[Potential barriers in xenogenic organ transplantation].
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
C Hammer
Publications and source records attributed to C Hammer.
Impact of immunoadsorption on complement activation, immunopathology, and hepatic perfusion during xenogeneic pig liver perfusion.
BACKGROUND: The impact of antibody adsorption by immunoapheresis on liver damage, complement activation, and hepatic perfusion was evaluated against the background of an application in extracorporeal pig liver perfusion for hepatic coma. METHODS: Eighteen pig livers were ex vivo perfused close to physiological conditions with fresh human blood for 4 hr. The influence of the perfusion circuit was investigated by perfusions of the circuit in the absence of livers (group 1 [G1]; n=5). Livers were xenoperfused without modifications in group 2 (G2; n=6). In group 3 (G3; n=6), pure Sepharose columns were used prior to liver perfusion. Immunoapheresis with Ig-Therasorb 100 columns was used in group 4 (G4; n=6). RESULTS: IgG was reduced by 95%, IgM by 72%, and IgA by 82% in G4, but only by about 30% in G3 (P<0.05). C4d, Bb fragment, and C3a levels were significantly lower in G4 than in G3 and G2 (P<0.05) after 180 min. Immunoadsorption diminished antibody and complement deposition as well as hepatocellular damage in G4. Portal angiographies demonstrated improved hepatic perfusion in G4. CONCLUSION: Immunoapheresis reduced organ damage as well as complement activation and improved hepatic perfusion during xenogeneic pig liver perfusion.
Acute Disease↗
Monitoring of microhemodynamic changes during ex vivo xenogeneic liver perfusion using intravital microscopy.
The main targets of xenogeneic rejection mechanisms are the endothelial cells of the graft. Their activation and the consequent alteration of the organ's microcirculation lead to the destruction of the xenograft. Microhemodynamic changes occurring during this process are still poorly characterized. The aim of this study was to analyze the microcirculation during xenogeneic ex vivo hemoperfusion of rat livers and to monitor the impact of treatment strategies using intravital fluorescence microscopy. In contrast to the isogeneic control group, blood flow almost completely stopped within the first minutes of xenoperfusion. Simultaneously, perfusion pressure increased and bile production was reduced. Acetylsalicylate (Aspisol) and the platelet-activating factor antagonist WEB 2170 improved the microcirculation and function of the xenoperfused liver. The combination showed a synergistic effect. After apheresis of preformed xenogeneic antibodies, the parameters measured were comparable with those seen in isogeneic experiments. Complement degradation with cobra venom factor revealed a minor improvement in perfusion. A rapid, extensive, and irreversible leukocyte accumulation in terminal portal vessels was observed in all xenogeneic experiments. Blood counts of the perfusate confirmed the early trapping of leukocytes and platelets in the xenoperfused liver, indicating nonimmunological, cellular involvement in this rejection process.
Animals↗
Analysis of the microcirculation during xenogeneic liver perfusion in the guinea pig--rat model. The contribution of leukocytes to the rejection process.
Since the main feature of hyperacute rejection is a disturbance of the xenograft's microcirculation, we analyzed microhemodynamic parameters during xenogeneic hemoperfusion of the guinea pig (GP) liver and investigated the contribution of leukocytes to the rejection process using intravital fluorescence microscopy. Isolated GP livers were hemoperfused via the portal vein in a recirculating system with a constant flow of 1 ml/min per g liver. In contrast to isogeneic perfusion with heparinized GP blood, a disturbance in the microcirculation was observed during xenogeneic perfusion using heparinized rat blood, with significantly higher values of perfusion pressure, reduced sinusoidal perfusion rates, and a larger number of stagnant leukocytes. A complete breakdown of the microcirculation, with the highest values of perfusion pressure and the smallest perfusion index, was associated with 100% accumulated leukocytes when rat blood was anticoagulated with sodium citrate. Almost isogeneic perfusion values were obtained when fucoidin, which inhibits L-selectin-dependent cell interaction, was added to heparinized rat blood. These data indicate that leukocyte-endothelial cell interaction contributes to xenogeneic rejection.
Animals↗
Cytoimmunology in monitoring: the Munich experience.
Explore the source record for details and available documents.
CD4-Positive T-Lymphocytes↗
Organs from animals for man.
In the following review some of the problems of xenotransplantation shall be discussed, based on the few experimental data available so far and on reports in the literature describing investigations which may be of importance for xenotransplantation. The impact of gravity on the upright posture of man versus almost all other mammals, the dysfunction between enzymes and hormones in different species and the lack of interactions between interleukins, cytokines and vasoactive substances will be taken into consideration. The question must be asked whether different levels of carrier molecules or serum proteins play a role in the physiological network. Even though the development of transgenic animals or other imaginative manipulations may lead to the acceptance of any type of xenografted organ, it has to be established for how long the products of the xenografts are able to act in the multifactorial orchestra. We are far from understanding xenogeneic molecular mechanisms involved in toxicity, necrosis and apoptosis or even reperfusion injury and ischemia in addition to the immediate mechanisms of the hyperacute xenogeneic rejection. Here, cell adhesion, blood clotting and vasomotion collide and bring micro- and macrocirculation to a standstill. All types of xenogeneic immunological mechanisms studied so far were found to have a more serious impact than those seen in allogeneic transplantation. In addition we are now only beginning to understand that so-called immunological parameters in allogeneic mechanisms act also in a true physiological manner in the xenogeneic situation. These molecular mechanisms occur behind the curtain of hyperacute, accelerated, acute or chronic xenograft rejection of which only some folds have been lifted to allow glimpses of part of the total scene. Other obstacles are likely to arise when long-term survival is achieved. These obstacles include retroviral infections, transfer of prions and severe side effects of the massive immunosuppression which will be needed. Moral, ethical and religious concerns are under debate and the species-specific production of proteins of the foreign donor species developed for clinical use suddenly appears to be a greater problem than anticipated.
Animals↗
[Xenotransplantation: what is its future?].
The ultimate solution to the growing shortage of organs for transplantation could be the use of animal organs or tissues. Xenotransplantation of organs of all kinds, size and number would be available to be transplanted to patients with endstage diseases. Advances in technology, as well as the accelerating rate of progress in biochemical, biological and genetic fields, means that it will be possible to bridge times until allografts are available or that successful engraftment becomes possible. Open questions in immunology or metabolism as well as the transfer of infectious material from animal to man need, however, major efforts to be solved. An optimistic outlook can be given, that the problems of xenotransplantation are solved in the next decade, or certainly in the next generation.
Animals↗
[Transgenic swine as potential organ donors? Results of the ex-vivo hemoperfusion hDAF transgenic kidney with human blood].
Kidney xenotransplantation is not yet a realistic clinical treatment modality. However, during the last decades more than 30 kidneys from other species have been transplanted into humans; some of the kidneys sustained some function up to 60 days. Recent progress in genetic engineering has raised the possibility to create large transgenic animals which express human complement regulatory proteins (CRP). Since early complement activation is believed to be the main triggering event for xenograft destruction, complement regulation by species-specific CRP should avoid hyperacute rejection in transspecies transplantation. The perfusion of hDAF-transgenic pig kidneys with human blood was not associated with the morphological signs of hyperacute rejection when compared to non-transgenic control organs. Specific immunohistology could demonstrate that the transgene was sufficient to regulate complement activation beyond C3 despite the endothelial deposition of xenoantibodies. In the future, these organs could be further optimized and ultimately tested in a clinical pilot protocol under appropriate immunosuppression.
Animals↗
Immunopathological observations after xenogeneic liver perfusions using donor pigs transgenic for human decay-accelerating factor.
BACKGROUND: Donor pigs transgenic for human decay-accelerating factor (hDAF) were used in a xenogeneic ex vivo liver perfusion model to study the effect of this modification on the development of hyperacute rejection. METHODS: Three transgenic pigs were hepatectomized after hypothermic portal and transaortal gravity perfusion. Livers from six nontransgenic pigs served as controls. All livers were perfused for 3 hr with human blood from two donors diluted to a hematocrit of 30%. Particular importance was placed on the use of an optimal perfusion technique incorporating the floating suspension of the organs in a waterbath and intermittent external pressurization. Biochemical, physiological, and immunological parameters were assessed. Tissue specimens taken before and after perfusion were analyzed using routine histology, electron microscopy, and immunohistology. RESULTS: Complement activation was more pronounced in the control group. AP50 and CH50 values fell to about 60% of the initial levels in control experiments, whereas they remained at 80% of the initial levels during perfusion of hDAF livers. After 180 min, pig tumor necrosis factor alpha levels were 7862+/-1645 pg/ml for unmodified livers and 2830+/-734 pg/ml in the hDAF group. Human tumor necrosis factor alpha levels were similar in both groups. Control livers showed marked morphological alterations and distinct deposition of complement factors, whereas livers expressing hDAF showed no signs of hepatocellular necrosis and almost no complement deposition beyond C3 activation. CONCLUSIONS: These results confirm that the transgenic expression of the human complement regulatory protein hDAF reduces complement activation and prevents hyperacute rejection in a xenogeneic liver perfusion model over the 3-hr evaluation period used in this study.
Animals↗
Application of immunoapheresis for delaying hyperacute rejection during isolated xenogeneic pig liver perfusion.
BACKGROUND: Extracorporeal liver perfusion in hepatic coma, used to eliminate toxic metabolites causing hepatic encephalopathy, is limited by the antibody (Ab) and complement-mediated hyperacute rejection of discordant xenografts. Thus, the efficacy of highly selective immunoadsorption columns to deplete xenoreactive human anti-porcine antibodies before ex vivo liver perfusion was examined in this study. METHODS: Eighteen domestic pigs were hepatectomized according to standard techniques. The livers were ex vivo perfused for 4 hr. The perfusion protocol closely followed the physiological conditions of a human liver. Parameters of liver function and damage were analyzed. Three groups were formed differing in the treatment of the perfusate. As basic control, livers were perfused with heparinized human blood (group 1; n=6). Immunoapheresis was applied in group 3 (n=6). Immunoapheresis was performed using Ig Therasorb columns consisting of sheep-anti-human IgG Abs covalently coupled to Sepharose CL-4B. Additionally, the effect of pure Sepharose CL-4B without immobilized Ab was tested in group 2 (n=6). RESULTS: The use of Ig Therasorb 100 columns in group 3 resulted in a reduction of IgG, IgM, and IgA in the order of 95.0%, 72.3%, and 81.5%, respectively. In group 2, IgG, IgM, and IgA were lowered by 30% to 39%. Determination of liver-specific enzymes and tolerance tests revealed a significant reduction of cellular damage and functional restrictions in group 3 compared with the control groups. CONCLUSIONS: Immunoapheresis conducted according to this protocol appears to be an effective approach for delaying antibody-mediated hyperacute xenogeneic rejection.
Acute Disease↗
Fluorescence videomicroscopic assessment of xenogeneic microcirculation and impact of antibody removal by immunoadsorption.
BACKGROUND: Alterations in microcirculation are considered central to the pathogenesis of hyperacute xenogeneic rejection (HXR) of vascularized xenografts, but currently there exist no data describing these microhemodynamic alterations. METHODS: Rat livers were perfused in situ with either isogeneic rat blood or xenogeneic human blood. The microcirculation of these xenoperfused livers was investigated directly using intravital fluorescence microscopy, and compared with that of isogeneic hemoperfused livers. In addition, the impact of antibody depletion by immunoadsorption was investigated. RESULTS: Although a homogenous microcirculation was found during isogeneic liver perfusion (index of acinar perfusion 90.4%/sinusoidal perfusion rate 93.6%), xenoperfusion resulted in a rapid breakdown of the microcirculation (47.5%/67.1%, respectively). Perfusion deficits were found predominantly in the periportal areas. Immunoadsorption reduced the total amount of IgM and IgG by 75.2% and 96.2%, respectively, and caused a significantly improved liver perfusion (80.2%/84.4%) and liver function, as indicated by bile production. In contrast, the massive hepatic leukocyte and platelet accumulation observed during perfusion with untreated xenogeneic blood was not altered by antibody depletion. CONCLUSIONS: Thus, the combination of isolated rat liver perfusion and intravital fluorescence microscopy enables the observation and quantification of the early phase of HXR. This is an important step forward for sensitive characterization of the rejection process and will enable the mechanisms involved in HXR to be elucidated. Antibody depletion was shown to improve liver function and perfusion, but did not reconstitute liver viability to the level of the isogeneic perfusion. These findings highlight the need for additional therapeutic regimens in xenografting.
Acute Disease↗
Morphology of hDAF (CD55) transgenic pig kidneys following ex-vivo hemoperfusion with human blood.
Discordant xenotransplantation of pig kidneys into man may be possible in the future using transgenic organs which regulate complement activity. It was the aim of this experimental study to characterize morphologic alterations of organs transgenic for human decay accelerating factor (hDAF/CD55) perfused with human blood since no data on function of these organs after exposure to human blood are available. An ex-vivo system was developed that allows computer driven pressure-controlled perfusion of kidneys including a separate cartridge oxygenator circuit. Following cold ischemia time of 1-4 hr, 8 kidneys from heterozygote transgenic animals (TG) and 9 control kidneys (C) were perfused with 500 ml freshly drawn heparinized human blood at physiological conditions. A histologic grading system from 0 to +4 was used to describe the histologic findings. Using a mouse antihuman DAF moAB, hDAF was stained on all TG kidneys both on glomerular capillary (4+) and vascular endothelium (2+), but there was no detectable hDAF-expression on controls. No difference in xenoantibody deposition on vascular endothelium was seen between both groups. There was comparable staining for complement fraction C4 in both groups, but significant reduction of C3 and C9 staining on glomerular and vascular endothelium in TG. P-selectin was expressed on a higher level in C (+4) compared with TG (+2). Neutrophil extravasation [NP-57 elastase] was higher in C (80.2 vs. 32.2 C vs. TG [values as n/high power field]). Tubular epithelial cell swelling and mild necrosis was paralleled by glomerular hemorrhage and platelet microthrombus formation in both groups as seen in transmission electron microscopy. The observed results allow the conclusion that hDAF expression on transgenic pig kidneys was sufficient to inhibit complement activation beyond C3 during xenoperfusion with human blood despite xenoantibody deposition.
Animals↗
Role of leukocyte adhesion molecules during ex vivo kidney xenoperfusion.
Explore the source record for details and available documents.
Animals↗
[Proliferative potential of nasal septum chondrocytes for in vitro culture of cartilage transplants].
BACKGROUND: Recent developments in the field of tissue engineering provide novel approaches in tissue repair and reconstructive surgery using the patients own cells. Isolated chondrocytes form new cartilage when seeded in appropriate scaffolds. Usually the number of cells from a cartilage biopsy is not sufficient. The present study investigates the potential of cell amplification of human nasal chondrocytes in monolayer culture. METHODS: Nasal cartilage cells from seven healthy patients with age between 16 and 60 years were enzymatically isolated with collagenase and hyaluronidase. Subsequently, cells were seeded in 75 cm2 culture flasks. After confluency, cultures were trypsinized, counted, and again seeded at a concentration of 5 x 10(4) cells/ml. Dulbecco's MEM supplemented with 10% FCS was used as culture medium. RESULTS: After enzymatic digest, an average of 5 x 10(5) cells per patient were isolated. At least 85% of the cells were vital. Within four to eight weeks, the cells number was increased 10(3) to 10(5) fold. No correlation between the proliferative activity and the age of the patient was observed in this study. DISCUSSION: The observed increase in cell number resembles about 10 to 20 cell doublings. Although the doubling time appears to be longer during the second month, no definite limit of proliferative activity was seen during the time of study. Proliferating chondrocytes in monolayer lose their tissue-specific phenotype. For the de novo formation of cartilage transplants, redifferentiation of the expanded cells has to be stimulated. CONCLUSION: This study shows that human nasal chondrocytes can be expanded sufficiently in monolayer for the engineering of autologous cartilage transplants.
Adolescent↗
[Transplantation of in vitro cultured cartilage materials: characterization of matrix synthesis].
BACKGROUND: Recently a three-dimensional model for the formation of cartilage in vitro was developed. The aim of this study was to investigate the amount and quality of newly synthesized matrix after graftig in vitro engineered cartilage into athymic nude mice. MATERIAL AND METHODS: Group I received transplants consisting of human chondrocytes, agarose, and E 200 (a bioabsorbable polymer fleece that offers mechanical stability. Ethicon Inc). Group II received chondrocytes and agarose only. At intervals of six, 12, and 24 weeks after subcutaneous transplantation we used azan blue staining and antibodies against collagen type I, collagen type II, and chondroitin-4sulfate to characterize the matrix synthesis. A quantitative analysis was performed using the computer image analyzing software photoshop (Adobe Inc). RESULTS: In group I, the amounts of newly synthesized cartilage specific collagen type II and chondroitin-4 sulfate increased progressively. Twenty-four weeks after transplantation, these amounts were comparable to the original human cartilage from which the chondrocytes were derived. Collagen type I was detected only in small quantities in the periphery of the transplants. Gross examination revealed sufficient mechanical stability and unremarkable changes in size and form. In contrast to this, group II transplants showed markedly smaller amounts of cartilage specific matrix components as collagen type II and chondroitin-4 sulfate and at the same time greater amounts of collagen type I. It was found both in the periphery and in central parts of the transplants. There was a remarkable loss of volume in all transplants and mechanical stability was poor. CONCLUSIONS: The absorbable cell carrier E 200 not only offers mechanical stability to in vitro engineered cartilage but also had a positive effect on the development of cartilage in our experiments. In conclusion, in vitro engineered cartilage is a promising pathway for the replacement of cartilage defects.
Adolescent↗
Transgenic human decay accelerating factor makes normal pigs function as a concordant species.
BACKGROUND: Increasing interest has focused on xenotransplantation as a potential solution to the organ shortage. To overcome hyperacute rejection, pigs have been produced that are transgenic for human decay accelerating factor (DAF). For the evaluation of the effects of human DAF, an ex vivo working heart model was used. METHODS: We compared hemodynamic performance of four transgenic pig hearts (group A) with that of four Landrace pig hearts (group B) and eight rhesus monkey hearts (group C). For perfusion fresh blood had been taken from healthy volunteers. From the coronary sinus effluent, samples were taken for the determination of 6-keto prostaglandin F1 alpha, prostaglandin E2, creatine phosphokinase, and lactate dehydrogenase, respectively. Hemodynamic parameters were measured continuously for 150 minutes after the start. After 15 minutes of reperfusion, the Langendorff-mode was switched to the working heart model. After hearts failed to pump against the afterload column, experiments were terminated, and tissue sections were taken for electron microscopy. RESULTS: Groups A and C showed superior cardiac performance as measured by stroke work index (SWI) that exceeded group B by 2.5 to 3 times (p < 0.05). In all three groups the SWI slowly decreased during perfusion. In group B, SWI decreased to a minimum as early as 90 minutes after the start. In all groups, 6-keto prostaglandin F1 alpha and prostaglandin E2 as indicators of endothelial cell activation increased. In group B, however, the levels exceeded those of groups A and C by six and nine times, respectively (p < 0.05). As markers of myocardial damage, creatine phosphokinase and lactate dehydrogenase increased in all groups. But again levels in group B exceeded those of groups A and C by four to five times (p < 0.05). Electron microscopy revealed single cell necrosis in group B, whereas groups A and C showed interstitial edema only. CONCLUSIONS: Our experiments indicate a crucial role of DAF in preventing rejection in discordant species combinations. Transgenic human DAF seems to inhibit successfully complement-mediated damage to the endothelial cell, thus preventing endothelial activation and consequently myocardial damage. Transgenic human DAF makes a discordant species (pig) function as a concordant species, that is, hyperacute rejection does not occur.
6-Ketoprostaglandin F1 alpha↗
Xenotransplantation: facts and fiction.
Xenotransplantation will certainly solve the problem of the allogeneic organ shortage if long-term organ function can be achieved. Acceptance or rejection of the grafts depends not only on immunological mechanisms but, as already recognized in chronic rejection (i.e. transplant atherosclerosis), on many other biochemical, physiological, and even morphological characteristics including inflammatory events originating from infections. The way in which signals in the form of hormones, neurotransmitters, enzymes and growth factors are translated in xenogeneic systems has not received adequate attention, if any at all. Central questions still to be answered are: How many xenogeneic hormonal and enzymatical interactions are possible? Are they able to maintain the metabolism in a foreign organ or body and for how long? In addition, transport molecules, the most important of which is albumin, must be considered when trying to achieve long-term survival time using organs from a widely divergent species. Large quantities of foreign proteins, mediators and enzymes are released into the circulation when, for example, xenogeneic livers or other organs suffer from HXAR. The function of the liberated enzymes, hormones and inhibitors is far from being understood or even investigated. The amount of potassium released from a deteriorating xenogeneic liver is able to mimic a sometimes lethal cardioplegic solution. The first data coming from intra-vital microscopy herald that adhesion molecules, the newest but not last in the line of trouble-shooters, play a major role in microcirculation together with interleukins and eicosanoids (32). These adhesion molecules may, as a falsely activated system or as an incompatible system, be unable to protect the endothelial cells from the attack, adhesion and migration of white blood cells. Even when transgenic animals or other imaginative manipulations lead to the acceptance of any type of xenograft, it has to be established how far, and for how long, the products of the xenograft will be able to interact in the multifactorial orchestra. Despite this rather careful if not pessimistic appraisal, xenotransplantation has to be investigated vigorously even though the short-term outlook does not seem to be promising.
Animals↗
Prevention of hyperacute rejection by human decay accelerating factor in xenogeneic perfused working hearts.
As a potential source of organs for xenotransplantation, pigs that are transgenic for human decay accelerating factor (DAF) have been bred in order to overcome hyperacute rejection. We investigated the protective effect of human DAF in a porcine working heart model perfused by human blood. Hearts of normal landrace pits served as controls. The following parameters were measured: stroke work index, coronary flow and arteriovenous oxygen consumption, 6-keto prostaglandin F1alpha and prostaglandin E2 as markers of endothelial cell activation; creatine phosphokinase and lactate dehydrogenase for evaluation of the extent of myocardial damage; TNFalpha and IL-6 as markers of mononuclear cell activation. Histological and ultrastructural investigations from myocardial tissue sections were done at the end of perfusion. Human (h) DAF appeared to inhibit complement-mediated endothelial cell activation of transgenic pig hearts successfully. This was in contrast to landrace pig hearts, which had a sixfold increase of prostaglandin levels during perfusion with human blood. The cardiac weight increase during perfusion time due to interstitial edema tended to be less in the hDAF group. Myocardial damage was minimal in transgenic hearts, whereas normal pig hearts produced a threefold increase of creatine phosphokinase and lactate dehydrogenase levels. In these hearts, electron microscopy revealed single cell necrosis of myocytes and vacuolization of mitochondria with cristae rupture. According to the results obtained in the working heart model, the breeding of pigs that are transgenic for hDAF represents a promising step to making heart xenotransplantation a clinical reality in the future.
Acute Disease↗