschiedlicher Patchmaterialien verschlossen. Initial wurden vor allem lyophilisierte Dura und ,,einfache" Kunststoffe verwendet. Mit diesen Materialien wurden zwar recht zufrieden stellende Ergebnisse in Bezug auf ihre Einheilung und Belastbarkeit erzielt, sie wurden aber wegen verschiedener Infektions-bzw. Adhäsionsprobleme recht bald zugunsten neuer alloplastischer Materialien verlassen. Einige dieser Materialien erwiesen sich in Bezug auf Handling, Einheilung und Belastbarkeit als sehr adäquat; da sie jedoch eine mehr oder weniger ausgeprägte Fremdkörperreaktion hervorrufen und ein Leben lang mit unbekannten Späteffekten in situ verbleiben, wurde nach neuen xenogenen Grafts gesucht. Auflagen waren ein fehlendes Infektionsrisiko, eine minimale Fremdkörperreaktion und vor allem Durch-bzw. Abbaubarkeit der Patches. Während der letzten Jahre wurden diverse solcher Materialien vorgestellt. Da bezüglich ihrer Sicherheit sehr unterschiedliche Erfahrungen publiziert wurden, entschlossen wir uns erst im letzten Jahr, ein solches Produkt, konkret Permacol TM , ein porcines Mesh, zu verwenden. Dies deshalb, da, wie aus den wenigen Literaturangaben hervorging, dieses azelluläre Mesh die eben angeführten Auflagen zu erfüllen schien. Wir entschlossen uns deshalb, dieses Material bei ausgesuchten Patienten mit zu erwartenden schwierigen Defektverschlüssen zu benutzen -Kriterien waren relativ große Bauchwanddefekte und vorgeschädigte Bauchhaut mit einer sehr dünnen Subkutanschichte, bei denen mit einer gestörten Wundheilung zu rechnen war. Das bisherige Ergebnis scheint uns recht zu geben, auch wenn die Durch-und Abbauprozesse noch auf sich warten lassen. Allerdings sind die Beobachtungszeiträume (aktuell zwischen 5 und 13 Monaten) noch relativ kurz.Schlussfolgerungen. Permacol TM ist ein neues xenogenes Patch-Material für spezielle Weichteilrekonstruktionen, das sich, wie unsere eigenen kurzzeitigen Erfahrungen bzw. die unserer viszeralchirurgischen Kollegen zeigen, gut handeln lässt und auch bei schwierigen Verhältnissen (,,ausgedünnte" Hautlappen, fehlendes Subkutangewebe, lokale Infektionen) ohne Gewebsverluste einheilt. Die Durch-und Abbauprozesse dauern allerdings, zumindest bei unseren Patienten, deutlich länger als vom Hersteller angegeben. Demzufolge ist derzeit nicht abzuschätzen, welche Spätprobleme dieses Material (immunologische Prozesse, toxische Einflüsse, kanzero-und=oder mutagene Effekte, Haltbarkeit des Narbengewebes) bedingen wird. Nicht von ungefähr gibt es deshalb neue Entwicklungen auf dem Sektor künstlicher Werkstoffe, die ebenfalls durch-bzw. abbaubar sind, aber durch kein herstellungsbedingtes Infektionsrisiko sowie praktisch keine Fremdkörperreaktion belastet und vor allem -im Gegensatz zu den bisher angebotenen Materialien -auch stabil und belastbar sein sollen. Background. Endoscopic stenting for palliative treatment of malignant gastric outlet obstruction or malignant obstruction in the upper part of the jejunum is increasingly replacing gastro-enteric bypass surgery.