Hyperacute rejection in the xenogenic transplanted rat liver is triggered by the complement system only in the presence of leukocytes and free radical species.

BACKGROUND: Reactive oxygen species (ROS) and nitric oxide species (NOS) are pivotal after ischemia-reperfusion. However, the role of different cells on the formation of free radical species after xenotransplantation remains elusive. We hypothesized that ROS and NOS formed during hyperacute rejection are dependent on leukocytes, erythrocytes, activated thrombocytes, and Kupffer cells (KCs). To address this issue, we developed a model of xenoperfused rat liver and assessed the relationship between free radical production and graft dysfunction. METHODS: Livers from Sprague-Dawley rats were isolated, flushed with cold Ringer solution, and perfused at physically flow rates for 120 min after 1 h of ischemia. The control group was perfused with rat whole blood (n = 9). In the study groups, the livers were perfused with human whole blood, human plasma with erythrocytes, and plasma with erythrocytes and isolated thrombocytes (n = 9/group). In an additional group, gadolinium chloride (GdCl3), a selective Kupffer cell (KC) toxic agent, was applied. Liver damage, hyperacute rejection, and the depletion of KCs were monitored histologically. Liver damage and function were determined by means of liver enzymes, portal pressure, and bile production. Malondialdehyde (MDA), nitric oxide formation, and peroxynitrite concentration, as well as total glutathione (tGSH) level, were measured as indicators for free radical formation and anti-oxidative status. RESULTS: Significant differences in the MDA, NO, peroxynitrite levels, and GSH levels after reperfusion with various cell populations were observed. Markedly high ROS/RNS production was evident in the KCs and the xenogeneic whole-blood group. The oxidative stress was mainly caused by leukocytes and to lower extent by KCs, but only in combination with leukocytes. Neither erythrocytes, thrombocytes, nor hepatocytes had an effect on the release of ROS and RNS, as we could not observe significant differences in the MDA, peroxynitrite, and NO levels in these groups compared with control. Tissue injury and hyperacute rejection were more evident in the KC and whole-blood livers. No sign of damage was observed for the control, erythrocyte, and thrombocyte group. Removal of leukocytes from the perfusate by filtration had a major protective effect on the liver function and the grade of hyperacute rejection, whereas KC depletion reduced the ROS production, but did not have an impact on the hyperacute rejection and liver damage. In all xenogeneic perfused groups, the activation of the complement was histologically observed by positive C3c and C9b. Neither KC depletion nor the removal of leukocytes or thrombocytes from the perfusate had an effect on the activation of the complement system. Damage of the rat liver by the complement system was only observed in association with leukocytes. CONCLUSION: Our data revealed that various cell populations contribute to the formation of free radicals in our model. The production of free radicals was mainly linked to leukocytes and to a minor extent to KCs, but only in combination with leukocytes. Free radicals critically contribute to injury, rejection, and dysfunction of the xenotransplanted liver. Furthermore, hyperacute rejection in the xenogeneic perfused liver is triggered by the complement system only in the presence of leukocytes and free radical formation.

Reduced fibrin deposition and intravascular thrombosis in hDAF transgenic pig hearts perfused with tirofiban.

BACKGROUND: Solid organ xenograft rejection is associated with vascular injury resulting at least in part in platelet activation, and rejected xenografts invariably demonstrate intravascular thrombosis and interstitial hemorrhage. Complement activation plays a prominent role in platelet-endothelial interaction. We tested the effects of platelet GPIIb/IIIa inhibitor tirofiban during perfusion of hDAF pig hearts. METHODS: Using a working-heart model, nontransgenic and hDAF pig hearts were perfused with tirofiban or human blood only. Myocardial damage was determined by hemodynamic parameters (cardiac output, stroke work index) and creatine phosphokinase. Further monitoring included the assessment of complement factors (C3, C4), platelets, fibrinogen, ATIII, and graft histology. RESULTS: Tirofiban increased cardiac output (CO) and stroke work index (SWI) of nontransgenic pig hearts and improved superior CO and SWI of hDAF pig hearts. Although perfusion time of nontransgenic pig hearts was prolonged by tirofiban (196+/-65 min vs. 162+/-122 min), a similar effect in hDAF pig hearts (218+/-116 min vs. 222+/-30 min) could not be demonstrated. Tirofiban reduced consumption of C3 and C4 independently from hDAF. Depletion of fibrinogen was equally diminished by tirofiban and hDAF; the combination of both agents obtained no further reduction. ATIII consumption was most effectively inhibited by this combination. Intravascular fibrin deposition was reduced by tirofiban and hDAF, but particularly by the combination of the two agents. CONCLUSIONS: Improvement of heart performance and reduction of myocardial damage and intravascular thrombosis confirm a role of the GPIIb/IIIa inhibitor tirofiban for the prevention of hDAF pig heart rejection and xenograft function.

In vivo visualization of the effect of polyclonal antithymocyte globulins on the microcirculation after ischemia/reperfusion in a primate model.

BACKGROUND: Ischemia-reperfusion injury (IRI) leads to increased leukocyte adherence enhancing acute cellular rejection and microvascular dysfunction. Polyclonal antithymocyte globulins (ATGs) induce T-cell depletion and functional impairment of nondepleted lymphocytes in peripheral blood. ATGs represent an important option in the treatment of acute cellular rejection but little is known about their effects on the microcirculation in IRI. METHODS: In a perfusion system, 19 cynomolgus monkeys were used to evaluate the influence of three different ATGs on the leukocyte-endothelium interaction after cold ischemia. ATGs were administered to human blood 30 min prior to reperfusion of primate extremities. Using intravital fluorescence microscopy the postreperfusion microcirculation of skeletal muscle was visualized. RESULTS: Significant differences were found between ATG-treated and ATG-free groups concerning blood flow velocity, leukocyte count, and leukocyte-endothelium interaction. ATGs reduced microvascular leukocyte adhesion, count, and blood flow impairment. CONCLUSION: ATGs have a favorable impact on early mechanisms of IRI. Due to reduced leukocyte adherence to the antigen-presenting endothelial cells, recognition events cannot take place in the posttransplant period of reperfusion. In addition to inhibiting acute transplant rejection, increase of posttransplant blood flow supports the use of ATGs as pretransplant induction therapy.

Influence of polyclonal anti-thymocyte globulins upon ischemia-reperfusion injury in a non-human primate model.

BACKGROUND: Polyclonal anti-thymocyte globulins (ATGs) are used to induce immunosuppression and to treat acute rejection after transplantation. ATGs induce apoptosis and in peripheral T-lymphocytes having the potential to inhibit leukocyte adhesion. We analysed the influence of three different ATGs upon the microvasculature and the different cell-subpopulations after ischemia/reperfusion (IRI). MATERIALS AND METHODS: Extremities of cynomolgus monkeys were surgically isolated and flushed with Ringer's lactate at 4 degrees C. After 60 min of ischemia the limbs were reperfused with matching human blood. ATGs were added to the blood 30 min prior to the reperfusion. Four groups were generated: Tecelac-ATG group, Fresenius(S)-ATG group, Thymoglobulin-ATG group and a control group. Blood analyses were performed in blood samples taken after the beginning of the reperfusion. Biopsies from muscular tissue were obtained after the experiments. RESULTS: The number of circulating leukocytes was lower in the ATG-groups than in control. Morpho-cytological analyses showed depletion of peripheral lymphocytes. Histological examination showed less tissue damage, reduced presence of fibrin and adherent thrombocytes in the ATG-treated groups. Leukocyte infiltration, both in muscle and vascular structures, was significantly diminished in the ATG-groups in comparison to control. DISCUSSION: Our results show that ATGs have a favourable impact on early mechanisms of IRI. ATGs showed a reduction of the number of adherent leukocytes and muscle infiltrates suggesting that preoperative therapy with ATGs may have an advantageous effect on primary non-function and on chronic rejection as well as a positive influence upon IRI.

A new animal model to assess angiogenesis and endocrine function of parathyroid heterografts in vivo.

BACKGROUND: It is still a matter of investigation how angiogenesis and restoration of gland perfusion determine graft function after free parathyroid autotransplantation. We provide a new animal model allowing simultaneous and repetitive in vivo assessment of angiogenesis and endocrine function of parathyroid transplants. METHODS: Fresh human parathyroid tissue from patients with secondary hyperparathyroidism was grafted into dorsal skinfold chamber preparations of athymic nude mice (CD1-nu; n=8). Equivalent pieces of the same human donor specimens were heat-inactivated and served as control grafts (n=7). RESULTS: In all animals receiving parathyroid transplants, intact human parathyroid hormone levels were detectable by species-specific enzyme-linked immunosorbent assay analysis of plasma samples on day 5 after transplantation and increased by 2.5-fold over the observation period (19 days) in contrast with controls. Plasma Ca levels revealed no differences between the groups. On day 5 after transplantation, intravital fluorescence microscopy revealed murine angiogenic microvessels sprouting along nonperfused human donor vessels, and 1 week later functional microvasculature was established in all parathyroid transplants. Histologic analysis revealed well-vascularized endocrine tissue. In contrast, control grafts were necrotic and partly resorbed; they exhibited no angiogenic activity or well-vascularized fat cells indicating fatty degeneration. In addition, species-specific Western blot analysis revealed vascular endothelial growth factor expression of parathyroid transplants rather than functional vessel density as the functional parameter of angiogenesis determining transplant function in vivo. CONCLUSION: This model may serve to understand mechanisms associated with specific parathyroid transplant angiogenesis and its significance for transplant function to optimize clinical success of autotransplantation in therapy-resistant patients.

Synergistic effects of brain death and liver steatosis on the hepatic microcirculation.

BACKGROUND: The routine transplantation of steatotic livers could potentially mitigate the donor shortage, but so far is associated with a high rate of graft dysfunction. Steatosis and brain death have been perceived as independent risk factors, but they may synergistically target the hepatic microcirculation. This study compares the effects of brain death on the microcirculation of steatotic and normal livers. METHODS: Brain death was induced in obese and lean Zucker rats. Lean and obese sham-operated animals served as controls. Liver microcirculation was investigated using intravital fluorescence microscopy. Serum liver enzyme and reduced glutathione, expression of P-selectin, ICAM-1 and VCAM-1 mRNA in the liver were determined. The ultrastructural alterations were compared by electron microscopy. RESULTS: In nonbrain-dead animals, liver steatosis was associated with smaller sinusoidal diameters, but did not impair sinusoidal perfusion. During brain death, sinusoidal diameter and perfusion were reduced in normal and, to a greater extent, in steatotic livers. Also, more leukocytes were recruited to the microvasculature of steatotic livers than to normal livers in brain-dead state. The highest liver enzyme activities and the lowest hepatic GSH concentrations were measured in brain-dead animals with steatotic livers; only in these organs was endothelial cell swelling regularly observed. In brain-dead state, only the P-selectin mRNA expression was increased in steatotic livers as compared to normal livers. CONCLUSIONS: Brain death amplifies the adverse effects of steatosis on the hepatic microcirculation. Our results underline the need for therapeutic intervention in brain-dead state when steatotic livers are to be used for transplantation.

In vitro influence of polyclonal anti-thymocyte globulins on leukocyte expression of adhesion molecules.

OBJECTIVES: Polyclonal anti-thymocyte globulins (ATGs) are drugs used in the induction of immunosuppression, in the treatment of acute rejection, and in the therapy of hematologic disorders. Treatment with ATGs can produce adverse effects due to cross-reacting antibodies directed against nonmyeloid cells. This study sought to evaluate the interaction of ATGs and some adhesion molecules expressed on the surface of neutrophils and lymphocytes. MATERIALS AND METHODS: We determined the effects of different doses of 3 polyclonal ATGs on the activation and expression of lymphocyte and neutrophil adhesion molecules in whole blood by means of flow cytometry. RESULTS: ATG treatment reduced the percentage of lymphocytes gated for CD18 and CD62L, as well as the expression of CD11b, CD18, and CD62L in a dose-dependent manner. ATGs modulated the percentage of gated neutrophils for CD18. Although ATG treatment did not affect CD11b or CD62L gating in neutrophils, it did regulate expression of these adhesion markers. CONCLUSIONS: Our results show that ATGs can modify the expression levels of some of the main leukocyte adhesion molecules that are responsible for the characteristic cellular adhesion after ischemia/ reperfusion. These properties of ATGs may contribute to reduced leukocyte infiltration after solid-organ transplantation.

In vitro assessment of dose-dependent platelet activation by polyclonal antithymocyte globulins: a flow-cytometric analysis.

Polyclonal antithymocyte globulins (ATGs) are immunosuppressive drugs widely used in transplantation and hematologic disorders. Treatment with ATGs can induce side effects such as neutropenia and thrombocytopenia because of unspecific antibodies directed against nonmyeloid cells present in these preparations. Depletion, activation, and expression of adhesion molecules on platelets in vitro were studied in the whole blood of healthy volunteers by means of flow cytometry after incubation with different doses of three polyclonal ATGs. Our data show no ATG-mediated cytotoxic activity against platelets. ATGs are able to induce activation of platelets through increased expression of P-selectin and hLAMP-1 and higher percentages of gated thrombocytes expressing these molecules. Furthermore, increased expression of hLAMP-1 presented a dose-dependent pattern. ATGs induced activation and enhanced expression of adhesion molecules in unstimulated platelets. Increased adhesion may be responsible for undesirable side effects such as thrombocytopenia and reticulopenia.

Polyclonal antithymocyte globulins reduce the expression of IL-4 in a non-human primate model of ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) is a non-specific, antigen independent event, which significantly influences the outcome of transplanted organs. Interleukin-4 (IL-4) is an immunological mediator belonging to the interleukin family that mainly regulates the differentiation of T-helper lymphocytes into Th2 phenotype as well as enhances cellular activation, both are important features in IRI. The influence of polyclonal antithymocyte globulins (ATGs) on expression of IL-4 in reperfused tissues of cynomolgus monkeys (n=18) after 60 min of ischemia was assessed by immunohistochemical methods. Our results show an inhibition of the production and release of IL-4 by activated lymphocytes in the groups treated with ATGs in comparison to control, although a causal relationship between IL-4 and tissue damage was not demonstrated. Implication of IL-4 as an inflammatory mediator upon IRI must be further investigated.

Prevention of hyperacute xenograft rejection through direct thrombin inhibition with hirudin.

BACKGROUND: Hyperacute xenograft rejection (HXR) is characterized by complement activation and intravascular thrombosis. The pathogenesis of HXR is attributed to antibodies binding to α-Gal-epitopes on the endothelial cells (EC) of the xenograft, activating complement and thrombin-mediated coagulation mechanisms. Our aim was to evaluate the influence of thrombin inhibition upon HXR and tissue integrity in an ex-vivo working heart model. MATERIAL/METHODS: Eighteen isolated porcine hearts were perfused with human whole blood in a working heart model. The blood was treated with heparin (n=9) in group G-I and with heparin and additionally recombinant hirudin (0.012 mg/ml bolus, afterwards 4.5 µg/ml/h continuously) in group G-II (n=9). The experiments were terminated at end of cardiac output. Histological analysis was performed after the experiments. RESULTS: Working heart time of G-II was significantly longer (712.0±37.8 vs. 125.0±31.4 min, p<0.01). Heart weight increase in G-II was lower (0.05±0.01 vs. 0.30±0.06%/min, p<0.01). Stroke work index and specific coronary flow improved significantly in G-II after 120 minutes. Histological analysis revealed increased tissue damage and thrombosis phenomena in G-I. Moreover, immunohistochemistry showed increased C3 and C5b-C9 upon EC of G-I. CONCLUSIONS: Direct thrombin inhibition with Hirudin could be a successful strategy in primate xenotransplantation experiments to prevent tissue damage thus improving the graft survival.

Soluble Galalpha(1,3)Gal conjugate combined with hDAF preserves morphology and improves function of cardiac xenografts.

BACKGROUND: Cytotoxic anti-Galalpha(1,3)Gal antibodies play a key role in the rejection of pig organs transplanted into primates. Regimens reducing anti-Galalpha(1,3)Gal antibodies were associated with severe side effects unable to prevent antibody rebound until soluble synthetic oligosaccharides with terminal Galalpha(1,3)Gal inhibiting antigen binding became available. We displayed kinetics of anti-pig and anti-Galalpha(1,3)Gal IgM and IgG antibody levels using GAS914, a Galalpha(1,3)Gal trisaccharide conjugated to poly-l-lysine, and investigated corresponding changes of parameters of heart function. METHODS: Using a working heart model, hDAF pig hearts were perfused with human blood containing GAS914 (group 1). As controls hDAF pig hearts (group 2) and landrace pig hearts (group 3) were perfused with human blood only. Levels of anti-Galalpha(1,3)Gal (IgM, IgG) and anti-pig antibodies were assessed to prove the effectiveness of GAS914. As parameters of heart function, cardiac output (CO), stroke work index (SWI), coronary blood flow (CBF) and coronary resistance were measured. Creatine phosphokinases, lactate dehydrogenase and aspartate aminotransferase were evaluated as markers of myocardial damage. Histological and immunohistochemical investigations were performed at the end of perfusion. RESULTS: In group 1 an immediate and extensive reduction in both IgM and IgG anti-Galalpha(1,3)Gal was found. Anti-pig antibodies were eliminated accordingly. Antibody binding to GAS914 was complete before the start of organ perfusion. Corresponding to rapid antibody elimination in group 1 GAS914 not only was able to significantly prolong the beating time of the heart in hDAF pigs, but also to clearly improve functional parameters. When switching to the working heart mode hDAF pig hearts perfused with human blood containing GAS914 (group 1) revealed a CO starting at a significantly higher level than hDAF (group 2) and non-transgenic pig hearts (group 3) perfused with human blood only. Similarly, in group 1 SWI was significantly increased at the beginning of perfusion compared to that of group 2 and group 3. The increase in CBF during perfusion and the corresponding fall of coronary resistance occurred without significant differences between the groups revealing the independence of hDAF and GAS914. CONCLUSIONS: Due to an immediate and profound reduction in Galalpha(1,3)Gal-specific antibodies, soluble Galalpha(1,3)Gal conjugates not only prolong survival, but also improve the hemodynamic performance of the heart in DAF pigs.

Administration of GAS914 in an orthotopic pig-to-baboon heart transplantation model.

BACKGROUND: Long-term survival of transgenic cardiac xenografts is currently limited by a form of humoral rejection named acute vascular rejection. Preformed and elicited cytotoxic antibodies against Galalpha(1,3)Gal terminating carbohydrate chains, known as the primary cause of hyperacute rejection, are crucial for this process. We investigated whether GAS914, a soluble, polymeric form of a Galalpha(1,3)Gal trisaccharide would sufficiently minimize xenograft rejection of hDAF-transgenic pig hearts orthotopically transplanted into baboons. METHODS: Orthotopic heart transplantations were performed using hDAF transgenic piglets as donors and four non-splenectomized baboons as recipients. Baseline immunosuppression consisted of tacrolimus, sirolimus, ATG, steroids. In addition two animals received low-dose GAS914, and two animals high-dose GAS914. One of these baboons received high dose GAS914 and cyclophosphamide induction therapy. Serum levels of anti-Galalpha(1,3)Gal IgM and IgG antibodies, and anti-pig antibodies were controlled daily by anti-Galalpha(1,3)Gal enzyme-linked immunosorbant assay and anti-pig hemolytic assays. Histomorphological (hematoxylin and eosin, elastic van Gieson) and immunohistochemical (IgM, IgG) evaluations were performed on tissue specimens. RESULTS: Following low-dose GAS914 therapy survival time was 1 and 9 days, respectively. In baboons treated with high dosages of GAS914 a survival of 30 h and 25 days could be obtained. GAS914 caused an immediate and significant reduction of both anti-Galalpha(1,3)Gal IgM and IgG antibodies. However, sufficient antibody reduction was independent of dosage and form of application of GAS914. A pre-transplant GAS914 treatment was not necessary to effectively reduce antibody levels and prevent hyperacute rejection. In the early postoperative period preformed anti-pig antibodies corresponded predominantly to anti-Galalpha(1,3)Gal antibodies making them susceptible to GAS914. Subsequently, while anti-Galalpha(1,3)Gal antibodies remained low, anti-pig antibodies increased despite of GAS914 application. Corresponding to increased anti-pig antibody titers depositions of IgM and IgG immunoglobulins were detected, which were possibly non-Galalpha(1,3)Gal-specific. CONCLUSIONS: Following orthotopic transplantation of hDAF-transgenic pig hearts into baboons, GAS914 is able to maintain a sufficient reduction of Galalpha(1,3)Gal-specific cytotoxicity to the graft. GAS914 therefore is able to prevent not only hyperacute rejection, but also acute vascular rejection at its beginning, when serum cytotoxicity to the pig heart appears to be predominantly Galalpha(1,3)Gal-specific. A sustained prevention of acute vascular rejection, however, still requires the identification of antibody specificities other than to Galalpha(1,3)Gal.

hDAF porcine cardiac xenograft maintains cardiac output after orthotopic transplantation into baboon--a perioperative study.

BACKGROUND: Only limited data are available on the physiological functional compatibility of cardiac xenografts after orthotopic pig to baboon transplantation (oXHTx). Thus we investigated hemodynamic parameters including cardiac output (CO) before and after oXHTx. METHODS: Orthotopic xenogeneic heart transplantation from nine hDAF transgeneic piglets to baboons was performed. We used femoral arterial thermodilution for the invasive assessment of CO and stroke volume. RESULTS: Baseline CO of the baboons after induction of anesthesia was 1.36 (1.0-1.9) l/min. 30 to 60 min after termination of the cardiopulmonary bypass, CO of the cardiac xenograft was significantly increased to 1.72 (1.3-2.1) l/min (P < 0.01). The stroke volumes of the baboon heart before transplantation and the cardiac xenograft was comparable [14.9 (11-26) vs. 11.8 (10-23) ml]. Thus the higher CO was achieved by an increase in heart rate after oXHTx [75.0 (69-110) vs. 140.0 (77-180)/min; P < 0.01]. Despite the increased CO, oxygen delivery was reduced [256 (251-354) vs. 227 (172-477) ml/min; P < 0.01] due to the inevitable hemodilution during the cardiopulmonary bypass and the blood loss caused by the surgical procedures. CONCLUSION: Our results demonstrate that in the early phase after orthotopic transplantation of hDAF pig hearts to baboons, cardiac function of the donor heart is maintained and exceeds baseline CO. However, in the early intraoperative phase this was only possible by using inotropic substances and vasopressors due to the inevitable blood loss and dilution by the priming of the bypass circuit.

Extracorporeal liver perfusion as hepatic assist in acute liver failure: a review of world experience.

BACKGROUND: There are almost no prospective, controlled and randomized clinical trials comparing different approaches towards hepatic assist. In order to create a basis for comparing the value of the existing different hepatic assist methods this article offers a systematic review of the world experience with allogeneic or xenogeneic extracorporeal liver perfusion (ECLP). METHODS: An Internet-assisted search was conducted in the international literature published from 1964 to 2000. Only articles with a clear description of methodology and outcome of patients were included. For multivariate analysis of variance the general linear method (GLM) procedure was used. Differences within the groups were analyzed by chi-square test. Data of 198 patients were included into the statistical analysis for systematic review. RESULTS: The long-term survival rate (SVR) of these patients was 26%, thus not exceeding published data concerning SVR under standard intensive care. Age below 40 years (P<0.029), coma stage lower than III-IV (P<0.003), total perfusion time over 10 hours (P<0.024), hepatitis B as cause for acute liver failure (ALF) (P<0.05) as well as use of baboon and human livers (P<0.02) were identified as independent positive prognostic markers for improved survival. ECLP as bridging therapy to liver transplantation was successful in 12 of 14 patients. CONCLUSION: ECLP using pig livers did not surpass the success of conventional intensive care treatment. An additional effect of transgenic expression of human regulators of complement regulation in porcine livers has not yet been proven. ECLP with human livers not suitable for liver transplantation might prove effective and practicable for temporary hepatic support. Bridging to liver transplantation by long-term ECLP using porcine and human livers appears to have comparable efficacy as bioartificial support methods.

Polyclonal anti-thymocyte globulins influence apoptosis in reperfused tissues after ischaemia in a non-human primate model.

Reperfusion triggers the expression of inflammatory cytokines and adhesion molecules that increase the rate of apoptosis in the reperfused tissues after ischaemia, thus worsening the outcome of the grafts. Polyclonal anti-thymocyte globulins (pATGs) are able to reduce the number of lymphocytes as well as block adhesion molecules and induce apoptosis in T-lymphocytes through Fas-ligand. The aims of this study were to investigate the influence of pATGs on the prevention of apoptosis of reperfused tissues after ischaemia and to monitor their capability to enhance lymphocyte apoptosis thus decreasing the deleterious effects of ischaemia/reperfusion injury (IRI). Extremities of cynomolgus monkeys ( n=8) were flushed via either the femoral or the brachial artery. After 60 min of ischaemia the limbs were reperfused with human blood. ATG was added to the blood in a therapeutic dose 20 min prior to reperfusion of the extremities. Surgically available limbs ( n=20) were assigned to the following groups: ATG group ( n=10) and control group (without ATG; n=10). DNA fragmentation analysis was performed in situ to detect apoptosis at the single-cell level. Our study shows an increased rate of muscle and connective tissue apoptosis in the control group compared with the ATG-treated group. Cells found in the vascular areas present different rates of apoptosis, with enhanced cellular death of endothelium and connective perivascular areas being observed in the control group. The group treated with ATG shows an increased rate of white blood cell (WBC) apoptosis in vascular and perivascular areas. Previous studies have shown that pATGs are able to induce apoptosis as well as complement-mediated cell death in peripheral T-lymphocytes in vitro. Our results confirm that pATGs not only increase the rate of apoptosis of WBCs in vivo but also have a protective effect on the reperfused tissue. This may alleviate the damage after reperfusion of solid-organ transplantation.

Heat-shock preconditioning protects fatty livers in genetically obese Zucker rats from microvascular perfusion failure after ischemia reperfusion.

Reduced tolerance of steatotic livers to ischemic injury is considered to correlate with impaired microcirculation. The aim of this study was to investigate the impact of heat-shock preconditioning (HSPC) on microcirculatory failure after ischemia/reperfusion (I/R) in steatotic livers by means of intra-vital fluorescence microscopy. Obese Zucker rats were used. In the HS group, rats underwent whole-body hyperthermia followed by 60-min partial liver ischemia. In group IR, rats were exposed only to ischemia. Microcirculation parameters (sinusoidal perfusion rate, sinusoidal diameter, leukocyte-endothelial interaction) were significantly better preserved in the HS group than in the IR group. Liver enzymes, oxygenated glutathione/reduced glutathione (GSSG/GSH) ratio, and electron microscopy showed less damage in the HS group. A marked expression of heat shock protein 72 (HSP72) and heme oxygenase (HO-1) was found only in the livers of group HS. HSPC mitigated the I/R injury of steatotic livers by preventing post-ischemic failure of microcirculation. This beneficial effect was found to be associated with the induction of HSP72 and HO-1.