Prevention Of Hyperacute Rejection Research Articles

(369) - A-Antigen Re-Expression and Prevention of Hyperacute Rejection Following In Vivo Enzymatic Treatment in a Model of ABO-Incompatible Transplantation

(369) - A-Antigen Re-Expression and Prevention of Hyperacute Rejection Following In Vivo Enzymatic Treatment in a Model of ABO-Incompatible Transplantation

Identification of peptide mimetics of xenoreactive α-Gal antigenic epitope by phage display

The prevention of hyperacute rejection (HAR) triggered by interaction between the human natural antibody and xenoreactive antigenic epitope (Gal-α1, 3Gal) present on pig cells is the key to success in pig-to-human xenotransplantation. The phage display technology offers an effective strategy for screening peptides which can interact with the anti-Gal antibody to block α-Gal antigen binding site. Two peptide libraries, linear 7 peptide library and C7C library, were panned on the anti-B monoclonal antibody which has the characteristic of binding to the α-Gal antigenic epitope. After four rounds of panning, 22 positive phage clones were selected. Highly homologous sequence PT and STL existed among these selected peptides. Stachyose competitive ELISAs revealed that these peptides specifically bound to α-Gal antigen binding site. Eight peptide mimics of α-Gal antigenic epitope could inhibit the agglutination of pig red blood cells mediated by human sera in a dose-dependent manner. These results demonstrated that the selected peptides can mimic the conformational structure of α-Gal antigenic epitope and have the therapeutic potential in xenotransplantation.

Prevention of Hyperacute Rejection in a Model of Orthotopic Liver Xenotransplantation From Pig to Baboon Using Polytransgenic Pig Livers (CD55, CD59, and H-Transferase)

Introduction The search for alternative sources for transplant organs leads us to the search for animals as an inexhaustible source of organs. The objective of this study was to analyze whether livers from polytransgenic pigs expressing the human complement regulatory proteins CD55 (hDAF), CD59, and alfa α1,2-fucosyltransferase (H-transferase), protected against hyperacute rejection after orthotopic liver xenotransplantation to a baboon and also to study pig liver function in a nonhuman primate. Materials and methods Nine liver transplants from pig to baboon were divided into two groups: a control group ( n = 4) of genetically unmodified pigs and an experimental group ( n = 5) of pigs transgenic for CD55, CD59, and H-transferase as donors. All the donating piglets obtained through hysterectomy were maintained in specific pathogen-free conditions. The selection of transgenic pig donors followed demonstration of transgene expression using monoclonal antibodies (antiCD55, antiCD59) and immunohistological studies on liver biopsies. Results All animals in the control group developed hyperacute rejection with survival rates less than 16 hours without function of transplanted livers. In the experimental group none of the animals suffered hyperacute rejection. Survival in this group was between 13 and 24 hours. The livers were functional, producing bile and maintaining above 35% prothrombin activity. Only in one case was there primary dysfunction of the xenograft. Conclusion Polytransgenic livers for complement regulatory proteins prevent hyperacute rejection when xenotransplanted into a baboon.

Pathology of xenograft rejection: a commentary

Trends in solid organ xenograft pathology are presented, with the focus on pig-to-nonhuman primate models. A simplified classification of rejection is followed, including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), and acute cellular xenograft rejection (ACXR). The main components in HAR are natural xenoreactive antibodies in combination with complement activation. This is evident from the prevention of HAR in recipients in whom either antibodies or complement activation is depleted or inhibited. However, these strategies generally fail to prevent AHXR, which occurs later. AHXR is a multifactorial process in which natural and elicited antibodies may play roles, possibly in conjunction with complement, coagulation factors, and white blood cells. A main target appears to be the microvasculature which, in kidney grafts, is associated with a glomerular thrombotic microangiopathy. It is not clear to what extent species-specific physiologic disparities in complement and coagulation processes may play a role, separate from antibody-initiated processes. As rejection of solid organ xenografts is currently from AHXR, ACXR has not yet received close attention. In addition to intragraft rejection events, systemic complications following host-graft interactions have emerged, including (often fatal) consumptive coagulopathy and immune complex disease. It is anticipated that rejection processes will change when pigs with new genetic modifications become available. For instance, the precise role of natural antibodies to Galalpha1,3Gal will be able to be distinguished from other factors when pigs that lack the target antigen are available, and their organs can be evaluated in large animal xenotransplantation models.

Prevention of hyperacute rejection by phosphatidylinositol-anchored factor I

Prevention of hyperacute rejection by phosphatidylinositol-anchored factor I

Analysis of human serum antibody–carbohydrate interaction using biosensor based on surface plasmon resonance

Surface plasmon resonance (SPR) was used to monitor the interaction of αGal-antibodies from human blood group O serum with linear blood group B-saccharides, employing Galα1-3Galβ1-4GlcNAc-HSA immobilised on a sensor chip surface. Strong binding of antibodies, as evident from high relative response values exceeding 200 RU, was observed. The interaction was influenced by the nature of the oligosaccharide that was added to the antibody sample prior to measurement. For example, the addition of either of the linear B-saccharides Galα1-3Gal and Galα1-3Galβ1-4GlcNAc produced complete inhibition of antibody binding to the sensor surface, whereas the addition of the related but non-specific blood group A saccharide, GalNAcα1-3(Fucα1-2)Gal, had little effect on binding. The technique was used for the rapid monitoring of the removal of αGal-antibodies from human serum by affinity columns, which contained either Galα1-3Gal or Galα1-3Galβ1-4GlcNAc as ligand. The above carbohydrates are currently evaluated as inhibitors or as affinity ligands, in the prevention of hyperacute rejection during xenotransplantation.

A reliable, rapid and inexpensive two-color fluorescence assay to monitor serum cytotoxicity in xenotransplantation

Removal and/or neutralization of preformed anti-pig antibodies in non-human primate blood have been shown to prevent the hyperacute rejection of transplanted pig organs. The purpose of this study was to establish a suitable in vitro method that would allow for screening and comparison of various agents and methods potentially useful in the prevention of hyperacute rejection. The pig kidney cell line (PK15), pig aortic endothelial cell line (AG08472), and a primary culture of endothelial cells explanted from a pig aorta were incubated with either human or baboon sera. Complement-dependent cytotoxic activity of human and baboon sera was determined on all three types of pig cells using a two-color fluorescence assay and compared with the conventional 51 Chromium ( 51 Cr )-release assay. The assay was also performed on PK15 cells as a 2-chambered slide assay and compared with a microcytotoxicity assay performed in Terasaki trays. Using the microcytotoxicity assay, a 1-step assay utilizing endogenous complement was compared with a 2-step assay where rabbit complement was added. Of the three types of cells studied, PK15 cells were the most sensitive to cytotoxic injury, followed by AG cells and the primary endothelial culture. Good correlation between the 51 Cr -release and the two-color fluorescence method was documented. There was good agreement between the results obtained using the 2-chambered slide method and the microcytotoxicity assay, as there was between the 1- and the 2-step assays. The 1- and 2-step assays provided information on the level and efficacy of endogenous complement. We conclude that the two-color fluorescence assay is suitable for the rapid and inexpensive screening of therapeutic interventions that might be useful in the prevention of hyperacute xenograft rejection, and that PK15 cells are suitable for use in this assay.

Capture of anti-(Galalpha1-3Gal) antibodies by immobilized Galalpha1-3Gal oligomers derived from carrageenan.

An immobilized Galalpha1-3Gal-bearing affinity ligand was prepared as a possible prophylactic binding site of human anti-(Galalpha1-3Gal) antibodies for the prevention of hyperacute rejection of pig xenografts. lambda-Carrageenan, a natural polysaccharide known to possess alternately alpha1-3 and beta1-4-linked D-galactopyranose sulphate residues, was selectively degraded by acetolysis and subsequently deacetylated to obtain lambda-carrageenan oligosaccharides. The resulting syrup was ultrafiltered to remove higher polymers and chromatographed on Sephadex G-15 to produce a series of sized oligosaccharide fractions. An immuno-dot-blot method was established on the basis of the competition between the lambda-carrageenan oligosaccharides in solution and bovine thyroglobulin (BTG), a glycoprotein known to express the Galalpha1-3Gal marker, as the solid-phase antigen. Increasing amounts of lambda-carrageenan fractions added to normal human plasma resulted in a progressive decrease in Galalpha1-3Gal-specific human IgM and IgG binding to BTG. Complete inhibition of Galalpha1-3Gal-specific human immunoglobulin was attained at concentrations less than 0.6 mg of carbohydrate/ml of human plasma with the more active fractions. These bioactive lambda-carrageenan oligosaccharides were immobilized on hydrazide-modified microporous nylon membranes, yielding capacities between 2.18 and 2.86 mg/ml of membrane. A subsequent decrease in the human anti-(Galalpha1-3Gal) antibody level in normal human plasma was demonstrated and proved with the established immuno-dot-blot procedure.

Prevention of hyperacute rejection by phosphatidylinositol-anchored mini-complement receptor type 1

Complement receptor type 1 (CR1, CD35) contains both factor I cofactor activity and convertase decay accelerating activity. but is, in general, thought to be an extrinsicregulator of complement activation. In this study, we prepared a phosphatidylinositol (PI)-anchored mini-CR1, which is composed of the short consensus repeat (SCR) 8–11 of CR1 and the PI anchor of DAF, and expressed it stably on a swine endothelial cell (SEC) line. We then examined the intrinsic regulatory activity of the mini-CR1, with respect to complement-mediated cell lysis as an in vitro hyperacute rejection model of a swine to human discordant xenograft. Flowcytometric profiles of the stable SEC lines with mini-CR showed a moderate level of expression for this molecule. Mini-CR1 blocked human complement-mediated cell lysis by approximately 50–70% on SEC. From the data of this study and our previous studies, mini-CR1 was more effective than membrane cofactor protein (MCP, CD46), and as effective as decay accelerating factor (DAF, CD55) in this system. The results suggest that PI-anchored mini-CR1 represents a useful molecule for clinical xenotransplantation.

Prevention of hyperacute rejection by PI-anchored mini-factor H

Prevention of hyperacute rejection by PI-anchored mini-factor H

Pig to monkey bone marrow and kidney xenotransplantation

Background . The intensity of discordant xenograft cellular rejection makes it unlikely that safe doses of immunosuppressive drugs will alone be sufficient to permit long-term survival. We have therefore concentrated our efforts on establishing tolerance to xenogeneic organs through lymphohematopoietic chimerism and the elimination of preformed natural antibodies (nAbs). Methods . Here we report the most recent series of 11 technically successful porcine to nonhuman primate transplantation procedures. In eight experimental animals induction therapy consisted of (1) 3 × 100 cGy nonlethal whole body irradiation (day -6 and day -5) to all animals, (2) horse anti-human thymocyte globulin (day -2, day -1, and day 0) to seven of the animals, (3) 700 cGy thymic irradiation (day -1) to five of the animals, and (4) pig bone marrow infused on day 0 (2–9 × 10 8/cells/kg). On day 0, just before the renal xenograft, the recipient was splenectomized, and antipig nAbs were removed by means of perfusion of the monkey's blood through either a pig liver (n = 6) or a Gal-α(1,3)-Gal adsorption column (n = 5). Three control animals did not receive this pretransplantation induction therapy but did undergo hemoperfusion and posttransplantation immunosuppression identical to the experimental animals. All 11 recipients were treated after transplantation with cyclosporin A and 15-deoxyspergualin. Recombinant pig-specific growth factors (interleukin-3 and stem cell factor) were given to six experimental animals from day 0 until the termination of the experiment. Results . Analysis of recipients' sera by means of flow cytometry indicated the effective removal of immunoglobulin M and immunoglobulin G nAbs by either liver perfusion or column adsorption. In the eight experimental animals, nAb titers remained low until death (up to 15 days), but in the three control animals nAb titers increased substantially with time. The longest surviving recipient maintained excellent kidney function with creatinine levels at 0.8 to 1.3 mg/dl throughout its course. Death occurred at day 15 from complications caused by a urinary leak and pancytopenia. Histologic examination of the xenograft revealed only focal tubular necrosis and cytoplasmic vacuolization, with trace amounts of fibrin and C3 in peritubular capillaries. In this animal a fraction of the peripheral blood cells (3%) at day 7 were of pig origin as detected by pig-specific monoclonal antibodies. In addition, colony-forming assays performed on a bone marrow biopsy specimen taken at day 14 indicated that approximately 30% of the relatively few myeloid progenitors detected were of swine origin. Conclusions . We have demonstrated that our protocol is effective in the prevention of hyperacute rejection and in the maintenance of excellent function of the renal xenograft for up to 15 days. These results also indicate that at least short-term engraftment of the xenogeneic donor bone marrow cells is possible to achieve in this discordant large animal combination. Longer survivals will be required to assess the possible effect of this engraftment on induction of tolerance.

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.

Glycans derived from porcine stomach mucin are effective inhibitors of natural anti-alpha-galactosyl antibodies in vitro and after intravenous infusion in baboons.

The current shortage of donor organs has stimulated investigation of pig-to-human xenotransplantation as a practical alternative to allotransplantation. However, a major obstacle to this xenotransplantation is hyperacute rejection, which is believed to be initiated by the interaction of natural anti-alpha-galactosyl (alphaGal) antibodies with alphaGal epitopes on pig vascular endothelium. Previously, we reported that neutral oligosaccharides derived from porcine stomach mucin (PSM) are effective inhibitors of human anti-alphaGal IgG in vitro. We now report that O-glycans derived from PSM by beta-elimination (PSMO) reduce the cytotoxicity of both baboon and human sera to pig kidney (PK15) cells in vitro. Crude PSM had some inhibitory effect in vitro, but PSMO were more than 100 times more potent. Moreover, 1 microg/ml of beta-eliminated PSMO that bound to an immunoaffinity column of anti-alphaGal antibodies were four times more efficient than total PSMO in protecting PK15 cells from the cytotoxic effect of baboon or human sera. Blood recovered from baboons after intravenous infusion of PMSO also showed significant protection of PK15 cells. We conclude that PSMO eluted from an anti-alphaGal immunoaffinity column demonstrate potent inhibitory effects against baboon and human serum cytotoxicity to PK15 cells in vitro and when administered intravenously. PSM may provide a cheap and readily available source of glycans that will be of therapeutic value in the prevention of hyperacute rejection.

The Effect of Total Blood Exchange with PHP Solution on Cardiac Xenotransplantation

Prevention of hyperacute rejection is a difficult and unsolved problem in xenotransplantation. Natural antibodies and complement activation have been known to play an important role in the xenotransplantation between discordant species pairs. In the present study, total blood exchange (TBE) was performed with pyridoxalated-hemoglobin-polyoxyethylene conjugate (PHP) solution (Ajinomoto Co., Inc., Kawasaki, Japan) before cardiac xenotransplantation in order to remove the immunoglobulins and prolong xenograft survival time. Guinea pigs and rats were used as the discordant species combination for donor and recipient. Two groups were established: Group 1, untreated control (n = 8) and Group 2, TBT with PHP solution (n = 8). The exchange blood transfusion was carried out at the rate of 15-20 ml/h utilizing PHP solution using a blood pump. After the blood exchange was processed, hematocrit (Ht) levels dropped to 4 or 5%, and a cardiac xenotransplantation was performed within 24 h. The levels of serum IgA, IgM, and IgG were decreased to less than 25, 25, and 10% of the base line, respectively, after blood exchange. A mean xenograft survival time in Group 2 was prolonged to 472 +/- 74 min and to 10.4 +/- 1.8 min in Group 1 (p < 0.01). A titer of the anti-guinea pig lymphocytotoxic antibody in rat serum was decreased to almost nil. The data from this study suggest that total blood exchange with PHP solution may be useful in preoperative removal of xenograft antibodies in xenotransplantation.