In clinical reality the drugs used for H1/H2-prophylaxis are not restricted to the combination of dimetinden/cimetidine, also only for this combination the effectiveness for preventing severe cardiorespiratory disturbances is proven in a randomised controlled clinical trial. However, it is almost impossible to conduct such an extended clinical trial in order to check all possible combinations. Instead of this, we developed a complex animal model featuring clinical variability and the principles of a well conducted randomised controlled clinical trial (CMRT = clinic modelling randomised trials), to evaluate different H1/H2 combinations. In this CMRT in pigs (four groups of 15 animals), the H1/H2 combination of dimetinden/cimetidine and dimetinden/famotidine showed an effectiveness similar to the randomised clinical trial. With dimetinden/ranitidine no significant prophylactic effect was observed. Two conclusions can be drawn: (1) CMRTs in animals are able to answer relevant clinical and research questions that otherwise only could be solved by clinical studies and may be a successful intermediate between basic research and clinical trials. (2) Drugs, even of the same substance class, may not be simply exchangeable. Hence, before changing a proven medication, trials in an adequate complex animal model (CMRT) should be mandatory.

The prognosis of acute necrotizing pancreatitis is dependent on systemic complications. The endothelial adhesion molecule ICAM-1 mediates both leukocyte adhesion and migration. Expression of ICAM-1 was investigated at various time points in mild and severe necrotizing pancreatitis in the pancreas, lungs and intestine. A possible therapeutic effect of monoclonal antibodies against ICAM-1 was evaluated in severe necrotizing disease. The expression of ICAM-1 preceded the fulminant leukocyte Infiltration of the organs. In contrast to mild pancreatitis, ICAM-1 expression was increased at an earlier time point and systemically in severe necrotizing disease. Treatment with antibodies against ICAM-1 improved microcirculation and reduced local and systemic organ damage in severe pancreatitis.

The aim of the present study was to determine the changes in permeability and mucosal function after ileo-pouchanal anastomosis (IPAA) in patients with ulcerative colitis. We examined 24 patients (m: f = 15:9; Age = 35.2 +/- 12.5) prior to colectomy (pre IPAA), prior to closure of ileostomy (pre CIS), after closure of ileostomy (post CIS), in case of pouchitis and 5 controls. With a miniaturizised Ussing-chamber electrophysiological parameters of permeability and resorptive isotope-fluxes for mannitol were measured. Total resistance could be differenciated into epithelial and subepithelial resistance by using alternating current impedance measurements. Active Na(+)-glucose-Cotransport was measured by detection of the maximal short-curcuit-current under stepwise addition of glucose and active Cl(-)-secretion was measured by the increase in short-curcuit-current after stimulation with Theophillin and PGE2. Epithelial resistance of pouchitis was slightly raised towards controls but did not differ from the others. Subepithelial resistance post CIS and pouchitis was significantly increased versus controls and pre CIS (21.2 +/- 22.4 vs. 10.7 +/- 13.3 omega cm2) Whereas Cl-secretion of pouchitis was significantly reduced vs. post CIS and controls (38.6 vs. 120.9 +/- 113.9 microA cm-2). In the same way Na(+)-glucose Cotransport of pouchitis was significantly reduced vs. post CIS and controls(41.8 vs. 138.4 +/- 264.4 microA cm-2). Increasing subepithelial resistance post CIS and in pouchitis is a hint for an adaptive thickening of the subepitheal layer. Concerning the functional analysis pouchitis and terminal ileum prior to IPAA show a reduced secretion and resorption whereas the mucosal barrier towards Mannitol remains unchanged.

The role of parathyroid transplantation for the therapy of permanent hypoparathyroidism is undisputed. Because the parathyroid hormone deficiency syndromee rarely every is a vital thread to patients affected, systemic immunosuppression for transplant recipients is not justified. A technique of microencapsulation was modified for transplantation of parathyroid tissue. Using a core substance suitable for clinical use (amitogenic alginate), we accomplished allotransplantation of functioning parathyroid tissue in the long-term animal model and, very recently, reported first clinical cases without postoperative immunosuppression. In a controlled animal model of totally parathyroidectomized rats (PTX, two groups of n = 40), we investigated the ability of microencapsulation with the amitogenic alginate to enable transplantation across the highest immunological barrier (xenotransplantation: human-rat); to ensure intact transplant function and to protect from rejection. Rat parathyroid hormone (PTHRA i.S.) and serum calcium levels served as parameters of completeness of PTX; intact human PTH (PTHRA i.S.) and serum calcium levels of recipient animals were used to assess graft function. Also, tissue integrity within explanted capsules was assessed by histology. Cultured and microencapsulated parathyroid tissue resumes and maintains function in vivo, even if transplanted across the highest immunological barrier. Functionally, PTHHU i.S. replaced (PTHRA i.S.) in PTX animals entirely and restored normocalcemia. These results suggest, that xeno-transplantation of the parathyroids can be achieved without postoperative immuno-suppression in a long term animal model. These data also imply the possibility of clinical heterotransplantation of parathyroid glands.

In the pulmonary circulation, endotoxemia induces leukocyte/endothelium interaction in pulmonary arterioles and venules. Thus, leukocytes may contribute to the pathogenesis of acute lung injury. In order to investigate, whether this interaction can be inhibited by early blockade of the adhesion cascade, we studied the effect of the humanized anti-L-selectin antibody HuDreg 200 on leukocyte kinetics in pulmonary arterioles and venules following i.v.-infusion of endotoxin. In endotoxemia HuDreg 200 reduces sticking of leukocytes in both pulmonary arterioles and venules. Thus, we were able to demonstrate that early blockade of the adhesion cascade effectively prevents leukocyte accumulation in pulmonary microvessels in endotoxemia. Therefore, HuDreg 200 may exhibit protective effects on the manifestation of leukocyte-mediated acute lung injury.

The experimental study investigates the resuscitation of livers procured from non heart beating donors by venous systemic oxygen persufflation in a pig transplantation model. In group 1 livers were harvested from donor animals after 60 min of potassium induced cardiac arrest. Livers were then flushed and preserved in 4 degrees C cold University of Wisconsin solution for 4 hrs. Then liver transplantation was performed in recipient animals. Animals of group 2 received the same treatment, but during hypothermic storage we applied venous systemic oxygen gas persufflation (VSOP) via the supra-hepatic vena cava. Livers of group 3 were harvested without warm ischemia and without oxygen treatment during cold ischemic storage and were then transplanted. All recipient animals of group 2 and 3 survived the five day follow-up time. All recipient animals of group 1 died a few hours after liver reperfusion due to circulatory shock or liver failure. The liver quality of group 2 recipient animals was similar to group 3 livers, because serum transaminase levels and blood coagulation test showed no significantly different values between those groups. Our data suggest that livers procured from non heart beating donors after 1 hr of cardiac arrest can be resuscitated and successfully transplanted by therapeutic treatment with VSOP.

The process of chronic rejection (CR) threatens long-term organ graft survival and is the major remaining barrier preventing successful clinical transplantation. The etiology of CR remains speculative, but its correlation with acute rejection episodes, HLA mismatch and immunosuppressive non-compliance suggests that active immune attack is responsible. The authors hypothesize that transforming growth factor beta-1 (TGF beta-1) plays a causal role in regulating and modulating both the acute and the chronic rejection. To investigate this hypothesis in rats, recombinant adenoviruses (rAdv)-mediated gene transfer encoding downregulating antisense--or upregulating bioactive TGF beta-1 transgene were used to infect orthotopic aortic grafts. In a high responder MHC class I histocompatibility difference (ACI to Lewis rat) and in syngeneic controls (Lewis to Lewis) both expression vectors were detected 1, 2 and 12 weeks following transplantation by intragraft cytokine transcription. Aortic segments were divided and processed for histology and RNA extraction. TGF beta-1 RNA expression was then evaluated by semi-quantitative RT-PCR (s26 standardized, HPLC quantitated). Histological evaluation was performed by a transplant pathologist in a blinded fashion. All analysis were prospective and repeated in triplicate. Untreated allografts were used as background controls for the acute and chronic rejection showing an endogenous up-regulation of TGF beta-1 at early time points (1 wk 2.7 +/- 0.5; 2 wk 9.2 +/- 6.1) and a decreased TGF beta-1/s26 ratio in chronic rejection (12 wk 1.2 +/- 0.11). Successful rAdv-transgene activity was, however, detected in low levels in all aortic layers showing a 25%-35% transfection rate whereas beta-galactosidase control gene expression was found as far as 35 days post transplant. Administration of down-regulating antisense TGF beta-1 gene into transplant segments significantly decreased the intragraft TGF beta-1 transcription (1 wk 0.8 +/- 0.2; 2 wk 1.9 +/- 0.5; 12 wk 0.7 +/- 0.15) and was correlated with absence of ongoing acute graft rejection in allografts (p < 0.01) during the first 2 weeks. The degree of intimal hyperplasia proliferation was also decreased by 40% in chronic allograft rejection. The transfection of upregulating bioactive TGF beta-1 vector led to clear increase of the TGF beta 1 gene expression but had no significant effect on immune response either on syngeneic nor allogeneic grafts. These data suggest that TGF beta-1 plays a key role in modulating the early stage of acute rejection and is a crucial mediator of the outcome of chronic rejection. Down regulation of intragraft TGF beta-1 gene expression shows immunosuppresisve property and could be used to develop clinically relevant strategies in transplantation.

Systemic adjuvant chemotherapy achieves unsatisfactory results for inoperable liver tumors and metastases. Various clinical studies have shown that locoregional chemotherapy increased the survival rate by a few months. The aim of this study was to investigate tumor targeting of the most frequently used cytostatic 5-fluouracil by modifying systemic and locoregional therapy by liposome encapsulation. The tumor concentration of 5-FU encapsulated in SUV-PEG liposomes increases by a factor of 27 in systemic therapy and a factor of 90 in locoregional therapy. The tumor concentration of 5-FU increases by a factor of 8000, if the blood flow is additionally slowed by starch microspheres (Spherex) during locoregional therapy with liposome-encapsulated 5-FU.

The accumulation of malignant tumors in a single family, as well as the early onset of the disease, are highly suggestive of a hereditary condition. The evidence of microsatellite instabilities suggest a disease that might be associated with the HNPCC-syndromee. In contrary to the favourable prognosis of colorectal cancer associated with the HNPCC-syndromee we found in our animal model a far more aggressive progression of the disease in patients with HNPCC-associated gastric carcinomas than in patients with sporadic gastric carcinomas. This observation is consistent with the clinical course. Therefore, gastric carcinomas that are associated with the HNPCC-syndromee seem to represent a separate group of HNPCC-associated carcinomas and may even represent another hereditary disease.

Gene therapy was tested for inducing functional angiogenesis in the superficial rat epigastric island flap to allow earlier pedicle division. Autologous rat fibroblasts were grown, harvested, cultured and retrovirally transfected to produce PDGF-AA, an angiogenetically active protein. Stable gene expression was monitored by PDGF-AA ELISA. 180 animals were divided into 3 groups (I-III) and a bilateral flap created in each animal. In all experiments, the rightsided flap was subjected to experimental treatment and the left-sided flap served as control (1 ml saline 0.9%). During flap elevation, group I received 5 x 10(6) GMFB (genetically modified fibroblasts) plus 1 ml DMEM as medium. Group II was treated with 5 x 10(6) NMFB (non modified fibroblasts) plus 1 ml medium and group III received 1 ml medium alone. The flaps were sutured back and the vascular pedicle was bilaterally ligated and divided in each 10 animals during the following 6 days. 7 days later, the flaps were harvested, the amount of necrosis measured and histologically examined. The GMFB produced up the 560-times more PDGF-AA than the NMFB, measured by ELISA. The GMFB-treated flaps tolerated surgical division of the vascular pedicle significantly earlier than groups II and III. Histologically, fibroblasts persisted in all flaps of groups I and II without major inflammatory reaction. In all GMFB-treated flaps, massive angiogenesis could be demonstrated. By means of retroviral gene transfer autologous rat fibroblasts can be genetically modified for stable expression of the PDGF-A gene to produce high amounts of PDGF-AA, which is angiogenetically active. After injection into the panniculus carnosus, these cells induce functional angiogenesis to permit earlier division of the vascular pedicle in this flap model.

Purpose of the present study was, to proof whether homologous recombinant GH has an stimulating effect on bone healing in distraction osteogenesis. Therefore the left tibiae of 20 micropigs were osteotomized and distracted over an external fixator 2 mm daily on each of ten consecutive days. Animals were sacrificed after a healing time of another ten days. The treatment group received 100 micrograms r-pGH per kg bodyweight per day. After sacrifice, the regenerates were embedded undecalcified and cut into 6 microns thick sagittal oriented slices. Callus area, bone area, cartilagee area and bone perimeter in the regenerates were measured using an image analysis system. The results revealed, that GH promotes bone formation and maturation of the regenerate without disturbing the callus structure.

Under in vitro conditions, the resistance to ischemia may be improved by hypoxic preconditioning. This study was designed to test the hypothesis, that hypoxic preconditioning can also be induced in vivo by exposure of an intact animal to simulated high altitude. Male Wister rats (n = 48) were exposed for 60 minutes to simulated high altitude at 3000 m or 5000 m above sea level in a hypobaric chamber. 30, 150 or 300 min following exposure, hearts were excised and perfused aerobically in the working heart model prior to 45 minutes of global ischemia at 30 degrees C and subsequent normothermic reperfusion. For control, hearts of unexposed rats were either preconditioned by one cycle of 5 minutes of normothermic ischemia and reperfusion prior to subsequent ischemia or (n = 8) or not (n = 8). Postischemic measurements included functional parameters such as aortic flow, maximum developed left ventricular pressure, creatine kinase leakage from collected samples of the coronary effluent and the morphometric assessment of the area of myocardium at risk. When compared to non-preconditioning, ischemic preconditioning significantly improved the recovery of aortic flow, left ventricular systolic pressure and reduced the postischemic creatine kinase leakage. Likewise, exposure to a simulated high altitude at 3000 m above sea level significantly improved the recovery of aortic flow and reduced creatinkinase leakage when the time interval between exposure to simulated high altitude and perfusion was set to 150 minutes. Further increase in hypoxic stress (5000 m above sea level) or the reduction of this time interval to 30 minutes reduced postischemic recovery of hearts. The exposure of an intact organism to simulated high altitude can improve the resistance of the heart to ischemia by hypoxic preconditioning. The induction of this beneficial effect depends on the level of hypoxic stress and the time interval between preconditioning and subsequent ischemia.

Using the bioreactor model developed by J. Gerlach, we examined the potential of normal human hepatocytes for application in bioartificial liver devices. From normal human donor livers 1.5 x 10(8) hepatocytes were isolated. Hepatocytes were perfused in a woven multi-compartment capillary system in serum-free culture medium containing ammoniachloride over a period of 2 weeks. These cells demonstrated a well differentiated ultrastructure with formation of junctional complexes and bile canaliculi between adjacent cells. During reactor run, a constant albumin synthesis with levels above 11 mg/ml and maintenance of urea production and lignocaine metabolism (MEGX-test) were detected. These initial results indicate that normal human hepatocytes express typical morphology and ultrastructure and are able to keep differentiated functions in suitable perfusion models. Combination of the distinct human liver cell populations might enable promotion of further specific functions (clotting factors) and induction of liver cell proliferation.

The loop-diuretic piretanide was used to study the influence of pharmacological donor pretreatment on immediate postischemic function in a pig model of kidney transplantation based on the results of a clinical pilot study [4]. Following laparotomy, both kidneys were flushed via a transaortal catheter with Eurocollins-solution and surgically removed. A cold ischemic period of 1 or 24 h was chosen. After that period, kidneys were reperfused with intraoperatively drawn heparinized blood for one hour. We used a special instrument for hemoperfusion of isolated organs which allows perfusion for several hours under steady-state conditions. Four groups were formed: Control and piretanide, 1 or 24 h cold ischemia. The perfusion of piretanide-treated organs resulted in a lower perfusion-resistance, calculated as pressure/flow-ratio or as pressure/glomerular filtrationratio. The flow in the pretreated group was higher, thus excluding a higher shunt-volume. In parallel, oxygen consumption as a parameter of postischemic function start and creatinine clearance were higher in the piretanide treated groups. The experiments demonstrate a superior postischemic function of pretreated kidneys in comparison to control organs after 1 and 24 hours of cold ischemia in this model.

The effects of COS7 cells which constitutively express the apoptosis-inducing human CD95 ligand (COSh95L cells) on in vitro alloimmune responses were investigated. In the presence of COSh95L cells a clear-cut inhibition of alloantigen-induced proliferation was observed. In these cultures fully activated blastoid T cells were absent suggesting that deletion of activated T cells is one mechanism of COSh95L-mediated inhibition of proliferation. Non-fully activated cells were not eliminated in MLR by COSh95L cells but achieved a hyporesponsive state as shown by their failure to proliferate in response to restimulation with alloantigen or mitogen. These data indicate that the effects of CD95L on T cells are heterogeneous. Further analysis of the actions of CD95L in T cells of different activation states and subsets will be crucial to estimate the therapeutical potential of this molecule as target for immunomodulation after allo- and xenotransplantation.