Expression of recipient cytomegalovirus in immunosuppressed and xenotransplanted Macaca fascicularis may be related to more severe gastrointestinal lesions.

BACKGROUND: Xenotransplantation is a potential answer to the current organ shortage, but the risk of infections related to overimmunosuppression is an important parameter that may predict the recipient's long-term survival. Cytomegalovirus (CMV) in xenotransplanted and immunosuppressed primates is a well-known cause of disease particularly affecting the gastrointestinal (GI) tract and a zoonotic concern. METHODS: Post-mortem sera and tissues from 45 immunosuppressed and xenografted Macaca fascicularis were evaluated for CMV using antisera specific for the immediate early 1 (IE1), anti-RhCMV, and QPCR for virus. RESULTS: Serological analysis showed 100% positivity for the presence of CMV antibodies following the application of a specific test designed for RhCMV. Five of 45 primates showed typical lesions of CMV infection in the GI tract, including neutrophilic enteritis and inclusion bodies. Molecular analysis confirmed the presence of recipient's CMV in the tissues with CMV histopathology. Porcine CMV from the donor animals was not found in any of the CMV-specific IHC-positive recipients. CONCLUSION: The presence of active CMV infection in animals intended for xenograft experiments can lead to severe gastrointestinal lesions that could impact the overall aims of the study. In such cases, the animals should be investigated using appropriate (non-human primate-specific) diagnostic tools prior to use and treated aggressively with state-of-the-art antiviral therapy.

Thromboelastographic evaluation of coagulative profiles in pig-to-monkey kidney xenotransplantation.

BACKGROUND: Activation of the clotting cascade is central in acute xenograft rejection (AHXR) that occurs when pig organs are transplanted into primates. The coagulopathy reported in this model is a very complex process that involves simultaneously coagulation factors, platelets and phospholipid-bearing cells (i.e., leukocytes, red blood cells, and endothelial cells). Choosing whole blood for coagulation analysis theoretically appears more favorable compared with plasma. Whole blood rotation thromboelastometry (ROTEM(®) ) is a point-of-care global coagulation analyzer able to evaluate the characteristics of clot formation and lysis by dynamic monitoring. The aim of this study was to record thromboelastographic profiles, performed by ROTEM(®) , in a series of immunosuppressed nephrectomized primates that received a life-supporting kidney. METHODS: Of the eight primates, n = 4 received a pig kidney transgenic for human decay-accelerating factor (hDAF/Gal+); n = 2, an α 1,3-galactosyltransferase gene-knockout (GT-KO) pig kidney transgenic for human CD39, CD55, CD59 and fucosyltransferase (HTF); and n = 2, a GT-KO pig kidney transgenic for hDAF. Blood samples were collected before and at least once per week after transplantation till euthanasia. Intrinsic (INTEM) and extrinsic (EXTEM) coagulation pathways and the function of fibrinogen (FIBTEM) were evaluated. Thromboelastographic parameters considered were clotting time (CT, seconds) and clot formation time (CFT, seconds) in INTEM and EXTEM and maximum clot firmness (MCF, mm) in FIBTEM. The correlations between CT in INTEM and activated partial thromboplastin time (aPTT), CT in EXTEM and PT, CFT in INTEM and EXTEM, and platelet counts and MCF in FIBTEM and fibrinogen plasma levels were also considered. RESULTS: In all animals, thromboelastographic profiles showed progressive prolongation of CT (activation of coagulative cascade) in INTEM. A close correspondence was observed between (i) the prolongation of the CFT values (propagation of clot formation), both in INTEM and EXTEM, and the decrease in platelet counts; (ii) the reduction in MCF values (clot firmness) ​​in FIBTEM and the decrease in fibrinogen plasma levels. No concordance between CT in INTEM and aPTT and between CT in EXTEM and PT was observed. CONCLUSIONS: Our study demonstrated that ROTEM(®) analyzer could be a useful and complementary tool to study the consumptive coagulopathy, either "compensated" or "non-compensated," that takes place when transgenic pig kidneys are transplanted into primates. Larger and prospective studies are needed to confirm our results and to evaluate the role of ROTEM(®) to guide the management of consumptive coagulopathy in order to prolong the survival of the transplanted organ.

In vitro and in vivo effects of the carbon monoxide-releasing molecule, CORM-3, in the xenogeneic pig-to-primate context.

BACKGROUND: Carbon monoxide (CO) interferes with inflammatory and apoptotic processes associated with ischemia-reperfusion injury and graft rejection. Here, the in vitro effects of carbon monoxide releasing molecule-3 (CORM-3), a novel water-soluble carbonyl CO carrier, have been investigated on porcine aortic endothelial cells (PAEC) and primate peripheral blood mononuclear cells (PBMC). Furthermore, the pharmacodynamics and pharmacotolerance of CORM-3 after administration of single and multiple doses in the primate have been assessed in view of its potential application in pig-to-primate xenotransplantation models. METHODS: For in vitro studies, PAEC and primate PBMC were exposed for 24, 48 and 72 h to CORM-3 (20 to 1000 microm) and viability was measured using an MTS assay. PAEC and primate PBMC proliferation after exposure to CORM-3 was assessed by CFSE labelling. Proliferation of primate PBMC against irradiated pig lymphocytes was also assessed. Tumor necrosis factor alpha (TNF-alpha) production and Caspase-3 and -7 activity in Concanavalin A (conA)-stimulated primate PBMC were measured following treatment with CORM-3. In vivo, CORM-3 was administered i.v. to cynomolgus monkeys at 4 mg/kg, as single or multiple doses for up to 30 days. The effect of CORM-3 was evaluated by the assessment of production of TNF-alpha and interleukin 1beta following PBMC stimulation with LPS by species-specific ELISA. Complete hematologic and biochemical analyses were routinely performed in treated primates. RESULTS: At concentrations <500 microm, CORM-3 did not alter the viability of PAEC or primate PBMC cultures in vitro, nor did it induce significant levels of apoptosis or necrosis. Interestingly, at concentrations of 300 and 500 microm, significant PAEC proliferation was observed, whilst concentrations > or =50 microm inhibited conA-activated primate lymphocyte proliferation (IC(50) of 345.8 +/- 51.9 microm) and the primate xenogeneic response against pig PBMC. Such responses were demonstrated to be CO-dependent. In addition, CORM-3 significantly inhibited caspase-3 and -7 activity at concentrations between 200 and 500 microm and caused a significant reduction in TNF-alpha production (IC(50) 332.8 +/- 33.9 microm). In vivo, following the administration of multiple doses, TNF-alpha production was significantly reduced in comparison to pre-treatment responses, with decreased levels maintained throughout the study. Moreover, a slight and transient increase in transaminases and bilirubin was observed in animals exposed to multiple doses of CORM-3. CONCLUSIONS: These studies suggest that CORM-3 has anti-inflammatory and immunomodulatory properties in primates that may result in clinical benefit to allo- and xenografted organs.

Endodontic treatment of canine teeth in a cynomolgus monkey ( Macaca fascicularis).

A case of bilateral superior canine tooth pulp necrosis in an 18-year-old male Macaca fascicularis is described . A skull computed tomography scan was performed to confirm the diagnostic suspicion. A successful bilateral endodontic treatment with coronotomy was performed using rotary nickel-titanium instruments. Post-operative dental radiographs confirmed the correct positioning of the sealant. A rapid and excellent clinical recovery was obtained after each surgery. This innovative approach may contribute to improving the quality of root canal treatments provided to non-human primates.

Effects of long-term administration of high-dose recombinant human antithrombin in immunosuppressed primate recipients of porcine xenografts.

BACKGROUND: Fibrin deposition is central to the acute humoral rejection process occurring in the presence of consumptive coagulopathy when pig organs are transplanted into primates. METHODS: To assess whether strategies aimed at preventing fibrin formation may extend xenograft survival, we administered high daily doses of recombinant human antithrombin (rhAT) (500 U/kg twice daily) to obtain both anticoagulant and anti-inflammatory effects in immunosuppressed primate recipients of porcine kidneys. RESULTS: Some degree of consumptive coagulopathy developed in both rhAT-treated (n=3) and untreated (n=3) primates. No major differences in the coagulation parameters analyzed were observed between the 2 groups. Similarly, no difference in survival was seen between rhAT-treated (20.6+/-4 days; range: 15-23 days) and untreated animals (17.3+/-11.6 days; range: 7-30 days), although the rhAT-treated primates had a higher bleeding tendency. Despite the high daily dose of rhAT, considerable fibrin deposition was observed in the graft as early as 2 weeks after transplantation. CONCLUSIONS: These results suggest that a high daily dose of rhAT fails to influence survival or prevent fibrin formation and deposition in the graft in our pig-to-primate model. However, the potential role of rhAT administered in combination with heparins or other clotting inhibitor concentrates in this model remains to be determined.

HO-1 induction improves the type-1 cardiorenal syndrome in mice with impaired angiotensin II-induced lymphocyte activation.

Type-1 cardiorenal syndrome, characterized by acute kidney dysfunction secondary to cardiac failure and renal arteriolar vasoconstriction, is mediated by the renin-angiotensin-aldosterone axis and sympathetic nervous system activation. Previous reports indicate that angiotensin II modulates immune function and causes recruitment and activation of T-lymphocytes. The goal of this study was to evaluate the effects of postischemic heart failure on renal morphology and circulation and the beneficial effects of heme oxygenase-1 (HO-1) induction in T-lymphocyte-suppressed severe combined immune deficiency (SCID) mice. Mice were divided into 4 groups: sham, myocardial infarction (MI), MI treated with an HO-1 inducer, cobalt protoporphyrin, and with or without stannous mesoporphyrin, an inhibitor of HO activity. Heart and kidney function were studied 30 days after surgery. Fractional area change was reduced 30 days after surgery in both the C57 and SCID MI-groups as compared with their respective controls (P<0.01). Renal Pulsatility Index and renal injury were increased in C57 and SCID MI-groups compared with the sham group. HO-1 induction improved renal vasoconstriction as well as ameliorated renal injury in both the SCID and C57 MI-groups (P<0.01). However, improvement was more evident in SCID mice. In addition, our results showed that plasma creatinine, angiotensin II, and renin were significantly increased in the C57 and SCID MI-groups as compared with their respective controls. HO-1 induction decreased these parameters in both MI groups. Stannous mesoporphyrin reversed the beneficial effect of cobalt protoporphyrin in both mouse strains. The study demonstrates that T-lymphocyte suppression facilitated the HO-1-dependent improvement in the attenuation of type-1 cardiorenal syndrome.

Effects of long-term administration of recombinant human protein C in xenografted primates.

BACKGROUND: The role potential of recombinant human activated protein C (rhaPC), a recently developed molecule with anticoagulant and antiinflammatory properties, in prolonging survival in immunosuppressed primate recipients of porcine renal xenografts has been evaluated. METHODS: rhaPC was administered daily for 5 days (24 µg/kg/hr; group A; n = 3) or throughout the postoperative period (8-24 µg/kg/hr; group B; n = 2; or 24-48 µg/kg/hr; group C; n = 4). Animals in group D (n = 2) received rhaPC daily (24 µg/kg/hr) combined with recombinant human antithrombin (84 U/kg every 8 hr). Two animals served as control (group E). RESULTS: The results indicate that rhaPC is protective against fibrin deposition early after transplantation but does not prevent fibrin deposition and the occurrence of acute humoral xenograft rejection (AHXR) later on. Animals in the study survived between 8 and 55 days. At the dose used, rhaPC is able to prevent fibrin deposition in the graft in the first 2 weeks after xenotransplantation, except when it is administered in conjunction with antithrombin. However, rhaPC did not prevent the eventual occurrence of AHXR in primate recipients of porcine xenografts. CONCLUSIONS: In this pig to primate model, rhaPC confers a short advantage in the prevention of early perioperative xenograft damage but does not represent an effective strategy for preventing AHXR.

Reference values for thromboelastometry (ROTEM®) in cynomolgus monkeys (Macaca fascicularis).

INTRODUCTION: The imbalance in clotting homeostasis, tending towards hypercoagulation, is recognized as the real barrier to the long-term survival of porcine xenografts in pig-to-primate xenotransplatation. The present study aimed to validate in primate blood the applicability of whole blood rotation thromboelastometry, performed by ROTEM®, which evaluates the characteristics of clot formation by dynamic monitoring. MATERIALS AND METHODS: ROTEM® (Pentapharm GmbH, Munich, Germany) was used to investigate native coagulation (NATEM®), the intrinsic (INTEM®) and extrinsic (EXTEM®) pathways, the function of fibrinogen (FIBTEM®), and the presence of fibrinolysis in 40 naïve cynomolgus monkeys. Using classic validation approaches, the normal thromboelastographic profile was defined and the influence of haematocrit (Hct,%), platelet count (x10(9)/L), fibrinogen (mg/dl), and factor VIII (FVIII,%) was evaluated. RESULTS: In all four (NATEM®, INTEM®, EXTEM®, FIBTEM®) assays considered, Clotting Time (CT, sec) and Clot Formation Time (CFT, sec) were shorter in primates than humans. Moreover, α-angle (°), Maximum Clot Firmness (MCF, mm), and MaxVel (mm/min) were also higher in primates than humans. No substantial difference was observed for Hct and platelet count between the two species. On the contrary, FVIII was higher in primates than in humans whereas, interestingly enough, fibrinogen levels were lower in monkeys than in humans. CONCLUSION: ROTEM® depicts a hypercoagulable profile in primates as compared to humans. Taken together these data suggest that, with regard to coagulation, xenotransplantation in cynos may represent a much more difficult situation than xenotransplantation in humans.

Alterations in the coagulation profile in renal pig-to-monkey xenotransplantation.

Five monkey recipients of a porcine renal xenograft were studied to determine the relationship between fibrin formation in acute humoral xenograft rejection (AHXR) and procoagulant and anticoagulant factor levels to establish whether changes in coagulation parameters could be used to predict AHXR and determine whether AHXR is associated with overt disseminated intravascular coagulopathy (DIC) in this model. Variable degrees of compensated consumptive coagulopathy were observed in each primate. Elevated thrombin-antithrombin (TAT), F1+2 and D-dimer levels consistent with thrombin generation and fibrin formation were recorded. There was no consumption of the main clotting inhibitors (including antithrombin) or a progressive, severe drop in fibrinogen levels and platelet counts, although grafts were left in situ. After transplantation, D-dimer levels remained persistently high, so they were of limited value in defining this coagulopathy. At post mortem, no cases of multiorgan involvement typical of overt DIC were observed. The lack of a rapid postoperative recovery of clotting inhibitor levels after transplantation was invariably associated with early poor outcome. This study shows that AHXR is associated with various degrees of compensated consumptive coagulopathy in our pig-to-primate model. No clear relationship was found between coagulation parameter levels and graft outcome.

Ureteral stenosis in HDAF pig-to-primate renal xenotransplantation: a phenomenon related to immunological events?

The aim of this study was to analyze the incidence of ureteral stenosis in a life-supporting human decay-accelerating factor (hDAF) transgenic pig-to-cynomolgus monkey kidney transplantation model and determine the role of possible immunological events in its pathogenesis. Thirty consecutive bi-nephrectomized cynomolgus monkeys received a kidney from hDAF transgenic pigs with or without a ureteral stent. Four monkeys were euthanized prematurely after transplantation. In the remaining 26 cases, the mean survival was 24 +/- 19 days. Except in one case, there was a close relationship between ureter and kidney in terms of type and severity of rejection. There were six ureteral stenoses; five were repaired by stent positioning and resurgery extended survival for an additional 16 +/- 10 days. The stenotic ureters showed diffuse acute humoral xenograft rejection (AHXR), while all cases with no or only focal signs of ureteral rejection never revealed ureteral obstruction. Use of a ureteral stent extends the survival of a xenografted primate, thereby helping to clarify the immunological events surrounding the onset of AHXR in kidneys in long-term xenograft recipients.

Methotrexate for immunosuppression in life-supporting pig-to-cynomolgus monkey renal xenotransplantation.

Methotrexate (MTX) has been used successfully as an immunosuppressant in rodent xenotransplantation models, but the data generated so far with MTX in pig-to-baboon cardiac transplantation studies have been disappointing. The potential of this agent was consequently explored in a life-supporting pig-to-primate renal model using the cynomolgus monkey as the recipient species. Introductory in vitro and in vivo pharmacokinetic and pharmacodynamic studies with MTX were conducted in three cynomolgus monkeys. Subsequently, 10 cynomolgus monkey recipients of a life-supporting kidney from human decay-accelerating factor transgenic pigs were administered MTX intravenously according to three different regimens. All the animals also received cyclosporine A and steroids. In addition, mycophenolate sodium (MPS) was administered post-operatively in two of the three groups of transplanted animals. At clinically relevant concentrations, MTX is able in vitro to inhibit the mixed lymphocyte reactions (MLR) in cynomolgus monkeys. After intravenous administration, moreover, exposure of cynomolgus monkeys to MTX appeared to be higher than had been previously reported in baboons. Graft function was observed in the transplanted animals, which survived from 0 to 41 days. All but two animals revealed acute humoral rejection in the explanted graft and developed diarrhea. Diarrhea was the cause of euthanasia in five cases. It was unrelated to the administration of MPS and associated with severe histopathological signs of enteritis. This study demonstrates that the pharmacokinetic and pharmacodynamic profiles if MTX vary substantially between non-human primate species. In vitro, MTX has immunosuppressive properties in the cynomolgus monkey at clinically relevant concentrations. In vivo, MTX has a very narrow therapeutic window in cynomolgus monkeys, however, as it does in baboons. We conclude that MTX is scarcely effective as an immunosuppressant, be it for induction or maintenance, in pig-to-cynomolgus monkey renal xenotransplantation.