Efficacy and Safety of Everolimus and Mycophenolic Acid With Early Tacrolimus Withdrawal After Liver Transplantation: A Multicenter Randomized Trial.

SIMCER was a 6-mo, multicenter, open-label trial. Selected de novo liver transplant recipients were randomized (week 4) to everolimus with low-exposure tacrolimus discontinued by month 4 (n = 93) or to tacrolimus-based therapy (n = 95), both with basiliximab induction and enteric-coated mycophenolate sodium with or without steroids. The primary end point, change in estimated GFR (eGFR; MDRD formula) from randomization to week 24 after transplant, was superior with everolimus (mean eGFR change +1.1 vs. -13.3 mL/min per 1.73 m2 for everolimus vs. tacrolimus, respectively; difference 14.3 [95% confidence interval 7.3-21.3]; p < 0.001). Mean eGFR at week 24 was 95.8 versus 76.0 mL/min per 1.73 m2 for everolimus versus tacrolimus (p < 0.001). Treatment failure (treated biopsy-proven acute rejection [BPAR; rejection activity index score >3], graft loss, or death) from randomization to week 24 was similar (everolimus 10.0%, tacrolimus 4.3%; p = 0.134). BPAR was more frequent between randomization and month 6 with everolimus (10.0% vs. 2.2%; p = 0.026); the rate of treated BPAR was 8.9% versus 2.2% (p = 0.055). Sixteen everolimus-treated patients (17.8%) and three tacrolimus-treated patients (3.2%) discontinued the study drug because of adverse events. In conclusion, early introduction of everolimus at an adequate exposure level with gradual calcineurin inhibitor (CNI) withdrawal after liver transplantation, supported by induction therapy and mycophenolic acid, is associated with a significant renal benefit versus CNI-based immunosuppression but more frequent BPAR.

Reduced-dose tacrolimus with mycophenolate mofetil vs. standard-dose tacrolimus in liver transplantation: a randomized study.

We conducted a multicenter randomized study in liver transplantation to compare standard-dose tacrolimus to reduced-dose tacrolimus with mycophenolate mofetil to reduce the occurrence of tacrolimus side effects. Two primary outcomes (censored criteria) were monitored during 48 weeks post-transplantation: occurrence of renal dysfunction or arterial hypertension or diabetes (evaluating benefit) and occurrence of acute graft rejection (evaluating risk). Interim analyses were performed every 40 patients to stop the study in the case of increased risk of graft rejection. One hundred and ninety-five patients (control: 100; experimental: 95) had been included when the study was stopped. Acute graft rejection occurred in 46 (46%) and 28 (30%) patients in control and experimental groups, respectively (HR = 0.59; 95% CI: [0.37-0.94]; p = 0.024). Renal dysfunction or arterial hypertension or diabetes occurred in 80 (80%) and 61 (64%) patients in control and experimental groups, respectively (HR = 0.68; 95% CI: [0.49-0.95]; p = 0.021). Renal dysfunction occurred in 42 (42%) and 23 (24%) patients in control and experimental groups, respectively (HR = 0.49; 95% CI: [0.29-0.81]; p = 0.004). Leucopoenia (p = 0.001), thrombocytopenia (p = 0.017) and diarrhea (p = 0.002) occurred more frequently in the experimental group. Reduced-dose tacrolimus with mycophenolate mofetil reduces the occurrence of renal dysfunction and the risk of graft rejection. This immunosuppressive regimen could replace full-dose tacrolimus in adult liver transplantation.

Increased expression of regulatory Tr1 cells in recurrent hepatitis C after liver transplantation.

Immune response failure during HCV infection has been associated with the activity of regulatory T cells. Hepatitis C-related cirrhosis is the main reason for liver transplantation. However, 80% of transplanted patients present an accelerated recurrence of the disease. This study assessed the involvement of regulatory T-cell subsets (CD4+CD25+ cells: 'Treg' and CD49b+CD18+ cells: 'T regulatory-1' cells), in the recurrence of HCV after liver transplantation, using transcriptomic analysis, ELISA assays on serum samples and immunohistochemistry on liver biopsies from liver recipients 1 and 5 years after transplantation. Three groups of patients were included: stable HCV-negative recipients and those with mild and severe hepatitis C recurrence. At 5 years, Treg markers were overexpressed in all HCV+ recipients. By contrast, Tr1 markers were only overexpressed in patients with severe recurrence. At 1 year, a trend toward the overexpression of Tr1 was noted in patients evolving toward severe recurrence. IL-10 production, a characteristic of the Tr1 subset, was enhanced in severe recurrence at both 1 and 5 years. These results suggest that Tr1 are enhanced during severe HCV recurrence after liver transplantation and could be predictive of HCV recurrence. High levels of IL-10 at 1 year could be predictive of severe recurrence, and high IL-10 producers might warrant more intensive management.

Safety and efficacy of raltegravir in HIV-infected transplant patients cotreated with immunosuppressive drugs.

Solid organ transplantations (SOT) are performed successfully in selected HIV-infected patients. However, multiple and reciprocal drug-drug interactions are observed between antiretroviral (ARV) drugs and calcineurin inhibitors (CNIs) through CYP450 metabolization. Raltegravir (RAL), a novel HIV-1 integrase inhibitor, is not a substrate of CYP450 enzymes. We retrospectively reviewed the outcomes of 13 HIV-infected transplant patients treated by an RAL + two nucleosidic reverse transcriptase inhibitor (NRTI) regimen, in terms of tolerability, ARV efficacy (plasma viral load, CD4 cell count), drug interactions, RAL pharmacokinetics and transplant outcome. Thirteen patients with liver (n = 8) or kidney (n = 5) transplantation were included. RAL was initiated (400 mg BID) either at time of transplantation (n = 6), or after transplantation (n = 7). Median RAL trough concentration was 507 ng/mL (176-890), which is above the in vitro IC95 for wild type HIV-1 strains (15 ng/mL). Target trough levels of CNIs were promptly obtained with standard dosages of tacrolimus or cyclosporine. RAL tolerability was excellent. There was no episode of acute rejection. HIV infection remained controlled. After a median follow-up of 9 months (range: 6-14), all patients were alive with satisfactory graft function. The use of an RAL + two NRTI-based regimen is a good alternative in HIV-infected patients undergoing SOT.

Microsporidiosis in solid organ transplant recipients: two Enterocytozoon bieneusi cases and review.

Microsporidiosis first came to prominence as an opportunistic infection in patients with acquired immunodeficiency syndrome. Microsporidia are now emerging pathogens responsible for severe diarrhea during solid organ transplantation. Two main clinical entities can be identified: infection by Enterocytozoon bieneusi, causing diarrhea with limited treatment options; and infection by Encephalitozoon intestinalis, which may disseminate and usually responds to albendazole treatment. We describe here 2 cases of microsporidiosis caused by E. bieneusi in a renal and a liver transplant recipient, respectively, in whom complete clinical efficacy of a short course of fumagillin therapy was obtained. Long-term microbiological eradication was assessed using classical methods and monitored using a real-time quantitative polymerase chain reaction-based method. Both patients experienced drug-induced thrombocytopenia, which resolved after withdrawal of the treatment. We also review the 18 other previously reported cases of microsporidiosis in transplant recipients. In case of persistent diarrhea in solid organ transplant patients, microsporidiosis should be considered. Based on the present experience, treating E. bieneusi infection with 7 days of fumagillin therapy is adequate to eradicate E. bieneusi in this context.

APRI and FIB-4 Scores Are Useful After Liver Transplantation Independently of Etiology.

Noninvasive liver fibrosis scores are evaluated in hepatitis C virus-infected patients but are less known in liver transplant recipients. Fibrosis is a frequent, multifactorial event in these patients. This preliminary retrospective study reviewed the diagnostic performance of 3 simple scores for liver fibrosis in transplant patients: namely, APRI (aspartate aminotransferase to platelet ratio index), FORNS (platelets, gamma-glutamyltransferase, patient age, and cholesterol), and FIB-4 (patient age, aspartate aminotransferase, alanine aminotransferase, and platelets). Ninety-four biopsies were collected from 50 liver transplant recipients at a mean period after orthotopic liver transplantation (OLT) of 30.7 months (range, 12-108 months). The indications for OLT were hepatitis C in 23% of cases, hepatitis B in 14%, alcoholic disease in 33%, cholestatic disease in 19%, and others in 11%. According to the Metavir classification, 72% of biopsies revealed no significant histological fibrosis (F0-1 = group 1) and 28% showed significant fibrosis (F2-4 = group 2). A correlation was observed between the histological stage of fibrosis and albumin, gamma-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, and hyaluronic acid levels. APRI and FIB-4 correlated significantly with the histological stage of fibrosis both globally and in the subgroup of nonhepatitis C liver recipients. When APRI and FIB-4 tests were applied to predict fibrosis (area under the receiver operating characteristic curve), the results were 0.87 and 0.78, respectively. Values were not significant with the FORNS test. In conclusion, APRI and FIB-4 enabled accurate prediction of significant fibrosis after OLT. In the nonhepatitis C subgroup, we found similar predictive performances. These simple scores may be applied in clinical practice in the context of follow-up after OLT independent of hepatitis C status.

Significance of serum tumor markers carcinoembryonic antigen, CA 19-9, CA 125, and CA 15-3 in pre-orthotopic liver transplantation evaluation.

Tumor markers are elevated in a variety of nonneoplastic clinical situations, including liver diseases. Their sensitivity and specificity are lower for tumor screening in these cases. In this study, we investigated the frequency and significance of elevated tumor markers in the pre-orthotopic liver transplantation (OLT) evaluation among patients with end-stage liver disease who did not develop tumors after a long follow-up post-OLT. We performed a retrospective analysis of clinical and biological parameters of 100 OLT candidates comparing data for CA 125, CA 19-9, CA 15-3, and carcinoembryonic antigen (CEA) levels. CA 125, CA 19-9, CA 15-3, and CEA levels were elevated in 59%, 53%, 29%, and 28% of cases, respectively. CA 125, CA 15-3, and CEA were associated with disease severity (Child-Pugh classification). CA 125 was also elevated among patients with ascites, esophageal varices, or alcohol-related cirrhosis. Elevated CA 19-9 levels were associated with increased CA 15-3 and CEA levels. CA 15-3 levels were also increased among patients with elevated alkaline phosphatase, while elevated CEA was related to ascites, bilirubin, and prothrombin time (PT) levels, as well as alcohol-related cirrhosis. There was no association between hepatocellular carcinoma and tumor markers. In conclusion, CA 125, CA 19-9, CA 15-3, and CEA were frequently elevated among end-stage liver disease patients. These elevations were not associated with tumor diseases in this population.

Everolimus de novo in liver transplantation.

The safety and tolerability of everolimus has been evaluated in a randomized, phase II trial, comparing 3 doses of everolimus to a placebo, in association with cyclosporine and corticosteroids, after liver transplantation. There were no significant differences between groups in the rates of the composite end point (graft failure, biopsy-proven acute rejection, graft loss, death, or loss to follow-up) or its individual components. Although there were lower rates of treated acute rejection and mortality with the higher dosages (2 and 4 mg/day), these did not reach statistical significance. Interestingly, freedom from rejection correlated with trough blood levels of everolimus: patients with levels of 3 ng/mL or less had rejection rates 3-fold higher than patients with levels exceeding 3 ng/mL. All graft losses and most deaths were associated with typical posttransplant complications, not with study medication and not due to hepatic artery thrombosis. There were no clear dose-related differences among groups for hematology parameters. After transplantation, renal function declined to a similar extent in all 4 groups. The overall incidence of infection was comparable between groups (61-77%). Although the interpretation of the results of this trial is hampered by the small sample sizes of patient groups (about 30 in each group) and the high dropout rates (about 50%), this study suggests that everolimus is an effective immunosuppressive agent with an acceptable patient tolerance and safety profile after liver transplantation.

[Vaccinations for the traveling adult solid organ transplant recipient (excluding hematopoietic stem cell transplant recipients)]. / Vaccinations du voyageur adulte transplanté d'organes (à l'exclusion des receveurs de cellules souches hématopoïétiques).

Progress in transplantation technique has offered a growing number of solid organ transplant recipients the opportunity to travel to tropical and low-income countries. The issue of vaccine-preventable diseases is a challenging question in immunocompromised patients including those with solid organ transplant. Since the response to vaccines is weakened in case of chronic organ failure, candidates should be vaccinated early in the course of the disease. Clinicians should implement a vaccinal strategy until the patient is scheduled for transplantation and monitor its efficacy by serological assays. Live attenuated vaccines (such as yellow fever, measles-mumps-rubella, or chicken pox) are contra-indicated in solid organ transplant recipients and, when indicated, should be administered prior to transplantation, particularly in foreign-born patients highly likely to visit friends and relatives in endemic areas. Vaccinations for transplant recipients considering international travel should be realized according to the risk of acquiring vaccine-preventable diseases but also on both tolerance and immune response which are affected by degree and duration of immunosuppression, comorbidities, and type of organ transplanted. Routine and specific vaccinations for solid organ transplant recipients, as well as travel-related vaccination (such as hepatitis A, typhoid, meningococcal meningitis, rabies, tick-born encephalitis, Japanese encephalitis, and cholera) should be considered during a specific pretravel medical consultation. However, vaccination should be avoided in the 6 months following transplantation when patients are usually receiving the highest doses of immunosuppressive drugs. In this comprehensive review, we provide vaccination schedules based on published studies and guidelines for vaccination of solid organ transplant recipients.

Regulatory T cell therapy: An option to induce operational tolerance in liver transplantation.

Regulatory T cells (Treg) may play an important role in operational (clinical) tolerance (OT), a stable graft function without immunosuppression in an otherwise immunocompetent host, that is spontaneously observed in some patients many years after transplantation. Several ongoing clinical trials are currently testing the effects of donor-specific or non-specific Treg infusion with the goal to induce this state of OT a few months after liver transplantation (LT). The preliminary results of two of these trials have been recently published, and raise a number of comments and issues: (1) These two papers demonstrate that a 100 to 1000-fold ex-vivo expansion of Treg is possible in humans after 2 weeks of culture. The optimal human Treg dose is however not clearly established, and might be higher than the dose that would be expected from translating murine data. (2) A lot of concerns are remaining regarding the Treg purity before expansion, the Treg stability during in vitro culture and the in vivo fate of infused cells. A strict monitoring of Treg should thus be done at each step. (3) Since Treg may play a detrimental role in some conditions, such as viral diseases and cancer, potential LT recipients with such diseases should probably be excluded from the initial trials of Treg infusion. (4) The follow-up of tolerant liver recipients should include repeated liver biopsies and detection of autoantibodies and humoral response, in addition to conventional liver graft assessment, in order to prevent the development of immune complications related to immunosuppression withdrawal. (5) The final issue raised by Treg therapy in LT is the choice of the immunosuppressive regimen used before tapering or withdrawal, appropriate to preserve OT establishment.

Response to antiviral treatment in hepatitis C virus-associated marginal zone lymphomas.

A link between chronic hepatitis C virus (HCV) infection and low-grade B-cell lymphomas has been suggested by epidemiological studies. Marginal zone lymphomas (MZLs) including splenic lymphomas with villous lymphocytes are among the most frequently reported subgroups in the setting of chronic HCV infection. In this study, we examined the effect of antiviral treatment in eight patients with HCV-associated MZL. We found that five out of eight patients have responded to interferon alpha and ribavirin. In some cases, hematologic responses were correlated to virologic responses. In addition, we report a case of large granular lymphocyte leukemia occurring in association with MZL and HCV, and responding to interferon and ribavirin. We suggest that there is an etiologic link between HCV and antigen-driven lymphoproliferative disorders.

Ursodeoxycholic acid (UDCA) in the treatment of chronic cholestatic diseases.

Several studies suggest that UDCA treatment has beneficial effects in chronic cholestatic diseases. We designed a controlled trial to assess the efficacy and tolerance of UCDA in primary biliary cirrhosis (PBC): 73 patients received UDCA (13-15 mg/kg per day) and 73 a placebo. One side-effect required interruption of therapy in each group. The relative risk of treatment failure (doubling of the bilirubin level or occurrence of a severe complication of cirrhosis) was 3 times higher in the placebo group. Pruritus resolved in 40% of the patients of UDCA group vs 19% in placebo group. Biological and histological parameters significantly improved in the patients receiving UDCA. Unexpectedly, immune parameters, including IgM levels and anti-mitochondrial antibody titers, also improved. The Mayo risk score was significantly different between the two groups at one and two years, suggesting that UDCA could prolong survival in PBC. Recent studies suggest that UDCA could have immunoregulating properties. Abnormal MHC class I expression by hepatocytes, observed in PBC, was dramatically reduced by UDCA treatment. Cholestasis itself induces hepatic MHC expression: hepatocyte MHC class I expression was present in 6/6 cholestatic patients vs 0/8 control subjects. Experimental cholestasis in the rat induced MHC class I expression. Cyclosporin or corticosteroids had no effect on this overexpression, suggesting that an immune mechanism is not involved in this phenomenon. To assess the effect of bile acids on MHC expression, human hepatocytes were incubated with bile acids. Chenodeoxycholic acid (CDCA) (an endogenous bile acid) but not UDCA induced a dose-dependent MHC class I hyperexpression. UDCA suppressed the CDCA-induced MHC hyperexpression.(ABSTRACT TRUNCATED AT 250 WORDS)

Target antigens of hyperacute xenogeneic rejection in the rat to guinea pig to rat discordant combinations.

The increasing shortage in allografts has led to a renewed interest in xenogeneic transplantation. Discordant combinations are characterized by hyperacute rejection partly due to the presence of natural antixenogeneic antibodies in the recipient. The aim of this work was to characterize the target antigens, using 2 discordant models. In the rat into guinea pig model, analysis of organ homogenates by immunoblotting revealed numerous bands. Some of these bands were organ specific, whereas others, namely in the 55-kDa region, were detected in liver, heart, lung, and kidney. Using membrane extracts of liver cells or of aortic endothelial cells, only bands of 55 kDa were revealed. No band could be seen using extracts of isolated hepatocytes. Two bands of 55 kDa disappeared after preabsorption of guinea pig sera on the various rat tissue homogenates, suggesting that they represent xenoantigens common to these tissues. In order to investigate the in vivo relevance of these 55-kDa antigens, isolated rat livers were perfused with decomplemented guinea pig sera. Eluates revealed one single print of 55 kDa on rat tissue homogenates. Finally, preincubation of rat mononuclear cells with various xenogeneic sera did not inhibit the binding of mAb specific for rat class I or class II MHC antigens, suggesting that the latter are not recognized by natural xenoantibodies. In the guinea pig to rat model, the antigens detected had a molecular mass ranging from 95 to 110 kDa. Absorption and perfusion experiments also showed that these antigens were common to various tissues and involved in the binding of rat natural antibodies ex vivo. In conclusion, our results indicate that rat xenoantigens of about 55 kDa are recognized by guinea pig natural antibodies, while guinea pig xenoantigens of 95-110 kDa are bound by rat natural antibodies. These antigens are common to liver, heart, lung, and kidney, are borne by endothelial cells, and cannot be found on hepatocytes.

Enhanced risk of steroid-resistant acute rejection following pretransplant steroid therapy in liver graft recipients.

Most episodes of acute rejection will resolve after steroid therapy without detrimental consequences on the liver allograft. However, steroid-resistant acute rejection may induce irreversible lesions of the graft and is associated with an increased risk of chronic rejection. The aim of this study was to determine whether there were predictive factors for steroid-resistant acute rejection after liver transplantation. A total of 108 adult liver recipients with a follow-up of at least 2 years have been analyzed; sixty-two (57%) patients had at least one episode of acute rejection. The rates of steroid resistance were 35%, 52% and 83% after a first (n = 62), second (n = 25), or third (n = 7) episode of acute rejection, respectively. Steroid resistance of acute rejection was significantly associated with a past history of pretransplant steroid therapy (P = 0.004). High levels of ALT (P = 0.03) and serum bilirubin (P = 0.002) were also predictive of steroid-resistant acute rejection. Eight (7%) patients eventually developed chronic rejection. Predictive factors for chronic rejection included steroid-resistant acute rejection (P = 0.01), recurrent acute rejection (P = 0.03), and CMV infection (P = 0.01). In conclusion, this study suggests that pretransplant steroid administration or high levels of ALT and bilirubin in rejecting patients are risk factors for steroid resistance and should lead to aggressive antirejection therapy without delay.

The role of natural IgM in the hyperacute rejection of discordant heart xenografts.

The mechanism of xenograft hyperacute rejection in discordant species combinations remains controversial. The purpose of this work was to study the role of natural antibodies in the hyperacute rejection of guinea pig hearts transplanted into rats, a highly discordant combination. This study was conducted in vitro, ex vivo, and in vivo. The endothelial cells of the graft being the first targets damaged in the process of hyperacute rejection, the binding of rat natural antibodies to guinea pig endothelial cells was studied by immunofluorescence. The study was carried out in vitro on guinea pig endothelial cells in culture, and ex vivo on isolated guinea pig hearts perfused with either rat serum or immunoglobulins or immunoglobulin fragments bearing the antigen-binding site. In vitro and ex vivo, rat natural IgM were found to bind specifically to guinea pig endothelial cells, since IgM fragments bearing the antigen-binding site (Fab mu and Fab' mu) could be detected on these cells. IgM fragments were able to inhibit the fixation of native IgM molecules. In contrast, rat IgG only bound to endothelial cells through Fc portions. Thus rat natural IgM might play a role in hyperacute rejection by binding to the graft endothelial cells and triggering the complement cascade activation. In order to test the role of natural IgM in vivo, isolated guinea pig hearts were first perfused with rat Fab' mu, which inhibit the binding of IgM and are unable to activate the complement cascade. These hearts were then transplanted into Lewis rats. The rejection time of Fab' mu-perfused guinea pig hearts was prolonged compared with hearts perfused with buffer or IgG F(ab')2. Therefore, in the guinea pig to rat combination, preventing the binding of the recipient's natural IgM to the graft endothelium delays the hyperacute rejection. In addition, natural IgM are likely to play a greater role than natural IgG.

Common bile duct stenosis caused by chronic pancreatitis after liver transplantation for alcoholic cirrhosis.

BACKGROUND: The prevalence of chronic pancreatitis in patients with alcoholic cirrhosis ranges from 7% to 11% and is not considered a contraindication for liver transplantation. METHODS: Among 59 liver transplant recipients grafted for alcoholic cirrhosis, we report two observations of common bile duct stenosis due to chronic pancreatitis. RESULTS: In both cases, pretransplant work-up disclosed no clinical or radiological evidence of chronic pancreatitis. The diagnosis of common bile duct stricture was made 6 and 60 months after liver transplantation. One patient was reoperated upon, and his choledochocholedochostomy was converted into a Rouxen-Y choledochojejunostomy. The second patient experienced metastatic laryngeal carcinoma and died before reoperation. CONCLUSIONS: These observations suggest that common bile duct stricture caused by chronic pancreatitis may occur after liver transplantation for alcoholic cirrhosis, even after a long-standing history of abstinence.

Evidence for survival and metabolic activity of encapsulated xenogeneic hepatocytes transplanted without immunosuppression in Gunn rats.

BACKGROUND: Hepatocyte transplantation could be an alternative to whole organ transplantation to correct enzymatic disorders. To this end, it would be of major importance to use xenogeneic cells without immunosuppression. The aim of this study was to investigate the survival and metabolic activity of encapsulated xenogeneic hepatocytes in the absence of immunosuppression. For this purpose, we used Gunn rats genetically incapable of bilirubin conjugation. METHODS: Xenogeneic (from guinea pigs) and allogeneic (from Lewis rats) hepatocytes (2x10(7)) were isolated, macroencapsulated in hydrogel hollow fibers made with an acrylonitrile-sodium methallyl-sulfonate copolymer, and transplanted into the peritoneum of Gunn rats without any immunosuppression. Plasma bilirubin levels were evaluated weekly. Bilirubin conjugates in bile and cell morphology were studied after 5 and 12 weeks, respectively. RESULTS: In Gunn rats transplanted with xenogeneic hepatocytes, a significant decrease in the serum bilirubin level was observed between 3 and 9 weeks after transplantation when compared with controls transplanted with empty hollow fibers: it fell to 62% of the initial level at weeks 5-7 (P < 0.01). A comparable result was observed in Gunn rats transplanted with encapsulated allogeneic cells. Bilirubin conjugates were observed in bile samples of rats transplanted with encapsulated hepatocytes. After explantation, hollow fibers appeared intact with minimal fibrosis. Cell viability and hepatocyte morphology were preserved. CONCLUSIONS: These results indicate that macroencapsulated xenogeneic hepatocytes can survive and remain functional for more than 2 months when transplanted in vivo in the absence of any immunosuppression.

Hyperacute xenograft rejection in the swine-to-human donor-recipient combination. In vitro analysis of complement activation.

Complement activation is central to the rejection of discordant xenografts. In order to assess the respective roles of direct and alternative pathways, an in vitro model of hyperacute rejection in the swine-to-human donor-recipient combination was designed, using a complement-dependent cytotoxicity test with swine endothelial cells in culture as targets, and fresh human serum as the source of xenogeneic antibodies and complement. The cytotoxic activity of the sera was evaluated by a colorimetric assay using (3-[4,5-dimethyldiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). Pure human serum lysed 58 +/- 5% of swine endothelial cells. Selective inhibition of the direct pathway by adding EGTA to the serum reduced cytolysis to 51 +/- 2% (P < 0.01 versus normal serum). Similarly, when using C1q-deficient human sera, only 37 +/- 7% of swine endothelial cells were killed (P < 0.001 versus normal serum). When the alternative pathway was selectively inhibited by heating for 20 min at 50 degrees C, the lytic activity of human serum dropped to 42 +/- 5% (P < 0.001 versus normal serum). Factor B-deficient human serum could only lyse 42 +/- 10% of porcine endothelial cells (P < 0.001 versus normal serum). Syngeneic normal swine serum and heat-inactivated serum were not cytotoxic. Mixing serum with deficient direct pathway and serum with deficient alternative pathway restored the cytotoxicity to normal levels. Similarly, the cytotoxic activity of deficient serum supplemented with purified C1q or factor B at physiological concentrations reached that of normal human serum. In this model of in vitro hyperacute rejection, both pathways of complement activation are involved, suggesting that regimens designed to inhibit hyperacute rejection of swine xenografts into humans should take into account the dual activation of complement in this donor-recipient combination.

Comparison between aortic and sinusoidal liver endothelial cells as targets of hyperacute xenogeneic rejection in the pig to human combination.

Endothelial cells of aortic origin are usually used in vitro as targets of hyperacute xenogeneic rejection, although endothelial cells from organs may have different properties. The sensitivities of aortic and liver endothelial cells to hyperacute xenogeneic rejection were compared in the pig to human combination. Sinusoidal liver endothelial cells were isolated and purified by collagenase perfusion of pig livers, sedimentation on a percoll gradient and selective adherence. Purity and viability of isolated liver endothelial cells after adherence were 85+/-6% and >95%, respectively. Endothelial cells from pig aortae (purity and viability >95%) were isolated by scraping. Immunoblotting analysis of xenoantigens on liver and aortic endothelial cell membranes preparations showed identical patterns. The strongest bands revealed by human IgM were located between 110 and 135 kD, while human IgG detected two major bands at 115 and 75kD. The membrane expression of xenoantigens recognized by human sera, analyzed by flow cytometry, was significantly lower on liver than on aortic endothelial cells (IgM: P=0.0006; IgG: P=0.0009). However, the complement-dependent cytotoxic activity of human sera was the same whether liver (54.5+/-1.4%) or aortic endothelial cells (50.0+/-4.2%) were used as targets. Taken together, those results allow the use of aortic instead of sinusoidal liver endothelial cells in the characterization of pig antigens recognized by human natural antibodies.

Reversal of the vasoconstrictor step of hyperacute xenogeneic rejection of the liver by endothelin antagonists.

The role of endothelin in the initial vasoconstrictor step of hyperacute xenogeneic rejection was investigated. Isolated rat livers were perfused in recirculation. Perfusion with human sera provided an ex vivo model of hyperacute rejection in a discordant combination. Perfusion of 10% xenogeneic serum induced a marked (70%) and sustained reduction of the liver flow and induced the release of endothelin into the perfusion medium. In contrast, perfusion of 10% allogeneic serum or of 10% decomplemented human serum induced a weak (25%) and transient reduction of the liver flow and induced the release of minimal amounts of endothelin. The simultaneous administration of BQ 123 and BQ 788, the respective antagonists of ET(A) and ET(B) endothelin receptors, or that of bosentan, a mixed ET(A)/ET(B) antagonist, antagonized the vasoconstrictor effect of 10% xenogeneic human serum, as well as that of 10(-9) M endothelin-1. The vasoconstrictor effects of xenogeneic serum on liver circulation are, at least partly, mediated through the release of endothelin by the graft.