Use of echocardiography to assess function of hDAF-transgenic pig cardiac xenografts.

The current standard of hand palpation may not be a sensitive method to detect rejection in heterotopic heart xenotransplants (HHTx). We sought to assess the use of echocardiography to detect rejection of pig heart xenografts. Four cynomolgus monkeys received HHTx from hDAF-transgenic pigs. Immunosuppression was cyclophosphamide induction, cyclosporine, steroids, sodium mycophenolate, alphaGal trisaccharide polymer, +/-soluble complement receptor type 1. Echocardiography was performed immediately after HHTx and three times a week postoperatively. Contractility on echo was scored as 1(none), 2(severely impaired), 3(moderate to severely impaired), 4(moderately impaired), 5(mild to moderately impaired), 6(mildly impaired), or 7(normal). Left ventricle wall thickness (LVWT) was measured in the anterior, inferior, posterior, lateral, and septal walls, the average was calculated. Impaired contractility or increase in LVWT were considered rejection and treated with steroids (solumedrol 15 mg/kg IV for 3-5 days). Palpation score (4-strong to 1-none) was recorded daily. Myocardial biopsies were obtained infrequently. At the time of first rejection, all four monkeys had an increase in LVWT and a decrease in contractility on echocardiography. Steroid treatment enhanced contractility in four monkeys and decreased LVWT in three monkeys. Palpation score remained at four of four during initial rejection episodes. Decrease in contractility and increase in LVWT on echocardiography appear to signify graft injury because steroid treatment results in improvement. Compared to palpation, echocardiography is more sensitive for assessing function of heterotopic pig heart xenografts. Echocardiography has, therefore, the potential to detect and treat early rejection episodes of heterotopic heart xenografts in nonhuman primates. This may help to achieve longer graft survival.

Genetic control of the humoral immune response to xenografts. II. Monoclonal antibodies that cause rejection of heart xenografts are encoded by germline immunoglobulin genes.

The early phases of the rejection of xenografts exchanged between closely related species are dominated by a vigorous humoral immune response. We have recently used a linker-mediated polymerase chain reaction (LM-PCR) to generate Ig heavy and light chain-specific cDNA libraries to examine the Ig gene control of a prototypic IgM monoclonal antibody, HAR-1, that causes the hyperacute rejection of hamster xenografts. Recombinant clones from the library were screened directly from bacterial colonies by PCR and the nucleic acid sequences of the clones established. Our results demonstrate that the HAR-1 hybridoma is encoded by Ig VH and JH genes in a germline configuration. Comparison of the cDNA sequence for HAR-1 VH with the germline equivalent of this gene isolated from newborn LEW liver (provisionally designated VHHAR-1) showed that the two VH sequences share a nucleic acid identity of 99.3%. Similarly, the HAR-1 monoclonal uses a Ig JH gene that is 98.2% identical with the JH1 nucleic acid sequence available in the GeneBank. The use of Ig VH and JH genes in a germline configuration is similar to that seen with polyreactive natural antibodies to infectious agents and autoantibodies. These humoral responses are thought to be the result of the stimulation of a T cell-independent subset of B cells, the B-1a/B-1b subset, that is responsible for producing antibodies that serve as a primitive humoral (natural antibody) defense mechanism against infectious diseases. Our results suggest that the humoral component of the rejection of xenografts in the hamster-to-rat model may represent the stimulation of this type of B cell antibody response by xenogeneic target antigens that share antigenic epitopes with bacteria and other infectious agents.

Functional metabolic characteristics of intact pig livers during prolonged extracorporeal perfusion: potential for a unique biological liver-assist device.

BACKGROUND: The clinical development of liver-support devices based on perfusion of either pig hepatocytes cartridges or whole pig livers has been hampered by the ability to use sufficient liver cell mass to provide adequate metabolic support, limited perfusion times, and the potential for patient exposure to pig zoonotic diseases. METHODS: We designed an original system in which an isolated intact pig liver was perfused extracorporeally under physiological conditions in a closed loop circuit with allogeneic pig blood and constant monitoring of major physiological and functional parameters. The perfusion circuit further included an interface membrane to provide for separation of patient and liver perfusion circulation. RESULTS: Prolonged (6-21 hr) liver perfusion did not produce significant liver damage as reflected by modest rises in the levels of the serum transaminases, stability of main biochemical parameters (including potassium), and the maintenance of normal cellular morphology. Optimal liver function was documented as measured by lactate consumption, control of glycemia, and the results of clotting studies and functional assays. The perfused liver cleared 82% and 79% of peak bilirubin and ammonia concentrations with clearing kinetics identical throughout perfusion. Indocyanine green clearance was identical to that observed in the living donor before explant surgery. CONCLUSIONS: In conclusion, the extracorporeal pig liver perfusion apparatus described here allows optimal pig liver function for prolonged periods of time. The microporous membrane to provide separation of donor organ and recipient and the high level of functional activity suggest that this form of liver metabolic support may have important clinical applications.

Microbiological hazards related to xenotransplantation of porcine organs into man.

Pigs are emerging as the most likely providers of genetically engineered organs and cells for the purpose of clinical xenotransplantation. Introduction of clinical trials has been delayed primarily by uncertainties regarding the risk of swine pathogen transmission that could harm the recipient. The concern that xenotransplantation carries the potential for a new epidemic has been highlighted by recent experiences with both bovine spongiform encephalopathy and human immunodeficiency diseases. As clinical trials have been postponed and xenotransplantation teams are working actively to gather data for an estimation of the risk, this review provides the reader with a state-of-the-art estimation of the microbiological hazards related to xenotransplantation of porcine organs to man. Particular emphasis is put on viral and retroviral hazards. Both current diagnostic tools and those under development are described, along with breeding strategies to provide donor animals that would not put the recipient or the general population at risk.

Hepatectomy with hypothermic perfusion of the liver.

Whereas most liver resections can be performed within 60 min, the period of vascular clamping and resulting ischemia may prove too short to allow complex major liver resections (MLR) especially on diseased livers. To overcome this problem, cooling of the liver with 4 degrees C preservations solution routinely used in liver transplantation may be used in three different approaches to MLR: I "In situ": the liver remains in the abdomen and integrity of afferent and efferent vessels is conserved. II "Ex situ-in vivo": the liver exteriorized from the abdomen by transecting all hepatic veins, remains connected to the porta hepatis. III "Ex vivo": the liver being removed from the abdomen, the MLR is performed extracorporeally. Of 15 MLR reported here, 11 were performed "in situ" and 4 "ex situ-in vivo"/Nowadays, the liver surgeon's "toolbox" must contain hypothermic liver perfusion. In carefully selected cases, these techniques allow MLR on diseases livers or mandating complex vascular procedures.

[Vascular clamping of the liver. Indications and limits]. / Les clampages vasculaires du foie. Indications et limites.

Control of bleeding during liver surgery is an essential prognostic factor for postoperative morbidity and mortality. Several well defined methods are currently available to ensure vascular occlusion, ranging from selective clamping of a segmental pedicle to total vascular exclusion of the liver. These methods of vascular control each have specific indications. However, they can induce ischaemia of the liver whose functional consequences, such as postoperative liver failure, are particularly severe in the case of prolonged ischaemia, affecting the remaining liver and in the presence of histological or functional alterations of the hepatic parenchyma. Selective methods of vascular control, only affecting the part of the liver to be resected, can be used systematically. In contrast, when the occlusion is not selective, they must be used sparingly, essentially in the case of bleeding from the parenchymal section, adopting the principal objective of the briefest possible total ischaemia. Minimization of bleeding must be weighed up against the consequences of ischaemia on the remaining liver, especially in the case of extensive hepatectomy, prolonged clamping and pathological non-neoplastic liver.

[Role of surgery in the treatment of refractory ascites in cirrhotic patients]. / Place de la chirurgie dans le traitement de l'ascite réfractaire du cirrhotique.

Ascites, generally directly reflecting portal hypertension, is the commonest cause of hospitalisation in patients with cirrhosis. In almost 10% of patients with ascites, optimal medical treatment combining bed rest, salt and water restriction, and diuretic treatment, is unable to induce sodium excretion and decrease the volume of the ascites, corresponding to the definition of refractory ascites. In other cases, it is the treatment of ascites itself (salt and water restriction and diuretics) which induce complications: water and electrolyte disturbances, functional renal failure, encephalopathy, the development of which also corresponds to refractory ascites. The therapeutic armamentarium for the management of refractory ascites remains varied, with the use of aspiration of ascites with compensation, peritoneovenous shunts, transhepatic or surgical porto-systemic anastomoses, and finally, liver transplantation. At the present time, each therapeutic measure must be taken while keeping in mind the possibility of subsequent liver transplantation and the potential risk of compromising liver transplantation by inappropriate treatments. In this context, the authors review and analyse the respective places of the various therapeutic modalities in the management of refractory ascites in cirrhotic patients.

Pharmacology of AMG 181, a human anti-α4 ß7 antibody that specifically alters trafficking of gut-homing T cells.

BACKGROUND AND PURPOSE: AMG 181 is a human anti-α4 ß7 antibody currently in phase 1 and 2 trials in subjects with inflammatory bowel diseases. AMG 181 specifically targets the α4 ß7 integrin heterodimer, blocking its interaction with mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the principal ligand that mediates α4 ß7 T cell gut-homing. EXPERIMENTAL APPROACH: We studied the in vitro pharmacology of AMG 181, and the pharmacokinetics and pharmacodynamics of AMG 181 after single or weekly i.v. or s.c. administration in cynomolgus monkeys for up to 13 weeks. KEY RESULTS: AMG 181 bound to α4 ß7 , but not α4 ß1 or αE ß7 , and potently inhibited α4 ß7 binding to MAdCAM-1 (but not vascular cell adhesion molecule-1) and thus inhibited T cell adhesion. Following single i.v. administration, AMG 181 Cmax was dose proportional from 0.01 to 80 mg·kg(-1) , while AUC increased more than dose proportionally. Following s.c. administration, dose-proportional exposure was observed with single dose ranging from 5 to 80 mg·kg(-1) and after 13 weekly doses at levels between 20 and 80 mg·kg(-1) . AMG 181 accumulated two- to threefold after 13 weekly 80 mg·kg(-1) i.v. or s.c. doses. AMG 181 had an s.c. bioavailability of 80%. The linear elimination half-life was 12 days, with a volume of distribution close to the intravascular plasma space. The mean trend for the magnitude and duration of AMG 181 exposure, immunogenicity, α4 ß7 receptor occupancy and elevation in gut-homing CD4+ central memory T cell count displayed apparent correlations. CONCLUSIONS AND IMPLICATIONS: AMG 181 has in vitro pharmacology, and pharmacokinetic/pharmacodynamic and safety characteristics in cynomolgus monkeys that are suitable for further investigation in humans.

[Genetic control of humoral immune response to xenografts. A monoclonal antibody that causes the hyperacute rejection of cardiac xenografts uses the genes in a native form]. / Contrôle génétique de la réponse immune humorale aux xénogreffes. Un anticorps monoclonal qui provoque le reject hyperaigu de xénogreffes cardiaques utilise des gènes dans une forme native.

The early phases of the rejection of xenografts exchanged between closely-related species are dominated by a vigorous humoral immune response. We have recently used a linkermediated polymerase chain reaction to generate Ig heavy and light chain specific cDNA libraries to examine the Ig gene control of a prototypic IgM monoclonal antibody, HAR-1, that causes the hyperacute rejection of hamster xenografts. Recombinant clones from the library were screened directly from bacterial colonies by PCR and the nucleic acid sequences of the clones established. Our results demonstrate that the HAR-1 hybridoma is encoded by Ig VH and JH genes in a germline configuration. Comparison of the cDNA sequence for HAR1-VH with the germline equivalent of the gene isolated from newborn LEW liver (provisionally designated VH1.1) showed that the two VH sequences share a nucleic acid identity of 99,3%. Similarly, the HAR-1 monoclonal uses a Ig JH gene that is 98,2% identical with the JH1 nucleic acid sequence available in the GenBank. The use of Ig VH and JH genes in a germline configuration is similar to that seen with polyreactive natural antibodies to infectious agents and autoantibodies. These humoral responses are thought to be the results of the stimulation of a T cell-independent subset of B cells, the B-1a/B-1b subset, that are responsible for producing antibodies that serve as a primitive humoral (natural antibody) defense mechanism against infectious diseases. Our results suggest that the humoral component of the rejection of xenografts in the hamster-to-rat model may represent the stimulation of this type of B cell antibody response by xenogeneic target antigens that share antigenic epitopes with bacteria and other infectious agents.