Achieving tolerance modifies cancer susceptibility profiles in liver transplant recipients.

Long-term survival of transplant recipients is significantly impacted by malignancy. We aimed to determine whether calcineurin inhibitor (CNI)-treated recipients converted to and weaned off molecular target of rapamycin inhibitor (mTOR-I) therapy have favorable changes in their molecular profiles in regard to malignancy risk. We performed gene expression profiling from liver biopsy and blood (PBMC) specimens followed by network analysis of key dysregulated genes, associated diseases and disorders, molecular and cellular functions using IPA software. Twenty non-immune, non-viremic patients were included, and 8 of them achieved tolerance. Two comparisons were performed: (1) tolerance time point vs tacrolimus monotherapy and (2) tolerance time point vs sirolimus monotherapy. Upon achieving tolerance, IPA predicted significant activation of DNA damage response (p = 5.40e-04) and inhibition of DNA replication (p = 7.56e-03). Conversion from sirolimus to tolerance showed decrease in HCC (p = 1.30e-02), hepatic steatosis (p = 5.60e-02) and liver fibrosis (p = 2.91e-02) associated genes. In conclusion, this longitudinal study of patients eventually achieving tolerance reveals an evolving molecular profile associated with decreased cancer risk and improved hepatic steatosis and liver fibrosis. This provides a biological rationale for attempting conversion to mTOR-I therapy and tolerance following liver transplantation particularly in patients at higher risk of cancer incidence and progression post-transplant.

Renal Function at Discharge Among Kidney Recipients Experiencing Delayed Graft Function and Its Associations With Long-term Outcomes.

Delayed graft function (DGF) after kidney transplantation is associated with higher rates of acute rejection and poor graft survival and outcomes. Current DGF definitions based on posttransplant need for dialysis are not standardized and there are no objective methodologies for quantifying DGF severity. Methods: Using Organ Procurement and Transplantation Network data, we examined DGF, and used recipient serum creatinine at discharge as a correlate of renal function and DGF severity (mild: <2.5 mg/dL; severe: ≥2.5 mg/dL). The associations between donor and recipient factors and DGF severity were quantified using logistic regression. We also examined the associations between DGF severity and long-term recipient outcomes, adjusting for potential confounders. Results: A predictive model using donor and recipient factors had a reasonably good ability to discriminate mild (low creatinine) versus severe (high creatinine) DGF (c-statistic of 0.70). In Cox regression, DGF and creatinine at discharge were both independently associated with long-term outcomes, yet their effects differed depending on the outcome (graft function, death-censored graft function, recipient mortality). Our findings suggest that having DGF, but with relatively good renal function (creatinine <2.5) at discharge, may be less deleterious on graft and recipient survival compared with severe, prolonged DGF, which was associated with a decreased median graft survival of ~2.6 y compared with no DGF with low creatinine at discharge. Conclusions: Our novel DGF severity stratification identified unique factors associated with DGF severity, along with DGF's association with long-term graft and patient survival. The adverse cost and outcome implications of severe DGF warrant additional investigation to improve kidney transplantation practice.

DAMPs Released From Injured Renal Tubular Epithelial Cells Activate Innate Immune Signals in Healthy Renal Tubular Epithelial Cells.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) predictably causes acute kidney injury after shock and major cardiovascular procedures in all kidneys procured for transplantation. The earliest events of IRI are triggered by molecules released from injured cells, damage-associated molecular patterns (DAMPs), that bind pattern recognition receptors (PRRs) constitutively expressed on many cells within the kidney. Activation of PRR signaling leads to production of proinflammatory molecules, which incite a cascade of inflammatory events leading to acute kidney injury. Renal tubular epithelial cells (RTECs) are particularly susceptible to ischemic injury, and proximal RTEC injury is pathognomonic of renal IRI. To better understand how injured RTECs contribute to the cycle of deleterious inflammation in the setting of renal IRI, this study asked whether DAMPs released from injured RTECs induced PRR signals in healthy RTECs. METHODS: Human RTECs were necrosed ex vivo to release intracellular DAMPs and resulting necrotic supernatant used to stimulate healthy RTECs, T lymphocytes, and monocytes. RESULTS: DAMPs released from necrosed RTECs upregulated PRRs known to be associated with renal IRI and activated mitogen-activated protein kinase signaling pathways. Proinflammatory cytokines were upregulated in response to necrotic supernatant, and this upregulation was abrogated by MEK-1 inhibition. The RTEC-derived DAMPs were also potent inducers of T-cell activation/proliferation and monocyte migration. CONCLUSIONS: This is the first study to our knowledge to show that endogenous DAMPs released from injured RTECs directly activate PRR signaling in healthy RTECs. These findings provide new insights directed to therapeutics for renal IRI.

Combining Blood Gene Expression and Cellfree DNA to Diagnose Subclinical Rejection in Kidney Transplant Recipients.

BACKGROUND AND OBJECTIVES: Subclinical acute rejection is associated with poor outcomes in kidney transplant recipients. As an alternative to surveillance biopsies, noninvasive screening has been established with a blood gene expression profile. Donor-derived cellfree DNA (cfDNA) has been used to detect rejection in patients with allograft dysfunction but not tested extensively in stable patients. We hypothesized that we could complement noninvasive diagnostic performance for subclinical rejection by combining a donor-derived cfDNA and a gene expression profile assay. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We performed a post hoc analysis of simultaneous blood gene expression profile and donor-derived cfDNA assays in 428 samples paired with surveillance biopsies from 208 subjects enrolled in an observational clinical trial (Clinical Trials in Organ Transplantation-08). Assay results were analyzed as binary variables, and then, their continuous scores were combined using logistic regression. The performance of each assay alone and in combination was compared. RESULTS: For diagnosing subclinical rejection, the gene expression profile demonstrated a negative predictive value of 82%, a positive predictive value of 47%, a balanced accuracy of 64%, and an area under the receiver operating curve of 0.75. The donor-derived cfDNA assay showed similar negative predictive value (84%), positive predictive value (56%), balanced accuracy (68%), and area under the receiver operating curve (0.72). When both assays were negative, negative predictive value increased to 88%. When both assays were positive, positive predictive value increased to 81%. Combining assays using multivariable logistic regression, area under the receiver operating curve was 0.81, significantly higher than the gene expression profile (P<0.001) or donor-derived cfDNA alone (P=0.006). Notably, when cases were separated on the basis of rejection type, the gene expression profile was significantly better at detecting cellular rejection (area under the receiver operating curve, 0.80 versus 0.62; P=0.001), whereas the donor-derived cfDNA was significantly better at detecting antibody-mediated rejection (area under the receiver operating curve, 0.84 versus 0.71; P=0.003). CONCLUSIONS: A combination of blood-based biomarkers can improve detection and provide less invasive monitoring for subclinical rejection. In this study, the gene expression profile detected more cellular rejection, whereas donor-derived cfDNA detected more antibody-mediated rejection.

UNOS/OPTN Data-guided Assessment of Focal Segmental Glomerulosclerosis After Kidney Transplantation and Evaluation of Immunosuppressive Protocols in a Steroid-free Center.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a common recurrent glomerulopathy associated with graft loss and patient survival after kidney transplantation (KT). However, its natural history, clinical predictors, and treatment response are still poorly understood. Steroid withdrawal regimens in KT have been associated with improvements in cardiovascular risk and patient outcomes. The Scripps Center for Organ Transplantation (SCOT) uses a rapid low-dose steroid withdrawal immunosuppression (IS) protocol for KT maintenance. METHODS: We assessed the impact of our protocol on FSGS disease recurrence over a 10-y period to reassess our steroid and IS protocols and to evaluate if our patient outcomes diverge from published data. We compared 4 groups: steroids always, steroid free, steroid switch on, and steroid weaned off. We used IS and induction-matched retrospective data from United Network for Organ Sharing (UNOS) to investigate patient and graft survival for FSGS at SCOT. RESULTS: Our analysis results differ from earlier studies showing that FSGS was associated with a higher risk of graft loss, perhaps because of selection of a UNOS data set filtered to match the SCOT IS protocol for making direct comparisons. Overall outcomes of graft failure and recipient death did not differ between SCOT patients and steroid-free transplant patient data from the UNOS data for FSGS. SCOT recurrence rate for FSGS was 7.5%, which was lower than in most published single-center studies. CONCLUSIONS: Based on our results, we believe that it is safe to continue the steroid avoidance protocols at SCOT and the steroid-free protocol may not be detrimental when the adverse effects and toxicities associated with steroid use are considered.

Mitigation of radiation exposure during surgical hepatectomy after yttrium-90 radioembolization.

Yttrium-90 (Y-90) radioembolization for the treatment of hepatocellular carcinoma can present safety challenges when transplanting recently treated Y-90 patients. To reduce surgeons' contact with radioactive tissue and remain within occupational dose limits, current guidelines recommend delaying transplants at least 14 days, if possible. We wanted to determine the level of radiation exposure to the transplant surgeon when explanting an irradiated liver before the recommended decay period. Anex-vivoradiation exposure analysis was conducted on the explanted liver of a patient who received Y-90 therapy 46 h prior to orthotopic liver transplant. To estimate exposure to the surgeon's hands, radiation dosimeter rings were placed inside three different surgical glove configurations and exposed to the explanted liver. Estimated radiation doses corrected for Y-90 decay were calculated. Radiation safety gloves performed best, with an average radiation exposure rate of 5.36 mSV h-1in the static hand position, an 83% reduction in exposure over controls with no glove (31.31 mSv h-1). Interestingly, non-radiation safety gloves also demonstrated reduced exposure rates, well below occupational regulation limits. Handling of Y-90 radiated organs within the immediate post-treatment period can be done safely and does not exceed federal occupational dose limits if appropriate gloves and necessary precautions are exercised.

The Advantage of Multiple Listing Continues in the Kidney Allocation System Era.

BACKGROUND: Transplant candidates can be listed at multiple transplant centers to increase the probability of receiving an organ. We evaluated the association between multilisting (ML) status and access to a deceased donor kidney transplant (DDKT) to determine if ML provides a long-term advantage regarding wait-list mortality and recipient outcomes. MATERIALS AND METHODS: Candidates between January 2010 and October 2017 were identified as either singly or multiply listed using Organ Procurement and Transplantation Network data and cohorts before and after implementation of the Kidney Allocation System (KAS). Cross-sectional logistic regression was used to assess relationships between candidate factors and ML prevalence (5.4%). RESULTS: Factors associated with ML pre-KAS included having blood type B (reference, type O; odds ratio [OR], 1.20; P < .001), having private insurance (OR, 1.5; P < .001), wait time (OR, 1.28; P < .001), and increasing calculated panel-reactive antibody (cPRA) (reference, cPRA 0-100; OR for cPRA 80-98, 2.83; OR for cPRA 99, 3.47; OR for cPRA 100, 5.18; P < .001). Transplant rates were double for multilisted vs singly listed recipients (adjusted hazard ratio [aHR], 2.16; P < .001). Extra-donor service area ML candidates received transplants 2.5 years quicker than single-listing (SL) candidates, conferring a 42% wait-list advantage. Recipient death (aHR, 0.94; P = .122) and graft failure (aHR, 0.91; P = .006) rates were also lower for ML recipients. CONCLUSIONS: In the KAS era, ML continues to increase the likelihood of receiving a DDKT and lower the incidence of wait-list mortality, and it confers a survival advantages over SL.

Safety and Efficacy of a Steroid Avoidance Immunosuppression Regimen in Renal Transplant Patients With De Novo or Preformed Donor-Specific Antibodies: A Single-Center Study.

Although interest in the role of donor-specific antibodies (DSAs) in kidney transplant rejection, graft survival, and histopathological outcomes is increasing, their impact on steroid avoidance or minimization in renal transplant populations is poorly understood. Primary outcomes of graft survival, rejection, and histopathological findings were assessed in 188 patients who received transplants between 2012 and 2015 at the Scripps Center for Organ Transplantation, which follows a steroid avoidance protocol. Analyses were performed using data from the United Network for Organ Sharing. Cohorts included kidney transplant recipients with de novo DSAs (dnDSAs; n = 27), preformed DSAs (pfDSAs; n = 15), and no DSAs (nDSAs; n = 146). Median time to dnDSA development (classes I and II) was shorter (102 days) than in previous studies. Rejection of any type was associated with DSAs to class I HLA (P < .05) and class II HLA (P < .01) but not with graft loss. Although mean fluorescence intensity (MFI) independently showed no association with rejection, an MFI >5000 showed a trend toward more antibody-mediated rejection (P < .06), though graft loss was not independently associated. Banff chronic allograft nephropathy scores and a modified chronic injury score were increased in the dnDSA cohort at 6 months, but not at 2 years (P < .001 and P < .08, respectively). Our data suggest that dnDSAs and pfDSAs impact short-term rejection rates but do not negatively impact graft survival or histopathological outcomes at 2 years. Periodic protocol post-transplant DSA monitoring may preemptively identify patients who develop dnDSAs who are at a higher risk for rejection.

Systems biology approaches in solid organ transplantation.

PURPOSE OF REVIEW: Organ transplantation research has led to the discovery of several interesting individual mechanistic pathways, molecules and potential drug targets but there are still no comprehensive studies that have addressed how these varied mechanisms work in unison to regulate the posttransplant immune response that drives kidney rejection and dysfunction. RECENT FINDINGS: Systems biology is a rapidly expanding field that aims to integrate existing knowledge of molecular concepts and large-scale genomic and clinical datasets into networks that can be used in cutting edge computational models to define disease mechanisms in a holistic manner. Systems biology approaches have brought a paradigm shift from a reductionist view of biology to a wider agnostic assessment of disease from several lines of evidence. Although the complex nature of the posttransplant immune response makes it difficult to pinpoint mechanisms, systems biology is enabling discovery of unknown biological interactions using the cumulative power of genomic data sets, clinical data and endpoints, and improved computational methods for the systematic deconvolution of this response. SUMMARY: An integrative systems biology approach that leverages genomic data from varied technologies, such as DNA sequencing, copy number variation, RNA sequencing, and methylation profiles along with long-term clinical follow-up data has the potential to define a framework that can be mined to provide novel insights for developing therapeutic interventions in organ transplantation.

Feasibility and Comparison Study of Fecal Sample Collection Methods in Healthy Volunteers and Solid Organ Transplant Recipients Using 16S rRNA and Metagenomics Approaches.

The human microbiome encompasses a variety of microorganisms that change dynamically and are in close contact with the body. The microbiome influences health and homeostasis, as well as the immune system, and any significant change in this equilibrium (dysbiosis) triggers both acute and chronic health conditions. Microbiome research has surged, in part, due to advanced sequencing technologies enabling rapid, accurate, and cost-effective identification of the microbiome. A major prerequisite for stool sample collection to study the gut microbiome in longitudinal prospective studies requires standardized protocols that can be easily replicated. However, there are still significant bottlenecks to stool specimen collection that contribute to low patient retention rates in microbiome studies. These barriers are further exacerbated in solid organ transplant recipients where diarrhea is estimated to occur in up to half the patient population. We sought to test two relatively easy sample collection methods (fecal swab and wipes) and compare them to the more cumbersome "gold" standard collection method (scoop) using two different sequencing technologies (16S ribosomal RNA sequencing and shotgun metagenomics). Our comparison of the collection methods shows that both the swabs and the wipes are comparable to the scoop method in terms of bacterial abundance and diversity. The swabs, however, were closer in representation to the scoop and were easier to collect and process compared to the wipes. Potential contamination of the swab and the wipe samples by abundant skin commensals was low in our analysis. Comparison of the two sequencing technologies showed that they were complementary, and that 16S sequencing provided enough coverage to detect and differentiate between bacterial species identified in the collected samples. Our pilot study demonstrates that alternative collection methods for stool sampling are a viable option in clinical applications, such as organ transplant studies. The use of these methods may result in better patient retention recruitment rates in serial microbiome studies.

Discovery and cross-validation of peripheral blood and renal biopsy gene expression signatures from ethnically diverse kidney transplant populations.

We determined peripheral blood (PB) and biopsy (Bx) RNA expression signatures in a Brazilian and US cohort of kidney transplant patients. Phenotypes assigned by precise histology were: acute rejection (AR), interstitial fibrosis/tubular atrophy/chronic rejection (CR), excellent functioning transplants (TX), and glomerulonephritis recurrence (GN). Samples were analyzed on microarrays and profiles from each cohort were cross-validated on the other cohort with similar phenotypes. We discovered signatures for each tissue: (1) AR vs TX, (2) CR vs TX, and (3) GN vs TX using the Random Forests algorithm. We validated biopsies signatures of AR vs TX (area under the curve [AUC] 0.97) and CR vs TX (AUC 0.87). We also validated both PB and Bx signatures of AR vs TX and CR vs TX with varying degrees of accuracy. Several biological pathways were shared between AR and CR, suggesting similar rejection mechanisms in these 2 clinical phenotypes. Thus, we identified gene expression signatures for AR and CR in transplant patients and validated them in independent cohorts of significantly different racial/ethnic backgrounds. These results reveal that there are strong unifying immune mechanisms driving transplant diseases and identified in the signatures discovered in each cohort, suggesting that molecular diagnostics across populations are feasible despite ethnic and environmental differences.

Development and clinical validity of a novel blood-based molecular biomarker for subclinical acute rejection following kidney transplant.

Noninvasive biomarkers are needed to monitor stable patients after kidney transplant (KT), because subclinical acute rejection (subAR), currently detectable only with surveillance biopsies, can lead to chronic rejection and graft loss. We conducted a multicenter study to develop a blood-based molecular biomarker for subAR using peripheral blood paired with surveillance biopsies and strict clinical phenotyping algorithms for discovery and validation. At a predefined threshold, 72% to 75% of KT recipients achieved a negative biomarker test correlating with the absence of subAR (negative predictive value: 78%-88%), while a positive test was obtained in 25% to 28% correlating with the presence of subAR (positive predictive value: 47%-61%). The clinical phenotype and biomarker independently and statistically correlated with a composite clinical endpoint (renal function, biopsy-proved acute rejection, ≥grade 2 interstitial fibrosis, and tubular atrophy), as well as with de novo donor-specific antibodies. We also found that <50% showed histologic improvement of subAR on follow-up biopsies despite treatment and that the biomarker could predict this outcome. Our data suggest that a blood-based biomarker that reduces the need for the indiscriminate use of invasive surveillance biopsies and that correlates with transplant outcomes could be used to monitor KT recipients with stable renal function, including after treatment for subAR, potentially improving KT outcomes.

Transcriptomic studies in tolerance: Lessons learned and the path forward.

Immunosuppression after solid organ transplantation is a delicate balance of the immune response and is a complex phenomenon with many factors involved. Despite advances in the care of patients receiving organ transplants the adverse effects associated with immunosuppressive agents and the risks of long-term immunosuppression present a series of challenges and the need to weigh the risks and benefits of either over or under-immunosuppression. Ideally, if all transplant recipients could develop donor-specific immunological tolerance, it could drastically improve long-term graft survival without the need for immunosuppressive agents. In the absence of this ideal situation, the next best approach would be to develop tools to determine the adequacy of immunosuppression in each patient, in a manner that would individualize or personalize therapy. Despite current genomics-based studies of tolerance biomarkers in transplantation there are currently, no clinically validated tools to safely increase or decrease the level of IS that is beneficial to the patient. However, the successful identification of biomarkers and/or mechanisms of tolerance that have implications on long-term graft survival and outcomes depend on proper integration of study design, experimental protocols, and data-driven hypotheses. The objective of this article is to first, discuss the progress made on genomic biomarkers of immunological tolerance and the future avenues for the development of such biomarkers specifically in kidney transplantation. Secondly, we provide a set of guiding principles and identify the pitfalls, advantages, and drawbacks of studies that generate genomic data aimed at understanding transplant tolerance that is applicable to all solid transplants.

Gene expression signature predicts human islet integrity and transplant functionality in diabetic mice.

There is growing evidence that transplantation of cadaveric human islets is an effective therapy for type 1 diabetes. However, gauging the suitability of islet samples for clinical use remains a challenge. We hypothesized that islet quality is reflected in the expression of specific genes. Therefore, gene expression in 59 human islet preparations was analyzed and correlated with diabetes reversal after transplantation in diabetic mice. Analysis yielded 262 differentially expressed probesets, which together predict islet quality with 83% accuracy. Pathway analysis revealed that failing islet preparations activated inflammatory pathways, while functional islets showed increased regeneration pathway gene expression. Gene expression associated with apoptosis and oxygen consumption showed little overlap with each other or with the 262 probeset classifier, indicating that the three tests are measuring different aspects of islet cell biology. A subset of 36 probesets surpassed the predictive accuracy of the entire set for reversal of diabetes, and was further reduced by logistic regression to sets of 14 and 5 without losing accuracy. These genes were further validated with an independent cohort of 16 samples. We believe this limited number of gene classifiers in combination with other tests may provide complementary verification of islet quality prior to their clinical use.

Peripheral Blood Cell Gene Expression Diagnostic for Identifying Symptomatic Transthyretin Amyloidosis Patients: Male and Female Specific Signatures.

BACKGROUND: Early diagnosis of familial transthyretin (TTR) amyloid diseases remains challenging because of variable disease penetrance. Currently, patients must have an amyloid positive tissue biopsy to be eligible for disease-modifying therapies. Endomyocardial biopsies are typically amyloid positive when cardiomyopathy is suspected, but this disease manifestation is generally diagnosed late. Early diagnosis is often difficult because patients exhibit apparent symptoms of polyneuropathy, but have a negative amyloid biopsy. Thus, there is a pressing need for an additional early diagnostic strategy for TTR-aggregation-associated polyneuropathy and cardiomyopathy. METHODS AND FINDINGS: Global peripheral blood cell mRNA expression profiles from 263 tafamidis-treated and untreated V30M Familiar Amyloid Neuropathy patients, asymptomatic V30M carriers, and healthy, age- and sex-matched controls without TTR mutations were used to differentiate symptomatic from asymptomatic patients. We demonstrate that blood cell gene expression patterns reveal sex-independent, as well as male- and female-specific inflammatory signatures in symptomatic FAP patients, but not in asymptomatic carriers. These signatures differentiated symptomatic patients from asymptomatic V30M carriers with >80% accuracy. There was a global downregulation of the eIF2 pathway and its associated genes in all symptomatic FAP patients. We also demonstrated that the molecular scores based on these signatures significantly trended toward normalized values in an independent cohort of 46 FAP patients after only 3 months of tafamidis treatment. CONCLUSIONS: This study identifies novel molecular signatures that differentiate symptomatic FAP patients from asymptomatic V30M carriers as well as affected males and females. We envision using this approach, initially in parallel with amyloid biopsies, to identify individuals who are asymptomatic gene carriers that may convert to FAP patients. Upon further validation, peripheral blood cell mRNA expression profiling could become an independent early diagnostic. This quantitative gene expression signature for symptomatic FAP could also become a biomarker to demonstrate significant disease-modifying effects of drugs and drug candidates. For example, when new disease modifiers are being evaluated in a FAP clinical trial, such surrogate biomarkers have the potential to provide an objective, quantitative and mechanistic molecular diagnostic of disease response to therapy.

Genome-wide expression profiling in the peripheral blood of patients with fibromyalgia.

OBJECTIVES: Fibromyalgia (FM) is a common pain disorder characterized by nociceptive dysregulation. The basic biology of FM is poorly understood. Herein we have used agnostic gene expression as a potential probe for informing its underlying biology and the development of a proof-of-concept diagnostic gene expression signature. METHODS: We analyzed RNA expression in 70 FM patients and 70 healthy controls. The isolated RNA was amplified and hybridized to Affymetrix® Human Gene 1.1 ST Peg arrays. The data was analyzed using Partek Genomics Suite version 6.6. RESULTS: Fibromyalgia patients exhibited a differential expression of 421 genes (p<0.001), several relevant to pathways for pain processing, such as glutamine/glutamate signaling and axonal development. There was also an upregulation of several inflammatory pathways and downregulation of pathways related to hypersensitivity and allergy. Using rigorous diagnostic modeling strategies, we show "locked" gene signatures discovered on Training and Test cohorts, that have a mean Area Under the Curve (AUC) of 0.81 on randomized, independent external data cohorts. Lastly, we identified a subset of 10 probesets that provided a diagnostic sensitivity for FM of 95% and a specificity of 96%. We also show that the signatures for FM were very specific to FM rather than common FM comorbidities. CONCLUSIONS: These findings provide new insights relevant to the pathogenesis of FM, and provide several testable hypotheses that warrant further exploration and also establish the foundation for a first blood-based molecular signature in FM that needs to be validated in larger cohorts of patients.

Genomic biomarkers correlate with HLA-identical renal transplant tolerance.

The ability to achieve immunologic tolerance after transplantation is a therapeutic goal. Here, we report interim results from an ongoing trial of tolerance in HLA-identical sibling renal transplantation. The immunosuppressive regimen included alemtuzumab induction, donor hematopoietic stem cells, tacrolimus/mycophenolate immunosuppression converted to sirolimus, and complete drug withdrawal by 24 months post-transplantation. Recipients were considered tolerant if they had normal biopsies and renal function after an additional 12 months without immunosuppression. Of the 20 recipients enrolled, 10 had at least 36 months of follow-up after transplantation. Five of these 10 recipients had immunosuppression successfully withdrawn for 16-36 months (tolerant), 2 had disease recurrence, and 3 had subclinical rejection in protocol biopsies (nontolerant). Microchimerism disappeared after 1 year, and CD4(+)CD25(high)CD127(-)FOXP3(+) regulatory T cells and CD19(+)IgD/M(+)CD27(-) B cells were increased through 5 years post-transplantation in both tolerant and nontolerant recipients. Immune/inflammatory gene expression pathways in the peripheral blood and urine, however, were differentially downregulated between tolerant and nontolerant recipients. In summary, interim results from this trial of tolerance in HLA-identical renal transplantation suggest that predictive genomic biomarkers, but not immunoregulatory phenotyping, may be able to discriminate tolerant from nontolerant patients.

Genomic meta-analysis of growth factor and integrin pathways in chronic kidney transplant injury.

BACKGROUND: Chronic Allograft Nephropathy (CAN) is a clinical entity of progressive kidney transplant injury. The defining histology is tubular atrophy with interstitial fibrosis (IFTA). Using a meta-analysis of microarrays from 84 kidney transplant biopsies, we revealed growth factor and integrin adhesion molecule pathways differentially expressed and correlated with histological progression. A bioinformatics approach mining independent datasets leverages new and existing data to identify correlative changes in integrin and growth factor signaling pathways. RESULTS: Analysis of CAN/IFTA Banff grades showed that hepatocyte growth factor (HGF), and epidermal growth factor (EGF) pathways are significantly differentially expressed in all classes of CAN/IFTA. MAPK-dependent pathways were also significant. However, the TGFß pathways, albeit present, failed to differentiate CAN/IFTA progression. The integrin subunits ß8, αv, αµ and ß5 are differentially expressed, but ß1, ß6 and α6 specifically correlate with progression of chronic injury. Results were validated using our published proteomic profiling of CAN/IFTA. CONCLUSIONS: CAN/IFTA with chronic kidney injury is characterized by expression of distinct growth factors and specific integrin adhesion molecules as well as their canonical signaling pathways. Drug target mapping suggests several novel candidates for the next generation of therapeutics to prevent or treat progressive transplant dysfunction with interstitial fibrosis.