Release of pig leukocytes and reduced human NK cell recruitment during ex vivo perfusion of HLA-E/human CD46 double-transgenic pig limbs with human blood.

BACKGROUND: In pig-to-human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non-classical human leukocyte antigen-E (HLA-E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood. METHODS: Six pig forelimbs per group, respectively, stemming from either wild-type (wt) or HLA-E/hCD46 double-transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12 hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells. RESULTS: Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60 minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10 minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12 hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA-E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells. CONCLUSIONS: Ex vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell-mediated rejection mechanisms of vascularized pig-to-human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA-E/hCD46 in pig limbs provides partial protection from human NK cell-mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.

Endoluminal laser-assisted vascular anastomosis-an in vivo study in a pig model.

Microvascular surgery is time consuming and requires high expertise. Laser-assisted vascular anastomosis (LAVA) is a promising sutureless technique that has the potential to facilitate this procedure. In this study, we evaluate the handling of our soldering material and the 1-week patency rate in a porcine model. Six pigs were subjected to LAVA. For each pig, the saphenous artery on one side was transected while the contralateral side was used as control. A porous polycaprolactone scaffold soaked in 40% (w/w) bovine serum albumin solution in combination with 0.1% (w/w) indocyanine green was wrapped at the anastomosis site and at the control site. Both sides were then soldered with a diode laser coupled into a light diffuser fiber emitting radiation with a wavelength of 808 nm and a power of 2-2.2 W. Vessels were successfully soldered with a 100% immediate patency rate. The 1-week patency rate was 83% for the anastomoses versus 67% for the control side. Vessels irradiated for 80 to 90 s tended to maintain the highest patency rate. Macroscopically, there was no difference between the two sides. The patch was easy to handle provided that the environment could be kept dry. This study shows the potential and the limitations of endoluminal LAVA as a one-step procedure without the use of stay sutures. Further studies are needed to improve the soldering material, the long-term patency rate, and standardized irradiation parameters. The long-term effects of laser soldering on the vessel wall remain to be determined.

Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA-E double transgenic pig forelimbs with human blood.

BACKGROUND: Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model. METHODS: α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples. RESULTS: No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1ß, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions. CONCLUSION: Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection.

Ischemia/reperfusion injury of porcine limbs after extracorporeal perfusion.

BACKGROUND: Revascularization of amputated extremities after prolonged ischemia is complicated by reperfusion injury. We assessed ischemia/reperfusion (I/R) injury of porcine extremities after prolonged preservation using extracorporeal circulation (ECC). METHODS: Forelimbs of 32 pigs were divided into four groups based on ischemia times: group I: 6 h, group II: 12 h, group III: 0 h plus replantation, and group IV: 6 h plus replantation. Limbs were perfused with autologous blood using ECC for 12 h except group II with only 5 h perfusion. Limbs from groups III and IV were heterotopically replanted with a 7-d follow-up. Contralateral limbs served as controls in all groups. Tissue, plasma, and serum were analyzed for the extent of I/R injury. RESULTS: No significant differences in tissue wet/dry ratios were found within or between groups. This finding was confirmed by histology, except for an increased damage in group IV muscles compared with baseline (P = 0.016). Complement C3 deposition was only increased in group IV muscle (P = 0.031), group II nerves (P = 0.046), and group II vessels (P = 0.037). Group IV muscle and nerve tissues were the only ones with significant IgM antibody deposition (P = 0.031) at end of perfusion. Values were normal again after replantation. Reduced complement activity and elevated IL-6, IL-8, MCP-1, VEGF, PDGF-bb, bFGF, and complement split products were found during perfusion but were normal again after replantation. Staining for heparin sulfate proteoglycans and von Willebrand factor confirmed minimal activation of endothelial cells. CONCLUSION: The results demonstrate that prolonged limb preservation using ECC has minimal impact on I/R-induced tissue injury. Extracorporeal perfusion is a potential limb-preserving technique encouraging further studies for use in limb revascularization.

Evaluation of a porcine whole-limb heterotopic autotransplantation model.

BACKGROUND: Recently performed vascularized composite tissue allotransplantations (CTAs) stimulate the ongoing research in the area of whole-limb transplantation. A reliable in vivo animal model is required for investigations in vascularized whole-limb CTA. The model should allow in vivo assessment in whole-limb preservation, allograft and xenograft response, and host immunomodulation. The goal of this study is to describe and evaluate the in vivo feasibility and reproducibility of a whole-limb porcine model as a basis for future research in this field. MATERIALS AND METHODS: In seven large white pigs, one forelimb was amputated under anesthesia and autotransplanted heterotopically with an arc of rotation of 180° and partially placed in a subcutaneous pocket. Clinical parameters were monitored and muscle biopsies were analyzed using ultrastructural morphological assessment of mitochondria quality after an observation period of 7 days. RESULTS: All animals could fully mobilize postoperatively without restrictions. At sacrifice, the anastomosed pedicle vessels of the limb were patent in six animals. In one pig, venous thrombosis could be observed. Muscle response was triggered following direct electrostimulation in six replanted limbs. The replanted extremities gained 12.97% weight within 7 days postreplantation compared with the amputation baseline values (P = 0.464 while maintaining normal compartment pressures at sacrifice (8.25 ± 5.31 cmH(2)O, P = 0.60). The ultrastructural evaluation of mitochondria morphology revealed intact mitochondria without signs of ischemia/reperfusion damage. CONCLUSION: This porcine model proved feasible, reliable, and reproducible for whole-limb autotransplantation. It presents significant potential in future preclinical research of whole-limb CTA transplantation.

7-year follow-up after open reduction and internal screw fixation in Bennett fractures.

BACKGROUND: Bennett fractures are unstable, and, with inadequate treatment, lead to osteoarthritis, weakness and loss of function of the first carpometacarpal joint. This study focuses on long-term functional and radiological outcomes after open reduction and internal fixation. METHODS: Between June 1997 and December 2005, 24 patients with Bennett fractures were treated with open reduction and internal fixation with screws at our center. Radiological and functional assessments including range of motion of the thumb and pinch and grip strength were performed 4 months post-procedure and at the long-term follow-up, on average 83 months after surgery. RESULTS: Reduction of the Bennett fracture was maintained as it was at the time of the procedure in 96 % of the cases when fixation with two lag screws was performed. At the 4-month follow-up, mean pinch and grip strength reached 92 ± 3 and 89 ± 4 % of the contralateral side, respectively. Long-term follow-up demonstrated no correlation between the accuracy of the fracture reduction and the development of post-traumatic arthritis. CONCLUSION: Good clinical results could be observed, if successful reduction of the fracture was achieved and maintained. However, there was no correlation between the accuracy of the fracture reduction considering a gap and step <2 mm and the development of arthritis.

Preservation of amputated extremities by extracorporeal blood perfusion; a feasibility study in a porcine model.

BACKGROUND: Successful extremity transplantations and replantations have to be performed within 6 h of amputation to avoid irreversible tissue loss. This study investigates ex vivo the technical feasibility and the limb preservation potential of extracorporeal whole blood perfusion in a porcine model. METHODS: Forelimbs of eight large white pigs were divided into paired groups: I perfusion group, II contralateral cold ischemia controls. In group I axillary arteries and veins were cannulated and perfusion with anticoagulated autologous blood was performed for 12 h; O(2), CO(2), Hb, lactate, potassium, pH, and muscle contractility were monitored. Tissue biopsies were examined by histology and immunofluorescence. Group II was stored at 4°C. RESULTS: Continuous limb perfusion could be performed in all extremities of group I for 12 h. pH was maintained normal and potassium controlled with insulin and glucose. Lactate levels increased initially during perfusion due to the lack of a metabolizing liver. Muscle stimulation was possible throughout the entire perfusion, whereas a complete loss of response was noted in cold ischemia controls. Minor tissue damage was observed histologically and by immunofluorescence in group I, whereas the samples of group II were apparently preserved with the exception of a loss of endothelial heparan sulfate. CONCLUSIONS: The tissue preserving potential and the feasibility of extremity perfusion using common extracorporeal blood circulation techniques was demonstrated in this ex vivo study. The results encourage further investigations in prolonged perfusion followed by limb replantation. This approach harbors promising clinical potential for extremity preservation in extremity transplantation and replantation.

Porcine extrahepatic vascular endothelial asialoglycoprotein receptor 1 mediates xenogeneic platelet phagocytosis in vitro and in human-to-pig ex vivo xenoperfusion.

BACKGROUND: Asialoglycoprotein receptor-1 (ASGR1) mediates capture and phagocytosis of platelets in pig-to-primate liver xenotransplantation. However, thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1-mediated xenogeneic platelet phagocytosis in vitro and ex vivo on porcine aortic, femoral arterial, and liver sinusoidal endothelial cells (PAEC/PFAEC/PLSEC). METHODS: Porcine forelimbs were perfused with whole, heparinized human or autologous pig blood. Platelets were counted at regular intervals. Pig limb muscle and liver, as well as PAEC/PFAEC/PLSEC, were characterized for ASGR1 expression. In vitro, PAEC cultured on microcarrier beads and incubated with non-anticoagulated human blood were used to study binding of human platelets and platelet-white blood cell aggregation. Carboxyfluorescein diacetate succinimidyl ester-labeled human platelets were exposed to PAEC/PFAEC/PLSEC and analyzed for ASGR1-mediated phagocytosis. RESULTS: Human platelet numbers decreased from 102 ± 33 at beginning to 13 ± 6 × 10/µL (P < 0.0001) after 10 minutes of perfusion, whereas no significant decrease of platelets was seen during autologous perfusions (171 ± 26 to 122 ± 95 × 10/µL). The PAEC, PFAEC, and PLSEC all showed similar ASGR1 expression. In vitro, no correlation was found between reduction in platelet count and platelet-white blood cell aggregation. Phagocytosis of human carboxyfluorescein diacetate succinimidyl ester-labeled platelets by PAEC/PFAEC/PLSEC peaked at 15 minutes and was inhibited (P < 0.05 to P < 0.0001) by rabbit anti-ASGR1 antibody and asialofetuin. CONCLUSIONS: The ASGR1 expressed on aortic and limb arterial pig vascular endothelium plays a role in binding and phagocytosis of human platelets. Therefore, ASGR1 may represent a novel therapeutic target to overcome thrombocytopenia associated with vascularized pig-to-primate xenotransplantation.

Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions.

BACKGROUND: Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions. METHODS: Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion. RESULTS: No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions. CONCLUSIONS: In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models.

BACKGROUND: Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. However, in ischemia-reperfusion injury, IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose-binding lectin (MBL) in pig-to-human xenotransplantation models. METHODS: Activation of the different complement pathways was assessed by cell enzyme-linked immunosorbent assay using human serum on wild-type (WT) and α-galactosyl transferase knockout (GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed serum. RESULTS: Activation of all the three complement pathways was observed in vitro as indicated by IgM, C1q, MBL, and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders' coefficient [3D] r=0.93), C3b/c (r=0.84), C4b/c (r=0.86), and C6 (r=0.80). IgM colocalized with MBL (r=0.87) and MASP2 (r=0.83). Human IgM led to dose-dependently increased deposition of MBL, C3b/c, and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's coefficient [2D] rp=0.88), C3b/c (rp=0.82), C4b/c (rp=0.63), and C6 (rp=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46-PAEC. CONCLUSION: Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti-Gal IgM and may play a pathophysiologic role in pig-to-human xenotransplantation.

Sciatic neuropathy after body contouring surgery in massive weight loss patients.

To date, obesity affects a substantial population in industrialised countries. Due to the increased awareness of obesity-related morbidity, efficient dietary regimens and the recent successes with bariatric surgery, there is now a high demand for body contouring surgery to correct skin abundancies after massive weight loss. The known risks for this type of surgery are mainly wound-healing complications, and, more rarely, thromboembolic or respiratory complications. We present two female patients (23 and 39 years of age) who, in spite of standard positioning and precautions, developed sciatic neuropathy after combined body contouring procedures, including abdominoplasty and inner thigh lift. Complete functional loss of the sciatic nerve was found by clinical and electroneurographic examination on the left side in patient one and bilaterally in patient two. Full nerve conductance recovery was obtained after 6 months in both patients. Although the occurrence of spontaneous neuropathies after heavy weight loss is well documented, this is the first report describing the appearance of such a phenomenon following body contouring surgery. One theoretical explanation may be the compression of the nerve during the semirecumbent positioning combined with hip flexion and abduction, which was required for abdominal closure and simultaneous access to the inner thighs. We advise to avoid this positioning and to include the risk of sciatic neuropathy in the routine preoperative information of patients scheduled for body contouring surgery after heavy weight loss.