Ian P. J. Alwayn

Omega-3 fatty acid supplementation prevents hepatic steatosis in a murine model of nonalcoholic fatty liver disease.

Prolonged use of total parenteral nutrition can lead to nonalcoholic fatty liver disease, ranging from hepatic steatosis to cirrhosis and liver failure. It has been demonstrated that omega-3 fatty acids are negative regulators of hepatic lipogenesis and that they can also modulate the inflammatory response in mice. Furthermore, they may attenuate hepatic steatosis even in leptin-deficient ob/ob mice. We hypothesized that omega-3 fatty acid supplementation may protect the liver against hepatic steatosis in a murine model of parenteral nutrition in which all animals develop steatosis and liver enzyme disturbances. For testing this hypothesis, groups of mice received a fat-free, high-carbohydrate liquid diet ad libitum for 19 d with enteral or i.v. supplementation of an omega-3 fatty acid emulsion or a standard i.v. lipid emulsion. Control mice received food alone or the fat-free, high-carbohydrate diet without lipid supplementation. Mice that received the fat-free, high-carbohydrate diet only or supplemented with a standard i.v. lipid emulsion developed severe liver damage as determined by histology and magnetic resonance spectroscopy as well as elevation of serum liver function tests. Animals that received an i.v. omega-3 fatty acid emulsion, however, showed only mild deposits of fat in the liver, whereas enteral omega-3 fatty acids prevented hepatic pathology and led to normalization of liver function tests. In conclusion, whereas standard i.v. lipid emulsions fail to improve dietary-induced steatotic injury to the liver, i.v. supplementation of omega-3 fatty acids partially and enteral supplementation completely protects the liver against such injury.

Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates.

Background. Efforts to achieve tolerance to transplanted pig organs in nonhuman primates by the induction of a state of mixed hematopoietic chimerism have been associated with disorders of coagulation and thrombosis. Activation of recipient vascular endothelium and platelets by porcine hematopoietic cells and/or activation of donor organ vascular endothelium and/or molecular differences between the species may play roles. Irradiation or drug therapy could possibly potentiate endothelial cell activation and/or injury. Methods. We have investigated parameters of coagulation and platelet activation in nonhuman primates after (1) a regimen aimed at inducing mixed hematopoietic chimerism and tolerance (TIR that included total body irradiation, T cell depletion, and splenectomy; (2) pig bone marrow or pig peripheral blood mobilized progenitor cell transplantation (PCTx); and/or (3) pig organ transplantation (POTx). Five experimental groups were studied. Baboons were the recipient subjects in all groups except Group 1. Gp 1 Cynomolgus monkeys (n=6) underwent TIR + allotransplantation of hematopoietic cells and a kidney or heart or TIR + concordant xenotransplantation (using baboons as donors) of cells and a kidney; Gp 2 Baboons (n=4) underwent TIR with or without (±) autologous hematopoietic cell infusion; Gp 3 (n=12) PCTx±TIR; Gp 4 (n=5) POTx±TIR; Gp 5 (n=4) TIR + PCTx + POTx. Platelet counts, with plasma prothrombin time, partial thromboplastin time, fibrinogen levels, fibrin split products and/or D-dimer were measured. Results. In the absence of a discordant (porcine) cellular or organ transplant (Groups 1 and 2), TIR resulted in transient thrombocytopenia only, in keeping with bone marrow depression from irradiation. PCTx alone (Group 3) was associated with the rapid development of a thrombotic thrombocytopenic (TTP)-like microangiopathic state, that persisted longer when PCTx was combined with TIR. POTx (±TIR) (Group 4) was associated with a gradual fall (over several days) in platelet counts and fibrinogen with disseminated intravascular coagulation (DIC); after graft excision, the DIC generally resolved. When TIR, PCTx and POTx were combined (Group 5), an initial TTP-like state was superseded by a consumptive picture of DIC within the first week, necessitating graft removal. Conclusions. Both PCTx and POTx lead to profound alterations in hemostasis and coagulation parameters that must be overcome if discordant xenotransplantation of hematopoietic cells and organs is to be fully successful. Disordered thromboregulation could exacerbate vascular damage and potentiate activation of coagulation pathways after exposure to xenogeneic cells or a vascularized xenograft.

Donor-derived mesenchymal stem cells suppress alloreactivity of kidney transplant patients.

Background. Human mesenchymal stem cells (MSC) have immunosuppressive capacities. Although their efficacy is currently studied in graft-versus-host disease, their effect on alloreactivity in solid organ transplant patients is unknown. In this study, the immunosuppressive effect of MSC on recipient anti-donor reactivity was examined before and after clinical kidney transplantation. Methods. Anti-donor reactivity was established in pretransplant and posttransplant mixed lymphocyte reactions (MLR) of 14 living-kidney donor-recipient pairs. MSC from donors and third-party controls were added to the MLR in a ratio of 1:5. Results. MSC were isolated from donor perirenal fat and showed multilineage differentiation potential and the capacity to inhibit lymphocyte proliferation. The immunosuppressive effect of MSC was dose dependent and mediated by cell-membrane contact and soluble factors, including interleukin-10 and indoleamine 2,3-dioxygenase. Donor-derived MSC significantly inhibited the recipient anti-donor reactivity before and 1 month after transplantation. This effect was independent of human leukocyte antigen background of MSC. Flow cytometric analysis showed that MSC inhibited the proliferation of CD4+ and CD8+ T-lymphocyte subsets in pretransplant and posttransplant donor-directed MLR, whereas MSC had no effect on B- or natural killer-cell proliferation. Conclusion. Donor MSC significantly inhibited the proliferation of alloactivated recipient T cells before and after kidney transplantation. We believe these findings should encourage MSC-based intervention in clinical organ transplantation.

Pig kidney transplantation in baboons: anti-Gal(alpha)1-3Gal IgM alone is associated with acute humoral xenograft rejection and disseminated intravascular coagulation

BACKGROUND Kidneys harvested from miniature swine or pigs transgenic for human decay-accelerating factor (hDAF) were transplanted into baboons receiving an anti-CD154 monoclonal antibody (mAb) and either a whole body irradiation (WBI)- or cyclophosphamide (CPP)-based immunosuppressive regimen. METHODS Group 1 baboons (n=3) underwent induction therapy with WBI and thymic irradiation, pretransplantation antithymocyte globulin, and immunoadsorption of anti-Gal(alpha)1-3Gal (Gal) antibody (Ab). After transplantation of a miniature swine kidney, maintenance therapy comprised cobra venom factor, mycophenolate mofetil, and an anti-CD154 mAb (for 14-28 days). In group 2 (n=2), WBI was replaced by CPP in the induction protocol. Group 3 (n=3) animals received the group 2 regimen, but underwent transplantation with hDAF pig kidneys. RESULTS Group 1 and 2 animals developed features of disseminated intravascular coagulation (DIC), with reductions of fibrinogen and platelets and increases of prothrombin time, partial thromboplastin time, and fibrin split products. Graft survival was for 6-13 days. Histology showed mild acute humoral xenograft rejection (AHXR) of the kidneys, but severe rejection of the ureters. Group 3 animals developed features of DIC in two of three cases during the fourth week, with AHXR in the third case. Graft survival was for 28 (n=1) or 29 (n=2) days. Histology of day 15 biopsy specimens showed minimal focal mononuclear cellular infiltrates, with predominantly CD3+ cells. By days 28 and 29, kidneys showed mild-to-moderate features of AHXR. In all groups, the humoral response was manifest by reappearance of anti-Gal IgM below baseline level, with no or low return of anti-Gal IgG. All excised kidneys showed IgM deposition, but no complement and no or minimal IgG deposition. No baboon showed a rebound of anti-Gal Ab immediately after excision of the graft, and anti-Gal Ab increased over pretransplantation levels only when anti-CD154 mAb was discontinued. CONCLUSIONS DIC was observed with WBI- or CPP-based therapy, and after miniature swine or hDAF kidney transplantation. AHXR+/-DIC was observed in all recipients even in the absence of complement and no or low levels of anti-Gal IgG, but was significantly delayed in the hDAF recipients. These results confirm our earlier observation that CD154 blockade prevents T cell-dependent sensitization in baboons to pig antigens, but that baseline natural anti-Gal Ab production is not inhibited. We suggest that IgM deposition, even in the absence of IgG and complement, leads to endothelial cell activation with the development of DIC, even when there are only minimal histologic changes of AHXR.

Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade

The aim of the present study was to assess the survival of adult porcine islets transplanted into baboons receiving either (1) conventional triple drug immunosuppressive therapy or (2) a non‐myeloablative regimen and an anti‐CD154 monoclonal antibody (mAb) aimed at tolerance‐induction.

The problem of anti-pig antibodies in pig-to-primate xenografting: current and novel methods of depletion and/or suppression of production of anti-pig antibodies

The role of antibodies directed against Galα1–3Gal (α‐Gal) epitopes in porcine‐to‐primate xenotransplantation has been widely studied during the past few years. These antibodies (anti‐α‐Gal) have been associated with both hyperacute rejection and acute vascular rejection of vascularized organs. Depletion and (temporary or permanent) suppression of production of anti‐α‐Gal seem to be essential to the long‐term survival of these organs, even when the ultimate aim is accommodation or tolerance. Although more than 95% depletion of anti‐α‐Gal can be achieved by the use of immunoaffinity column technology, to date no regimen has been successful in preventing the return of anti‐α‐Gal from continuing production. In this review, we discuss current and novel methods for achieving depletion or inhibition (i.e. extracorporeal immunoadsorption, anti‐idiotypic antibodies, the intravenous infusion of immunoglobulin or oligosaccharides) and suppression of production (i.e. irradiation, pharmacologic agents, specific monoclonal antibodies, immunotoxins) of anti‐α‐Gal antibodies.

Prevention of intra-abdominal adhesions using the antiangiogenic COX-2 inhibitor celecoxib.

Objective:To determine the effects of COX-2 specific inhibitors on postoperative adhesion formation. Summary and Background Data:Intra-abdominal adhesions are the major cause of intestinal obstruction and secondary infertility after surgical procedures. Because adhesion synthesis requires angiogenesis, and cyclooxygenase-2 enzyme (COX-2) inhibitors have antiendothelial activity, we tested COX-2 inhibitors in a murine model of intra-abdominal adhesion formation. Methods:A silicone patch was secured to the lateral abdominal wall of groups of C57BL/6 mice, followed by cecal abrasion to promote adhesion formation. Beginning on the day of surgery, mice were treated with the selective COX-2 agents, celecoxib or rofecoxib, and the nonspecific COX inhibitors, aspirin, naproxen, ibuprofen, or indomethacin. Animals were treated for 10 days and killed. A second group (celecoxib, rofecoxib, aspirin) was treated for 10 days and observed for an additional 25 days. After treatment, intra-abdominal adhesions were scored using a standard method. The patch was subjected to immunohistochemistry with the endothelial-specific marker, CD31. Results:Animals treated with selective and nonselective COX-2 inhibitors, except aspirin, had significantly fewer adhesions than control animals. Celecoxib produced a maximal reduction in adhesion formation compared with rofecoxib and the nonselective COX-2 inhibitors at 10 days. After 25 days, celecoxib and rofecoxib, but not aspirin, had fewer adhesions than control mice. Adhesions from mice treated with celecoxib had reduced microvessel density compared with rofecoxib, the nonselective COX inhibitors, and control animals. Conclusions:Selective COX-2 inhibitors, in particular celecoxib, provide durable inhibition of intra-abdominal adhesions through an antiangiogenic mechanism.

Omega-3 Fatty Acids Improve Hepatic Steatosis in a Murine Model: Potential Implications for the Marginal Steatotic Liver Donor

The presence of nonalcoholic fatty liver disease often precludes potential organs from being used for transplantation. To date, there is no adequate treatment for hepatic steatosis, and it is expected that, because of increased obesity in Western society, the incidence of this disorder will increase. We investigated the effect of omega-3 polyunsaturated fatty acid supplementation on the treatment of hepatic steatosis in C57/Bl6 mice fed a high-carbohydrate, fat-free diet and in B6.V-Lep(ob) obese mice. Omega-3 fatty acid supplementation reversed hepatic steatosis in C57/Bl6 mice fed a high-carbohydrate, fat-free diet and converted macrovesicular to microvesicular steatosis in B6.V-Lep(ob) obese mice as determined by histology, magnetic resonance spectroscopy, and liver biochemistry We therefore conclude that omega-3 fatty acid supplementation improves hepatic steatosis in mice and may be used to increase the pool of potential live liver donors that are currently excluded because of the presence of macrovesicular steatosis.

The current practice of live donor nephrectomy in Europe

Background. The increasing number of live kidney donors in the last decade has stimulated interest in the surgical technique of donor nephrectomy. In this study, we evaluated the current status of the surgical approach in European transplant centers. Methods. A questionnaire was sent to 131 centers in 12 European countries. Questions included the number of donors, the technique used, and the inclusion and exclusion criteria for a technique. Results. Ninety-two replies (70%) were included. In the responding centers, approximately 1450 live donor nephrectomies were performed in 2004 (more than 80% of all live kidney donations in these countries). The number of living donors ranged from 0 to 95 per center. Nineteen institutions (21%) removed kidneys using endoscopic techniques only. Twenty-two centers (24%) performed both open and laparoscopic donor nephrectomy. Vessel length, difficult anatomy and right-sided donor nephrectomy were common reasons to choose an open technique. Twelve centers had performed laparoscopic donor nephrectomy but quit their program for various reasons. In 51 centers (55%), only open donor nephrectomy was carried out. Lack of evidence that endoscopic techniques provide better results was the main reason for these centers to stick to an open approach. Incisional hernias occurred after all types of open surgery in up to 30% of the donors per center. Twenty-nine clinics still carry out the classic flank incision. Conclusion. The surgical technique of live donor nephrectomy varies greatly between transplant centers in European countries. To define the optimal surgical approach a European registration of donor nephrectomies would be helpful.

Effects of specific anti-B and/or anti-plasma cell immunotherapy on antibody production in baboons: depletion of CD20- and CD22-positive B cells does not result in significantly decreased production of anti-alphaGal antibody.

Abstract: Anti‐Galα1–3Gal antibodies (antiαGal Ab) are a major barrier to clinical xenotransplantation as they are believed to initiate both hyperacute and acute humoral rejection. Extracorporeal immunoadsorption (EIA) with αGal oligosaccharide columns temporarily depletes antiαGal 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 antiαGal Ab following EIA.