Evidence Of Hyperacute Rejection Research Articles

Pig-to-human kidney transplantation using brain-dead donors as recipients: One giant leap, or only one small step for transplantkind?

Pig kidney xenotransplantation is increasingly regarded as a realistic solution to the current shortage of human organ donors for patients with end-stage organ failure. Recently, the news of three pig-to-human transplantation cases has awakened public interest. Notably, the case by the Alabama team reported detailed and important findings for the xenotransplantation field. Using a genetically modified pig, two porcine kidneys were transplanted into a brain-dead recipient. They applied several approaches established in the preclinical NHP study, including gene-edited pig kidney graft and preoperative laboratory inspection such as crossmatching and infection screening. The pig-to-human kidney xenotransplantation had no unexpected events during surgery or evidence of hyperacute rejection. Unfortunately, the grafts did not work appropriately, and the study had to be terminated due to the decompensation of the recipient. While this study demonstrated the outstanding achievement in this research area, it also revealed remaining gaps to move xenotransplantation to the clinic. While brain-dead human recipients could reinforce the compatibility achievements of gene-edited pigs in NHP, their pro-inflammatory and pro-coagulant environment, in combination with short-duration of experiments will limit the assessment of kidney function, infection and rejection risk post-transplant, in particular antibody-mediated rejection. The use of successful immunosuppressive protocols of non-human primates xenotransplant experiments including anti-CD154 antibody will be critical to maximize the success in the first in-human trials.

HLA Allosensitization in ECMO as a Bridge to Lung Transplantation

Purpose Use of extracorporeal membrane oxygenation (ECMO) as a bridge to transplantation (BTT) has raised concerns ECMO support leads to the development of serum antibodies due to multiple blood transfusions. We examined the development of HLA sensitization in a cohort of lung transplant (LT) recipients where ECMO BTT was employed. Methods and Materials A retrospective chart review of all LT recipients with ECMO BTT from 2006-2012 was conducted. Data collected included demographics, Luminex, blood product utilization and post-transplant outcomes. HLA sensitization was defined as PRA>10% in either HLA type I or II while highly sensitized was PRA>90%. Results 21 patients (12 males) were identified as BTT to LT on ECMO, median ECMO duration of 7.0 days (range:0-38 days). 6 patients (35%) were sensitized prior to ECMO, mean PRA HLA type I 36%±22.7 and type II 27%±2.2. No significant change from baseline PRA was noted, mean difference for HLA type I of 4.3±11.9 (p=0.19) or type II of 2.9±7.7 (p=0.16). During ECMO BTT, 1 sensitized patient became highly sensitized (HLA type I 94%) who received 47 PRBCs. The second highest PRBC usage (31) during BTT resulted in new sensitization (HLA type I 31%). Patients received on average of 14.0±2.7 (range:0-49) during ECMO BTT, which was not associated with the mean difference between baseline-ECMO for HLA type I (p=0.52) or type II (p=0.06). All patients were successfully transplanted, 8 received induction therapy, with no evidence of hyperacute rejection. Conclusions HLA sensitization does occur rarely in ECMO BTT and may be associated with large amounts of PRBCs received during ECMO BTT. Although sensitization may occur, successful transplantation is possible without evidence of hyperacute rejection. PRBCs Received during ECMO BTT Patient Days on ECMO PRBCs Received Δ Sensitization Status 1 10 27 No 2 3 21 No 3 13 15 Yes 4 10 49 Yes 5 1 2 No 6 4 9 Yes 7 7 18 Yes 8 2 4 No 9 38 31 Yes 10 1 0 No 11 8 15 Yes 12 5 11 No 13 17 11 No 14 4 3 No 15 3 7 No 16 12 15 Yes 17 1 0 No 18 4 0 No 19 22 23 No 20 20 27 No 21 15 5 No PRBCs-packed red blood cell units

Enhanced Survival of Bone–Marrow‐Derived Pluripotent Stem Cells in an Animal Model of Auditory Neuropathy

The loss of spiral ganglion neurons (SGNs) is one of the major causes of profound sensorineural hearing loss (SNHL). Stem cell replacement therapy, which is still in its infancy, has the potential to treat or cure those who suffer from an array of illnesses and degenerative neurologic disorders, including sensorineural deafness (SNHL). Little is known about the potentials of mesenchymal stem cells (MSCs) and their ability to take on properties of SGNs. The two main purposes of this study were to evaluate the survival of mouse MSCs transplanted into normal and ouabain-treated gerbil cochleae and to determine the migratory patterns of MSCs with two differing injection methods. Thirty-two Mongolian gerbils, 3 to 4 months old, were used as recipients, and four 6-week-old TgN(ACTbEGFP) mice that ubiquitously express green fluorescent protein (GFP) were used as donors. The animals were deafened by ouabain, which damaged SGNs while leaving hair cell systems intact. After 4 weeks of recovery, the animals received an intraperilymphatic transplantation of 1.0x10(6) GFP-positive undifferentiated MSCs via two different injection methods: scala tympani injection and modiolar injection. Seven days after the transplantation, the survival of MSCs was evaluated by microscopic examination of frozen sections cut through the cochleae of the recipient animals. The number of profiles was counted on the five most central modiolar sections. One-way analyses of variance (ANOVA) were used to determine any significantdifferences among mean profile counts across the experimental conditions. Our findings indicated that undifferentiated MSCs were able to survive in the modiolus both in the control and the ouabain-treated cochleae. The average number of profiles found in the modiolus was greater in the ouabain-treated cochleae than in the control cochleae. This difference was statistically significant (P<.01) as determined using a one-way ANOVA and an ad hoc Tukey-Kramer's test. With the scala tympani injection, there were no profiles found in the modiolus either in the control or ouabain-treated cochleae. This finding may indicate that donor MSCs need to be directly injected into the modiolus to replace injured SGNs. Finally, there was no evidence of hyperacute rejection in any of the gerbils despite the use of xenotransplantation. These findings may have important clinical implications as a means of delivering MSCs in the cochlea for stem-cell replacement therapy. Survival of transplanted MSCs into the modiolus of the cochlea may result in regeneration of damaged SGNs.

No functional benefit for hDAF-transgenic rat livers despite protection from tissue damage following perfusion with human serum.

Currently, xenogeneic extracorporeal liver perfusion is used in the treatment of acute liver failure. In order to determine whether transgeneity for human regulatory proteins could improve the functional outcome of the ex-vivo liver in relation to the histopathological changes, we studied the effect of the humoral mechanism in xenogeneic isolated rat liver perfusion in normal and transgenic rat livers. Isolated rat liver perfusion was performed for 2 h in normal rat livers with Krebs Henseleit (KH) and human serum (HS), and in livers transgenic for human decay accelerating factor (hDAF; Tg HS). Function of the liver was established by measurement of liver enzymes and bile production, and clearance of bromosulphophthalein (BSP). Tissue specimens taken after perfusion were analysed by routine histology and immunofluorescence staining for C3c deposition. No change in release of liver enzymes could be established throughout the perfusion period. In the 2nd hour, a higher level of bile production was seen for the transgenic group than for the HS group. The transgenic rat livers outperformed the normal livers perfused with HS, when BSP concentration in the bile was measured; however, clearance of BSP from the perfusate was not significantly different. Haematoxylin-eosin (HE) staining of the liver tissue showed evidence of hyperacute rejection in the HS group. There was only mild tissue injury in the transgenic liver. High-intensity fluorescent staining for C3c deposition was seen in the normal livers perfused with HS, and significantly less in the transgenic livers. Although histologically less tissue damage and less C3c deposition was shown, no significantly improved function of the livers transgenic for hDAF was established. These results suggest that for short-term extracorporeal liver perfusion transgenesis offers no functional benefit.

Transplant Accommodation in Highly Sensitized Patients: A Potential Role for Bcl-xL and Alloantibody

Transplantation of renal allografts into recipients with circulating anti-HLA antibodies results in hyperacute rejection. In some cases, however, antibodies return without causing harm; this phenomenon has been termed 'accommodation'. We have investigated this process in human allotransplantation. We removed anti-HLA antibodies by immunoadsorption in seven highly sensitized dialysis patients who subsequently underwent renal transplantation. Immunohistochemistry of renal biopsies for IgG and antiapoptotic proteins was performed. We also developed a model of 'accommodation' using anti-HLA antibodies eluted from sensitized patients and incubated with human umbilical vein endothelial cells (HUVECs) at different concentrations. Their effect on HUVEC phenotype was then analysed. Anti-donor antibody returned in 4/7 patients, without evidence of hyperacute rejection. Three out of four of these 'accommodated' grafts showed specific endothelial up-regulation of Bcl-xL and 2/2 tested positive for endothelial IgG deposition. HUVECs incubated with subsaturating concentrations of anti-HLA antibody showed increased expression of Bcl-xL, were rendered refractory to endothelial cell activation and became resistant to complement-mediated lysis. In contrast, HUVECs incubated with saturating concentrations underwent activation and expressed low levels of Bcl-xL. In conclusion, endothelial Bcl-xL expression defines the accommodation process in human allografts and this phenotype may be initiated by exposure of endothelium to low concentrations of anti-donor HLA antibodies.

Influence of cold ischemia time, pretransplant anti-porcine antibodies, and donor/recipient size matching on hyperacute graft rejection after discordant porcine to cynomolgus kidney transplantation.

Organs transplanted between phylogenetically disparate species, such as from the pig into the primate, are subject to hyperacute rejection (HAR). This form of xenograft rejection is mediated by preformed natural antibodies and is believed to occur invariably in discordant xenografts thus leading to rapid destruction and complete thrombosis of the graft. Recent data, however, have shown that in the porcine to cynomolgus monkey setting, HAR is not inevitably seen after porcine kidney transplantation. The influence of preoperative antiporcine antibody levels in the recipient, cold ischemia time, and donor organ weight on the onset of HAR was investigated by using unmodified large white pigs (aged 3-12 weeks) as organ donors and adult cynomolgus monkeys (aged 1.5-3.5 years) as recipients. Porcine kidney xenotransplantation was performed in either a non-life-supporting model (n=7) or in a life-supporting model (n=8). In both models, no correlation was found between cold ischemia time and HAR. When preoperative anti-porcine antibody levels were investigated, a significant increase in incidence of HAR was observed in animals with elevated anti-porcine IgM (P<0.05) but not IgG levels (P=NS). Interestingly, although 5 of 12 grafts with an organ weight of less than 50 g underwent HAR, none of three grafts with a donor organ weight of more than 70 g showed signs of HAR. In addition, all three larger grafts showed intraoperative and postoperative urine production, although only in 1 (48 g) of the 12 grafts weighing less than 50 g primary graft function was observed. In one animal, a second porcine kidney (23 g) was successfully transplanted (without HAR) immediately after HAR and subsequent removal of a first porcine kidney (20 g). These results indicate that in the porcine to cynomolgus monkey setting anti-porcine IgM rather than IgG anti-porcine antibody levels seem to be of predominant importance for the induction of HAR. By increasing the donor organ size and weight the frequency of the onset of HAR can be at least reduced. This is most likely due to immunoabsorption of the recipients preformed antibodies in the porcine kidney without lethal damage for the graft.

EXTRA-CORPOREAL PERFUSION OF NORMAL AND hDAF TRANSGENIC LIVERS WITH HUMAN BLOOD

212 The development of pigs transgenic for human decay accelerating factor (hDAF) has led to a resurgence of interest in extra-corporeal liver perfusion in the treatment of acute liver failure. We have developed an isolated perfusion system which incorporates oxygenation, temperature regulation and independent perfusion of the artery and portal vein. When pig livers were perfused with pig blood (n=5), reliable synthetic function and metabolic function were maintained for at least 3 days, at which stage essentially normal histological appearances were seen. Synthetic function was demonstrated by CH50 analysis (complement levels increased four-fold), urea production (increased 15 fold with unchanged creatinine levels) and factor V synthesis (stable factor V levels, whereas anhepatic perfusions showed no factor V activity after 36 hours). Metabolic function was demonstrated by acid base and potassium maintenance within the normal physiologic range. When connected to an anaesthetised pig in acute liver failure, stabilisation of metabolic (pH) and cardiovascular (arterial pressure, peripheral vascular resistance, inotrope requirement) parameters were maintained for the duration of perfusion. Unsupported control animals died with a mean survival of 17 hours (n=5). When normal (n=5) and hDAF transgenic (n=5) pig livers were perfused with fresh human blood, synthetic and metabolic function was demonstrated in both groups. Transgenic livers produced significantly more complement and urea (p<0.01) while factor V production was similar. Factor V levels stabilised at porcine rather than at human levels (400% vs. 100%). Metabolic function was significantly better (p <0.01) in transgenic livers than in nontransgenic livers as assessed by acid-base and potassium homeostasis. There was no evidence of hyperacute rejection. However, in both groups of xenoperfusions the duration of function was limited by progressive loss of red cells. The red cell loss appears to be of immunological origin and due to phagocytosis rather than haemolysis. The rate of red cell consumption, however, is compatible with a modest transfusion requirement and may not, therefore, restrict clinical application.

TRANSPLANT ACCOMMODATION IS ASSOCIATED WITH UP-REGULATION OF ANTI-APOPTOTIC PROTEINS: IN VITRO MODELS IMPLICATE iNOS INDUCTION BY LOW LEVELS OF ANTI-DONOR ANTIBODY IN ACCOMMODATED ENDOTHELIAL CELLS

798 Under certain, still ill-defined circumstances, anti-donor antibodies may not induce hyperacute rejection (HAR) but accommodation. This process has been reported following transplantation of ABO-incompatible allografts and in xenograft models. Active changes in the donor endothelium are thought to be responsible. We have investigated the molecular mechanisms involved in accommodation in vivo, in highly sensitised human allograft recipients, and in vitro using human and porcine endothelial cells. Seven highly sensitised patients were immunoadsorbed, to remove their anti-HLA antibodies prior to renal transplantation. Immunohistochemistry was performed on transplant biopsies from these highly sensitised recipients as well as on control native and transplant biopsies. The sections were stained for Bcl-xL, Bcl-2 and HO-1. 4/7 of the patients transplanted had a return of anti-donor antibodies without evidence of HAR. Prominent Bcl-xL expression was observed in the graft microvasculature, in 3/4 patients in whom anti-donor antibodies returned and in none in whom they did not, nor in any of the controls. Neither HO-1 nor Bcl2 were specific for the accommodated grafts, endothelial staining having been found in other inflammatory conditions. We went on to develop in vitro models of accommodation. Allogeneic or xenogeneic endothelial cells (EC) were cultured in the presence of anti-HLA antibodies or human IgG containing anti-porcine antibodies, respectively. Only following prolonged treatment with low doses of anti-donor antibodies did the anti-apoptotic proteins Bcl2 and Bcl-xL become up-regulated on the EC, in both systems. Following this antibody treatment the cells underwent phenotypic changes consistent with accommodation, including resistance to complement mediated lysis and attenuation of adhesion molecule expression, as judged by ICAM levels in the human EC and VCAM expression in the porcine EC. Furthermore the cells showed functional protection from apoptosis, as judged by Annexin V staining. In the porcine EC there was neo-expression of inducible nitric oxide synthase (iNOS). Interestingly up-regulation of Bcl2 and Bcl-xL could be mimicked by the nitric oxide donor detanonoate. Taken together, these data shed light on the mechanisms responsible for allograft endothelial cell accommodation, and implicate iNOS as an important contributor to the accommodated phenotype. Further insights into the mechanisms underlying accommodation may allow the development of specific therapies to promote accommodation in allografts and xenografts prior to transplantation.

Prolonged discordant xenograft survival and delayed xenograft rejection in a pig-to-baboon orthotopic cardiac xenograft model

Objective: Our objectives were to study delayed xenograft rejection and the effectiveness of pretransplantation total lymphoid irradiation combined with immunosuppression on rejection in a pig-to-baboon cardiac xenograft model. Methods: Baboons were treated with pretransplantation total lymphoid irradiation, cyclosporine A (INN: ciclosporin), and methotrexate. Orthotopic pig-to-baboon cardiac transplantations were performed after depletion of circulating xenoreactive natural antibody by pretransplantation donor organ hemoperfusion. Tissue samples were collected for immunologic and immunopathologic evaluation. Results: Pig cardiac xenografts survived more than 18 and 19 days without evidence of hyperacute rejection. Immunologic analysis of serum samples demonstrated that circulating xenoreactive natural antibody levels did not return to pretransplantation levels. The production of xenoreactive natural antibodies from the recipient's splenocytes was inhibited completely. Histologic examination of xenografts showed the feature of acute vascular rejection. Immunohistochemical studies demonstrated infiltration of cardiac xenografts by large numbers of macrophages, small numbers of natural killer cells, and a few T cells. The infiltrating macrophages also showed expression of interleukin-1 and tumor necrosis factor. Diffuse deposition of immunoglobulin G, C1Q, C3, and fibrin on xenograft vasculature was observed. Interleukin-2 expression was not found in rejected cardiac xenografts. Xenograft endothelial cells also showed evidence of activation (expression of cytokines interleukin-1 and tumor necrosis factor). Conclusions: This study demonstrates prolonged discordant cardiac xenograft survival and delayed xenograft rejection in a pig-to-baboon model. The delayed xenograft rejection is mediated by both humoral and cellular mechanisms. Pretransplantation total lymphoid irradiation combined with cyclosporine A and methotrexate can inhibit xenoreactive natural antibody production but not elicited antipig antibody production and the xenoreactivity of macrophages. (J Thorac Cardiovasc Surg 1998;115:1342-9)

Prevention of hyperacute rejection by removal of antibodies to HLA immediately before renal transplantation

Many patients with circulating antibodies to human leucocyte antigens (anti-HLA) are highly sensitised against renal transplantation and are liable to immediate graft loss through hyperacute rejection. Our aim was to find out whether removal of anti-HLA immediately before renal transplantation prevented hyperacute graft rejection. 13 highly sensitised patients underwent cadaveric renal transplants immediately after immunoadsorption (IA) treatment to remove anti-HLA. Before IA, 12 patients had a positive crossmatch against donor cells either by cytotoxic or flow-cytometric assay; results for one patient were equivocal. Renal biopsy samples were obtained 20 min after removal of the vascular clamps in nine patients. There was no evidence of hyperacute rejection in six of the nine patients; the other three patients showed glomerular thrombosis but no other evidence of hyperacute rejection. Two of these three grafts were functioning at 31 months of follow-up. Six episodes of acute rejection occurred in five patients during the first month after transplantation and overall there were 13 rejection episodes in nine patients. At latest follow-up (median 26 months, range 9-42), 12 of 13 patients were alive and seven of 13 grafts were surviving with a median plasma creatinine concentration of 185 mumol/L (range 106-296) in the functioning grafts. No graft was lost as a result of classic hyperacute rejection. Immediate pretransplant IA can prevent hyperacute rejection and provide an opportunity for successful transplantation in highly sensitised patients.

Key role of the alternate complement pathway in hyperacute rejection of rat hearts transplanted into fetal sheep.

Hyperacute rejection (HAR) mediated by xenoreactive natural antibodies (XNA), which are thought to develop in early infancy, is a major impediment to transplantation between widely disparate species. The ability to diagnose certain forms of congenital heart defects early in prenatal life suggests the potential for these defects to be corrected by cardiac transplantation, prior to the development of XNA and host immunocompetence. This study investigated whether discordant cardiac xenotransplantation into fetal and neonatal recipients might obviate HAR due to the relative lack of XNA. Six neonatal lambs at 3 days (n=3) or 7 days (n=3) of life, and two fetal lambs at 125 and 142 days of gestation (term = 145 days) received cardiac grafts from adult Wistar-Furth (275-350 g) rats. All eight cardiac xenografts showed clinical evidence of HAR, with rapid swelling, loss of contractility, and ecchymosis and a mean survival time of 12.5 +/- 6.4 min. Sections of explanted grafts showed classical histologic features of HAR, including interstitial hemorrhages, platelet microthrombi, and edema, without leukocyte infiltration. Immunopathology of grafts harvested from fetal recipients showed a lack of significant intragraft deposition of sheep IgM, IgG, or C1q, but widespread endothelial labeling for C3, factor B, and properdin. In contrast, grafts in neonatal recipients showed IgM, IgG, and C1q deposition, as well as C3, factor B, properdin, and terminal complement (C) components. Fibrin deposition and platelet thrombi were seen in both groups of recipients. Injection of cobra venom factor resulted in prolongation of cardiac xenograft survival in neonatal lambs (n=3) to 12 hr. Analysis by immunohistology showed that normal sera from neonatal and adult, but not fetal, sheep contained IgM and IgG XNA reactive with rat cells. In conclusion, rodent grafts transplanted into fetal sheep undergo HAR, likely through direct activation of the alternate pathway of C, whereas neonatal lambs acquire XNA in the very early postnatal period and reject rat hearts through activation of both the classical and alternate pathways of C. Thus, at least in some species combinations, cardiac transplantation during the early postnatal period, or even in utero, may still be subject to development of HAR.

Issues in xenotransplantation.

The shortage of organs for transplantation is especially severe for the critically ill newborn infant, for whom donors of the appropriate size are particularly scarce. One way to overcome this problem is to use animals in lieu of human as organ donors. The major limitation to using animals for this purpose is the susceptibility of animal organs to hyperacute rejection, a violent rejection reaction thought to be mediated by antidonor antibody and complement. To evaluate the potential application of xenotransplantation to newborns, we tested neonatal humans and neonatal baboons, and found that neither populatioh expressed significant levels of xenoreactive antipig antibodies. We transplanted heterotopically hearts from newborn pigs into unmanipulated newborn baboons (n = 4). There was no evidence of hyperacute rejection in any of the grafts; the animals were killed with functioning grafts at 15, 81, and 82 hour. This outcome contrasts with that of newborn pig-to-mature baboon and mature pig-to-mature baboon cardiac xenografts, which were rejected within one hour of transplantation. The histology of pig graft biopsies from the new born recipients was normal. Immunohistochemistry revealed only traces of IgM, C3, C4 and the membrane attack complex along graft endothelium. Fibrin deposition along endothelial surfaces was observed early after transplantation and became more extensive with time; in the absence of endothelial bound antibody or complement, this change may represent preservation injury. This study suggests that due to low levels of natural antibody, the newborn infant may permit prolonged xenograft survival.

Absence of hyperacute rejection in newborn pig-to-baboon cardiac xenografts.

The shortage of organs for transplantation is especially severe for the critically ill newborn infant, for whom donors of the appropriate size are particularly scarce. One way to overcome this problem is to use animals in lieu of humans as organ donors. The major limitation to using animals for this purpose is the susceptibility of animal organs to hyperacute rejection, a violent rejection reaction thought to be mediated by antidonor antibody and complement. To evaluate the potential application of xenotransplantation to newborns, we tested neonatal humans and neonatal baboons and found that neither population expressed significant levels of xenoreactive anti-pig antibodies. We transplanted heterotopically hearts from newborn pigs into unmanipulated newborn baboons (n = 4). There was no evidence of hyperacute rejection in any of the grafts; the animals were killed with functioning grafts at 15, 81, 82, and 82 hr. This outcome contrasts with that of newborn pig-to-mature baboon and mature pig-to-mature baboon cardiac xenografts, which were rejected within 1 hr of transplantation. The histology of pig graft biopsies from the newborn recipients was normal. Immunohistochemistry revealed only traces of IgM, C3, C4, and the membrane attack complex along graft endothelium. Fibrin deposition along endothelial surfaces was observed early after transplantation and became more extensive with time; in the absence of endothelial bound antibody or complement, this change may represent preservation injury. This study suggests that due to low levels of natural antibody, the newborn infant may permit prolonged xenograft survival.

Pretransplant donor specific blood transfusions for one haplotype stimulatory mixed lymphocyte culture living related donor transplants.

Three donor specific blood transfusions were given at 2-wk intervals pretransplantation, to those donor-recipient pairs who were one haplotype identical, but had stimulatory MLC. Two of 10 recipients who received donor specific blood transfusions developed T cell cytotoxic antibodies against their donor and were not transplanted. Eight recipients were successfully transplanted with no evidence of hyperacute rejection. All eight grafts are functioning from 2 to 15 mo post-transplant. Serum creatinines range from 1.0--3.9 mg%. There have been five patients who developed acute rejection episodes during the first 5 days post-transplant, and there have been a mean of 2.4 rejection episodes per patient. These results compare favorably to historical controls at the same institution who had a 40% incidence of graft success. The long-term graft survival remains to be elucidated.