Early prediction of renal graft function: Analysis of a multi-center, multi-level data set.

PURPOSE OF THE STUDY: Long-term graft survival rates after renal transplantation are still poor. We aimed to build an early predictor of an established long-term outcomes marker, the estimated glomerular filtration rate (eGFR) one year post-transplant (eGFR-1y). MATERIALS AND METHODS: A large cohort of 376 patients was characterized for a multi-level bio-marker panel including gene expression, cytokines, metabolomics and antibody reactivity profiles. Almost one thousand samples from the pre-transplant and early post-transplant period were analysed and employed for machine learning-assisted prediction. RESULTS: Pre-transplant data led to a prediction achieving a Pearson's correlation coefficient of r=0.38 between measured and predicted eGFR-1y. Two weeks post-transplant, the correlation was improved to r=0.63, and at the third month, to r=0.76. eGFR values were stable throughout the first post-transplant year. Several characteristics were predictive for eGFR, including age of donor and recipient, body mass index, HLA mismatch, cytomegalovirus mismatch and valganciclovir prophylaxis. Additionally, a subset of 19 nuclear magnetic resonance bins of the urine metabolome data was shown to have potential applications in non-invasive eGFR monitoring. Importantly, we identified the expression of the genes TMEM176B and HMMR as potential prognostic markers for changes in the eGFR after the second post-transplantation week. CONCLUSIONS: Our multi-center, multi-level data set represents a milestone in the efforts to predict transplant outcome. While an acceptable predictive capacity was achieved, we are still far from predicting changes in the eGFR precisely. Additional studies employing further marker panels are needed to establish predictors of eGFR-1y for clinical application; herein, gene expression markers seem to hold the most promise.

Sex-Associated Differences in Cytomegalovirus Prevention: Prophylactic Strategy is Potentially Associated With a Strong Kidney Function Impairment in Female Renal Transplant Patients.

Post-transplantation cytomegalovirus (CMV) syndrome can be prevented using the antiviral drug (val)ganciclovir. (Val)ganciclovir is typically administered following a prophylactic or a pre-emptive strategy. The prophylactic strategy entails early universal administration, the pre-emptive strategy, early treatment in case of infection. However, it is not clear which strategy is superior with respect to transplantation outcome; sex-specific effects of these prevention strategies are not known. We have retrospectively analyzed 540 patients from the multi-centre Harmony study along eight pre-defined visits: 308 were treated according to a prophylactic, 232 according to a pre-emptive strategy. As expected, we observed an association of prophylactic strategy with lower incidence of CMV syndrome, delayed onset and lower viral loads compared to the pre-emptive strategy. However, in female patients, the prophylactic strategy was associated with a strong impairment of glomerular filtration rate one year post-transplant (difference: -11.8 ± 4.3 ml min-1·1.73 m-2, p = 0.006). Additionally, we observed a tendency of higher incidence of acute rejection and severe BK virus reactivation in the prophylactic strategy group. While the prophylactic strategy was more effective for preventing CMV syndrome, our results suggest for the first time that the prophylactic strategy might lead to inferior transplantation outcomes in female patients, providing evidence for a strong association with sex. Further randomized controlled studies are necessary to confirm this potential negative effect.

A novel approach reveals that HLA class 1 single antigen bead-signatures provide a means of high-accuracy pre-transplant risk assessment of acute cellular rejection in renal transplantation.

BACKGROUND: Acute cellular rejection (ACR) is associated with complications after kidney transplantation, such as graft dysfunction and graft loss. Early risk assessment is therefore critical for the improvement of transplantation outcomes. In this work, we retrospectively analyzed a pre-transplant HLA antigen bead assay data set that was acquired by the e:KID consortium as part of a systems medicine approach. RESULTS: The data set included single antigen bead (SAB) reactivity profiles of 52 low-risk graft recipients (negative complement dependent cytotoxicity crossmatch, PRA < 30%) who showed detectable pre-transplant anti-HLA 1 antibodies. To assess whether the reactivity profiles provide a means for ACR risk assessment, we established a novel approach which differs from standard approaches in two aspects: the use of quantitative continuous data and the use of a multiparameter classification method. Remarkably, it achieved significant prediction of the 38 graft recipients who experienced ACR with a balanced accuracy of 82.7% (sensitivity = 76.5%, specificity = 88.9%). CONCLUSIONS: The resultant classifier achieved one of the highest prediction accuracies in the literature for pre-transplant risk assessment of ACR. Importantly, it can facilitate risk assessment in non-sensitized patients who lack donor-specific antibodies. As the classifier is based on continuous data and includes weak signals, our results emphasize that not only strong but also weak binding interactions of antibodies and HLA 1 antigens contain predictive information. TRIAL REGISTRATION: ClinicalTrials.gov NCT00724022 . Retrospectively registered July 2008.

BKV, CMV, and EBV Interactions and their Effect on Graft Function One Year Post-Renal Transplantation: Results from a Large Multi-Centre Study.

BACKGROUND: BK virus (BKV), Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) reactivations are common after kidney transplantation and associated with increased morbidity and mortality. Although CMV might be a risk factor for BKV and EBV, the effects of combined reactivations remain unknown. The purpose of this study is to ascertain the interaction and effects on graft function of these reactivations. METHODS: 3715 serum samples from 540 kidney transplant recipients were analysed for viral load by qPCR. Measurements were performed throughout eight visits during the first post-transplantation year. Clinical characteristics, including graft function (GFR), were collected in parallel. FINDINGS: BKV had the highest prevalence and viral loads. BKV or CMV viral loads over 10,000 copies·mL-1 led to significant GFR impairment. 57 patients had BKV-CMV combined reactivation, both reactivations were significantly associated (p = 0.005). Combined reactivation was associated with a significant GFR reduction one year post-transplantation of 11.7 mL·min-1·1.73 m-2 (p = 0.02) at relatively low thresholds (BKV > 1000 and CMV > 4000 copies·mL-1). For EBV, a significant association was found with CMV reactivation (p = 0.02), but no GFR reduction was found. Long cold ischaemia times were a further risk factor for high CMV load. INTERPRETATION: BKV-CMV combined reactivation has a deep impact on renal function one year post-transplantation and therefore most likely on long-term allograft function, even at low viral loads. Frequent viral monitoring and subsequent interventions for low BKV and/or CMV viraemia levels and/or long cold ischaemia time are recommended. FUND: Investigator Initiated Trial; financial support by German Federal Ministry of Education and Research (BMBF).

High-accuracy determination of internal circadian time from a single blood sample.

BACKGROUND: The circadian clock is a fundamental and pervasive biological program that coordinates 24-hour rhythms in physiology, metabolism, and behavior, and it is essential to health. Whereas therapy adapted to time of day is increasingly reported to be highly successful, it needs to be personalized, since internal circadian time is different for each individual. In addition, internal time is not a stable trait, but is influenced by many factors, including genetic predisposition, age, sex, environmental light levels, and season. An easy and convenient diagnostic tool is currently missing. METHODS: To establish a validated test, we followed a 3-stage biomarker development strategy: (a) using circadian transcriptomics of blood monocytes from 12 individuals in a constant routine protocol combined with machine learning approaches, we identified biomarkers for internal time; and these biomarkers (b) were migrated to a clinically relevant gene expression profiling platform (NanoString) and (c) were externally validated using an independent study with 28 early or late chronotypes. RESULTS: We developed a highly accurate and simple assay (BodyTime) to estimate the internal circadian time in humans from a single blood sample. Our assay needs only a small set of blood-based transcript biomarkers and is as accurate as the current gold standard method, dim-light melatonin onset, at smaller monetary, time, and sample-number cost. CONCLUSION: The BodyTime assay provides a new diagnostic tool for personalization of health care according to the patient's circadian clock. FUNDING: This study was supported by the Bundesministerium für Bildung und Forschung, Germany (FKZ: 13N13160 and 13N13162) and Intellux GmbH, Germany.

Is there a typical germinal center? A large-scale immunohistological study on the cellular composition of germinal centers during the hapten-carrier-driven primary immune response in mice.

Germinal centers (GCs) are complex, multicell-type, transient structures that form in secondary lymphatic tissues in response to T cell-dependent stimulation. This process is crucial to the adaptive immune response because it is the source of affinity maturation and long-lived B cell memory. Our previous studies showed that the growth of murine splenic GCs is nonsynchronized, involving broad-volume distributions of individual GCs at any time. This raises the question whether such a thing as a typical GC exists. To address this matter, we acquired large-scale confocal data on GCs throughout the course of the 2-phenyl-5-oxazolone chicken serum albumin-driven primary immune response in BALB/c mice. Semiautomated image analysis of 3457 GC sections revealed that, although there is no typical GC in terms of size, GCs have a typical cellular composition in that the cell ratios of resident T cells, macrophages, proliferating cells, and apoptotic nuclei are maintained during the established phase of the response. Moreover, our data provide evidence that the dark zone (DZ) and light zone (LZ) compartments of GCs are about the same size and led us to estimate that the minimal cell loss rate in GCs is 3% per hour. Furthermore, we found that the population of GC macrophages is larger and more heterogeneous than previously thought, and that despite enrichment of T cells in the LZ, the DZ of murine splenic GCs is not poor in T cells. DZ and LZ differ in the T cell-to-macrophage ratio rather than in the density of T cells.

Affinity maturation of B cells involves not only a few but a whole spectrum of relevant mutations.

Affinity maturation of B lymphocytes within germinal centers involves both diversification of their B-cell receptors (BCRs) by somatic hypermutation (SHM) and a crucial receptor-mediated selection step. However, in contrast to recent advances in revealing the molecular mechanism of SHM, the fundamentals of the selection process are still poorly understood, i.e. it is often not clear how and how many mutations contribute to improving a BCR during the response against a given antigen. A general drawback in assessing the mutations relevant to the selection process is the difficult task of rating the relative contributions of selection and intrinsic biases to the experimentally observed mutation patterns of BCRs. The approach proposed here is premised on statistical comparison of the frequency distributions of nucleotide substitutions as observed in datasets of hypermutated BCRs against their frequency distribution expected under the null hypothesis of no selection. Thereby, we show that the spectrum of mutations relevant to maturation of canonical anti-(4-hydroxy-3-nitrophenyl)acetyl BCRs is much broader than previously acknowledged, going beyond the scope of single key mutations. Moreover, our results suggest that maturation not only involves selection by means of affinity but likewise expression and stabilization of BCRs.

Broad volume distributions indicate nonsynchronized growth and suggest sudden collapses of germinal center B cell populations.

Immunization with a T cell-dependent Ag leads to the formation of several hundred germinal centers (GCs) within secondary lymphoid organs, a key process in the maturation of the immune response. Although prevailing perceptions about affinity maturation intuitively assume simultaneous seeding, growth, and decay of GCs, our previous mathematical simulations led us to hypothesize that their growth might be nonsynchronized. To investigate this, we performed computer-aided three-dimensional reconstructions of splenic GCs to measure size distributions at consecutive time points following immunization of BALB/c mice with a conjugate of 2-phenyl-oxazolone and chicken serum albumin. Our analysis reveals a broad volume distribution of GCs, indicating that individual GCs certainly do not obey the average time course of the GC volumes and that their growth is nonsynchronized. To address the cause and implications of this behavior, we compared our empirical data with simulations of a stochastic mathematical model that allows for frequent and sudden collapses of GCs. Strikingly, this model succeeds in reproducing the empirical average kinetics of GC volumes as well as the underlying broad size distributions. Possible causes of GC B cell population collapses are discussed in the context of the affinity-maturation process.

Recirculation of germinal center B cells: a multilevel selection strategy for antibody maturation.

Optimization of antibody affinity is a hallmark of the humoral immune response. It takes place in hundreds of transient microstructures called germinal centers (GCs). Their function and time-dependent behavior are subjects of active investigation. According to a generally accepted notion, their individual kinetics follows the average kinetics of all GCs present in the observed lymphatic tissue. In this review, we challenge this view and point out, with the help of mathematical simulations, that inferring the kinetics of individual GCs from cross-sectional evaluation of GC kinetics is virtually impossible. Thus, the time course of individual GCs is open to conjecture. For instance, one possible interpretation is that GCs exist for a time span considerably shorter than that of the observed average kinetics. We explore the implications of different temporal organizations of GCs in the light of the hypothesis that GC B-cell emigrants recolonize GC niches. This assumption leads to a view where GCs work in parallel but are linked by recirculation of B-cell emigrants. In this view, interleaved global and local competition provide for an implementation of multiple levels of B-cell selection in affinity maturation. The concepts of iteration, all-or-none behavior, and phasic mutation schedule are discussed in the light of this hypothesis.