Anti-Gal alpha1-3Gal antibodies (anti alpha Gal Ab) are a major barrier to clinical xenotransplantation as they are believed to initiate both hyperacute and acute humoral rejection. Extracorporeal immunoadsorption (EIA) with alpha Gal oligosaccharide columns temporarily depletes anti-alpha Gal Ab, but their return is ultimately associated with graft destruction. We therefore assessed the ability of two immunotoxins (IT) and two monoclonal antibodies (mAb) to deplete B and/or plasma cells both in vitro and in vivo in baboons, and to observe the rate of return of anti alpha Gal Ab following EIA.The effects of the mouse anti-human IT anti-CD22-ricin A (proportional to CD22-IT, directed against a B cell determinant) and anti-CD38-ricin A (proportional to CD38-IT, B and plasma cell determinant) and the mouse anti-human anti-CD38 mAb (proportional to CD38 mAb) and mouse/human chimeric anti-human anti-CD20 (alpha CD20 mAb, Rituximab, B cell determinant) on B and plasma cell depletion and anti alpha Gal Ab production were assessed both in vitro and in vivo in baboons (n=9) that had previously undergone splenectomy. For comparison, two baboons received nonmyeloablative whole body irradiation (WBI) (300 cGy), and one received myeloablative WBI (900 cGy). Depletion of B cells was monitored by flow cytometry of blood, bone marrow (BM) and lymph nodes (LNG, staining with anti-CD20 and/or anti-CD22 mAbs, and by histology of LN. EIA was carried out after the therapy and anti alpha Gal Ab levels were measured daily.In vitro proportional to CD22-IT inhibited protein synthesis in the human Daudi B cell line more effectively than proportional to CD38-IT. Upon differentiation of B cells into plasma cells, however, less inhibition of protein synthesis after proportional to CD22-IT treatment was observed. Depleting CD20-positive cells in vitro from a baboon spleen cell population already depleted of granulocytes, monocytes, and T cells led to a relative enrichment of CD20-negative cells, that is plasma cells, and consequently resulted in a significant increase in anti alpha Gal Ab production by the remaining cells, whereas depleting CD38-positive cells resulted in a significant decrease in anti alpha Gal Ab production. In vivo, WBI (300 or 900 cGy) resulted in 100% B cell depletion in blood and BM,> 80% depletion in LN, with substantial recovery of B cells after 21 days and only transient reduction in anti alpha Gal Ab after EIA. proportional to CD22-IT depleted B cells by > 97% in blood and BM, and by 60% in LN, but a rebound of B cells was observed after 14 and 62 days in LN and blood, respectively. At 7 days, serum anti alpha Gal IgG and IgM Ab levels were reduced by a maximum of 40-45% followed by a rebound to levels up to 12-fold that of baseline anti alpha Gal Ab by day 83 in one baboon. The results obtained with proportional to CD38-IT were inconclusive. This may have been, in part, due to inadequate conjugation of the toxin. Cell coating was 100% with proportional to CD38 mAb, but no changes in anti alpha Gal Ab production were observed. proportional to CD20 mAb resulted in 100% depletion of B cells in blood and BM and 80% in LN, with recovery of B cells starting at day 42. Adding 150cGy WBI at this time led to 100% depletion of B cells in the BM and LN. Although B cell depletion in blood and BM persisted for >3 months, the reduction of serum anti alpha Gal IgG or IgM Ab levels was not sustained beyond 2 days.proportional to CD20 mAb+WBI totally and efficiently depleted CD20- and CD22-positive B cells in blood, BM, and LN for >3 months in vivo, but there was no sustained clinically significant reduction in serum anti alpha Gal Ab. The majority of antibody secretors are CD38-positive cells, but targeting these cells in vitro or in vivo with alpha CD38-IT was not very effective. These observations suggest that CD20-and CD22-positive B cells are not the major source of anti alpha Gal Ab production. Future efforts will be directed towards suppression of plasma cell function.

Background: Anti-Gal alpha 1-3Gal (Gal) antibodies (Gal Ab) contribute to the rejection of porcine organs transplanted into primates. Extracorporeal immunoadsorption (EIA) has been developed to eliminate Gal Ab from the circulation.Methods: Between 1995 and 1999 we performed 320 EIAs in baboons using a COBE-Spectra apheresis unit incorporating a synthetic Gal immunoaffinity column. Three plasma volumes were immunoadsorbed on each occasion. The 221 consecutive EIAs performed in 41 immunosuppressed baboons between January 1997 and April 1999 form the basis of this review. Of these 41 baboons, 29 underwent a series of three or four EIAs at daily intervals, seven had multiple series of three EIAs, and the remainder underwent single or double EIAs. Serum Gal Ab levels were monitored by ELISA before and at intervals after the course of EIA.Results: There were two fatal complications, one from a respiratory mishap (unrelated to the EIA) and one from persistent hypotension unresponsive to therapeutic interventions. Seven procedures (3%) were terminated early owing to technical difficulties and/or persistent hypotension. Mean pre-EIA Gal Ab levels in naive baboons were 33.1 mu g/ml (IgM) and 14.5 mu g/ml (IgG). Immediately after three consecutive EIAs, IgM was depleted by a mean of 97.3% and IgG by 99.4%. By 18 to 24 h later, Gal Ab was returning but depletion remained at 80.1% (IgM) and 84.7% (IgG). The subsequent rate of return of Gal Ab depended on the immunomodulatory protocol used.Conclusions: (1) With appropriate monitoring, EIA is an acceptably safe procedure, even in small(<10 kg) baboons. (2) Three consecutive EIAs are effective in removing >97% of Gal Ab. (3) In the majority of cases, return of Gal Ab begins within 24 h, irrespective of the immunomodulatory protocol.

Despite the success of desensitization protocols, antibody-mediated rejection (AMR) remains a significant contributor to renal allograft failure in patients with donor-specific antibodies. Plasmapheresis and high-dose intravenous immunoglobulin have proved to be effective treatments to prevent and treat AMR, but irreversible injury in the form of transplant glomerulopathy can commonly manifest months to years later. There is an unmet need to improve the outcomes for patients at risk for AMR. Updated Banff criteria now take into account the increasing understanding of the complex and heterogeneous nature of AMR phenotypes, including the timing of rejection, subclinical and chronic AMR, C4d-negative AMR, and antibody-mediated vascular rejection. Treatment for AMR is not standardized, and there is little in the way of evidence-based treatment guidelines. Refining more precisely the mechanisms of injury responsible for different AMR phenotypes and establishing relevant surrogate endpoints to facilitate more informative studies will likely allow for more accurate determination of prognosis and efficacious intervention using new therapeutic approaches. In addition to plasma exchange and intravenous immunoglobulin, a number of other add-on therapies have been tried in small studies without consistent benefit, including anti-CD20, proteasome inhibitors, complement inhibitors, anti-interleukin-6 receptor blockers, and immunoglobulin G-degrading enzyme of Streptococcus pyogenes (called IdeS).

Background. Xenotransplantation would provide a solution to the current shortage of organs for transplantation. Our group has been successful in inducing tolerance in mice and monkey models of allogeneic transplantation. The present study attempts to extend the same tolerance-inducing regimen to a pig-to-baboon organ transplantation model.Methods. Nine baboons underwent a conditioning regimen (consisting of nonmyeloablative or myeloablative whole body and thymic irradiation, splenectomy, antithymocyte globulin, pharmacologic immunosuppression and porcine bone marrow transplantation [BMTx]), which has previously been demonstrated to induce donor-specific allograft tolerance in monkeys. In addition, immunoadsorption of anti-alpha Gal antibody (Ab) was performed. Four of the nine baboons received pig kidney transplants (KTx), and one also underwent repeat transplantation with an SLA-matched kidney. Two received heterotopic pig heart transplants (HTx). Three baboons underwent conditioning without organ transplantation for long-term studies of natural Ab kinetics.Results, In the three baboons that received the conditioning regimen without an organ transplant, immunoadsorption reduced Ab by approximately 90%, but recovery of Ab to pretreatment level or higher occurred within 7 days. In contrast, the level of Ab remained low after organ transplant. No Ab to pig antigens other than alpha Gal was detected in any baboon before or after BMTx, KTx, or HTx. No graft succumbed to hyperacute rejection. KTx function began to deteriorate within 3-6 days, with oliguria and hematuria progressing to anuria, and the kidneys were excised after 3, 6, 9, 11, and 14 days, respectively. One HTx ceased functioning at 8 days; the second baboon died with a contracting HTx at 15 days. Features of coagulopathy and thrombocytopenia developed in all six transplanted baboons thigh D dimer, prolonged prothrombin time and partial thromboplastin time, and falling fibrinogen) resulting in serious bleeding complications in two baboons, one of which died on day 9, Donor organs showed progressive acute humoral rejection with deposits of IgM, IgG, and complement; a focal mononuclear cellular infiltrate was also observed. The ureter was the earliest structure of the KTx affected by rejection, with progression to necrosis.Conclusions. This conditioning regimen prevented hyperacute rejection but was ineffective in preventing the return of Ab, which was associated with the development of acute humoral rejection with features of coagulopathy. No baboon developed anti-pig Ab other than alpha Gal Ab. Further modifications of the protocol directed toward suppression of production of Ab are required to successfully induce tolerance to pig organs in baboons.

The pig-to-primate model is increasingly being utilized as the final preclinical means of assessing therapeutic strategies aimed at allowing discordant xenotransplantation. We review here the world experience of both pig-to-human and pig-to-nonhuman primate organ transplantation. Eight whole organ transplants using discordant mammalian donors have been carried out in human recipients; only one patient was reported (in 1923) to have survived for longer than 72 hr. Therapeutic approaches in the experimental laboratory setting have included pharmacologic immunosuppression, antibody and/or complement depletion or inhibition, the use of pig organs transgenic for human complement regulatory proteins, and conditioning regimens aimed at inducing a state of tolerance or specific immunologic hyporesponsiveness. The greatest success to date has been obtained with methods that inhibit complement-mediated injury, either by the administration of cobra venom factor or soluble complement receptor I to the recipient (with organ survival up to 6 weeks) or by the use of donor organs transgenic for human decay-accelerating factor (with organ survival up to 2 months). The future of xenotransplantation may lie in the judicious combination of current approaches.

Background. A state of tolerance may be more easily achieved if fully vascularized and functional donor thymus is transferred to the recipient at the time of whole organ transplantation.Methods. A composite "thymoheart" allograft was created by implanting autologous thymus into a donor heart 60-90 days before organ procurement. Successful intracardiac engraftment of autologous thymus was documented by histology and by flow cytometric analysis.Results. Histology of the thymic autografts at explantation revealed viable thymus with preservation of normal thymic architecture. Cells retrieved from thymic autografts 60 days after implantation exhibited the same MHC class I and class II staining profiles by flow cytometry as cells taken from the residual native thymus.Conclusion. We have created a novel composite organ that confers vascularized and functional donor thymus to heart allograft recipients at the time of transplantation without affecting cardiac function.

In the last few years, transplantation was an area of intense research activity. However, there is a worldwide shortage of donor organs for clinical transplantations. Currently, interest in xenotransplantation research is growing not only because of the increased demand for organs but also because of advances in molecular biology techniques that make possible the genetic or immunological manipulations of the animal donor rather than the human recipient. The better definitions of the mechanisms responsible for xenograft rejection should facilitate appropriate therapeutic strategies for long xenograft survival.

Ongoing studies at our center on facilitating transplantation of discordant xenogeneic organs are focused on tolerance induction. To abrogate hyperacute rejection, we have used adsorption methods to eliminate natural anti-Gal(alpha)1-3Gal (alphaGal) antibodies from the circulation of baboons. We have analyzed data concerning antibody removal in baboons that were 1) immunologically naive, 2) receiving conventional pharmacologic immunosuppressive therapy (IS), and 3) treated with a conditioning regimen for tolerance induction. We compared the efficiency of removing alphaGal antibody 1) by perfusion of whole blood through an alphaGal affinity column (CP; n=5) with 2) perfusion of plasma (separated from cellular components by apheresis) through an alphaGal column (CPA; n=39). Our studies demonstrate that 1) CP and CPA are equally effective in removing anti-alphaGal antibody, 2) CPA is the method of choice if multiple adsorptions are required, 3) CPA in naive animals transiently affects levels of total IgG and IgM, 4) four CPAs repeated at 2-4 day intervals in association with heavy IS reduce the pool of anti-alphaGal antibody and total Ig, and 5) splenectomy and/or IS delay the return of anti-alphaGal antibody.

The aim of this study was to deplete baboons of anti-(alpha)galactosyl (alphaGal] antibody and attempt to maintain depletion by pharmacologic immunosuppressive therapy (PI). In 12 experiments, involving nine baboons, repeated extracorporeal immunoadsorption (EIA) was carried out by plasma perfusion through immunoaffinity columns of synthetic alphaGal trisaccharide type 6. Five of the baboons were immunologically naive and four had undergone various procedures at least 6 months previously. All, however, had recovered lymphohematopoietic function and (with one exception) had levels of anti-alphaGal antibody within the normal range. Eleven protocols included continuous i.v. cyclosporine (to maintain whole blood levels of approximately 1,600 ng/ml). In addition, in ten protocols, the baboon received one or more of the following drugs: cyclophosphamide (1-20 mg/kg/day), mycophenolate mofetil (70-700 mg/ kg/day), brequinar sodium (1-12 mg/kg/day), prednisolone (1 mg/kg/day), melphalan (0.15-0.6 mg/kg/day), methylprednisolone (125 mg/day x3), and antilymphocyte globulin (ATG) (50 mg/kg/day x3). EIA was carried out on 1-9 occasions in each study and was temporarily successful in removing all antibody. When no PI was administered, antibody returned close to pre-EIA levels within 48 hr. Cyclosporine alone delayed the rate of antibody return only slightly. While EIA was continuing on a daily or alternate day schedule, antibody levels (both IgM and IgG) were maintained at 20-45% of pre-EIA levels. Once EIA was discontinued but PI maintained, IgM rose to 40-90% and IgG to 30-60% of pre-EIA levels. In vitro testing demonstrated significant cytotoxicity to pig cells at these antibody levels. We conclude that i) EIA utilizing columns of alphaGal trisaccharide is successful in temporarily depleting baboons of anti-alphaGal antibody, but ii) none of the PI regimens tested suppressed antibody production to levels which would be expected to prevent antibody-mediated rejection of pig xenografts. Additional strategies will therefore be required if xenotransplantation is to become a clinical reality.

During winter 1994-95, four and three sperm whales (Physeter macrocephalus) were stranded along the Belgian and the Dutch coasts, respectively. Necropsies and tissue samplings were collected 24 hrs post mortem. Lesions on several whales included round and linear skin scars, ventral skin abrasions, acute skin ulcers, acute ulcerative stomatitides, acute to chronic external otitides, and passive visceral congestion. In addition, these sperm whales appeared to be debilitated with severe weight deficit, had blubber thickness reduction, the absence of abdominal fat, and the intestinal tracts were almost empty. Three categories of lesions and their possible relation with the stranding were evaluated. Cutaneous scars observed on the seven whales appeared to have no relation with the stranding. The poor body condition and acute integument ulcerative lesions were present before the stranding. Ventral skin abrasions and visceral passive congestion were caused by the strandings. Absence of food in the alimentary tracts, evidence of weight loss and blubber thickness reduction were compatible with an extended presence of the sperm whales in the North Sea, where adequate food is not available. This might lead to progressive weakness, predisposing the animals to secondary pathogens such as viral diseases. Finally, the coastal configuration of the southern North Sea makes it a trap for sperm whales which have entered the area during their wanderings.

Abstract: Ten piglets, 7 to 16 weeks old, were partially thymectomised and 1 to 4 cm3 of minced thymic fragments autografted under the renal capsule. They were sacrificed, respectively, after 2, 4, 6, 8, 12, and 20 weeks. After 2 weeks, irregular whitish zones are present under the renal capsule. They were composed principally of two cell types: the first type was characterized by small round basophilic nuclei and little cytoplasm typical of lymphocytes; the second cell type had larger ovoid nuclei and a large vacuolised cytoplasm. Each cell type could be found in separate lobules or mixed in variable proportion in the same structure. The thymic autografts grew to form a layer up to 4 mm thick after 20 weeks. In the meantime, at the beginning of 4th week, the lobular structure became well organized with the cell type presenting large nuclei and cytoplasm being restricted to the center of the lobules while lymphocytes composed a peripheral layer. Hassal corpuscles (HC) appeared in the center of the lobules. Immunohistochemical labeling with anti‐cytokeratin mono‐ and poly‐ clonal Ab and with anti‐neurophysin polyclonal Ab displayed all the characteristics of normal functional thymic microenvironment. It is proposed that this novel experimental preparation ending up as a neo‐organ (thymo‐kidney) be used for xenotransplantation in an attempt to produce specific xenotolerance.

The veterinary medicine in rescue to the transplantation: Brief review of the history of transplantation from the early days of the mythology to the actual renal transplantation. Actual situation of the organs transplantation. History, advantages, risks and perspectives of research in xenotransplantation are reviewed.