Cherry on Top or Real Need? A Review of Explainable Machine Learning in Kidney Transplantation.

Research on solid organ transplantation has taken advantage of the substantial acquisition of medical data and the use of artificial intelligence (AI) and machine learning (ML) to answer diagnostic, prognostic, and therapeutic questions for many years. Nevertheless, despite the question of whether AI models add value to traditional modeling approaches, such as regression models, their "black box" nature is one of the factors that have hindered the translation from research to clinical practice. Several techniques that make such models understandable to humans were developed with the promise of increasing transparency in the support of medical decision-making. These techniques should help AI to close the gap between theory and practice by yielding trust in the model by doctors and patients, allowing model auditing, and facilitating compliance with emergent AI regulations. But is this also happening in the field of kidney transplantation? This review reports the use and explanation of "black box" models to diagnose and predict kidney allograft rejection, delayed graft function, graft failure, and other related outcomes after kidney transplantation. In particular, we emphasize the discussion on the need (or not) to explain ML models for biological discovery and clinical implementation in kidney transplantation. We also discuss promising future research paths for these computational tools.

Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles.

Purpose: Kidney transplantation is the optimal treatment for patients with end-stage kidney disease. Donor-specific urinary extracellular vesicles (uEVs) hold potential as biomarkers for assessing allograft status. We aimed to develop a method for identifying donor-specific uEVs based on human leukocyte antigen (HLA) mismatching with the kidney transplant recipients (KTRs). Patients and Methods: Urine and plasma were obtained from HLA-A2+ donors and HLA-A2- KTRs pre-transplant. CD9 (tetraspanin, EV marker) and HLA-A2 double-positive (CD9+ HLA-A2+) EVs were quantified using isolation-free imaging flow cytometry (IFCM). Healthy individuals' urine was used to investigate CD9+ HLA-class-I+ uEV quantification using IFCM, time-resolved fluoroimmunoassay (TR-FIA), and immunogold staining cryo-electron microscopy (cryo-EM). Culture-derived CD9+ HLA-class-I+ EVs were spiked into the urine to investigate urine matrix effects on uEV HLA detection. Deceased donor kidneys and peritumoral kidney tissue were used for HLA class I detection with histochemistry. Results: The concentrations of CD9+ HLA-A2+ EVs in both donor and recipient urine approached the negative (detergent-treated) control levels for IFCM and were significantly lower than those observed in donor plasma. In parallel, universal HLA class I+ uEVs were similarly undetectable in the urine and uEV isolates compared with plasma, as verified by IFCM, TR-FIA, and cryogenic electron microscopy. Culture supernatant containing HLA class I+ vesicles from B, T, and human proximal tubule cells were spiked into the urine, and these EVs remained stable at 37°C for 8 hours. Immunohistochemistry revealed that HLA class I was predominantly expressed on the basolateral side of renal tubules, with limited expression on their urine/apical side. Conclusion: The detection of donor-specific uEVs is hindered by the limited release of HLA class I+ EVs from the kidney into the urine, primarily due to the polarized HLA class I expression on renal tubules. Identifying donor-specific uEVs requires further advancements in recognizing transplant-specific uEVs and urine-associated markers.

CoPoP liposomes displaying stabilized clade C HIV-1 Env elicit tier 2 multiclade neutralization in rabbits.

One of the strategies towards an effective HIV-1 vaccine is to elicit broadly neutralizing antibody responses that target the high HIV-1 Env diversity. Here, we present an HIV-1 vaccine candidate that consists of cobalt porphyrin-phospholipid (CoPoP) liposomes decorated with repaired and stabilized clade C HIV-1 Env trimers in a prefusion conformation. These particles exhibit high HIV-1 Env trimer decoration, serum stability and bind broadly neutralizing antibodies. Three sequential immunizations of female rabbits with CoPoP liposomes displaying a different clade C HIV-1 gp140 trimer at each dosing generate high HIV-1 Env-specific antibody responses. Additionally, serum neutralization is detectable against 18 of 20 multiclade tier 2 HIV-1 strains. Furthermore, the peak antibody titers induced by CoPoP liposomes can be recalled by subsequent heterologous immunization with Ad26-encoded membrane-bound stabilized Env antigens. Hence, a CoPoP liposome-based HIV-1 vaccine that can generate cross-clade neutralizing antibody immunity could potentially be a component of an efficacious HIV-1 vaccine.

Polyfunctional donor-reactive T cells are associated with acute T-cell-mediated rejection of the kidney transplant.

Acute T-cell-mediated rejection (aTCMR) still remains a clinical problem after kidney transplantation despite significant improvements in immunosuppressive regimens. Polyfunctional T cells, i.e. T cells producing multiple pro-inflammatory cytokines, are believed to be the most relevant T cells in an immune response. The aim of this study was to determine whether polyfunctional donor-reactive T cells are associated with aTCMR. In a case-control study, 49 kidney transplant recipients with a biopsy-proven aTCMR in the first year after transplantation were included, as well as 51 controls without aTCMR. Circulating donor-reactive T cells were identified by the expression of CD137 after short-term co-culture with donor antigen-presenting cells. Polyfunctional donor-reactive T cells were further characterized by dissection into different T-cell subsets encompassing the spectrum of naïve to terminally differentiated effector T cells. Prior to kidney transplantation, proportions of donor-reactive CD4+ (0.03% versus 0.02%; P < 0.01) and CD8+ (0.18% versus 0.10%; P < 0.01) CD137++ T cells were significantly higher in recipients with a biopsy-proven aTCMR versus non-rejectors. Polyfunctionality was higher (P = 0.03) in this subset of CD137-expressing T cells. These cells were predominantly of the EM/EMRA-phenotype, with polyfunctional donor-reactive CD137++CD4+ T cells predominantly co-expressing CD28 whereas approximately half of the polyfunctional CD137++CD8+ T cells co-expressed CD28. In addition, at the time of aTCMR, polyfunctional donor-reactive CD137++ CD4+, but not CD8+, T cells, were specifically decreased by 75% compared to before transplantation in recipients with as well as those without an aTCMR. Prior to transplantation, the proportion of polyfunctional donor-reactive CD137++ T cells is associated with the occurrence of a biopsy-proven aTCMR within the first year after transplantation.

Booster with Ad26.COV2.S or Omicron-adapted vaccine enhanced immunity and efficacy against SARS-CoV-2 Omicron in macaques.

Omicron spike (S) encoding vaccines as boosters, are a potential strategy to improve COVID-19 vaccine efficacy against Omicron. Here, macaques (mostly females) previously immunized with Ad26.COV2.S, are boosted with Ad26.COV2.S, Ad26.COV2.S.529 (encoding Omicron BA.1 S) or a 1:1 combination of both vaccines. All booster vaccinations elicit a rapid antibody titers increase against WA1/2020 and Omicron S. Omicron BA.1 and BA.2 antibody responses are most effectively boosted by vaccines including Ad26.COV2.S.529. Independent of vaccine used, mostly WA1/2020-reactive or WA1/2020-Omicron BA.1 cross-reactive B cells are detected. Ad26.COV2.S.529 containing boosters provide only slightly higher protection of the lower respiratory tract against Omicron BA.1 challenge compared with Ad26.COV2.S-only booster. Antibodies and cellular immune responses are identified as complementary correlates of protection. Overall, a booster with an Omicron-spike based vaccine provide only moderately improved immune responses and protection compared with the original Wuhan-Hu-1-spike based vaccine, which still provide robust immune responses and protection against Omicron.

Alloreactive T cells to Assess Acute Rejection Risk in Kidney Transplant Recipients.

Memory T cells are important mediators of transplant rejection but are not routinely measured before or after kidney transplantation. The aims of this study were as follows: (1) validate whether pretransplant donor-reactive memory T cells are reliable predictors of acute rejection (AR) (2) determine whether donor-reactive memory T cells can distinguish AR from other causes of transplant dysfunction. Methods: Samples from 103 consecutive kidney transplant recipients (2018-2019) were obtained pretransplantation and at time of for-cause biopsy sampling within 6 mo of transplantation. The number of donor-reactive interferon gamma (IFN-γ) and interleukin (IL)-21-producing memory T cells was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay. Results: Of the 63 patients who underwent a biopsy, 25 had a biopsy-proven acute rejection (BPAR; 22 aTCMR and 3 aAMR), 19 had a presumed rejection, and 19 had no rejection. Receiver operating characteristic analysis showed that the pretransplant IFN-γ ELISPOT assay distinguished between patients who later developed BPAR and patients who remained rejection-free (area under the curve [AUC] 0.73; sensitivity 96% and specificity 41%). Both the IFN-γ and IL-21 assays were able to discriminate BPAR from other causes of transplant dysfunction (AUC 0.81; sensitivity 87% and specificity 76% and AUC 0.81; sensitivity 93% and specificity 68%, respectively). Conclusions: This study validates that a high number of donor-reactive memory T cells before transplantation is associated with the development of AR after transplantation. Furthermore, it demonstrates that the IFN-γ and IL-21 ELISPOT assays are able to discriminate between patients with AR and patients without AR at the time of biopsy sampling.

Booster vaccination with Ad26.COV2.S or an Omicron-adapted vaccine in pre-immune hamsters protects against Omicron BA.2.

Since the original outbreak of the SARS-CoV-2 virus, several rapidly spreading SARS-CoV-2 variants of concern (VOC) have emerged. Here, we show that a single dose of Ad26.COV2.S (based on the Wuhan-Hu-1 spike variant) protects against the Gamma and Delta variants in naive hamsters, supporting the observed maintained vaccine efficacy in humans against these VOC. Adapted spike-based booster vaccines targeting Omicron variants have now been authorized in the absence of human efficacy data. We evaluated the immunogenicity and efficacy of Ad26.COV2.S.529 (encoding a stabilized Omicron BA.1 spike) in naive mice and in hamsters with pre-existing immunity to the Wuhan-Hu-1 spike. In naive mice, Ad26.COV2.S.529 elicited higher neutralizing antibody titers against SARS-CoV-2 Omicron BA.1 and BA.2, compared with Ad26.COV2.S. However, neutralizing titers against the SARS-CoV-2 B.1 (D614G) and Delta variants were lower after primary vaccination with Ad26.COV2.S.529 compared with Ad26.COV2.S. In contrast, we found comparable Omicron BA.1 and BA.2 neutralizing titers in hamsters with pre-existing Wuhan-Hu-1 spike immunity after vaccination with Ad26.COV2.S, Ad26.COV2.S.529 or a combination of the two vaccines. Moreover, all three vaccine modalities induced equivalent protection against Omicron BA.2 challenge in these animals. Overall, our data suggest that an Omicron BA.1-based booster in rodents does not improve immunogenicity and efficacy against Omicron BA.2 over an Ad26.COV2.S booster in a setting of pre-existing immunity to SARS-CoV-2.

Physiologically-Based Kinetic Modeling of Intravenously Administered Gold (Au) Nanoparticles.

Physiologically-based kinetic (PBK) modeling is a valuable tool to understand the kinetics of nanoparticles (NPs) in vivo. However, estimating PBK parameters remains challenging and commonly requires animal studies. To develop predictive models to estimate PBK parameter values based on NP characteristics, a database containing PBK parameter values and corresponding NP characteristics is needed. As a first step toward this objective, this study estimates PBK parameters for gold NPs (AuNPs) and provides a comparison of two different NPs. Two animal experiments are conducted in which varying doses of AuNPs attached with polyethylene glycol (PEG) are administered intravenously to rats. The resulting Au concentrations are used to estimate PBK model parameters. The parameters are compared with PBK parameters previously estimated for poly(alkyl cyanoacrylate) NPs loaded with cabazitaxel and for LipImage 815. This study shows that a small initial database of PBK parameters collected for three NPs is already sufficient to formulate new hypotheses on NP characteristics that may be predictive of PBK parameter values. Further research should focus on developing a larger database and on developing quantitative models to predict PBK parameter values.

Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry.

Urinary extracellular vesicles (uEVs) are promising biomarkers for various diseases. However, many tools measuring uEVs rely on time-consuming uEV isolation methods, which could induce sample bias. This study demonstrates the detection of single uEVs without isolation using imaging flow cytometry (IFCM). Unstained urine samples contained auto-fluorescent (A-F) particles when characterized with IFCM. Centrifugation successfully removed A-F particles from the unprocessed urine. Based on the disappearance of A-F particles, a gate was defined to distinguish uEVs from A-F particles. The final readouts of IFCM were verified as single EVs based on detergent treatment and serial dilutions. When developing this protocol to measure urine samples with abnormally high protein levels, 25 mg/mL dithiothreitol (DTT) showed improved uEV recovery over 200 mg/mL DTT. This study provides an isolation-free protocol using IFCM to quantify and phenotype single uEVs, eliminating the hindrance and influence of A-F particles, protein aggregates, and coincidence events.

P-glycoprotein, FK-binding Protein-12, and the Intracellular Tacrolimus Concentration in T-lymphocytes and Monocytes of Kidney Transplant Recipients.

BACKGROUND: . Transplant recipients may develop rejection despite having adequate tacrolimus whole blood predose concentrations (C 0 ). The intra-immune cellular concentration is potentially a better target than C 0 . However, little is known regarding intracellular tacrolimus concentration in T-lymphocytes and monocytes. We investigated the tacrolimus concentrations in both cell types and their relation with the expression and activity of FK-binding protein (FKBP)-12 and P-glycoprotein (P-gp). METHODS: . T-lymphocytes and monocytes were isolated from kidney transplant recipients followed by intracellular tacrolimus concentration measurement. FKBP-12 and P-gp were quantified with Western blot, flow cytometry, and the Rhodamine-123 assay. Interleukin-2 and interferon-γ in T-lymphocytes were measured to quantify the effect of tacrolimus. RESULTS: . Tacrolimus concentration in T-lymphocytes was lower than in monocytes (15.3 [8.5-33.4] versus 131.0 [73.5-225.1] pg/million cells; P < 0.001). The activity of P-gp (measured by Rhodamine-123 assay) was higher in T-lymphocytes than in monocytes. Flow cytometry demonstrated a higher expression of P-gp (normalized mean fluorescence intensity 1.5 [1.2-1.7] versus 1.2 [1.1-1.4]; P = 0.012) and a lower expression of FKBP-12 (normalized mean fluorescence intensity 1.3 [1.2-1.7] versus 1.5 [1.4-2.0]; P = 0.011) in T-lymphocytes than monocytes. Western blot confirmed these observations. The addition of verapamil, a P-gp inhibitor, resulted in a 2-fold higher intra-T-cell tacrolimus concentration. This was accompanied by a significantly fewer cytokine-producing cells. CONCLUSIONS: . T-lymphocytes have a higher activity of P-gp and lower concentration of the FKBP-12 compared with monocytes. This explains the relatively lower tacrolimus concentration in T-lymphocytes. The addition of verapamil prevents loss of intracellular tacrolimus during the cell isolation process and is required to ensure adequate intracellular concentration measurement.

Direct detection of circulating donor-derived extracellular vesicles in kidney transplant recipients.

Extracellular vesicles (EVs) are tissue-specific particles containing valuable diagnostic information. However, single EV analysis in blood is challenging due to their physical properties, the molecular complexity of plasma, and a lack of robust data interpretation methods. We assess the applicability of our recently-developed calibrated Imaging Flow Cytometry (IFCM)-based methodology to detect/characterize circulating tissue-specific EV subsets in the clinical setting of kidney transplantation. Platelet-poor plasma was generated from 36 HLA-A3 mismatched donor (HLA-A3 +) and kidney transplant recipients (KTRs; HLA-A3-). Samples taken before transplantation, 3 days, 7 days, and 6 months after transplantation as well as before 'for-cause' kidney transplant biopsies were stained with anti-CD9 (plasma EV-marker) and anti-HLA-A3. Before transplantation, no significant differences in total CD9 + EV concentrations were detected between donor and KTR samples. Tissue-specific EVs were identified as CD9 + HLA-A3 + . Serial dilution experiments of HLA-A3 + in HLA-A3- PPP showed that single CD9 + HLA-A3 + EVs were detectable down to ~ 1% above the recipient 'self-signal'. After transplantation, CD9 + HLA-A3 + EVs were detected above pre-transplantation concentrations in individuals with stable allograft function, but not in individuals with allograft dysfunction. These results demonstrate the applicability of our calibrated IFCM-based methodology in the direct detection of tissue-specific EV subsets in clinical samples. We believe that this EV methodology is applicable in a variety of clinical contexts.

Sequential use of Ad26-based vaccine regimens in NHP to induce immunity against different disease targets.

The adenovirus (Ad)26 serotype-based vector vaccine Ad26.COV2.S has been used in millions of subjects for the prevention of COVID-19, but potentially elicits persistent anti-vector immunity. We investigated if vaccine-elicited immunity to Ad26 vector-based vaccines significantly influences antigen-specific immune responses induced by a subsequent vaccination with Ad26 vector-based vaccine regimens against different disease targets in non-human primates. A homologous Ad26 vector-based vaccination regimen or heterologous regimens (Ad26/Ad35 or Ad26/Modified Vaccinia Ankara [MVA]) induced target pathogen-specific immunity in animals, but also persistent neutralizing antibodies and T-cell responses against the vectors. However, subsequent vaccination (interval, 26-57 weeks) with homologous and heterologous Ad26 vector-based vaccine regimens encoding different target pathogen immunogens did not reveal consistent differences in humoral or cellular immune responses against the target pathogen, as compared to responses in naïve animals. These results support the sequential use of Ad26 vector-based vaccine regimens targeting different diseases.

An imaging flow cytometry-based methodology for the analysis of single extracellular vesicles in unprocessed human plasma.

Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we present a clinically feasible, imaging flow cytometry (IFCM)-based methodology to phenotype and determine the concentration of EVs with a diameter ≤400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs. Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) we found that >90% of double-positive fluorescent events represented single EVs. Through this work, we provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore corners of EVs as cellular messengers in healthy and pathological conditions.

Extracellular Vesicles Released During Normothermic Machine Perfusion Are Associated With Human Donor Kidney Characteristics.

BACKGROUND: Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. The characterization of EVs released by kidney grafts during normothermic machine perfusion (NMP) may present a promising avenue to assess graft status before transplantation. METHODS: We phenotyped and determined the concentrations of EVs in the perfusate of 8 discarded expanded-criteria donor human kidneys during 6 h of NMP. Perfusate samples were taken at 0/60/180/360 min and examined with nanoparticle tracking analysis and imaging flow cytometry (IFCM). Using IFCM, EVs were identified by their expression of common EV markers CD9, CD63, and CD81 (tetraspanins) in combination with either platelet endothelial cell adhesion molecule (CD31), pan-leukocyte protein (CD45), or carboxyfluorescein succiminidyl ester (CFSE) fluorescence. RESULTS: Nanoparticle tracking analysis measurements revealed the release of nanoparticles <400 nm into the perfusate during NMP. With IFCM, tetraspanin protein signatures of the released nanoparticles were characterized, and the majority (~75%) of CFSE+ EVs were found to be CD81+, whereas ~16% were CD9+ and ~8% CD63+. Correlation analysis of concentrations of identified EV subsets with crude donor characteristics and NMP viability characteristics revealed significant correlations with cold ischemia time, donor age, and renal flow. CONCLUSIONS: Our findings demonstrate that discarded expanded-criteria donor kidney grafts release distinct EV subsets during NMP. Because these subsets correlate with well-established indicators of transplant outcome, EVs might represent new potential candidates for assessment of kidney graft quality.

Donor-Derived Cell-Free DNA for the Detection of Heart Allograft Injury: The Impact of the Timing of the Liquid Biopsy.

Background: In heart transplant recipients, donor-derived cell-free DNA (ddcfDNA) is a potential biomarker for acute rejection (AR), in that increased values may indicate rejection. For the assessment of ddcfDNA as new biomarker for rejection, blood plasma sampling around the endomyocardial biopsy (EMB) seems a practical approach. To evaluate the effect of the EMB procedure on ddcfDNA values, ddcfDNA values before the EMB were pairwise compared to ddcfDNA values after the EMB. We aimed at evaluating whether it matters whether the ddcfDNA sampling is done before or after the EMB-procedure. Methods: Plasma samples from heart transplant recipients were obtained pre-EMB and post-EMB. A droplet digital PCR method was used for measuring ddcfDNA, making use of single-nucleotide polymorphisms that allowed both relative quantification, as well as absolute quantification of ddcfDNA. Results: Pairwise comparison of ddcfDNA values pre-EMB with post-EMB samples (n = 113) showed significantly increased ddcfDNA concentrations and ddcfDNA% in post-EMB samples: an average 1.28-fold increase in ddcfDNA concentrations and a 1.31-fold increase in ddcfDNA% was observed (p = 0.007 and p = 0.03, respectively). Conclusion: The EMB procedure causes iatrogenic injury to the allograft that results in an increase in ddcfDNA% and ddcfDNA concentrations. For the assessment of ddcfDNA as marker for AR, collection of plasma samples before the EMB procedure is therefore essential.

Association Between the Intracellular Tacrolimus Concentration in CD3 + T Lymphocytes and CD14 + Monocytes and Acute Kidney Transplant Rejection.

BACKGROUND: Intracellular tacrolimus concentration in peripheral blood mononuclear cells (PBMCs) (TAC [PBMC] ) has been proposed to better represent its active concentration than its whole blood concentration. As tacrolimus acts on T lymphocytes and other white blood cells, including monocytes, we investigated the association of tacrolimus concentration in CD3 + T lymphocytes (TAC [CD3] ) and CD14 + monocytes (TAC [CD14] ) with acute rejection after kidney transplantation. METHODS: From a total of 61 samples in this case-control study, 28 samples were obtained during biopsy-proven acute rejection (rejection group), and 33 samples were obtained in the absence of rejection (control group). PBMCs were collected from both cryopreserved (retrospectively) and freshly obtained (prospectively) samples. CD3 + T lymphocytes and CD14 + monocytes were isolated from PBMCs, and their intracellular tacrolimus concentrations were measured. RESULTS: The correlation between tacrolimus whole-blood and intracellular concentrations was poor. TAC [CD3] was significantly lower than TAC [CD14] (median 12.8 versus 81.6 pg/million cells; P < 0.001). No difference in TAC [PBMC] (48.5 versus 44.4 pg/million cells; P = 0.82), TAC [CD3] (13.4 versus 12.5 pg/million cells; P = 0.28), and TAC [CD14] (90.0 versus 72.8 pg/million cells; P = 0.27) was found between the rejection and control groups. However, freshly isolated PBMCs showed significantly higher TAC [PBMC] than PBMCs from cryopreserved samples. Subgroup analysis of intracellular tacrolimus concentrations from freshly isolated cells did not show a difference between rejectors and nonrejectors. CONCLUSIONS: Differences in TAC [CD3] and TAC [CD14] between patients with and without rejection could not be demonstrated. However, further optimization of the cell isolation process is required because a difference in TAC [PBMC] between fresh and cryopreserved cells was observed. These results need to be confirmed in a study with a larger number of patients.

A Novel High-throughput Droplet Digital PCR-based Indel Quantification Method for the Detection of Circulating Donor-derived Cell-free DNA After Kidney Transplantation.

BACKGROUND: Donor-derived cell-free DNA (ddcfDNA) is a promising minimally invasive biomarker for acute rejection (AR) in kidney transplant recipients. To assess the diagnostic value of ddcfDNA as a marker for AR, ddcfDNA was quantified at multiple time points after kidney transplantation with a novel high-throughput droplet digital PCR indel method that allowed for the absolute quantification of ddcfDNA. METHODS: In this study, ddcfDNA in plasma samples from 223 consecutive kidney transplant recipients was analyzed pretransplantation; at 3, 7, and 180 d after transplantation; and at time of for-cause biopsies obtained within the first 180 d after transplantation. RESULTS: Median (interquartile range) ddcfDNA concentration was significantly higher on day 3 (58.3 [17.7-258.3] copies/mL) and day 7 (25.0 [10.4-70.8] copies/mL) than on day 180 after transplantation (4.2 [0.0-8.3] copies/mL; P < 0.001 and P < 0.001, respectively). At time of biopsy-proven AR (BPAR), between day 11 and day 180 after transplantation, ddcfDNA concentration was significantly higher (50.0 [25.0-108.3] copies/mL) than those when biopsies showed non-AR (0.0 [0.0-15.6] copies/mL; P < 0.05). ddcfDNA concentration within the first 10 d after transplantation showed no significant difference between recipients with BPAR and those with non-AR in their biopsy or between recipients with BPAR and ddcfDNA measured at day 3 and day 7. CONCLUSIONS: Unfortunately, ddcfDNA concentration is not a good biomarker to detect AR within the first 10 d after transplantation; however, BPAR occurring after 10 d after transplantation can be detected in kidney transplant recipients by ddcfDNA using a novel and unique high-throughput droplet digital PCR indel method.

The applications of DNA methylation as a biomarker in kidney transplantation: a systematic review.

BACKGROUND: Although kidney transplantation improves patient survival and quality of life, long-term results are hampered by both immune- and non-immune-mediated complications. Current biomarkers of post-transplant complications, such as allograft rejection, chronic renal allograft dysfunction, and cutaneous squamous cell carcinoma, have a suboptimal predictive value. DNA methylation is an epigenetic modification that directly affects gene expression and plays an important role in processes such as ischemia/reperfusion injury, fibrosis, and alloreactive immune response. Novel techniques can quickly assess the DNA methylation status of multiple loci in different cell types, allowing a deep and interesting study of cells' activity and function. Therefore, DNA methylation has the potential to become an important biomarker for prediction and monitoring in kidney transplantation. PURPOSE OF THE STUDY: The aim of this study was to evaluate the role of DNA methylation as a potential biomarker of graft survival and complications development in kidney transplantation. MATERIAL AND METHODS: A systematic review of several databases has been conducted. The Newcastle-Ottawa scale and the Jadad scale have been used to assess the risk of bias for observational and randomized studies, respectively. RESULTS: Twenty articles reporting on DNA methylation as a biomarker for kidney transplantation were included, all using DNA methylation for prediction and monitoring. DNA methylation pattern alterations in cells isolated from different tissues, such as kidney biopsies, urine, and blood, have been associated with ischemia-reperfusion injury and chronic renal allograft dysfunction. These alterations occurred in different and specific loci. DNA methylation status has also proved to be important for immune response modulation, having a crucial role in regulatory T cell definition and activity. Research also focused on a better understanding of the role of this epigenetic modification assessment for regulatory T cells isolation and expansion for future tolerance induction-oriented therapies. CONCLUSIONS: Studies included in this review are heterogeneous in study design, biological samples, and outcome. More coordinated investigations are needed to affirm DNA methylation as a clinically relevant biomarker important for prevention, monitoring, and intervention.