Daniel S. Warren

Multiple-laboratory comparison of microarray platforms

Microarray technology is a powerful tool for measuring RNA expression for thousands of genes at once. Various studies have been published comparing competing platforms with mixed results: some find agreement, others do not. As the number of researchers starting to use microarrays and the number of cross-platform meta-analysis studies rapidly increases, appropriate platform assessments become more important. Here we present results from a comparison study that offers important improvements over those previously described in the literature. In particular, we noticed that none of the previously published papers consider differences between labs. For this study, a consortium of ten laboratories from the Washington, DC–Baltimore, USA, area was formed to compare data obtained from three widely used platforms using identical RNA samples. We used appropriate statistical analysis to demonstrate that there are relatively large differences in data obtained in labs using the same platform, but that the results from the best-performing labs agree rather well.

Desensitization in HLA-Incompatible Kidney Recipients and Survival

BACKGROUND More than 20,000 candidates for kidney transplantation in the United States are sensitized to HLA and may have a prolonged wait for a transplant, with a reduced transplantation rate and an increased rate of death. One solution is to perform live-donor renal transplantation after the depletion of donor-specific anti-HLA antibodies. Whether such antibody depletion results in a survival benefit as compared with waiting for an HLA-compatible kidney is unknown. METHODS We used a protocol that included plasmapheresis and the administration of low-dose intravenous immune globulin to desensitize 211 HLA-sensitized patients who subsequently underwent renal transplantation (treatment group). We compared rates of death between the group undergoing desensitization treatment and two carefully matched control groups of patients on a waiting list for kidney transplantation who continued to undergo dialysis (dialysis-only group) or who underwent either dialysis or HLA-compatible transplantation (dialysis-or-transplantation group). RESULTS In the treatment group, Kaplan-Meier estimates of patient survival were 90.6% at 1 year, 85.7% at 3 years, 80.6% at 5 years, and 80.6% at 8 years, as compared with rates of 91.1%, 67.2%, 51.5%, and 30.5%, respectively, for patients in the dialysis-only group and rates of 93.1%, 77.0%, 65.6%, and 49.1%, respectively, for patients in the dialysis-or-transplantation group (P<0.001 for both comparisons). CONCLUSIONS Live-donor transplantation after desensitization provided a significant survival benefit for patients with HLA sensitization, as compared with waiting for a compatible organ. By 8 years, this survival advantage more than doubled. These data provide evidence that desensitization protocols may help overcome incompatibility barriers in live-donor renal transplantation. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and the Charles T. Bauer Foundation.).

Plasmapheresis, CMV hyperimmune globulin, and anti-CD20 allow ABO-incompatible renal transplantation without splenectomy

The majority of preconditioning protocols developed to allow ABO‐incompatible (ABOi) renal transplantation include concurrent splenectomy as a prerequisite to successful engraftment. Our center has developed a preconditioning protocol that includes plasmapheresis (PP), low‐dose CMV hyperimmune globulin (CMVIg), and anti‐CD20 monoclonal antibody (rituximab) to allow ABOi renal transplantation without splenectomy. Our initial experience has included treatment of six recipients and successful transplantation from blood group A1, A2, and group B living donors. Mean (± SD) serum creatinine was 1.3 ± 0.1 mg/dL among the six recipients and no episodes of antibody‐mediated rejection (AMR) occurred at a median follow‐up of 12 months. ABO antibody titers have remained below pretreatment levels. The absence of AMR and stable allograft function in this series show the potential of this preconditioning protocol to increase ABOi renal transplantation. The use of rituximab, allowing avoidance of splenectomy, may further remove one of the significant disincentives to ABOi transplantation, and eliminate the risk of post‐splenectomy infections.

The Use of Antibody to Complement Protein C5 for Salvage Treatment of Severe Antibody‐Mediated Rejection

Desensitized patients are at high risk of developing acute antibody‐mediated rejection (AMR). In most cases, the rejection episodes are mild and respond to a short course of plasmapheresis (PP) / low‐dose IVIg treatment. However, a subset of patients experience severe AMR associated with sudden onset oliguria. We previously described the utility of emergent splenectomy in rescuing allografts in patients with this type of severe AMR. However, not all patients are good candidates for splenectomy. Here we present a single case in which eculizumab, a complement protein C5 antibody that inhibits the formation of the membrane attack complex (MAC), was used combined with PP/IVIg to salvage a kidney undergoing severe AMR. We show a marked decrease in C5b‐C9 (MAC) complex deposition in the kidney after the administration of eculizumab.

C4d and C3d staining in biopsies of ABO- and HLA-incompatible renal allografts: correlation with histologic findings.

Biopsies of ABO‐incompatible and positive crossmatch (HLA‐incompatible) renal allografts were retrospectively examined to compare results of C4d and C3d staining, and the correlation between such staining and histologic findings suggestive of antibody‐mediated rejection (AMR). A total of 75 biopsies (55 protocol, 17 for graft dysfunction, 3 for other indications) of 24 ABO‐incompatible grafts and 244 biopsies (103 protocol, 129 for graft dysfunction, 12 for other indications) of 66 HLA‐incompatible grafts were examined; all were stained for C4d and ∼40% for C3d.

ABO incompatible renal transplantation: A paradigm ready for broad implementation

The requirements for potent immunosuppression coupled with the formidable risk of irreversible antibody-mediated rejection (AMR) have thus far limited the expansion of ABO incompatible (ABOi) kidney transplantation. We present a retrospective review of our single-center experience with 60 consecutive ABOi kidney transplants and describe the evolution of our treatment protocol to one that consists only of a brief escalation in immunosuppression without long-term B-cell suppression from splenectomy or anti-CD20. The 1-, 3-, and 5-year graft survival rates for the cohort were 98.3%, 92.9%, and 88.7%, respectively, which is comparable with United Network for Organ Sharing data for compatible live donor transplants. No instances of hyperacute rejection were observed, and no grafts were lost secondary to AMR. In fact, fewer than 15% of the patients experienced a clinical episode of AMR, and rejections were mild. Elimination of B-cell ablative therapies did not result in an increased incidence of AMR. Excellent graft function persists with a current median creatinine clearance of 60 mL/min. The findings of this study and the relatively simple therapeutic regimen used should facilitate widespread application of ABOi kidney transplantation resulting in one of the most rapid escalations in access to organs in the modern era of kidney transplantation.

Outcomes of kidneys from donors after cardiac death: Implications for allocation and preservation

Although donation after cardiac death (DCD) kidneys have a high incidence of delayed graft function (DGF) and have been considered marginal, no tool for stratifying risk of graft loss nor a specific policy governing their allocation exist. We compared outcomes of 2562 DCD, 62 800 standard criteria donor (SCD) and 12 812 expanded criteria donor (ECD) transplants reported between 1993 and 2005, and evaluated factors associated with risk of graft loss and DGF in DCD kidneys. Donor age was the only criterion used in the definition of ECD kidneys that independently predicted graft loss among DCD kidneys. Kidneys from DCD donors <50 had similar long‐term graft survival to those from SCD (RR 1.1, p = NS). While DGF was higher among DCD compared to SCD and ECD, limiting cold ischemia (CIT) to <12 h decreased the rate of DGF 15% among DCD <50 kidneys. These findings suggest that DCD <50 kidneys function like SCD kidneys and should not be viewed as marginal or ECD, and further, limiting CIT <12 h markedly reduces DGF.

The EAR-motif of the Cys2/His2-type Zinc Finger Protein Zat7 Plays a Key Role in the Defense Response of Arabidopsis to Salinity Stress

Cys2/His2-type zinc finger proteins, which contain the EAR transcriptional repressor domain, are thought to play a key role in regulating the defense response of plants to biotic and abiotic stress conditions. Although constitutive expression of several of these proteins was shown to enhance the tolerance of transgenic plants to abiotic stress, it is not clear whether the EAR-motif of these proteins is involved in this function. In addition, it is not clear whether suppression of plant growth, induced in transgenic plants by different Cys2/His2 EAR-containing proteins, is mediated by the EAR-domain. Here we report that transgenic Arabidopsis plants constitutively expressing the Cys2/His2 zinc finger protein Zat7 have suppressed growth and are more tolerant to salinity stress. A deletion or a mutation of the EAR-motif of Zat7 abolishes salinity tolerance without affecting growth suppression. These results demonstrate that the EAR-motif of Zat7 is directly involved in enhancing the tolerance of transgenic plants to salinity stress. In contrast, the EAR-motif appears not to be involved in suppressing the growth of transgenic plants. Further analysis of Zat7 using RNAi lines suggests that Zat7 functions in Arabidopsis to suppress a repressor of defense responses. A yeast two-hybrid analysis identified putative interactors of Zat7 and the EAR-domain, including WRKY70 and HASTY, a protein involved in miRNA transport. Our findings demonstrate that the EAR-domain of Cys2/His2-type zinc finger proteins plays a key role in the defense response of Arabidopsis to abiotic stresses.

Domino paired kidney donation: a strategy to make best use of live non-directed donation

Current models for allocation of kidneys from living non-directed donors Living non-directed (LND) donors, also known as altruistic, good Samaritan, anonymous, or benevolent community donors, are a new and rapidly growing source of solid organs for transplantation. The willingness of individuals to donate organs without a designated recipient has been unexpected, but has probably developed as a societal response to the growing crisis in organ availability. In the context of this shortage, health professionals have attempted to make the best use of kidneys from LND donors. We present a novel application of paired donation that has the potential to multiply the number of recipients who can benefi t from each LND donation. At present, there is no universally accepted system for allocation of organs from LND donors. Selection of recipients has been at the discretion of the transplant centres where LND donors have presented and has generally been guided by one of three models: donorcentric, recipient-centric, or sociocentric allocation. Each of these models is supported by valid ethical arguments. The main goal of donor-centric allocation is to ensure a successful outcome for the recipient. A good outcome provides justifi cation for medical professionals to assist a person who is not ill to put themselves in harm’s way to aid another. A positive result also gives an LND donor a sense that their eff ort was fruitful and worthwhile. However, this model dictates allocation to the healthiest patient on the transplant waiting list. These recipients are the most likely to have good outcomes on dialysis or with organs from deceased donors, and therefore are arguably the least in need. Recipient-centric allocation is based on the belief that society has a responsibility to protect its most vulnerable and disadvantaged members. Under this model, organs from LND donors are given to those patients in the greatest need, those for whom a kidney transplant might be truly life saving, or those disadvantaged under the existing system for allocation of kidneys from deceased donors. This model mainly benefi ts children, patients who have no vascular access, highly sensitised patients, and those with life-threatening medical illnesses related to dialysis. However, because the recipient-centric model accords priority to such patients, it tends to yield unacceptably poor transplant outcomes, and could lead to a negative public perception of LND donation. Under the third model, of sociocentric allocation, the LND donated organ is treated as a public resource that should be allocated in the fairest and most equitable way, irrespective of outcome or need. This rationale dictates that the recipient should be the patient at the top of the transplant waiting list administered by the United Network of Organ Sharing (UNOS). UNOS oversees the allocation of deceased donor organs in the USA, using a so-called match run algorithm that ranks potential recipients according to agreed criteria. The limitations of this model are that a patient at the top of the list will probably receive a kidney from a deceased donor in the near future, and that they will have already incurred the costs, and exposure to comorbidity, that result from a long period on dialysis. The waiting list for deceased donor kidneys can be circumvented by patients who fi nd a willing live donor. But direct donation might be complicated by diff erences in blood type and by HLA sensitivity. Some incompatible donor-recipient pairs enter into programmes that facilitate paired donation, also known as kidney paired donation. A donor and recipient who have incompatible blood groups or HLA sensitivity can be matched with another incompatible pair, to result in two compatible transplants (fi gure). Although there are many ways to match up a pool of incompatible pairs, the mathematical technique of optimisation helps to fi nd out which matches will yield the best results. Nevertheless, even in paired-donation programmes in which mathematical optimisation is applied, more than 50% of the incompatible pairs in the pool remain unmatched. In many cases, pools of incompatible donor-recipient pairs have a high proportion of patients with blood types that are hard to match and those with HLA sensitisation.

Subclinical Acute Antibody‐Mediated Rejection in Positive Crossmatch Renal Allografts

Subclinical antibody‐mediated rejection (AMR) has been described in renal allograft recipients with stable serum creatinine (SCr), however whether this leads to development of chronic allograft nephropathy (CAN) remains unknown.