Targeted Quantification of Proteoforms in Complex Samples by Proteoform Reaction Monitoring.

Existing mass spectrometric assays used for sensitive and specific measurements of target proteins across multiple samples, such as selected/multiple reaction monitoring (SRM/MRM) or parallel reaction monitoring (PRM), are peptide-based methods for bottom-up proteomics. Here, we describe an approach based on the principle of PRM for the measurement of intact proteoforms by targeted top-down proteomics, termed proteoform reaction monitoring (PfRM). We explore the ability of our method to circumvent traditional limitations of top-down proteomics, such as sensitivity and reproducibility. We also introduce a new software program, Proteoform Finder (part of ProSight Native), specifically designed for the easy analysis of PfRM data. PfRM was initially benchmarked by quantifying three standard proteins. The linearity of the assay was shown over almost 3 orders of magnitude in the femtomole range, with limits of detection and quantification in the low femtomolar range. We later applied our multiplexed PfRM assay to complex samples to quantify biomarker candidates in peripheral blood mononuclear cells (PBMCs) from liver-transplanted patients, suggesting their possible translational applications. These results demonstrate that PfRM has the potential to contribute to the accurate quantification of protein biomarkers for diagnostic purposes and to improve our understanding of disease etiology at the proteoform level.

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.

Transplant regimen adherence for kidney recipients by engaging information technologies (TAKE IT): Rationale and methods for a randomized controlled trial of a strategy to promote medication adherence among transplant recipients.

BACKGROUND: Several studies report a high prevalence of non-adherence to prescribed immunosuppressive (IS) medications among kidney transplant recipients (KTRs), yet few interventions have been effective for helping patients sustain appropriate post-transplant adherence. We describe a multifaceted, evidence-based, medication adherence monitoring strategy ('TAKE IT') that leverages available transplant center resources to identify potential medication non-adherence and other concerns earlier to prevent complications that could result from inadequate IS adherence. METHODS: The TAKE IT strategy includes: 1) medication adherence mobile application; 2) routine, online patient self-reported adherence assessments; 3) care alert notifications via the electronic health record (EHR) directed to transplant coordinators; 4) quarterly adherence reports to monitor IS values and summarize adherence trends; 5) deployment of adherence support tools tailored to specific adherence concerns. To test the TAKE IT intervention, we will conduct a two-arm, patient-randomized controlled trial at two large, diverse transplant centers (Northwestern University, Mayo Clinic, AZ) with planned recruitment of 450 KTRs (n = 225 per site) within 2 years of transplantation and 2 years of follow-up. Study assessments will take place at baseline, 6 weeks, 6, 12, 18 and 24 months. The primary effectiveness outcome is medication adherence via pill count, secondary outcomes include self-reported adherence and clinical outcomes. Process outcomes and cost-effectiveness will also be examined. CONCLUSION: The TAKE IT trial presents an innovative approach to monitoring and optimizing medication adherence among a population taking complex medication regimens. This trial seeks to evaluate the effectiveness and feasibility of this strategy compared to usual care.

Acute murine cytomegalovirus disrupts established transplantation tolerance and causes recipient allo-sensitization.

We have previously shown that acute cytomegalovirus (CMV) infection disrupts the induction of transplantation tolerance. However, what impact acute CMV infection would have on the maintenance of established tolerance and on subsequent recipient allo-sensitization is a clinically important unanswered question. Here we used an allogeneic murine islet transplantation tolerance model to examine the impact of acute CMV infection on: (a) disruption of established transplantation tolerance during tolerance maintenance; and (b) the possibility of recipient allo-sensitization by CMV-mediated disruption of stable tolerance. We demonstrated that acute CMV infection abrogated transplantation tolerance during the maintenance stage in 50%-60% recipients. We further demonstrated that acute CMV infection-mediated tolerance disruption led to recipient allo-sensitization by reverting the tolerant state of allo-specific T cells and promoting their differentiation to allo-specific memory cells. Consequently, a second same-donor islet allograft was rejected in an accelerated fashion by these recipients. Our study therefore supports close monitoring for allo-sensitization in previously tolerant transplant recipients in whom tolerance maintenance is disrupted by an episode of acute CMV infection.

Kidney-intrinsic factors determine the severity of ischemia/reperfusion injury in a mouse model of delayed graft function.

Delayed graft function due to transplant ischemia/reperfusion injury adversely affects up to 50% of deceased-donor kidney transplant recipients. However, key factors contributing to the severity of ischemia/reperfusion injury remain unclear. Here, using a clinically relevant mouse model of delayed graft function, we demonstrated that donor genetic background and kidney-intrinsic MyD88/Trif-dependent innate immunity were key determinants of delayed graft function. Functional deterioration of kidney grafts directly corresponded with the duration of cold ischemia time. The graft dysfunction became irreversible after cold ischemia time exceeded six hours. When cold ischemia time reached four hours, kidney grafts displayed histological features reflective of delayed graft function seen in clinical kidney transplantation. Notably, kidneys of B6 mice exhibited significantly more severe histological and functional impairment than kidneys of C3H or BALB/c mice, regardless of recipient strains or alloreactivities. Furthermore, allografts of B6 mice also showed an upregulation of IL-6, neutrophil gelatinase-associated lipocalin, and endoplasmic reticulum stress genes, as well as an increased influx of host neutrophils and memory CD8 T-cells. In contrast, donor MyD88/Trif deficiency inhibited neutrophil influx and decreased the expression of IL-6 and endoplasmic reticulum stress genes, along with improved graft function and prolonged allograft survival. Thus, kidney-intrinsic factors involving genetic characteristics and innate immunity serve as critical determinants of the severity of delayed graft function. This preclinical murine model allows for further investigations of the mechanisms underlying delayed graft function.

Correction to: Trends and predictors of multidimensional health-related quality of life after living donor kidney transplantation.

In its original publication, an erroneous version of Fig. 2d was included in the manuscript. The corrected figure has now been added.

MCMV Dissemination from Latently-Infected Allografts Following Transplantation into Pre-Tolerized Recipients.

Transplantation tolerance is achieved when recipients are unresponsive to donor alloantigen yet mobilize against third-party antigens, including virus. After transplantation, cytomegalovirus (CMV) reactivation in latently-infected transplants reduces allograft viability. To determine if pre-tolerized recipients are resistant to viral dissemination in this setting, we transfused chemically-fixed donor splenocytes (1-ethyl-3- (3'-dimethyl-aminopropyl)-carbo-diimide (ECDI)-treated splenocytes (ECDIsp)) to induce donor antigen tolerance without immunosuppression. In parallel, we implanted donor islet cells to validate operational tolerance. These pre-tolerized recipients were implanted with murine CMV (MCMV) latently-infected donor kidneys (a validated model of CMV latency) to monitor graft inflammation and viral dissemination. Our results indicate that tolerance to donor islets was sustained in recipients after implantation of donor kidneys. In addition, kidney allografts implanted after ECDIsp and islet implantation exhibited low levels of fibrosis and tubulitis. In contrast, kidney cellular and innate immune infiltrates trended higher in the CMV group and exhibited increased markers of CD8+ T cell activation. Tolerance induction was unable to prevent increases in MCMV-specific CD8+ T cells or dissemination of viral IE-1 DNA. Our data suggest that latently-infected allografts are inherently more susceptible to inflammation that is associated with viral dissemination in pre-tolerized recipients. Thus, CMV latently-infected allografts require enhanced strategies to protect allograft integrity and viral spread.

Kidney Graft Surveillance Biopsy Utilization and Trends: Results From a Survey of High-Volume Transplant Centers.

An e-mail-based market research survey focused on high-volume US adult transplant centers was developed and implemented to assess surveillance based on United Network for Organ Sharing/Scientific Registry of Transplant Recipients data: 51 to 100 transplants, 101 to 200 transplants, and more than 200 transplants. Eighty-three centers responded to the survey. Respondent centers represented 13,837/21,167 (65%) of the total kidney transplants in 2018. In total, 38/83 (46%) centers reported the use of surveillance biopsies-20 centers in all patients and 18 in select patients. Surveillance biopsies were performed in 37% (7/19) of centers performing 51 to 100 transplants annually, in 44% (15/34) doing 101 to 200 transplants, and in 53% (16/30) of centers doing more than 200 transplants. Of the 20 centers doing surveillance biopsies in all patients, 17/20 (85%) perform more than 100 annual transplants, and 3/20 (15%) perform less than 100 annual transplants. Of the 45 centers not currently doing surveillance biopsies, 13 (29%) used surveillance biopsies in the past; discontinuation was primarily due to patient inconvenience, adverse events, and cost. Using survey percentages, it is estimated that surveillance biopsies are performed in approximately 34% of kidney transplant recipients and that 74% of all surveillance biopsies occur in centers performing more than 100 kidney transplants per year.

Trends and predictors of multidimensional health-related quality of life after living donor kidney transplantation.

PURPOSE: Living donor kidney transplant (LDKT) imparts the best graft and patient survival for most end-stage kidney disease (ESKD) patients. Yet, there remains variation in post-LDKT health-related quality of life (HRQOL). Improved understanding of post-LDKT HRQOL can help identify patients for interventions to maximize the benefit of LDKT. METHODS: For 477 LDKT recipients transplanted between 11/2007 and 08/2016, we assessed physical, mental, social, and kidney-targeted HRQOL pre-LDKT, as well as 3 and 12 months post-operatively using the SF-36, Kidney Disease Quality of Life-Short Form (KDQOL-SF), and the Functional Assessment of Cancer Therapy-Kidney Symptom Index 19 item version (FKSI-19). We then examined trajectories of each HRQOL domain using latent growth curve models (LGCMs). We also examined associations between decline in HRQOL from 3 months to 12 months post-LDKT and death censored graft failure (DCGF) using Cox regression. RESULTS: Large magnitude effects (d > 0.80) were observed from pre- to post-LDKT change on the SF-36 Vitality scale (d = 0.81) and the KDQOL-SF Burden of Kidney Disease (d = 1.05). Older age and smaller pre- to post-LDKT decreases in serum creatinine were associated with smaller improvements on many HRQOL scales across all domains in LGCMs. Higher DCGF rates were associated with worse physical [e.g., SF-36 PCSoblique hazard ratio (HR) 1.18; 95% CI 1.01-1.38], mental (KDQOL-SF Cognitive Function HR 1.27; 95% CI 1.00-1.62), and kidney-targeted (FKSI-19 HR: 1.18; 95% CI 1.00-1.38) HRQOL domains. CONCLUSION: Clinical HRQOL monitoring may help identify patients who are most likely to have failing grafts and who would benefit from post-LDKT intervention.

New Insights Into the Molecular Mechanisms and Immune Control of Cytomegalovirus Reactivation.

Cytomegalovirus (CMV) is a ß-herpesvirus that establishes lifelong latency in infected hosts. Following transplantation of a latently infected organ, reactivation can occur and consists of a spectrum of clinically apparent syndromes from mild symptoms to tissue-invasive, resulting in both direct and indirect sequelae. Before the advent of effective antiviral agents, the primary treatment was reduction in immunosuppression (IS). While antiviral agents provide effective prophylaxis, there are several important caveats associated with their use, including drug toxicity and resistance. The traditional view attributes CMV reactivation and the ensuing clinical disease primarily to IS, either intrinsic to disease-related immune compromise or from the extrinsic administration of IS agents. However, previous data from both animal models and human subjects showed that inflammatory signals could induce upregulation of latent viral gene expression. New data demonstrate that ischemia/reperfusion is necessary and sufficient to induce CMV reactivation following murine transplantation of a latently infected graft. In this article, we review a growing body of evidence that suggests that reactivation of both human CMV and murine CMV is first triggered by molecular events that activate CMV gene expression and lytic infection and viral dissemination are then facilitated by IS. The initial activation of viral gene expression may be mediated by oxidative stress, DNA damage, or inflammatory cytokines, and these factors may act synergistically. New therapeutic approaches are needed to capture this complex array of targets.

A Practical Guide to the Clinical Implementation of Biomarkers for Subclinical Rejection Following Kidney Transplantation.

Noninvasive biomarkers are needed to monitor stable patients following kidney transplantation (KT), as subclinical rejection, currently detectable only with invasive surveillance biopsies, can lead to chronic rejection and graft loss. Several biomarkers have recently been developed to detect rejection in KT recipients, using different technologies as well as varying clinical monitoring strategies defined as "context of use (COU)." The various metrics utilized to evaluate the performance of each biomarker can also vary, depending on their intended COU. As the use of molecular biomarkers in transplantation represents a new era in patient management, it is important for clinicians to better understand the process by which the incremental value of each biomarkers is evaluated to determine its potential role in clinical practice. This process includes but is not limited to an assessment of clinical validity and utility, but to define these, the clinician must first appreciate the trajectory of a biomarker from bench to bedside as well as the regulatory and other requirements needed to navigate this course successfully. This overview summarizes this process, providing a framework that can be used by clinicians as a practical guide in general, and more specifically in the context of subclinical rejection following KT. In addition, we have reviewed available as well as promising biomarkers for this purpose in terms of the clinical need, COU, assessment of biomarker performance relevant to both the need and COU, assessment of biomarker benefits and risks relevant to the COU, and the evidentiary criteria of the biomarker relevant to the COU compared with the current standard of care. We also provide an insight into the path required to make biomarkers commercially available once they have been developed and validated so that they used by clinicians outside the research context in every day clinical practice.

A novel murine model of differentiation-mediated cytomegalovirus reactivation from latently infected bone marrow haematopoietic cells.

CD34+ myeloid lineage progenitor cells are an important reservoir of latent human cytomegalovirus (HCMV), and differentiation to macrophages or dendritic cells (DCs) is known to cause reactivation of latent virus. Due to its species-specificity, murine models have been used to study mouse CMV (MCMV) latency and reactivation in vivo. While previous studies have shown that MCMV genomic DNA can be detected in the bone marrow (BM) of latently infected mice, the identity of these cells has not been defined. Therefore, we sought to identify and enrich for cellular sites of MCMV latency in the BM haematopoietic system, and to explore the potential for establishing an in vitro model for reactivation of latent MCMV. We studied the kinetics and cellular characteristics of acute infection and establishment of latency in the BM of mice. We found that while MCMV can infect a broad range of haematopoietic BM cells (BMCs), latent virus is only detectable in haematopoietic stem cells (HSCs), myeloid progenitor cells, monocytes and DC-enriched cell subsets. Using three separate approaches, MCMV reactivation was detected in association with differentiation into DC-enriched BMCs cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) followed by lipopolysaccharide (LPS) treatment. In summary, we have defined the kinetics and cellular profile of MCMV infection followed by the natural establishment of latency in vivo in the mouse BM haematopoietic system, including the haematopoietic phenotypes of cells that are permissive to acute infection, establish and harbour detectable latent virus, and can be stimulated to reactivate following DC enrichment and differentiation, followed by treatment with LPS.

A clinically relevant murine model unmasks a "two-hit" mechanism for reactivation and dissemination of cytomegalovirus after kidney transplant.

Reactivation of latent cytomegalovirus remains an important complication after transplant. Although immunosuppression (IS) has been implicated as a primary cause, we have previously shown that the implantation response of a kidney allograft can lead to early transcriptional activation of latent murine cytomegalovirus (MCMV) genes in an immune-competent host and to MCMV reactivation and dissemination to other organs in a genetically immune-deficient recipient. We now describe a model that allows us to separately analyze the impact of the implantation effect vs that of a clinically relevant IS regimen. Treatment with IS of latently infected mice alone does not induce viral reactivation, but transplant of latently infected allogeneic kidneys combined with IS facilitates MCMV reactivation in the graft and dissemination to other organs. The IS regimen effectively dampens allo-immune inflammatory pathways and depletes recipient anti-MCMV but does not affect ischemia-reperfusion injury pathways. MCMV reactivation similar to that seen in allogeneic transplants combined with also occurs after syngeneic transplants. Thus, our data strongly suggest that while ischemia-reperfusion injury of the implanted graft is sufficient and necessary to initiate transcriptional reactivation of latent MCMV ("first hit"), IS is permissive to the first hit and facilitates dissemination to other organs ("second hit").

Immunometabolism of Phagocytes and Relationships to Cardiac Repair.

Cardiovascular disease remains the leading cause of death worldwide. Myocardial ischemia is a major contributor to cardiovascular morbidity and mortality. In the case of acute myocardial infarction, subsequent cardiac repair relies upon the acute, and coordinated response to injury by innate myeloid phagocytes. This includes neutrophils, monocytes, macrophage subsets, and immature dendritic cells. Phagocytes function to remove necrotic cardiomyocytes, apoptotic inflammatory cells, and to remodel extracellular matrix. These innate immune cells also secrete cytokines and growth factors that promote tissue replacement through fibrosis and angiogenesis. Within the injured myocardium, macrophages polarize from pro-inflammatory to inflammation-resolving phenotypes. At the core of this functional plasticity is cellular metabolism, which has gained an appreciation for its integration with phagocyte function and remodeling of the transcriptional and epigenetic landscape. Immunometabolic rewiring is particularly relevant after ischemia and clinical reperfusion given the rapidly changing oxygen and metabolic milieu. Hypoxia reduces mitochondrial oxidative phosphorylation and leads to increased reliance on glycolysis, which can support biosynthesis of pro-inflammatory cytokines. Reoxygenation is permissive for shifts back to mitochondrial metabolism and fatty acid oxidation and this is ultimately linked to pro-reparative macrophage polarization. Improved understanding of mechanisms that regulate metabolic adaptations holds the potential to identify new metabolite targets and strategies to reduce cardiac damage through nutrient signaling.

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.

Updated follow-up of a tolerance protocol in HLA-identical renal transplant pairs given donor hematopoietic stem cells.

Kidney transplant recipients given donor hematopoietic stem cells from their HLA-identical living related donors have now been followed between 5 and 9½ years post-operatively. Recipients who were designated as tolerant (Tol) have remained so since the last report when the 5 year (biopsy associated) milestone was reached. There has been 1 mortality of a Tol patient, unrelated to the study protocol, while 5 (of 15) have remained Tol between 7 and 8½ years post-operatively. There has been continuing elevated T-regulatory (CD4+CD25HighCD127-FOXP3+) cells in PBMC previously reported on. Ten year renal transplant biopsies are tentatively planned.

Incidence and Risk Factors of Intracranial Hemorrhage in Liver Transplant Recipients.

BACKGROUND: Intracranial hemorrhage after liver transplantation is an infrequently reported complication but one which can have devastating consequences. METHODS: We performed a retrospective cross-sectional analysis of all liver transplants performed between January 2010 and June 2015 at a single high-volume institution using a prospectively maintained electronic database and query of the electronic medical record. Cases of intracranial hemorrhage were adjudicated as either spontaneous intraparenchymal hemorrhage(IPH) or extra-axial hemorrhage (EAH). Patients with confirmed intracranial hemorrhage were compared with all other liver transplant recipients. Risk factors were identified by univariate analysis and logistic regression models for IPH and EAH. RESULTS: Thirty-one (5.2%) of 595 liver transplant recipients developed an intracranial hemorrhage within 12 months of transplantation, 15 IPH and 16 EAH. The majority of intracranial hemorrhages were diagnosed within 1 month of transplantation. Eight (26%) intracranial hemorrhage patients died during hospitalization. Fourteen (45%) intracranial hemorrhage patients died within 1 year of transplantation and 1-year mortality was greater than in patients without intracranial hemorrhage (11.2%, P < 0.01). Female sex (adjusted odds ratio [OR], 3.291; 95% confidence interval [CI], 1.092-9.924; P = 0.034), higher pretransplant bilirubin (adjusted OR, 1.037; 95% CI, 1.006-1.070; P = 0.020), and greater increase in pretransplant to posttransplant systolic blood pressure (adjusted OR, 1.029; 95% CI, 1.006-1.052; P = 0.012) were associated with posttransplant IPH. Lower pretransplant serum fibrinogen level (adjusted OR, 0.988; 95% CI, 0.979-0.998; P = 0.017) was associated with posttransplant EAH. CONCLUSIONS: Postoperative blood pressure control and pretransplant fibrinogen levels may be modifiable risk factors for preventing posttransplant intracranial hemorrhage.