Renal Transplant Patients Biopsied for Cause and Tested for C4d, DSA, and IgG Subclasses and C1q: Which Humoral Markers Improve Diagnosis and Outcomes?

The association between donor specific antibodies (DSA) and renal transplant rejection has been generally established, but there are cases when a DSA is present without rejection. We examined 73 renal transplant recipients biopsied for transplant dysfunction with DSA test results available: 23 patients diffusely positive for C4d (C4d+), 25 patients focally positive for C4d, and 25 patients negative for C4d (C4d-). We performed C1q and IgG subclass testing in our DSA+ and C4d+ patient group. Graft outcomes were determined for the C4d+ group. All 23 C4d+ patients had IgG DSA with an average of 12,500 MFI (cumulative DSA MFI). The C4d- patients had average DSA less than 500 MFI. Among the patients with C4d+ biopsies, 100% had IgG DSA, 70% had C1q+ DSA, and 83% had complement fixing IgG subclass antibodies. Interestingly, IgG4 was seen in 10 of the 23 recipients' sera, but always along with complement fixing IgG1, and we have previously seen excellent function in patients when IgG4 DSA exists alone. Cumulative DSA above 10,000 MFI were associated with C4d deposition and complement fixation. There was no significant correlation between graft loss and C1q positivity, and IgG subclass analysis seemed to be a better correlate for complement fixing antibodies in the C4d+ patient group.

Effects of mycophenolic acid on highly sensitized patients awaiting kidney transplant.

BACKGROUND: 10-30% of dialysis population awaiting renal transplantation is sensitized. Mycophenolic acid (MPA) has been shown to reduce panel reactive antibody (PRA) formation in kidney transplant recipients. Our aim was to investigate whether MPA could effectively reduce anti-HLA antibody levels and allow successful transplantation. METHODS: A total of 40 highly sensitized patients were treated orally with MPA. All patients had T-cell PRA values greater than 30% (73% of patients were ≥ 75%). The PRAs, T-cell/B-cell flow cytometry crossmatch (FCXM) mean channel shift (MCS), patient/graft survival, acute rejection, and serum creatinine (SCr) were recorded. RESULTS: All 40 patients showed a decrease in PRA levels. Eighteen of the 40 patients (40%) received a transplant. All four living donor recipients converted to a negative crossmatch. There was a significant decrease in FCXM MCS in all 18 transplanted patients. The mean SCr at 24 months was 1.00 ± 0.25mg/dL. Five patients (28%) experienced acute rejection. The overall one year actuarial patient and graft survival were 94% and 88%, respectively. CONCLUSIONS: These findings suggest that MPA therapy is effective in reducing PRAs and increases the likelihood of successful transplantation in sensitized recipients in a potentially simpler and more cost effective manner than the current regimens employed.

Reduction of HLA class I expression by ribonucleic acid interference mitigates allogenicity of human primary and Immortalized cells.

Unmatched human leukocyte antigens (HLA) expressed by allogeneic donor cells are the major target for immunological rejection. In order to reduce the immunogenicity of allograft cells, we have developed lentiviral vectors for delivery of short hairpin ribonucleic acid (shRNA) against Class I HLA. This approach was evaluated in both an established human embryonic kidney cell line and primary human CD34+ hematopoietic stem/progenitor cells. Target cells transduced with lentiviral vectors expressing either HLA-A*0201 allele-specific or HLA-A, -B, -C consensus sequence-specific shRNA showed effective knockdown of cell surface HLA expression. Mixed lymphocyte-target cell reactions showed significantly reduced interferon-gamma production from alloreactive cytotoxic T lymphocytes and significantly reduced levels of target cell apoptosis after shRNA-mediated knockdown of HLA expression and target cell survival correlated with vector transduction efficiency. Furthermore, increasing resistance to complement-dependent cytotoxicity mediated by anti-HLA antibodies was observed to correlate with increasing levels of shRNA vector transduction in primary human CD34+ cells. Notably, non-HLA restricted killing by lymphokine-activated killer cells was not incurred after HLA knockdown. These data demonstrate the potential for genetic engineering strategies targeting incompatible HLA alleles to reduce both cellular and humoral responses and enable graft survival after transplantation of allogeneic cells and tissues.

Immunoglobulin G subclass analysis of HLA donor specific antibodies in heart and renal transplant recipients.

Immunoglobulin G (IgG) subclasses IgG1 (G1) and lgG3 (G3) can induce complement dependent cytotoxicity (CDC) and bind to Fc receptors (FcR), which induces phagocytosis and antibody dependent cellular cytotoxicity. In contrast, IgG2 has low CDC activity, lgG4 (G4) has no CDC activity, and neither binds high affinity FcR. Seven transplant recipients were analyzed for G1- G4 human leukocyte antigen (HLA) donor-specific antibodies (DSAs); six had active rejection and one had stable function. Patients with rejection had equal numbers of DSAs, which were G1 and G3, but no G4. The predominant DSAs were directed against HLA Class II proteins. Even with successful anti-rejection therapy, DSA persisted, albeit in several instances with lowered levels. Our findings are consistent with the presence of CDC-inducing G1 and G3 subclass DSAs during rejection. One heart transplant recipient followed for over 42 months had consistent, continuous G4 HLA DSA and stable function. We hypothesize that the presence of G4 DSA in the heart transplant recipient is akin to the allergen specific G4 that has been found in allergic desensitization tolerance, controlled by regulatory T-cells, and that manipulating G4 class switching through HLA antigen desensitzation could produce a tolerant state.

Impact of hepatitis C infection on outcomes after heart transplantation.

BACKGROUND: Prevalence of hepatitis C infection (HCV) among heart transplant (OHT) recipients ranges from 7% to 18%. Despite the paucity of data regarding the outcomes of heart transplant recipients who are HCV positive before transplant, many transplant centers are declining to perform OHT in HCV-seropositive patients. METHODS: We assessed the clinical outcome of HCV-seropositive compared with HCV-seronegative heart transplant recipients using the Organ Procurement and Transplant Network/the United Network for Organ Sharing database. Between January 1, 2000, and December 31, 2005, 224 HCV-seropositive and 10,406 HCV-seronegative recipients who received HCV-seronegative donor organs were identified. RESULTS: Overall patient survival rates of HCV-seropositive recipients were significantly lower than those of HCV-seronegative recipients (84.8% at 1 year, 77.1% 3 years, 68.9% 5 years for HCV-seropositive group vs. 87.9% at 1 year, 80.7% 3 years, and 74.1% 5 years for HCV-negative recipients, log rank P=0.036). However, adjusted relative risk of recipient HCV-seropositive versus HCV-seronegative status did not reach to statistical significance level (relative risk=1.23 with P=0.087) after adjusting for other donor and recipient factors. Causes of death among HCV-seropositive and HCV-seronegative groups were similar. Cumulative incidence of an acute rejection episode in the first year after transplantation among HCV-seropositive recipients was 35.7% versus 32.6% HCV-seronegative recipients (P=0.32). CONCLUSIONS: A more rational approach should be developed for the management of HCV-seropositive heart transplant candidates. Carefully selected HCV-seropositive patients should not be excluded from OHT.

Long-range linkage on chromosome 6p of VEGF, FKBP5, HLA and TNF alleles associated with transplant rejection.

Polymorphisms in several genes on the short arm of chromosome 6 (6p), among them, VEGF, FKBP5, HLA-DR and TNF-alpha, have been associated with inflammation and transplant outcome, such as acute rejection. Independent segregation of these genes is unproven, so we investigated linkage between distant genes on 6p and the putative existence of evolutionarily conserved long-range 6p haplotypes. SNPs studied were VEGF-2578*C/A (rs69947), VEGF-1154*G/A (rs1570360) TNF-alpha-308*G/A (rs1800629) and FKBP5*C/T (rs1360780) in 206 random and 80 selected HLA-DR52 positive individuals. To simplify the analysis, the HLA-DR genotypes were collapsed to the five human ancestral supertypes, namely: HLA-DR51, DR52, DR53, DR1 and DR8. Gametic phase and linkage between paired genotypes were determined using Arlequin 3.01 software, and significance was determined by Chi-square and Fisher's exact test analysis. Significant allelic associations were evident across the 6p region examined. Two putative haplotypes were identified, associated with DR52 and DR1. Within the HLA-DR52 supertype, TNF-alpha -308*A was associated with DR3, while FKBP5*T was associated with DR6. The interval between VEGF and TNF-alpha is 12.31Mb. Therefore, allelic associations are surprising considering expected recombination and the evolutionary time (c10MY) since divergence of DR supertypes. This suggests that DR1 and DR52 haplotypes may have a survival advantage. Within the DR52 supertype, VEGF*C-DR3-TNF -308*A is a 'high inflammatory' haplotype associated with acute and chronic rejection, while the FKBP5*T-DR6 haplotype is associated with resistance to endogenous and exogenous glucocorticoids. Conversely, the DR1 haplotype is a 'low inflammatory' haplotype.

ASSOCIATION BETWEEN DE NOVO DONOR SPECIFIC HLA ANTIBODY, C4D STAINING IN RENAL GRAFT BIOPSY AND GRAFT OUTCOME: A SINGLE CENTER EXPERIENCE.

In 69 renal transplant recipients (RTR), all had a functioning graft (SCr < 2.0) at one year. After one year, transplant dysfunction was observed and these 69 RTR were biopsied, tested for C4d deposition and donor specific antibodies (DSA). Of these 69 RTR, 29 (42%) showed C4d negativity, 27 (39%) were C4d positive and 13 (19%) were not diagnostic. Forty-nine (71%) recipients had HLA antibodies and 41 (59%) had DSA. The proportion of C4d positivity was significantly higher in patients with DSA (HLA Class I only, II only, and I & II) in comparison to patients without post-transplant HLA antibodies. The incidence of graft failure (including current SCr > 4.0) in RTR with HLA Class II antibodies (Class II only or I & II) was significantly higher than in RTR without post-transplant HLA antibodies (P=0.03).Even after amelioration of rejection, the RTR with Class II DSA group continued to fail beyond 2 years after transplantation when compared with the other 2 groups (None/NDSA or HLA Class I only), however, the difference in graft survival between HLA Class II and None/NDSA groups did not reach statistical significance (log-rank P=0.32). Significant association between C4d staining, post-transplant HLA Class II antibodies and graft failure strongly suggests the importance of post-transplant HLA antibodies. HLA Class II DSAs may be an indicator of chronic allograft nephropathy (CAN) proceeding to graft loss. We propose that amelioration of CAN graft loss may be affected by monitoring and identification of DSA with appropriate immunosuppression of these antibodies.

Comparison of renal allograft outcomes in combined liver-kidney transplantation versus subsequent kidney transplantation in liver transplant recipients: Analysis of UNOS Database.

BACKGROUND: There may be an allograft-enhancing effect by the liver on the renal allograft in the setting of simultaneous combined liver-kidney transplantation (CLKT) from the same donor. This study was performed to investigate whether an existing liver allograft could protect a kidney allograft from immunologic injury due to histoincompatibility in liver transplant recipients who received sequential kidney transplantation (KALT). METHODS: Using the United Network for Organ Sharing database covering January 1996 to December 2003, outcomes of 352 KALT were compared to 1,136 CLKT. Incidence of acute and chronic rejection and rejection-free renal graft survival was compared between two groups. RESULTS: Renal half-life of KALT allografts was shorter than CLKT group (6.6+/-0.9 vs. 11.7+/-1.3 years, P < 0.001). Incidence of chronic rejection in KALT group was higher than CLKT group (4.6 vs. 1.2%, P < 0.001). One and three-year rejection-free renal graft survival of KALT and CLKT groups were different (77% and 67% KALT vs. 85% and 78% CLKT, respectively; P < 0.001). Among human leukocyte antigen mismatched and sensitized patients, rejection-free renal graft survival of KALT group was inferior to the CLKT group (75% at 1 year and 61% 3 years vs. 86% at 1 year and 79% 3 years, P < 0.001). CONCLUSION: Liver allograft provided renal graft immunoprotection if both organs are transplanted simultaneously (immunogenetic identity), but not for kidneys transplanted subsequently.

Risk factors for short- and long-term survival of primary cadaveric renal allografts in pediatric recipients: a UNOS analysis.

BACKGROUND: Pediatric kidney graft survival rates have improved in the United States. This study evaluates early and late risk factors for cadaveric graft loss in pediatric recipients. METHODS: From January 1994 to December 2002, 2,597 primary cadaveric kidney-alone transplants (donor age 5-45 years, recipient age 2-20 years) were reported to the United Network for Organ Sharing (UNOS). The analysis includes follow-up information based on OPTN data as of October 14, 2003. Odds ratio of early graft loss and relative risk of late graft loss are estimated using logistic regression and Cox proportional hazards model, respectively. RESULTS: Graft survival rates significantly improved during 1999-2002 (95% and 79% at 1-year and 3-years, respectively) compared with those of 1994-1998 (88% and 76% at 1-year and 3-years, respectively) (log rank P=0.02). After adjusting for other variables, the factors that significantly affected early transplant outcome adversely within 3 months posttransplant were prolonged cold ischemia time (>36 hours, odds ratio [OR]=3.38 vs. 0-36 hours) and young recipient age (2-5 years old, OR=2.02 vs. 6-12 years). Beyond 3 months, significant risk factors were African-American recipients (relative risk [RR]=1.93 vs. others), teenage recipients (13-20 yrs, RR=1.50 vs. 6-12 yrs), and patients with focal glomerulosclerosis (FGS) (RR=1.27 vs. others). CONCLUSIONS: The short-term graft survival rate of pediatric cadaveric kidney transplants has significantly improved, yet the long-term outcome has changed little. The long-term outcomes for teenagers (13-20 yrs), patients with FGS, and African-Americans lag significantly behind other groups. In order to improve long-term graft survival in these high-risk patients, newer preventive or treatment strategies must be developed.