Xenotransplantation-the current status and prospects.

Introduction: There is a continuing worldwide shortage of organs from deceased human donors for transplantation into patients with end-stage organ failure. Genetically engineered pigs could resolve this problem, and could also provide tissues and cells for the treatment of conditions such as diabetes, Parkinson's disease and corneal blindness. Sources of data: The current literature has been reviewed. Areas of agreement: The pathobiologic barriers are now largely defined. Research progress has advanced through the increasing availability of genetically engineered pigs and novel immunosuppressive agents. Life-supporting pig kidneys and islets have functioned for months or years in nonhuman primates. Areas of controversy: The potential risk of transfer of a pig infectious microorganism to the recipient continues to be debated. Growing points: Increased attention is being paid to selection of patients for initial clinical trials. Areas timely for developing research: Most of the advances required to justify a clinical trial have now been met.

The impact of proposed changes in liver allocation policy on cold ischemia times and organ transportation costs.

Changes to the liver allocation system have been proposed to decrease regional variation in access to liver transplant. It is unclear what impact these changes will have on cold ischemia times (CITs) and donor transportation costs. Therefore, we performed a retrospective single center study (2008-2012) measuring liver procurement CIT and transportation costs. Four groups were defined: Local-within driving distance (Local-D, n = 262), Local-flight (Local-F, n = 105), Regional-flight <3 h (Regional <3 h, n = 61) and Regional-Flight >3 h (Regional >3 h, n = 53). The median travel distance increased in each group, varying from zero miles (Local-D), 196 miles (Local-F), 384 miles (Regional <3 h), to 1647 miles (Regional >3 h). Increasing travel distances did not significantly increase CIT until the flight time was >3 h. The average CIT ranged from 5.0 to 6.0 h for Local-D, Local-F and Regional <3 h, but increased to 10 h for Regional >3 h (p < 0.0001). Transportation costs increased with greater distance traveled: Local-D $101, Local-F $1993, Regional <3 h $8324 and Regional >3 h $27 810 (p < 0.0001). With proposed redistricting, local financial modeling suggests that the average liver donor procurement transportation variable direct costs will increase from $2415 to $7547/liver donor, an increase of 313%. These findings suggest that further discussion among transplant centers and insurance providers is needed prior to policy implementation.

Transmission of Balamuthia mandrillaris through solid organ transplantation: utility of organ recipient serology to guide clinical management.

A liver, heart, iliac vessel and two kidneys were recovered from a 39-year-old man who died of traumatic head injury and were transplanted into five recipients. The liver recipient 18 days posttransplantation presented with headache, ataxia and fever, followed by rapid neurologic decline and death. Diagnosis of granulomatous amebic encephalitis was made on autopsy. Balamuthia mandrillaris infection was confirmed with immunohistochemical and polymerase chain reaction (PCR) assays. Donor and recipients' sera were tested for B. mandrillaris antibodies. Donor brain was negative for Balamuthia by immunohistochemistry and PCR; donor serum Balamuthia antibody titer was positive (1:64). Antibody titers in all recipients were positive (range, 1:64-1:512). Recipients received a four- to five-drug combination of miltefosine or pentamidine, azithromycin, albendazole, sulfadiazine and fluconazole. Nausea, vomiting, elevated liver transaminases and renal insufficiency were common. All other recipients survived and have remained asymptomatic 24 months posttransplant. This is the third donor-derived Balamuthia infection cluster described in solid organ transplant recipients in the United States. As Balamuthia serologic testing is only available through a national reference laboratory, it is not feasible for donor screening, but may be useful to determine exposure status in recipients and to help guide chemotherapy.

What liver transplant outcomes can be expected in the uninsured who become insured via the Affordable Care Act?

Our study objective is to measure the survival impact of insurance status following liver transplantation in a cohort of uninsured "charity care" patients. These patients are analogous to the population who will gain insurance via the Affordable Care Act. We hypothesize there will be reduced survival in charity care compared to other insurance strata. We conducted a retrospective study of 898 liver transplants from 2000 to 2010. Insurance cohorts were classified as private (n = 640), public (n = 233) and charity care (n = 23). The 1, 3 and 5-year survival was 92%, 88% and 83% in private insurance, 89%, 80% and 73% in public insurance and 83%, 72% and 51% in charity care. Compared to private insurance, multivariable regression analyses demonstrated charity care (HR 3.11, CI 1.41-6.86) and public insurance (HR 1.58, CI 1.06-2.34) had a higher 5-year mortality hazard ratio. In contrast, other measures of socioeconomic status were not significantly associated with increased mortality. The charity care cohort demonstrated the highest incidence of acute rejection and missed clinic appointments. These data suggest factors other than demographic and socioeconomic may be associated with increased mortality. Further investigations are necessary to determine causative predictors of increased mortality in liver transplant patients without private insurance.

Successful reversal of streptozotocin-induced diabetes with stable allogeneic islet function in a preclinical model of type 1 diabetes.

The recent focus on islet transplantation as primary therapy for type 1 diabetes has heightened interest in the reversal of type 1 diabetes in preclinical models using minimal immunosuppression. Here, we demonstrated in a preclinical rhesus model a consistent reversal of all measured glycemic patterns of streptozotocin-induced type 1 diabetes. The model used single-donor islet transplantation with induction of operational tolerance. The term "operational tolerance" is used to indicate durable survival of single-donor major histocompatibility complex (MHC)-mismatched islet allografts without maintenance immunosuppressive therapy and without rejection or loss of functional islet mass or insulin secretory reserve. In this operational tolerance model, all immunosuppression was discontinued after day 14 posttransplant, and recipients recovered with excellent health. The operational tolerance induction protocol combined peritransplant anti-CD3 immunotoxin to deplete T-cells and 15-deoxyspergualin to arrest proinflammatory cytokine production and maturation of dendritic cells. T-cell deficiency was specific but temporary, in that T-cell-dependent responses in long-term survivors recovered to normal, and there was no evidence of increased susceptibility to infection. Anti-donor mixed lymphocyte reaction responses were positive in the long-term survivors, but all showed clear evidence of systemic T-helper 2 deviation, suggesting that an immunoregulatory rather than a deletional process underlies this operational tolerance model. This study provides the first evidence that operational tolerance can protect MHC nonhuman primate islets from rejection as well as loss of functional islet mass. Such an approach has potential to optimize individual recipient recovery from diabetes as well as permitting more widespread islet transplantation with the limited supply of donor islets.

17beta-estradiol differentially activates mitogen-activated protein-kinases and improves survival following reperfusion injury of reduced-size liver in mice.

Ischemia-reperfusion injury (I/R-I), which is unavoidable in liver transplantation, impairs liver regeneration and predisposes to liver failure. The three major mitogen-activated protein-kinases (MAPKs): ERK, p38, and JNK, are critical in the transmission of signals triggered by proinflammatory cytokines, by stress, and by growth factors. JNK and p38alpha activation have been associated with apoptosis; p38beta with cell survival; and ERK with proliferation. Previous studies have demonstrated gender dimorphism in hepatocellular dysfunction after experimental trauma and hemorrhage. Female mice are protected to a much greater extent from I/R-I than male mice. We assessed the effects of 17beta-estradiol (17beta-E) on liver function, host survival, and cellular activation of MAPK in a murine model of I/R-I in reduced-size livers. C57BL/6 mice were subjected to 45 minutes of warm ischemia (70% of the liver mass). After reperfusion, the nonischemic lobes were excised. Vehicle, 17beta-E or the estrogen receptor antagonist ICI-182780, was delivered 1 hour before the injury. We evaluated AST and apoptosis as well as activation of JNK, p38, and ERK. Female mice showed a lower level of hepatocellular injury (AST = 445 +/- 82 IU/L) after I/R-I compared with male mice (AST = 1400 +/- 210). 17beta-E decreased the liver injury in male mice (AST = 522 +/- 77), an effect that was partially reversed by ICI-182,780 (910 +/- 92). A higher rate of apoptosis was observed in male animals given saline (enrichment factor = 7.22 +/- 0.8) versus those treated with 17beta-E (5.85 +/- 0.3, P < .05). A significant increase in liver regeneration, as assessed by the percentage of liver weight/body weight was demonstrated in females (184% +/- 24%) and male mice given 17beta-E (168% +/- 22%) compared with male mice given vehicle (9% +/- 4%). 17beta-E significantly down-regulated JNK and p38alpha activities, whereas I/R-I promoted p38beta and ERK activation. These results suggest that the cytoprotective effects of 17beta-E on I/R-I to reduced-size livers are associated with selective modulation of MAPK kinases.

Elevated plasma levels of S-100b protein in schizophrenic patients.

BACKGROUND: In this study, we examined the possibility that structural damage to the brain may play a role in the pathogenesis of schizophrenia. METHODS: We compared plasma levels of S-100b protein in 20 patients with schizophrenic psychosis and 20 age- and gender-matched healthy blood donors. Concentrations of S-100 protein were determined by microtiter-based immunofluorometric assay detecting predominantly S-100b. RESULTS: Mean concentrations of S-100b protein in blood were significantly (p < or = .001) higher in schizophrenic patients (0.165 +/- 0.138 microgram/L) compared to control subjects (0.054 +/- 0.031 microgram/L). Levels did not correlate with age of onset or duration of psychosis. CONCLUSIONS: Our findings indicate that patients with schizophrenia may suffer ongoing structural damage to cells of the central nervous system, and that the concentration of S-100b protein in plasma may help to identify clinical subgroups in schizophrenia.

Selective enhancement of beta cell activity by preparation of fetal pancreatic proislets and culture with insulin growth factor 1.

A limited collagenase digestion was used to prepare human fetal pancreatic (HFP) proislets that maintained in vitro and in vivo functional viability. Digestion times, collagenase concentrations, and culture conditions were optimized by assessing insulin release in response to low glucose (3.3 mM), high glucose (16.7 mM), and high glucose plus theophylline (10 mM). A stimulation index (ng insulin release in low glucose/ng insulin in high glucose plus theophylline) and insulin production (ng insulin/mg tissue) were used to determine functional viability. A media change 24 hr after digestion resulted in a marked increase in mean stimulation index (6.18 +/- 1.2 vs. 3.12 +/- 0.9). IGF-1 was found to enrich beta-cell viability as demonstrated by insulin-specific immunoperoxidase staining and insulin release in response to HGT (36.3 +/- 7.7 ng/mg tissue vs. 18.1 +/- 5.2 ng/mg tissue). Immunohistologic staining of proislets suggested selective enrichment of beta cells. Diabetic BALB/c/nu/nu transplanted with proislets cultured in the presence of IGF-1 (100 ng/ml) returned to normoglycemia in 6.7 +/- 3.1 weeks (range 4-11) versus 12.0 +/- 4.0 (range 10-15) in controls. Oral glucose tolerance test demonstrated in vivo serum glucose control equivalent to nondiabetic control mice. These findings suggest that HFP proislet preparation and culture with endocrine growth factors, specifically IGF-1, may provide a source of tissue with enriched beta-cell activity that may have decreased immunogenic load and is more suitable for clinical transplantation.

RS-61443--a new, potent immunosuppressive agent.

The immunosuppressive activity of RS-61443, a semisynthetic derivative of mycophenolic acid, was examined in 33 canine renal allografts. Initial studies established that triple therapy consisting of 20 mg/kg RS-61443 in combination with 5 mg/kg cyclosporine and 0.1 mg/kg methylprednisolone was the optimal combination to prevent graft rejection. The median survival time was 8.1 +/- 1.2 days in dogs without treatment (n = 5), 8.5 +/- 1.7 days in the treatment control group (CsA 5 mg/kg, MP 0.1 mg/kg; n = 6), 36.0 +/- 9.6 days with RS-61443 monotherapy (40 mg/kg; n = 6); and 122.4 +/- 38.75 days with triple therapy (n = 16). Graft prolongation was statistically significant when compared with controls (P less than 0.05 and 0.002, respectively). Six recipients in the triple therapy group survived over 150 days without major adverse effects. Long-term administration of RS-61443 (20 mg/kg/day) did not cause nephrotoxicity, hematotoxicity, or hepatotoxicity, with the exception of a slight elevation of the alkaline phosphatase levels. Gastrointestinal symptoms including gastritis, diarrhea, and anorexia were common, especially under 40 mg/kg RS-61443 monotherapy, and appeared to be dose-related. Despite its immunosuppressive activity, an increased susceptibility to bacterial or viral infections was not observed. Histological studies of the kidney grafts revealed slight interstitial cell infiltration without vascular or glomerular damage.

Tacrolimus (FK506) and mycophenolate mofetil combination therapy versus tacrolimus in adult liver transplantation.

BACKGROUND: Mycophenolate mofetil (MMF) prolongs allograft survival in experimental animals, prevents acute rejection in humans, and has recently been approved for use in renal transplantation in combination with cyclosporine. Tacrolimus (Prograf) has been shown to be effective for the prevention and treatment of allograft rejection in liver transplantation. However, there has been limited experience with the combination of tacrolimus and MMF in liver transplantation. METHODS: This retrospective pilot study examined the results in 130 primary, consecutive, adult liver transplants under two separate immunosuppressive protocols. Patients in the study group received MMF (1 g p.o. b.i.d.), tacrolimus (0.1 mg/kg p.o. b.i.d.), and a standard steroid taper. MMF was also tapered and then discontinued within 3 months of transplantation. A historical control received tacrolimus (0.15 mg/kg p.o. b.i.d.) and the same steroid taper. RESULTS: Pretransplant demographics, including creatinine, were not significantly different between the groups. The 6-month patient and graft survivals of 96.3% (control) versus 92.0% (study) were not significantly different. The incidence of acute rejection was 45.0% in the control group versus 26.0% in the study group (P = 0.03). The study group had a lower incidence of rejection (mean episodes/patient +/- SEM): 0.28+/-0.07 vs. 0.61+/-0.10 (P = 0.007). All of the study group members responded to high-dose steroids. In the control group, three patients required monoclonal antibody therapy and two patients required the addition of MMF. The incidence of cytomegalovirus was similar in the study group and the control group (13.8% vs. 10.0%, P = NS). Early renal function was better preserved in the tacrolimus/MMF group (mean creatinine +/- SEM): 1.09 mg/dl +/- 0.05 vs. 1.51 mg/dl +/- 0.08 at 30 days, P = 0.0001. The study design required dosing with less tacrolimus (mean mg/day +/- SEM), which was achieved at 1 week (23.2+/-0.7 vs. 13.5+/-0.5); 1 month (18.7+/-0.8 vs. 11.4+/-0.5); 3 months (14.5+/-0.6 vs. 9+/-0.5); and 6 months (11.6+/-0.6 vs. 8.2+/-0.6); P = 0.0001, for all time points. CONCLUSION: Combination therapy with tacrolimus and MMF may significantly reduce the incidence of acute liver allograft rejection, allow a significant reduction in tacrolimus dosage, and decrease the incidence of nephrotoxicity. Long-term analysis will be necessary to assess any increased risk of opportunistic infections.

The safety and efficacy of a two-dose daclizumab (zenapax) induction therapy in liver transplant recipients.

BACKGROUND: Induction therapy with daclizumab has been shown to be efficacious in the prevention of acute rejection in kidney transplant patients. The routine use of antibody induction therapy in liver transplantation has not gained widespread acceptance, except in the cases of renal insufficiency. The recent approval of daclizumab prompted us to initiate this pilot study using induction therapy in those patients at risk for developing posttransplant renal insufficiency. METHODS: This nonrandomized study examined the use of daclizumab in 39 of the last 97 liver transplants performed at the University of Alabama in Birmingham. The daclizumab group received 2 mg/kg intravenously before organ engraftment, and 38 of the 39 received 1 mg/kg intravenously on postoperative day 5. The control group consisted of the remaining 58 contemporary patients. Additional immunosuppression consisted of steroids, tacrolimus, or microemulsion cyclosporine in all patients and mycophenolate mofetil in selected patients. RESULTS: Pretransplant demographics were not significantly different between the groups. In the induction group there were significantly fewer males, 14 (36%) vs. 34 (59%) (P=0.03). They had greater renal insufficiency at the time of transplant, serum creatine 1.9+/-0.37 mg/dl vs. 0.8+/-0.5; P=0.0009, and more patients were at higher acuity (status 1 and 2A): 12 (31%) vs. 3 (5%) P=0.0006 than in the noninduction group. By postoperative day 7, renal function improved in the induction group such that it was not significantly different from the noninduction group and remained similar throughout the rest of the follow-up. The induction group also experienced significantly less acute rejection, 7 (18%) vs. 23 (40%) (P=0.02) than in the noninduction group in the first 6 months. The 1-, 3-, and 6-month patient survival rates were similar in the induction group, 97.4%, 97.4%, and 97.4%, vs. non-induction 94.8%, 93.0%, and 93% (P=NS). The incidence of cytomegalovirus, in the first 6 months, in the induction group was four (10%) vs. five (9%) (P=NS) in the noninduction group. CONCLUSION: In the pilot study, induction therapy with daclizumab was safe, facilitated improvement in renal function, and appeared to reduce the incidence of acute rejection. Combination therapy with daclizumab may be an important adjunct in immunosuppressive strategies for liver transplant recipients.

Long-term functional islet mass and metabolic function after xenoislet transplantation in primates.

BACKGROUND: Pancreatic islet transplantation (PIT) is an attractive alternative for patients with type I diabetes mellitus. PIT is not yet an effective clinical reality due in part to the high incidence of rejection and early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mortality. In the present study, the effects of PIT on glycemic parameters were assessed in spontaneously diabetic primates. METHODS: Five insulinopenic nonhuman primates (three Macacca fascicularis, one Ceropithecus aethiops, and one Macacca mulatta) were studied. All required twice-daily treatment with 4-10 U of insulin. For immunosuppression, the animals received anti-CD3-immunotoxin (100 microg/kg(initially infused 2 hr before transplantation and again on day +1), cyclosporine (CsA) (20 mg/kg(i.v./2 hr before transplantation), cyclosporine microemulsion (Neoral) 60 mg/kg/b.i.d. on days +1 to +3 with dose adjusted by blood levels, and methylprednisolone (15 mg/kg day 0 to +3). Three recipients were given islets from a single donor (M mulatta). The islets were prepared by a semiautomated technique using Liberase. A mean of 13,136 islet equivalents/kg was infused into the portal vein. Two animals (M fascicularis and M mulatta) were used as a diabetic, nontransplanted control. Several metabolic parameters were evaluated. RESULTS: All monkeys that underwent transplantation experienced reversal of diabetes mellitus with normalization of all diabetic glycemic parameters. In the nontransplanted primates given the same immunosuppression but no PIT, diabetic metabolic parameters were unchanged after 9 months of follow-up. In contrast, all three PIT recipients established fasting and nonfasting euglycemia within 1-2 weeks, and none required exogenous insulin after day 10. Normal intravenous glucose tolerance tests were observed at day 15, and no significant differences in the glucose disappearance rate (Kg) were observed at days 15, 45, 190, and 365 days after transplantation. The acute insulin response to glucose indicated no significant reduction of functional islet mass. CONCLUSIONS: PIT in severely insulinopenic type I diabetes mellitus primates resulted in restoration of normal glycemic parameters and durable islet mass. Operational tolerance was achieved with only 4 days of drug administration, sparing the animals from chronic exposure to potentially diabetogenic immunosuppressive drugs. These results offer an exciting new potential for type I diabetes mellitus treatment.

Pretransplant status and patient survival following liver transplantation.

The current liver allocation system has been criticized, since available organs go to those who are the most critically ill. These recipients have the poorest overall survival. Identification of pretransplant risk factors for mortality would allow better allocation of donor livers. This study was a retrospective analysis of pretransplant clinical and laboratory parameters and subsequent postoperative liver transplant mortality to identify high-risk subgroups. Of 347 consecutive consecutive primary liver transplant recipients, 59 (17%) met United Network for Organ Sharing (UNOS) criteria for status 4. Pretransplant factors included liver function, coagulation, albumin and ammonia levels, renal function, the presence of ascites, and etiology of liver disease. Overall 1-year patient survival was significantly worse for the status 4 recipients (89.0% vs. 67.7%; P = 0.01). In a univariate analysis of pretransplant risk factors for all recipients, elevated creatinine (P = 0.008) and ammonia (P = 0.009), and UNOS status 4 (P = 0.01) significantly affected postoperative survival. In multivariate analysis of pretransplant risk factors for all recipients, elevated creatinine (P = 0.003) was the only factor to significantly affect postoperative survival. In UNOS status 4 patients, univariate analysis of pretransplant risk factors and their influence on patient survival demonstrated that prolonged coagulation partial thromboplastin time (P = 0.04) and a higher grade of encephalopathy (P = 0.02) significantly affected postoperative survival. Advanced encephalopathy (P = 0.009) and prolonged partial thromboplastin time (P = 0.01) were the only significant risk factors by multivariate analysis in status 4 patients. In status 4 and non-status 4 patients, we identified risk factors that adversely affected patient survival, but their predictive power was insufficient to deny transplantation. Despite the higher mortality in status 4 recipients, their long-term survival is only slightly worse than that of non-status 4 patients. Until better predictors of survival are ascertained, our data do not support limiting the use of donor livers in UNOS status 4 recipients.

Successful extrarenal transplantation from non-heart-beating donors.

The current organ shortage has made utilization of organs from less-than-ideal donors more common. Although several transplant centers use kidneys from non-heart-beating donors (NHBDs), there has been reluctance to extend the use of these donors to extrarenal organs. Of the 130 donors referred to our organ procurement organization between January 1993 and May 1994, 16 (12.3%) were NHBDs. Organ retrieval from 10 of these resulted in extrarenal donation, 5 resulted in renal donation only, and 1 resulted in no retrieval as a result of prolonged warm ischemia (> 2 hr). A total of 39 organs were transplanted from these NHBDs. A rapid en bloc retrieval technique was used for extrarenal NHBDs. The mean warm ischemic time was 15.4 min; preservation times were similar for both NHBDs and heart-beating donors. After liver transplantation (n = 5), one episode of primary nonfunction that was technical in origin required retransplantation. Following simultaneous pancreas-kidney transplantation (n = 6), all patients were insulin independent and free of graft pancreatitis; one patient required hemodialysis (16.7%). After isolated renal transplantation (n = 21), 3 patients (14.3%) required hemodialysis. Three of 4 liver recipients are alive after a mean follow-up period of 12.7 months; all simultaneous pancreas-kidney and renal transplant recipients are alive after a mean follow-up period of 8.4 and 8.3 months, respectively. Three liver allografts, 5 pancreas and kidney allografts, and 19 renal allografts are functioning. The lung allograft was lost to rejection 81 days after transplantation; however, the recipient is alive 3 months after retransplantation. Our results demonstrate that in controlled situations, extrarenal organs can be utilized from NHBDs and can be expected to function similarly to organs retrieved from heart-beating donors. We increased the number of transplanted organs by 8.6% using NHBDs for both renal and extrarenal donation. Continued application of these techniques will likely further increase the number of organs retrieved for transplantation.

Tolerability and side effects of anti-CD3-immunotoxin in preclinical testing in kidney and pancreatic islet transplant recipients.

INTRODUCTION: Anti-CD3-immunotoxin (alpha-CD3-IT) promotes allograft tolerance in nonhuman primates owing to efficient depletion of sessile and circulating T cells. Common side effects of vascular leak syndrome, hepatotoxicity, and nephrotoxicity have limited tolerability of other immunotoxins. We report on preclinical studies of alpha-CD3-IT-related side effects. METHODS: Normal rhesus monkeys received a kidney transplant and alpha-CD3-IT alone (on day -to +2) or in combination with brief peritransplant adjunctive immunosuppressive therapy. Some received donor CD34+ cells. Blood chemistries, complete blood count, weight, liver, and kidney biopsies were examined for immunotoxin-related changes. Five spontaneously diabetic primates also received alpha-CD3-IT, three of whom had a pancreas islet transplant. RESULTS: The main side effect of alpha-CD3-IT, vascular leak syndrome, was entirely prevented by adjunctive immunosuppressive therapy. Renal and liver function tests and biopsies revealed a lack of nephrotoxicity and hepatotoxicity. All had transient weight loss (14+/-5%). Without infusion of donor CD34+ cells, 97% had full weight recovery. Of those given donor CD34+ cells, 50% were euthanized for wasting. CONCLUSIONS: Side effects of alpha-CD3-IT are manageable and should not prevent therapeutic application.

Cytoprotection of pancreatic islets before and soon after transplantation by gene transfer of the anti-apoptotic Bcl-2 gene.

Isolated pancreatic islet transplantation is a promising alternative to conventional insulin-dependent diabetes treatment but is not yet a practical clinical therapy. In the first few days after pancreatic islet transplantation, substantial donor pancreatic islet dysfunction and apoptosis commonly occur. Islet apoptosis has been documented after extracellular matrix disruption and exposure to proinflammatory cytokines, and during hypoxia before islet revascularization and rejection. These studies show that targeting the apoptosis pathway by adenoviral-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective effect on isolated macaque pancreatic islets. Bcl-2 transfection ex vivo protects these islets from apoptosis induced by disruption of the islet extracellular matrix during pancreatic digestion. Additionally, overexpression of Bcl-2 confers long-term, stable protection and maintenance of functional islet mass after transplantation of macaque islets into diabetic severe combined immunodeficency mice. Notably, genetic modification of pancreatic islets also reduced the islet mass required to achieve stable euglycemia. Ex vivo gene transfer of anti-apoptotic genes has potential as a therapeutic approach to both minimize loss of functional islet mass after transplant and reduce the high donor islet requirement currently needed for successful stable reversal of insulin-dependent diabetes.

Durable donor-specific T and B cell tolerance in rhesus macaques induced with peritransplantation anti-CD3 immunotoxin and deoxyspergualin: absence of chronic allograft nephropathy.

Tolerance induction can prevent acute kidney allograft rejection without chronic immunosuppression. It is uncertain whether specific tolerance can prevent chronic allograft nephropathy (CAN), which involves both nonimmune and immune injury. This report provides evidence that immunologically tolerant macaques, induced with immunotoxin and deoxyspergualin, developed neither acute rejection nor CAN. Long survivors, bearing MHC-mismatched grafts without chronic immunosuppression for 0.8 to 3.4 years, exhibited general immune competence with donor-specific T and B cell tolerance and no functional or histological evidence of CAN. Stringent criteria for tolerance were satisfied by specific prolongation of donor skin grafts with rapid rejection of third-party skin, followed by indefinite acceptance of a second donor kidney graft and establishment of microchimerism. Primate tolerance with documented absence of CAN may give impetus to the clinical application of tolerance.

Preclinical studies of allograft tolerance in rhesus monkeys: a novel anti-CD3-immunotoxin given peritransplant with donor bone marrow induces operational tolerance to kidney allografts.

A major challenge in clinical transplantation today is to design a practical and effective protocol for tolerance induction compatible with cadaver organ transplantation. A preclinical rhesus monkey kidney allograft model using immediate peritransplant anti-CD3 immunotoxin (anti-CD3-IT) and donor bone marrow (DBM) is shown here to induce operational tolerance with prolonged graft survival in the absence of chronic immunosuppressive drugs. Bone marrow harvested from the kidney donor was depleted of mature alloantigen-presenting cells and T cells by removing DR(bright) cells and CD3(bright) cells, respectively. In outbred, major histocompatibility complex-incompatible donor-recipient pairs with high pretransplant mixed lymphocyte response and cytotoxic T lymphocyte precursor activity, four of six allografts survived for periods of 120 days to >1.5 years. Graft acceptance after peritransplant treatment followed robust elimination of both peripheral blood T cells and lymph node T cells. In most recipients given anti-CD3-IT and DBM infusion, anti-donor immunoglobulin G responses were completely inhibited. Microchimerism was observed in all recipients studied, including those not given DBM, but levels of microchimerism did not correlate with graft survival. Anti-CD3-IT induction in combination with modified DBM protocols such as the depletion of mature T cells and DR(bright) antigen-presenting cells may offer new opportunities to improve clinical tolerance protocols beyond those attempted in the clinic to date. Overall, these results with anti-CD3-IT show promise for development of cadaver transplant tolerance induction.

Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation.

BACKGROUND: Liver function after transplantation is determined by the quality of the donor organ and the influences of preservation, flush, and reperfusion injury. In this regard, cell death (apoptosis) plays an important role in organ preservation and rejection. Therefore, we examined the possibility of genetic modification of the liver graft with a recombinant adenovirus vector encoding the Bcl-2 gene to reduce apoptosis during the preservation time. METHODS: Liver grafts from C57B1/6 mice were procured and preserved using standard techniques. A replication defective adenovirus vector (deltaE1) containing the human Bcl-2 gene (AdCMVhBcl-2) was developed in our laboratory. An adenovirus vector encoding an irrelevant gene (Escherichia coli beta-galactosidase) was used as a control. Each mouse received 1 x 10(9) plaque forming units administered i.v. 48 hr before the liver procurement. Analyses of liver enzyme activities were determined in the preservation solution. Apoptosis in liver biopsies was determined by DNA fragmentation with an in situ histochemical assay. RESULTS: Immunohistochemical analysis and RT-PCR confirmed the expression of hBcl-2 in the grafts. Grafts from livers expressing hBcl-2 showed significant reduction of the aspartame amino transferase (AST) and lactate dehydrogenase (LDH) release compared with grafts from the control groups. After rewarming, significant cytoprotection was also observed in grafts from animals treated with AdCMVhBcl-2. Histological analysis correlated with the hepatocellular injury determined with transaminases and LDH in the preservation solution. Significant reduction in the number of apoptotic cells was observed in grafts expressing hBcl-2. CONCLUSIONS: We have demonstrated a novel approach to reducing the preservation injury to liver grafts with the human Bcl-2 gene. This approach may allow a longer preservation time, potentially reduce the incidence of primary nonfunction, decrease the immunogenicity of the cold injured organ, and increase the safer use of "marginal" liver grafts.