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.

Human primary gastric dendritic cells induce a Th1 response to H. pylori.

Adaptive CD4 T-cell responses are important in the pathogenesis of chronic Helicobacter pylori gastritis. However, the gastric antigen-presenting cells that induce these responses have not yet been identified. Here we show that dendritic cells (DCs) are present in the gastric mucosa of healthy subjects and are more prevalent and more activated in the gastric mucosa of H. pylori-infected subjects. H. pylori induced gastric DCs isolated from noninfected subjects to express increased levels of CD11c, CD86 and CD83, and to secrete proinflammatory cytokines, particularly interleukin (IL)-6 and IL-8. Importantly, gastric DCs pulsed with live H. pylori, but not control DCs, mediated T-cell secretion of interferon-gamma. The ability of H. pylori to induce gastric DC maturation and stimulate gastric DC activation of Th1 cells implicates gastric DCs as initiators of the immune response to H. pylori.

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.

Axial and rotational alignment of the leg.

The axial and rotational alignments of the lower extremity are commonly referenced independently, with minimal research on whether coexistent axial and rotational malalignment cause pathologies. The present study analyzed whether a correlation exists between the axial and rotational alignments of the leg. The methodology to measure both alignments was adapted for computer tomography. Fifty patients were analyzed at five reference images to determine axial and rotational alignment. The reference images included the femoral head, the femoral shaft (at the level of the lesser trochanter), the distal femur, the proximal tibia, and the ankle joint. Axial alignment was calculated by using horizontal and vertical measurements of the location of the femoral head, the distal femur, and the ankle joint. Rotational alignments of femur, knee, and tibia were calculated using four angles: proximal femoral, distal femoral, proximal tibial, and ankle joint angles defined relative to a fixed reference. Pearson correlation analysis between axial alignment and the three mentioned rotational alignments were calculated. The correlation coefficient values ranged between -0.15-0.07 when comparing axial to rotational alignment, indicating that a week correlation exists between the two alignments. Though these results were derived using highly reproducible methods, the hypothesis of an existing correlation between the axial and rotational alignments of the leg was rejected. These findings allow for an improved understanding of lower extremity mechanics, which merit importance when considering pathologies of the leg and the surgical techniques that could ultimately benefit patients suffering from these pathologies.

Radio-frequency ablation in cirrhotic patients with hepatocellular carcinoma.

Current surgical treatments for hepatocellular carcinoma (HCC) include radio-frequency ablation (RFA), resection, and orthotropic liver transplant (OLT). RFA is particularly attractive in these high-risk patients because surgery is associated with high mortality and there is a relative scarcity of organs available for those in need of transplants. This study was performed to evaluate the management of cirrhotic patients with HCC undergoing RFA at a single Western institution. A retrospective study from March 1999 to June 2002 was performed to evaluate the clinicopathologic and treatment-related variables in cirrhotic patients with HCC. Forty-nine lesions in 26 patients with HCC and cirrhosis underwent RFA. Data was analyzed for safety and overall survival as the main endpoints. The mean age was 60.4 +/- 11 years, 19 patients were male, 5 had hepatitis B virus, and 19 had hepatitis C virus. The Child classification was 26 per cent, 39 per cent, and 35 per cent for A, B, and C; the number of lesions was 1 in 62 per cent, 2 in 23 per cent, and more than 2 in 15 per cent. The approach was laparoscopic in 58 per cent, percutaneous in 15 per cent, and open in 27 per cent. There were no mortalities and only 1 complication. Average hospital stay was 2.7 +/- 2 days. Subsequent to RFA, 9 patients underwent an OLT within a median of 4.1 months. The median follow-up of the whole group was 13 months and the disease-free survival 9.3 months. Tumor recurrence was identified in 3 previously ablated lesions, nonablated liver in 11, and as pulmonary metastases in 3. Overall survival (P = 0.03) was prolonged for those treated with RFA + OLT over RFA alone. We conclude that RFA is a safe ablative technique in high-risk cirrhotic patients with HCC. This technique may provide a bridge to OLT; however, it remains to be proven whether it prolongs survival in those who do not undergo OLT.

Gene transfer of the Bcl-2 gene confers cytoprotection to isolated adult porcine pancreatic islets exposed to xenoreactive antibodies and complement.

BACKGROUND: Exposing adult porcine pancreatic islets (PI) to xenoreactive natural antibodies (XNA) induces brisk inflammatory injury that involves activation of the complement system. Gene transfer of Bcl-2 has been shown to protect PI from apoptosis and necrosis in several models. In this study, we investigated the effect of Bcl-2 gene transfer on protection of PI from primate XNA and complement-mediated injury. METHODS: The PI were isolated from adult female sows. Only islet preparations that exhibited >90% viability and purity were used. Fresh rhesus monkey serum served as the XNA source. Gene transfer of Bcl-2 was achieved with an adenoviral vector (AdBcl-2) at 500 particle forming units (pfu)/cell. The Bcl-2 expression was confirmed by Western blot technique. Untransfected and transfected PI were incubated in 50% fresh complete serum (CS) or heat-inactivated (HI) rhesus serum for 24 hours. The PI viability was analyzed with acridine orange and ethidium bromide staining. Antibody and complement-mediated cytotoxicity were tested by intracellular lactate dehydrogenase (LDH) release. The PI function was assessed in vitro by static incubation studies and in vivo after intraportal transplantation in diabetic severe combined immunodeficiency (SCID) mice. RESULTS: The AdBcl-2 gene transfer resulted in Bcl-2 gene expression in >90% of PI cells. Following exposure to XNA, <15% of the untransfected cells were viable. Similar results were obtained in PI transfected with a similar recombinant adenovirus encoding the reporter gene E coli beta-galactosidase (AdLacZ), an irrelevant gene. A significant increase in LDH release was observed in control PI after exposure to CS compared with PI that overexpressed Bcl-2 (82.89% +/- 7.78% vs 34.31% +/- 5.4%, P <.005). Higher insulin release was observed in vitro in PI transfected with Bcl-2 compared with untransfected PI or islets transfected with AdLacZ (stimulation index of 0.9 +/- 0.31, 0.9 +/- 0.3 vs 2.67 +/- 0.4, respectively). Only PI treated with AdBcl-2 were able to achieve euglycemia after exposure to XNA and complement after transplantation. CONCLUSIONS: Transfer of the antiapoptotic and antinecrotic Bcl-2 gene into PI can reduce primate XNA and complement-mediated lysis. Cytoprotection of PI with Bcl-2 has potential to improve survival of PI xenotransplants.

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.

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.

Combined cardiac surgery and liver transplantation.

During evaluation for liver transplantation, a 63-year-old man with cirrhosis secondary to hepatitis C was diagnosed with severe aortic stenosis (aortic valve area, 0.87 cm(2)) and coronary artery disease. A combined procedure involving aortic valve replacement (pericardial xenograft), coronary artery bypass surgery, and orthotopic liver transplantation was performed. Convalescence was uneventful, and at 2 years after the procedure, the patient has normal cardiac function, good prosthetic valve function, and biochemically normal liver function.

Multimodality treatment for patients with hepatocellular carcinoma: analysis of prognostic factors in a single Western institution series.

There are few Western studies evaluating prognostic factors for survival in patients with hepatocellular carcinoma (HCC) and the influence on survival of various therapeutic options including orthotopic liver transplantation (OLT). A retrospective analysis was performed of 122 patients with HCC treated at the University of Alabama at Birmingham from January 1990 through December 1999. Clinicopathologic and treatment factors were analyzed with overall survival as the main outcome variable. Median age was 62 years. Most patients were male (74%) and white (79%). Eighty patients (66%) had associated cirrhosis. Sixty-three percent of patients presented with American Joint Committee on Cancer (AJCC) stage III or IV tumors. The median follow-up for survivors was 22 months. The 1-, 3-, and 5-year actuarial survival rates for the entire cohort were 46%, 24%, and 17%, respectively. On multivariate analysis, ablative surgery (P = 0.003), AJCC stages I and II (P = 0.0012), and absence of vascular invasion (P = 0.0001) were found to be independent favorable characteristics. Forty-four patients underwent surgical resection (including OLT, n = 20) or a surgical ablative procedure. All but two nonsurgical patients died of disease. The actuarial 1-, 3-, and 5-year survival rates for this group were 80%, 71%, and 61%, respectively. On multivariate analysis of the surgical group, only vascular invasion was associated with poor prognosis (P = 0.001). OLT was associated with a favorable prognosis on univariate analysis (P = 0.02). Forty percent of patients who received transplants underwent local/regional treatment before transplantation and the outcome in these patients was no different from that in other transplant patients. Surgical treatment is the only potential curative option for HCC, and qualifying for liver transplantation may be a favorable prognostic factor in surgical patients. Local/regional therapy prior to transplantation may provide a bridge to OLT without an increase in tumor-related mortality.

Experience with renal transplantation in the nonhuman primate: a modified ureteroneocystostomy to prevent urologic complications.

Nonhuman primates provide an optimal model for the evaluation of tolerance in the preclinical setting. Transplantation and management of nonhuman primates are technically demanding, and the purpose of this article is to review our extensive experience in renal transplantation in non-human primates, with particular emphasis on modifications of surgical techniques on urologic complications. We retrospectively reviewed our results with 329 renal transplants in rhesus monkeys over an 18-year period. The surgical technique and, in particular, the ureteroneocystostomy have evolved over this period of time. This review extensively details our current technique, the surgical and urologic complications, and their management. There were 329 renal transplants performed. There were 85 early deaths, or animals euthanized, within 30 days of the transplant operation. In the first 15 years, there were 27 (10.68%) surgical complications that required euthanasia, and in the last 3 years the complication rate has been reduced to 5 (7.3%, p < .05). The routine use of microsurgical techniques has reduced the incidence of arterial thrombosis (6.2% vs. 2.9%, p < .05). The incidence of ureteral strictures (15 vs. 0, p < .005) has been reduced by a modification of the ureteroneocystostomy technique detailed in the text. Renal transplantation in small rhesus monkeys is technically demanding. The routine use of microsurgical techniques and a modified ureteroneocystostomy has reduced the incidence of surgical complications.

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.

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.