Quantitative perfusion and water transport time model from multi b-value diffusion magnetic resonance imaging validated against neutron capture microspheres.

Intravoxel Incoherent Motion (IVIM) is a non-contrast magnetic resonance imaging diffusion-based scan that uses a multitude of b-values to measure various speeds of molecular perfusion and diffusion, sidestepping inaccuracy of arterial input functions or bolus kinetics in quantitative imaging. We test a new method of IVIM quantification and compare our values to reference standard neutron capture microspheres across normocapnia, CO2 induced hypercapnia, and middle cerebral artery occlusion in a controlled animal model. Perfusion quantification in ml/100g/min compared to microsphere perfusion uses the 3D gaussian probability distribution and defined water transport time as when 50% of the molecules remain in the tissue of interest. Perfusion, water transport time, and infarct volume was compared to reference standards. Simulations were studied to suppress non-specific cerebrospinal fluid (CSF). Linear regression analysis of quantitative perfusion returned correlation (slope = .55, intercept = 52.5, $R^2$= .64). Linear regression for water transport time asymmetry in infarcted tissue was excellent (slope = .59, intercept = .3, $R^2$ = .93). Strong linear agreement also was found for infarct volume (slope = 1.01, $R^2$= .79). Simulation of CSF suppression via inversion recovery returned blood signal reduced by 82% from combined T1 and T2 effects. Intra-physiologic state comparison of perfusion shows potential partial volume effects which require further study especially in disease states. The accuracy and sensitivity of IVIM provides evidence that observed signal changes reflect cytotoxic edema and tissue perfusion. Partial volume contamination of CSF may be better removed during post-processing rather than with inversion recovery to avoid artificial loss of blood signal.

Major carbohydrate epitopes in tissues of domestic and African wild animals of potential interest for xenotransplantation research.

We investigated the main glycotopes expressed on the tissues of 44 animal species, including primates, nonprimate mammals, marsupials, birds, and a reptile. Paraffin-embedded tissue sections of kidney, heart, liver, pancreas, lung, brain and intestine of 24 domestic animal species were stained with seven fluorescent-labeled lectins. Testis sections of 20 African wild animal species were tested with the same lectins. Overall, three main immunofluorescence patterns were found in the vascular compartment. First, humans and Old World monkeys express genetically polymorphic ABH antigens and do not express alphaGal. Second, New World monkeys, other mammals, and marsupials do not express ABH antigens, but have large amounts of a genetically monomorphic alphaGal. Third, birds and reptiles do not express either ABH or alphaGal, but have monomorphic betaGal, probably different from the lactosamine precursor of ABH and alphaGal. Epithelial cells producing exocrine secretions also expressed carbohydrate epitopes. The fluorescence patterns of the cells of the exocrine compartment are similar, but not identical, to those expressed in the vascular compartment. All the animals tested have some ABH and betaGal in exocrine tissues, but New World monkeys and lower mammals are the only ones expressing alphaGal in exocrine tissues.

Comparative histopathology of hepatic allografts and xenografts in the nonhuman primate.

Liver transplantation was performed in the following groups: Group 1, baboon-to-baboon allografting (n=8) (control group); Group 2, ABO-compatible vervet monkey-to-baboon xenografting (n=8); Group 3, ABO-incompatible vervet monkey-to-baboon xenografting (n=6); Group 4, pig-to-baboon xenografting (n=2); and Group 5, pig-to-rhesus monkey xenografting (n=6). Immunosuppressive therapy (cyclosporine, cyclophosphamide, and methylprednisolone) was begun 2-7 days before liver transplantation (LTx) and continued indefinitely after LTx. The liver grafts were biopsied pre-LTx and subsequently post-LTx at approximately 1 hr, 2-3 hr, 7-10 days, 20-30 days, 60 days, 120 days, and at euthanasia or spontaneous death. There were 19 successful LTxs with grafts functioning from one hour to 123 days. No pig liver (Groups 4 and 5) survived more than 5.5 hr, as there was an immediate severe vascular response after reperfusion, typical of hyperacute rejection (congestion and hemorrhage). Vascular rejection was not seen in allografts (Group 1), but early mild-to-moderate congestion and neutrophil infiltration were present in concordant xenografts (Groups 2 and 3), which were associated with moderate deposition of immunoglobulin, C3, and fibrinogen. Lymphoid cell infiltration, bile duct damage, and portal vein endothelialitis in the portal zones occurred later in both allografts (Group 1) and concordant xenografts (Groups 2 and 3), developing earlier in the presence of ABO-incompatibility (Group 3). In concordant xenografts it was usually followed by fibrosis.

Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy.

BACKGROUND: This study sought to (i) investigate the efficacy of cobra venom factor (CVF) in preventing hyperacute rejection (HAR) after pig-to-baboon heart transplantation, (ii) examine the effect of additional splenectomy (Spx) and pharmacologic immunosuppression (IS), and (iii) study delayed graft rejection when HAR is avoided by complement depletion. METHODS: Eleven recipient baboons received heterotopic pig heart transplants. Three received either no therapy or IS (cyclosporine + methylprednisolone +/- cyclophosphamide +/- methotrexate) at clinically well-tolerated doses, with graft survival for only 40, 32, and 15 min, respectively. Two received CVF+/-Spx, which extended survival to 5 and 6 days, respectively. Six underwent Spx + CVF therapy + IS; graft survival was 3 hr (technical complication), 6 days (death from sepsis), 10, 12, and 22 days (vascular rejection), and <25 days (euthanized for viral pneumonia with a functioning graft that showed histopathologic features of vascular rejection). RESULTS: Dense deposition of IgM and, to a lesser extent, IgG and IgA were seen on the endothelial cells within 1 hr of transplantation, but only trace levels of complement deposition were present in CVF-treated recipients. Within approximately 5-12 days, cardiac xenografts showed progressive infiltration by mononuclear cells, consisting primarily of activated macrophages producing tumor necrosis factor-alpha and small numbers of natural killer cells; T and B cells were absent. CONCLUSIONS: We conclude that (i) CVF prevents HAR, (ii) the addition of Spx + IS delays rejection, but (iii) the early deposition of antibody leads to progressive graft injury, resulting in (iv) delayed vascular rejection. Our findings indicate that the features of delayed xenograft rejection described in small animal models also occur in the pig-to-baboon model, and that rejection may occur in a complement-independent manner from the effects of antibody and/or host macrophages.

In vivo immunoadsorption of antipig antibodies in baboons using a specific Gal(alpha)1-3Gal column.

The major role of anti-alphaGal antibodies in the hyperacute rejection of pig organs by humans and baboons has been clearly demonstrated. Spacered alpha-galactose disaccharide (Gal(alpha1)-3Gal) hapten was produced by chemical synthesis and covalently attached to a flexible, hydrophilic polymer (PAA), which in turn was covalently coupled to macroporous glass beads, forming an immunoadsorbent that is mechanically and chemically stable and can be sterilized. The extracorporeal immunoadsorption (EIA) of anti-alphaGal antibodies using this column has been investigated in vivo in 3 baboons. In Baboon 1 (which had hyperacutely rejected a pig heart transplant 4 months previously, was not splenectomized, and did not receive any pharmacologic immunosuppression) the levels of anti-alphaGal antibody and antipig IgM and IgG, as well as serum cytotoxicity, fell significantly after each of 3 EIAs but were not eliminated. Serum cytotoxicity, antipig immunoglobulin and anti-alphaGal antibody rose steeply within 24 hr of the final EIA, suggesting that the return of cytotoxicity was associated with anti-alphaGa1 antibody. In Baboons 2 and 3 (which were immunologically naive and splenectomized, and received triple drug immunosuppressive therapy) serum cytotoxicity was totally eliminated and anti-alphaGal antibody and antipig IgM and IgG levels were greatly reduced by courses of EIA. In Baboon 2, cytotoxicity and all antibody levels remained negligible for approximately one week after the final (fourth) daily EIA. In Baboon 3, cytotoxicity and antibody levels were maintained low by intermittent EIA (over a period of 13 days) for almost 3 weeks, although antipig IgM began to rebound 4 days after the final EIA. We conclude that, in an immunosuppressed, splenectomized baboon, repeated EIA using a specific alphaGal disaccharide column will reduce antipig and anti-alphaGal antibody levels and serum cytotoxicity significantly for several days. This reduction in cytotoxicity will almost certainly be sufficient to delay the hyperacute rejection of a transplanted pig organ, but further studies are required to investigate whether it will be sufficient to allow accommodation to develop.

Cobra venom factor stimulates anti-alpha-galactose antibody production in baboons. Implications for pig-to-human xenotransplantation.

Cobra venom factor (CVF) depletes complement and may therefore be of use in preventing the hyperacute rejection that follows discordant organ xenotransplantation. In two baboons studied, the intramuscular injection of CVF (0.25 mg/kg) was followed by a marked reduction in serum C3 and CH50, and serum cytotoxicity to pig kidney (PK15) cells. There was, however, a very rapid rise in the level of anti-alpha-galactose (alpha Gal) antibody, and a slower rise in anti-CVF antibody. A second intramuscular injection of CVF on day 14 was ineffective in reducing C3, CH50, and serum cytotoxicity. The major oligosaccharide of CVF is known to contain alpha Gal residues, which we suggest stimulate the major increase in anti-alpha Gal antibody level seen in the present study. In the clinical situation, this might lead to an increased immune response to a concomitantly transplanted pig organ.

Secondary organ allografting after a primary "bridging" xenotransplant.

It remains uncertain whether xenotransplantation can sensitize the recipient to alloantigens, rendering subsequent allotransplantation unsuccessful. This is of considerable importance if a xenograft is to be used as a "bridge" to support the patient until a suitable allograft becomes available. When sera from 9 baboons that had received pig or African green monkey heart or liver xenografts were tested against a panel of lymphocytes from 5 or 6 potential donor baboons, positivity was seen in only 1 baboon (and then to only 2 of the potential 5 donors). In 4 baboons that had undergone previous xenotransplants (1 from this series of 9 baboons and 3 others), subsequent organ allografting was not followed by hyperacute, antibody-mediated, or accelerated cellular rejection. We conclude that organ xenotransplantation using discordant or concordant donor species does not prohibit subsequent allotransplantation.

Auxiliary liver allografting and xenografting in the nonhuman primate.

Auxiliary liver transplantation has been performed in the baboon using allografts (n = 8) and concordant xenografts from donor African green monkeys (n = 8). The native portal vein was ligated in all cases and the native common bile duct was ligated in 5 cases. The immunosuppressive therapy used was identical in both the allografts and xenografts and consisted of triple drug therapy (cyclosporine, cyclophosphamide, and methylprednisolone), all at dosages consistent with clinical use. During the determination of the surgical technique to be applied, there were 5 early failures (3 allografts, 2 xenografts), and 2 deaths at 10 and 20 days from multiorgan failure and sepsis, respectively (xenografts). The remaining 9 baboons (5 allografts, 4 xenografts) were electively euthanized at 16-62 days (allografts) and 35-120 days (xenografts). Hyperacute rejection or antibody-mediated rejection was not seen in the grafted livers. Episodes of acute cellular rejection occurred in the majority of animals within the first 30 days and recurred in the longer-term survivors, but could be controlled by bolus therapy with intravenous methylprednisolone. Satisfactory donor liver function was confirmed using a number of tests, including scintigraphy in 3 cases. We conclude that auxiliary liver transplantation using a closely related donor species is feasible in baboons and might be extended to humans with terminal liver failure. A baboon-to-man auxiliary liver graft may serve as a "bridge" until either a human cadaver donor liver became available or native liver function recovers in patients with fulminant hepatic failure.

Failure of intrathymic inoculation of donor-specific splenocytes to prolong cardiac or renal allograft survival in dogs.

The intrathymic inoculation (ITI) of donor splenocytes into potential organ transplant recipients has been demonstrated to result in donor-specific unresponsiveness and greatly prolonged survival of subsequent organ allografts in rodents without the need for long-term pharmacological immunosuppressive therapy. We have studied the effect of the ITI of saline (controls) (groups 1 (n = 6) and 3 (n = 6)) or donor splenocytes (groups 2 (n = 10) and 4 (n = 8)) in dogs that received either pharmacological immunosuppression (with cyclosporine and prednisone, +/- azathioprine/cyclophosphamide) (groups 1 and 2) or rabbit anti-dog antithymocyte globulin (groups 3 and 4) at the time of ITI. Kidney or heart allografting (from the donor of the splenocytes) was carried out 16-74 days after ITI; all but four transplants were performed within 16-22 days after ITI. Mean kidney allograft survival was 6, 10, 9, and 9 days, respectively, in groups 1-4. Mean cardiac allograft survival was 7, 14, 8, and 7 days, respectively. There was no statistical difference in allograft survival between those dogs that received ITI of saline and those that received donor splenocytes. These results would suggest that the protocols developed to date using ITI in rodent species may not be successful in dogs.

Evidence that intravenously administered alpha-galactosyl carbohydrates reduce baboon serum cytotoxicity to pig kidney cells (PK15) and transplanted pig hearts.

Methods of inhibiting the hyperacute antibody-mediated rejection that occurs when pig organs are transplanted into primates have been investigated using the baboon as a potential recipient. Baboons were treated with different regimens that included combinations of (1) splenectomy, (2) pharmacologic immunosuppression (CsA, cyclophosphamide, corticosteroids +/- methotrexate), and (3) intravenous infusion of oligosaccharides. The cytotoxicity of the serum was then assessed on cultures of pig kidney cells (PK15). Unmodified serum caused approximate 65-100% pig cell destruction. Splenectomy and/or pharmacologic immunosuppression, and infusions of dextran, dextrose or mannitol, did not result in any reduction of cytotoxicity. Infusions of melibiose and/or arabinogalactan, both of which have terminal non-reducing alpha-galactose, however, decreased relative PK15 cell damage significantly in a dose-dependent manner. At high concentrations (< or = 50 g/hr), complete inhibition of cytotoxicity was achieved in 4 of 15 baboons. The extracorporeal immunoadsorption of baboon serum utilizing immunoaffinity columns of melibiose also resulted in a significant reduction (of approximately 80%) in cytotoxic effect. In 1 baboon, melibiose and arabinogalactan infusion delayed vascular rejection of a pig cardiac xenograft from 10 min to about 12 hr, at which time the baboon died from the toxic effects of the carbohydrate infusion. These observations (1) add further support to the role that anti-alpha-galactosyl antibodies play in the hyperacute rejection of pig tissues transplanted into primates, and (2) demonstrate that serum cytotoxicity can be reduced by the intravenous infusion of alpha-galactosyl oligosaccharides or by extracorporeal immunoadsorption using these carbohydrates.

Cardiac allotransplantation across the ABO-blood group barrier by the neutralization of preformed antibodies: the baboon as a model for the human.

The baboon, like the human, expresses A and/or B blood group antigens on its tissues. Anti-A and anti-B antibodies are directed against these antigens, the epitopes of which are carbohydrate structures. Portions of these carbohydrates have been synthesized (trisaccharides A and B, respectively). When infused intravenously, the synthetic trisaccharides form a complex with the specific antibodies and neutralize their activity preventing them from binding to the antigen targets on a transplanted organ. In nonimmunosuppressed, hyperimmunized baboons, the continuous intravenous infusion of the specific trisaccharide alone (for 6 days) inhibited rejection of ABO-incompatible cardiac allografts, extending survival from a mean of 19 min (n = 3) to 8 days (n = 2), at which time the grafts failed from cellular (not vascular) rejection. The combination of long-term pharmacologic immunosuppression plus trisaccharide infusion (for periods of 8 to 19 days) extended survival to a mean of > 28 days (n = 4) with one heart functioning > 52 days. Accommodation clearly occurred in three of the four cases. This form of therapy may permit cadaveric organ allotransplantation across the ABO blood-group barrier in the human.

The pig as a potential organ donor for man. A study of potentially transferable disease from donor pig to recipient man.

Ten pigs, reared in an unmodified laboratory animal house environment, have been investigated to ascertain the incidence of diseases or disorders, including infection, neoplasia, or metabolic abnormalities, that might preclude the transplantation of major organs from the pig to man. Noninvasive studies were performed in the second month of life (study 1) and repeated after an interval that varied between 3 and 5 1/2 months (study 2). Necropsy was then performed as a means of assessing the accuracy of the 2 screening examinations. A total of 150 tests were performed on each pig. At both studies the feces contained cysts and/or trophozoites of several parasites, all of which were considered commensals. No other organisms potentially infective for man were identified either at study or at necropsy. Neither congenital anomalies nor malignant neoplasia was found at necropsy. However, in 2 pigs a vasculitis of uncertain etiology was present in the kidneys on microscopic examination, and in one of these the same condition affected the heart. This pathology was suspected neither from the screening examinations nor from the macroscopic appearance of these organs. Biopsy and microscopic examination would therefore appear to be essential before any organ is transplanted into a human.

Specific intravenous carbohydrate therapy. A new concept in inhibiting antibody-mediated rejection--experience with ABO-incompatible cardiac allografting in the baboon.

Heterotopic allografting of ABO-incompatible donor hearts in recipient baboons "hyperimmunized" against the incompatible A or B antigen (n = 3) was followed by hyperacute antibody-mediated vascular rejection within a mean of 19 min. The A and B epitopes against which these antibodies are directed are carbohydrates that can be synthesized. The continuous i.v. infusion of the specific synthetic A or B trisaccharide, beginning immediately pre-transplant and continued posttransplant for several days, prolonged allograft survival to a mean of 8 days (n = 2) and prevented antibody-mediated rejection, graft failure resulting from acute cellular rejection. The addition of triple pharmacologic immunosuppressive therapy (n = 4) resulted in prolongation of graft survival to a mean of > 28 days, with one heart still beating at 52 days; all grafts showed features of cellular rejection. "Accommodation" would appear to have developed in several baboons as graft function continued for periods of up to 39 days after discontinuation of carbohydrate therapy. Specific i.v. carbohydrate therapy should allow organ allografting to be performed across the ABO blood group barrier in humans. Furthermore, if the carbohydrate epitopes on the organs of discordant animals (e.g., the pig) against which human xenoreactive antibodies are directed can be confirmed, then this form of therapy might allow successful discordant organ xenotransplantation in man.