Donor-specific alloantibodies are associated with fibrosis progression after liver transplantation in hepatitis C virus-infected patients.

Hepatitis C virus (HCV) fibrosis progression after liver transplantation (LT) is accelerated in comparison with fibrosis progression before transplantation. The vast majority of the risk factors for fibrosis progression after LT are not modifiable. With the goal of identifying modifiable risk factors for fibrosis progression, we evaluated the impact of preformed and de novo donor-specific human leukocyte antigen alloantibodies (DSAs) on fibrosis progression after LT in HCV-viremic patients. After blinding, we analyzed all 507 HCV-viremic patients who underwent primary LT from January 2000 to May 2009 and had pretransplant and posttransplant samples available for analysis (86% of the total) for preformed and de novo class I and class II DSAs with a mean fluorescence intensity ≥ 5000 with single-antigen bead technology. Fibrosis was assessed on the basis of indication and protocol liver biopsies; compliance with protocol liver biopsies at 1, 2, and 5 years was ≥80%. Preformed class I DSAs [hazard ratio (HR) = 1.44, P = 0.04] and class II DSAs (HR = 1.86, P < 0.001) were independent predictors of progression to stage 2-4 fibrosis, and de novo DSAs (HR = 1.41, P = 0.07) had borderline significance. In addition, preformed class I DSAs (HR = 1.63, P = 0.03) and class II DSAs (HR = 1.72, P = 0.03) were statistically significantly associated with an increased risk of death. In conclusion, after we controlled for donor and recipient characteristics in multivariate modeling, DSAs were independently associated with fibrosis progression and death after LT in HCV-viremic patients.

Antibody-mediated rejection as a contributor to previously unexplained early liver allograft loss.

We analyzed 60 patients with idiopathic early allograft loss (defined as death or retransplantation at <90 days) to determine the relative contribution of preformed donor-specific human leukocyte antigen alloantibodies (DSAs) to this endpoint, and we defined strict criteria for the diagnosis of antibody-mediated rejection (AMR) in liver allografts. The inclusion criteria encompassed the availability of a pretransplant serum sample and both postreperfusion and follow-up tissue specimens for a blinded, retrospective re-review of histology and complement component 4d (C4d) staining. AMR was diagnosed on the basis of the presence of all 4 of the following strict criteria: (1) DSAs in serum, (2) histopathological evidence of diffuse microvascular injury/microvasculitis consistent with antibody-mediated injury, (3) diffuse C4d staining in the portal microvasculature with or without staining in the sinusoids or central veins in at least 1 sample, and (4) the exclusion of other causes of a similar type of injury. Patients thought to be experiencing definite AMR on the basis of routine histopathology alone showed the highest levels of DSA sensitization. Forty percent of patients with pretransplant DSAs with a pattern of bead saturation after serial dilutions developed AMR. Another multiparous female developed what appeared to be a strong recall response, which resulted in combined AMR and acute cellular rejection (ACR) causing graft failure. A contribution of DSAs to allograft failure could not be excluded for 3 additional patients who received marginal grafts. In conclusion, liver allograft recipients with preformed DSAs with a high mean fluorescence intensity despite dilution seem to be at risk for clinically significant allograft injury and possibly for loss from AMR, often in combination with ACR.

Preformed class II donor-specific antibodies are associated with an increased risk of early rejection after liver transplantation.

Preformed donor-specific human leukocyte antigen antibodies (DSAs) are considered a contraindication to the transplantation of most solid organs other than the liver. Conflicting data currently exist on the importance of preformed DSAs in rejection and patient survival after liver transplantation (LT). To evaluate preformed DSAs in LT, we retrospectively analyzed prospectively collected samples from all adult recipients of primary LT without another organ from January 1, 2000 to May 31, 2009 with a pre-LT sample available (95.8% of the patients). Fourteen percent of the patients had preformed class I and/or II DSAs with a mean fluorescence intensity (MFI) ≥ 5000. Preformed class I DSAs with an MFI ≥ 5000 remained persistent in only 5% of patients and were not associated with rejection. Preformed class II DSAs with an MFI of 5000 to 10,000 remained persistent in 23% of patients, and this rate increased to 33% for patients whose MFI was ≥10,000 (P < 0.001). Preformed class II DSAs in multivariable Cox proportional hazards modeling were associated with an increased risk of early rejection [hazard ratio (HR) = 1.58; p = 0.004]. In addition, multivariate modeling showed that in comparison with no DSAs (MFI < 1000), preformed class I and/or II DSAs with an MFI ≥ 5000 were independently correlated with the risk of death (HR = 1.51; p = 0.02).

Donor-specific human leukocyte antigen antibodies of the immunoglobulin G3 subclass are associated with chronic rejection and graft loss after liver transplantation.

In a previous study, we found that 92% of patients with chronic rejection had donor-specific human leukocyte antigen antibodies (DSAs), but surprisingly, 61% of comparator patients without rejection also had DSAs. We hypothesized that immunoglobulin G (IgG) subclasses were differentially distributed between the 2 groups. A modified single-antigen bead assay was used to detect the presence of individual IgG subclasses against human leukocyte antigen in 39 chronic rejection patients and 66 comparator patients. DSAs of the IgG1 subclass were most common and were found in 45% of all patients; they were followed by IgG3 DSAs (21%), IgG4 DSAs (14%), and IgG2 DSAs (13%). The percentage of patients with multiple IgG subclasses was significantly higher in the chronic rejection group versus the comparator group (50% versus 14%, P < 0.001). Patients with normal graft function in the presence of DSAs mostly had isolated IgG1, whereas patients with chronic rejection had a combination of IgG subclasses. Patients who developed DSAs of the IgG3 subclass showed an increased risk of graft loss (hazard ratio = 3.35, 95% confidence interval = 1.39-8.05) in comparison with patients with DSAs of other IgG subclasses or without DSAs. Although further study is needed, the determination of the IgG subclass in DSA-positive patients may help us to identify patients with a higher risk of chronic rejection and graft loss.

Implications of a positive crossmatch in liver transplantation: a 20-year review.

Whether a positive crossmatch result has any relevance to liver transplantation (LT) outcomes remains controversial. We assessed the impact of a positive crossmatch result on patient and graft survival and posttransplant complications. During a 20-year period, 2723 LT procedures with crossmatch results were identified: 2479 primary transplants and 244 retransplants. The rates of positive B cell and T cell crossmatches were 10.1% and 7.4%, respectively, for primary transplants and 14.6% and 6.4%, respectively, for retransplants (P = 0.049 for a B cell crossmatch). Across all primary transplants, females (P < 0.001) and patients with autoimmune hepatitis (P < 0.001) had greater frequencies of positive crossmatches. There was no effect from race or age. For both primary transplants and retransplants, patient survival and graft survival were not affected by the presence of a positive crossmatch. With respect to posttransplant complications, there were no differences in rejection episodes (hyperacute, acute, or chronic) or technical complications (biliary and vascular) between negative and positive crossmatch groups. However, there were significant differences in the pathological findings of preservation injury (PI) on liver biopsy samples taken at the time of transplantation and within the first week of transplantation (P = 0.003 for B cells and P = 0.03 for T cells). In summary, a positive crossmatch had no significant impact on patient survival or graft outcomes. However, there was a significantly higher incidence of PI in primary LT recipients with a positive crossmatch. This finding is important for a broader understanding of PI, which may include a significant immunological component.

Indications for combined liver and kidney transplantation: propositions after a 23-yr experience.

The frequency of combined liver and kidney transplants (CLKT) persists despite the pronounced scarcity of organs. In this review, we sought to ascertain any factors that would reduce the use of these limited commodities. Seventy-five adult CLKT were performed over a 23-yr period at our center, 29 (39%) of which occurred during the Model for End-stage Liver Disease (MELD) era. Overall, patient survival rates were 82%, 73%, and 62% at one, three, and five yr, respectively. There was no difference in patient survival based either on pre-transplant hemodialysis status or by glomerular filtration rate (GFR) at the time of transplant. Patients undergoing a second CLKT or a liver retransplantation at the time of CLKT had a survival rate of 30% at three months. In the MELD era, patient survival was unchanged (p = NS) despite an older recipient population (p = 0.0029) and a greater number of hepatitis C patients (p = 0.0428). In summary, patients requiring liver retransplantation with concomitant renal failure should be denied CLKT. Renal allografts may also be spared by implementing strict criteria for renal organ allocation (GFR < 30 mL/min at the time of evaluation) and considering the elimination of preemptive kidney transplantation in CLKT.

Prediction of crossmatch outcome of highly sensitized patients by single and/or multiple antigen bead luminex assay.

BACKGROUND: We used a solid-phase assay to identify human leukocyte antigen (HLA) Class I and II specificities in highly reactive sera, and applied this information to predict crossmatch outcome with greater than 90% accuracy. METHODS: Sera from 20 highly sensitized end-stage renal disease patients reactive to 70-100% of HLA Class I and II antigen panel were analyzed by single and/or multiple antigen flow microparticle bead assay using Luminex platform (Luminex assay). These sera contained antibodies directed against high frequency public HLA class I and/or II epitopes accounting for 70-100% of serum's total reactivity. More than 2,000 complement dependent cytotoxicity (CDC) and 200 flow crossmatches (FLXM) were performed using sera from these patients and deceased donor T and B lymphocytes. RESULTS: Luminex serum analysis was able to correctly predict outcomes of 95% of T and B cell FLXM. In contrast, predictive values for the CDC T and B cell crossmatches by Luminex serum analysis were only 77% and 75%, respectively. The use of serum analysis in donor selection would have reduced the total number of required FLXM by more than 50%. The frequency of negative T cell FLXM was 56% when donors were selected randomly; however, if serum antibody analysis had been used to preselect the donors by excluding donors that were likely to be positive, the frequency of corresponding negative crossmatches would have increased up to 93%. CONCLUSION: This approach to donor selection may allow wider geographic sharing of cadaver donor organs without actually performing the crossmatch.

A significant role for histocompatibility in human islet transplantation.

BACKGROUND: In recent years, transplantation of islets and pancreas has become a viable option for patients debilitated with type I diabetes. The success of islet transplantation has been attributed to the ability to isolate high quality islets for transplantation and capacity to maintain the recipient's immunosuppressive levels within a specific target range following transplantation. The purpose of this study was to determine the role of pretransplant sensitization to human leukocyte antigen (HLA) in islet transplantation. METHODS: We retrospectively analyzed seven patients that were transplanted with islets under the auspices of the Juvenile Diabetes Research Foundation and Islet Cell Resource Center/National Institutes of Health. Humoral sensitization towards donor antigens both prior to and following islet transplantation was detected by FLOW panel reactive antibodies (PRA) and donor-specific cellular sensitization was detected by performing enzyme-linked immunospot assay analysis for cytokines interferon-gamma and interleukin-2. RESULTS: Our analysis demonstrates that humoral and cellular sensitization to histocompatibility antigens prior to and after islet transplantation are associated with the failure of transplanted islets CONCLUSION: Patient selection based on sensitization to donor HLA may be one of the factors crucial for the success of islet transplant. Further, in some patients, rejection of islets can be associated with sensitization to mismatched donor histocompatibility antigens.

MHC class I-specific antibody binding to nonhematopoietic cells drives complement activation to induce transfusion-related acute lung injury in mice.

Transfusion-related acute lung injury (TRALI), a form of noncardiogenic pulmonary edema that develops during or within 6 h after a blood transfusion, is the most frequent cause of transfusion-associated death in the United States. Because development of TRALI is associated with donor antibodies (Abs) reactive with recipient major histocompatibility complex (MHC), a mouse model has been studied in which TRALI-like disease is caused by injecting mice with anti-MHC class I monoclonal Ab (mAb). Previous publications with this model have concluded that disease is caused by FcR-dependent activation of neutrophils and platelets, with production of reactive oxygen species that damage pulmonary vascular endothelium. In this study, we confirm the role of reactive oxygen species in the pathogenesis of this mouse model of TRALI and show ultrastructural evidence of pulmonary vascular injury within 5 min of anti-MHC class I mAb injection. However, we demonstrate that disease induction in this model involves macrophages rather than neutrophils or platelets, activation of complement and production of C5a rather than activation of FcγRI, FcγRIII, or FcγRIV, and binding of anti-MHC class I mAb to non-BM-derived cells such as pulmonary vascular endothelium. These observations have important implications for the prevention and treatment of TRALI.

A lifetime versus a graft life approach redefines the importance of HLA matching in kidney transplant patients.

INTRODUCTION: Human leukocyte antigen (HLA) matching has been de-emphasized in the allocation of renal allografts and further discounting is planned in the new United Network of Organ Sharing kidney allocation model. An unforeseen consequence of poorer matching could be increased sensitization for candidates pursuing retransplantation. METHODS: We examined candidates listed in the United States from 1988 to 2007 from the Scientific Renal Transplant Registry (SRTR) database that were relisted after loss of a primary kidney transplant (n=15,980). The primary outcome was change in panel reactive antibody (PRA) from prior to recipient's initial transplant to the subsequent listing. Absolute change in PRA levels were examined in general linear models and the likelihood of becoming newly sensitized in logistic models. RESULTS: There was no appreciable change in PRA for patients receiving a first 0 HLA-A, -B, -DR, or 0 HLA-A, -B-mismatched kidney transplant; contrariwise, there was a significant increase in PRA by increasing HLA mismatch of the first transplant. Only 10% of patients became sensitized after a 0 HLA-A, -B-mismatched transplant, whereas the proportion rose up to 37% with increasing HLA mismatches. Other factors, notably younger age and African American race, also contributed to a higher PRA at relisting. CONCLUSIONS: Although there might be a limited impact of HLA matching on acute rejection and graft survival, many patients might be negatively impacted from poor HLA matching of their first kidney transplant when needing a second transplant. This might be particularly important in patients with a long life expectancy because of the high likelihood of needing a second transplant during their lifetime.

Bortezomib provides effective therapy for antibody- and cell-mediated acute rejection.

BACKGROUND: Current antihumoral therapies in transplantation and autoimmune disease do not target the mature antibody-producing plasma cell. Bortezomib is a first in class proteosomal inhibitor, that is Food and Drug Administration approved, for the treatment of plasma cell-derived tumors that is multiple myeloma. We report the first clinical experience with plasma cell-targeted therapy (bortezomib) as an antirejection strategy. METHODS: Eight episodes of mixed antibody-mediated rejection (AMR) and acute cellular rejection (ACR) in six transplant recipients were treated with bortezomib at labeled dosing. Monitoring included serial donor-specific antihuman leukocyte antigen antibody (DSA) levels and repeated allograft biopsies. RESULTS: Six kidney transplant patients received bortezomib for AMR and concomitant ACR. In each case, bortezomib therapy provided (1) prompt rejection reversal, (2) marked and prolonged reductions in DSA levels, (3) improved renal allograft function, and (4) suppression of recurrent rejection for at least 5 months. Moreover, immunodominant DSA (iDSA) (i.e., the antidonor human leukocyte antigen antibody with the highest levels) levels were decreased by more than 50% within 14 days and remained substantially suppressed for up to 5 months. One or more additional DSA were present at lower concentrations (non-iDSA) in each patient and were also reduced to nondetectable levels. Bortezomib-related toxicities (gastrointestinal toxicity, thrombocytopenia, and paresthesias) were all transient. CONCLUSIONS: Bortezomib therapy: (1) provides effective treatment of AMR and ACR with minimal toxicity and (2) provides sustained reduction in iDSA and non-iDSA levels. Bortezomib represents the first effective antihumoral therapy with activity in humans that targets plasma cells.

Corticosteroid elimination: the Cincinnati experience.

Elimination of corticosteroid-related morbidity has been a goal of transplant clinicians from the earliest days of renal transplantation more than 50 years ago. Over the past decade, this goal has begun to be realized. Herein, we describe our efforts to eliminate corticosteroid therapy from maintenance immunosuppression-efforts that have spanned 15 years and have included design and conduct of five multicenter trials and over ten single center trials with over 650 patients at the University of Cincinnati. These efforts have led to a near complete elimination of corticosteroid-related morbidity, and, importantly, a more precise definition of the risk/benefit assessments of corticosteroid withdrawal in individual patient populations, which has allowed individualization and tailoring of corticosteroid-free immunosuppression.

Simultaneous corticosteroid avoidance and calcineurin inhibitor minimization in renal transplantation.

Steroids and calcineurin inhibitors (CNI) have been mainstays of immunosuppression but both have numerous side effects that are associated with substantial morbidity and mortality. This study was carried out to determine if steroids can be eliminated with early discontinuation of cyclosporine A (CsA) and later discontinuation of mycophenolate mofetil (MMF). Ninety-six patients with kidney transplants were entered into four subgroups of two pilot studies. All patients received Thymoglobulin induction, rapamycin (RAPA), and the immunonutrients arginine and an oil containing omega-3 fatty acids. Mycophenolate mofetil was started in standard doses and discontinued by 2 years. CsA was given in reduced doses for either 4, 6, or 12 months. Follow-up was 12-36 months. Thirteen first rejection episodes occurred during the first year (14%). Combining all patients, 86% were rejection-free at 1 year, 80% at 2 years and 79% at 3 years. No kidney has been lost to acute rejection. Ninety percent of the 84 patients at risk at the end of the study were steroid-free and 87% were off CNI. Fifty-seven percent of 54 patients with a functioning kidney at 3 years were receiving monotherapy with RAPA. We conclude that this therapeutic strategy is worthy of a prospective multi-center clinical trial.

African-American renal transplant recipients benefit from early corticosteroid withdrawal under modern immunosuppression.

African-Americans (AAs) have historically been considered high-risk renal transplant recipients due to increased rejection rates and reduced long-term graft survival. As a result, AAs are often excluded from corticosteroid withdrawal (CSWD) protocols. Modern immunosuppression has reduced rejections and improved graft survival in AAs and may allow successful CSWD. Outcomes in 56 AAs were compared to 56 non-AAs. All patients were enrolled in one of four early CSWD protocols. Results are reported as AA versus non-AA. Acute rejection at 1-year was 23% and 18%; (p = NS); creatinine clearance at 1-year was 75 versus 80 mL/min (p = NS); patient and graft survival was 96% versus 98% and 91% versus 91%; (p = NS). AAs benefit from early CSWD with significantly improved blood pressure, LDL < 130 mg/dL and HDL > 45 mg/dL at 1-year, post-transplant diabetes of 8.7%, and mean weight change at 1-year of 4.8 +/- 7.2 kg. In conclusion, early CSWD in AAs is associated with acceptable rejection rates, excellent patient and graft survival, and improved cardiovascular risk, indicating that the risks and benefits of early CSWD are similar between AAs and non-AAs. Additional follow-up is needed to determine long-term renal function, graft survival, and cardiovascular risk in AAs with early CSWD.