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OBJECTIVES: Rejection remains the most important factor limiting the survival of transplanted kidneys. Although a pathological biopsy of the transplanted kidney is the gold standard for diagnosing rejection, its limitations prevent it from being used as a routine monitoring method. Recently, peripheral blood lymphocyte subpopulation testing has become an important means of assessing the body's immune system, however, its application value and strategy in the field of kidney transplantation need further exploration. Additionally, the development and utilization of routine test parameters are also important methods for exploring diagnostic strategies and predictive models for kidney transplant diseases. This study aims to explore the correlation between peripheral blood lymphocyte subpopulations and T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR), as well as their diagnostic value, in conjunction with routine blood tests. METHODS: A total of 154 kidney transplant recipients, who met the inclusion and exclusion criteria and were treated at the Second Xiangya Hospital of Central South University from January to December, 2021, were selected as the study subjects. They were assigned into a stable group, a TCMR group, and an ABMR group, based on the occurrence and type of rejection. The basic and clinical data of these recipients were retrospectively analyzed and compared among the 3 groups. The transplant kidney function, routine blood tests, and peripheral blood lymphocyte subpopulation data of the TCMR group and the ABMR group before rejection treatment were compared with those of the stable group. RESULTS: The stable, TCMR group, and ABMR group showed no statistically significant differences in immunosuppressive maintenance regimens or sources of transplanted kidneys (all P>0.05). However, the post-transplant duration was significantly longer in the ABMR group compared with the stable group (P<0.001) and the TCMR group (P<0.05). Regarding kidney function, serum creatinine levels in the ABMR group were higher than in the stable group and the TCMR group (both P<0.01), with the TCMR group also showing higher levels than the stable group (P<0.01). Both TCMR and ABMR groups had significantly higher blood urea nitrogen levels than the stable group (P<0.01), with no statistically significant difference between TCMR and ABMR groups (P>0.05). The estimated glomerular filtration rate (eGFR) was lower in both TCMR and ABMR groups compared with the stable group (both P<0.01). In routine blood tests, the ABMR group had lower hemoglobin, red blood cell count, and platelet count than the stable group (all P<0.05). The TCMR group had higher neutrophil percentage (P<0.05) and count (P<0.05) than the stable group, and the ABMR group had a higher neutrophil percentage than the stable group (P<0.05). The eosinophil percentage and count in the TCMR group were lower than in the stable and ABMR groups (all P<0.05). Both TCMR and ABMR groups had lower basophil percentage and count, as well as lower lymphocyte percentage and count, compared with the stable group (all P<0.05). There were no significant differences in monocyte percentage and count among the 3 groups (all P>0.05). In lymphocyte subpopulations, the TCMR and ABMR groups had lower counts of CD45+ cells and T cells compared with the stable group (all P<0.05). The TCMR group also had lower counts of CD4+ T cells, NK cells, and B cells than the stable group (all P<0.05). There were no significant differences in the T cell percentage, CD4+ T cell percentage, CD8+ T cell percentage and their counts, CD4+/CD8+ T cell ratio, NK cell percentage, and B cell percentage among the stable, TCMR, and ABMR groups (all P>0.05). CONCLUSIONS: The occurrence of rejection leads to impaired transplant kidney function, accompanied by characteristic changes in some parameters of routine blood tests and peripheral blood lymphocyte subpopulations in kidney transplant recipients. The different characteristics of changes in some parameters of routine blood tests and peripheral blood lymphocyte subpopulations during TCMR and ABMR may help predict and diagnose rejection and differentiate between TCMR and ABMR.

Donor-derived cell-free DNA predicted allograft rejection and severe microvascular inflammation in kidney transplant recipients.

Introduction: The aim of this study is to investigate the clinical validity of donor-derived cell-free DNA (dd-cfDNA) in comparison with that of donor specific anti-HLA antibody (DSA) for predicting biopsy-proven rejection (BPR)and severe microvascular inflammation (severe MVI) in kidney transplant recipients (KTRs). Methods: In this prospective observational investigation, 64 KTRs who underwent the indicated biopsies were included. Blood samples collected prior to biopsy were tested for dd-cfDNA and DSA. Biopsy specimens were classified by a renal pathologist according to the Banff classification. The predictive performance of dd-cfDNA and DSA for histological allograft diagnosis was assessed. Results: KTRs were categorized into the high and low dd-cfDNA groups based on a level of 0.4%. Eighteen patients (28.1%) had positive DSA at biopsy, exhibiting higher dd-cfDNA levels than the DSA-negative patients. BPR and severe MVI incidences were elevated in the high dd-cfDNA group (BPR: 42.9% vs. 3.4%, P <0.001; severe MVI: 37.1% vs. 3.4%, P = 0.001). Also, elevated glomerulitis and MVI scores were observed in the high dd-cfDNA group. DSA showed the highest predictive value for BPR (AUC = 0.880), whereas dd-cfDNA alone excelled in predicting severe MVI (AUC = 0.855). Combination of DSA and dd-cfDNA (>0.4%) yielded sensitivities of 80.0% and 50.0% with specificities of 90.7% and 88.0% for antibody-mediated rejection and severe MVI detection, respectively. Conclusion: The dd-cfDNA test is a predictive tool for BPR and severe MVI, and it can improve the performance, especially when combined with DSA for BPR.

Lack of Association Between Donor-Derived Cell-Free DNA and Cardiac Allograft Vasculopathy.

Cardiac allograft vasculopathy (CAV) is a leading cause of death after heart transplantation (HT). We evaluated donor-derived cell-free DNA (dd-cfDNA) as a noninvasive biomarker of CAV development after HT. The INSPIRE registry at the Intermountain Medical Center was queried for stored plasma samples from HT patients with and without CAV. At Stanford University, HT patients with CAV (cases) and without CAV (controls) were enrolled prospectively, and blood samples were collected. All the samples were analyzed for dd-cfDNA using the AlloSure assay (CareDx, Inc.). CAV was defined per the ISHLT 2010 standardized classification system. Univariate associations between patient demographics and clinical characteristics and their CAV grade were tested using chi-square and Wilcoxon rank sum tests. Associations between their dd-cfDNA levels and CAV grades were examined using a nonparametric Kruskal-Wallis test. A total of 69 pts were included, and 101 samples were analyzed for dd-cfDNA. The mean age at sample collection was 58.6 ± 13.7 years; 66.7% of the patients were male, and 81% were White. CAV 0, 1, 2, and 3 were present in 37.6%, 22.8%, 22.8%, and 16.8% of included samples, respectively. The median dd-cfDNA level was 0.13% (0.06, 0.33). The median dd-cfDNA level was not significantly different between CAV (-) and CAV (+): 0.09% (0.05%-0.32%) and 0.15% (0.07%-0.33%), respectively, p = 0.25 and with similar results across all CAV grades. In our study, dd-cfDNA levels did not correlate with the presence of CAV and did not differ across CAV grades. As such, dd-cfDNA does not appear to be a reliable noninvasive biomarker for CAV surveillance.

Unveiling systemic responses in kidney transplantation: interplay between the allograft transcriptome and serum proteins.

Immunity, as defined by systems biology, encompasses a holistic response throughout the body, characterized by intricate connections with various tissues and compartments. However, this concept has been rarely explored in kidney transplantation. In this proof-of-concept study, we investigated a direct association between the allograft phenotype and serum protein signatures. Time-matched samples of graft biopsies and blood serum were collected in a heterogeneous cohort of kidney-transplanted patients (n = 15) for bulk RNA sequencing and proteomics, respectively. RNA transcripts exhibit distinct and reproducible, coregulated gene networks with specific functional profiles. We measured 159 serum proteins and investigated correlations with gene expression networks. Two opposing axes-one related to metabolism and the other to inflammation-were identified. They may represent a biological continuum between the allograft and the serum and correlate with allograft function, but not with interstitial fibrosis or proteinuria. For signature validation, we used two independent proteomic data sets (n = 21). Our findings establish a biological link between the allograft transcriptome and the blood serum proteome, highlighting systemic immune effects in kidney transplantation and offering a promising framework for developing allograft-linked biomarkers.

Diagnosis and classification of kidney transplant rejection using machine learning-assisted surface-enhanced Raman spectroscopy using a single drop of serum.

The quest to reduce kidney transplant rejection has emphasized the urgent requirement for the development of non-invasive, precise diagnostic technologies. These technologies aim to detect antibody-mediated rejection (ABMR) and T-cell-mediated rejection (TCMR), which are asymptomatic and pose a risk of potential kidney damage. The protocols for managing rejection caused by ABMR and TCMR differ, and diagnosis has traditionally relied on invasive biopsy procedures. Therefore, a convergence system using a nano-sensing chip, Raman spectroscopy, and AI technology was introduced to facilitate diagnosis using serum samples obtained from patients with no major abnormality, ABMR, and TCMR after kidney transplantation. Tissue biopsy and Banff score analysis were performed across the groups for validation, and 5 µL of serum obtained at the same time was added onto the Au-ZnO nanorod-based Surface-Enhanced Raman Scattering sensing chip to obtain Raman spectroscopy signals. The accuracy of machine learning algorithms for principal component-linear discriminant analysis and principal component-partial least squares discriminant analysis was 93.53% and 98.82%, respectively. The collagen (an indicative of kidney injury), creatinine, and amino acid-derived signals (markers of kidney function) contributed to this accuracy; however, the high accuracy was primarily due to the ability of the system to analyze a broad spectrum of various biomarkers.

Multi-centre analytical performance verification of an IVD assay to quantify donor-derived cell-free DNA in solid organ transplant recipients.

Donor-derived cell-free DNA (dd-cfDNA) has been widely studied as biomarker for non-invasive allograft rejection monitoring. Earlier rejection detection enables more prompt diagnosis and intervention, ultimately improving patient treatment and outcomes. This multi-centre study aims to verify analytical performance of a next-generation sequencing-based dd-cfDNA assay at end-user environments. Three independent laboratories received the same experimental design and 16 blinded samples to perform cfDNA extraction and the dd-cfDNA assay workflow. dd-cfDNA results were compared between sites and against manufacturer validation to evaluate concordance, reproducibility, repeatability and verify analytical performance. A total of 247 sample libraries were generated across 18 runs, with completion time of <24 h. A 96.0% first pass rate highlighted minimal failures. Overall observed versus expected dd-cfDNA results demonstrated good concordance and a strong positive correlation with linear least squares regression r2 = 0.9989, and high repeatability and reproducibility within and between sites, respectively (p > 0.05). Manufacturer validation established limit of blank 0.18%, limit of detection 0.23% and limit of quantification 0.23%, and results from independent sites verified those limits. Parallel analyses illustrated no significant difference (p = 0.951) between dd-cfDNA results with or without recipient genotype. The dd-cfDNA assay evaluated here has been verified as a reliable method for efficient, reproducible dd-cfDNA quantification in plasma from solid organ transplant recipients without requiring genotyping. Implementation of onsite dd-cfDNA testing at clinical laboratories could facilitate earlier detection of allograft injury, bearing great potential for patient care.

Insights into IL-1 family cytokines in kidney allograft transplantation: IL-18BP and free IL-18 as emerging biomarkers.

Proinflammatory cytokines and their inhibitors are involved in the regulation of multiple immune reactions including response to transplanted organs. In this prospective study, we evaluated changes in serum concentrations of six IL-1 family cytokines (IL-1 alpha, IL-1 beta, IL-1RA, IL-18, IL-18BP, and IL-36 beta) in 138 kidney allograft recipients and 48 healthy donors. Samples were collected before transplantation and then after one week, three months and one year, additional sera were obtained at the day of biopsy positive for acute rejection. We have shown, that concentrations of proinflammatory members of the IL-1 family (IL-1ß, IL-18, IL-36 ß) and anti-inflammatory IL-18BP decreased immediately after the transplantation. The decline of serum IL-1RA and IL-1α was not observed in subjects with acute rejection. IL-18, including specifically its free form, is the only cytokine which increase serum concentrations in the period between one week and three months in both groups of patients without upregulation of its inhibitor, IL-18BP. Serum concentrations of calculated free IL-18 were upregulated in the acute rejection group at the time of acute rejection. We conclude that IL-1 family cytokines are involved mainly in early phases of the response to kidney allograft. Serum concentrations of free IL-18 and IL-18BP represent possible biomarkers of acute rejection, and targeting IL-18 might be of therapeutic value.

Efficacy of Integrated Risk Score Using Omics-Based Biomarkers for the Prediction of Acute Rejection in Kidney Transplantation: A Randomized Prospective Pilot Study.

Acute rejection (AR) is critical for long-term graft survival in kidney transplant recipients (KTRs). This study aimed to evaluate the efficacy of the integrated risk score of omics-based biomarkers in predicting AR in KTRs. This prospective, randomized, controlled, multicenter, pilot study enrolled 40 patients who recently underwent high-immunologic-risk kidney transplantation (KT). Five omics biomarkers were measured, namely, blood mRNA (three-gene signature), urinary exosomal miRNA (three-gene signature), urinary mRNA (six-gene signature), and two urinary exosomal proteins (hemopexin and tetraspanin-1) at 2 weeks and every 4 weeks after KT for 1 year. An integrated risk score was generated by summing each biomarker up. The biomarker group was informed about the integrated risk scores and used to adjust immunosuppression, but not the control group. The outcomes were graft function and frequency of graft biopsy. Sixteen patients in the biomarker group and nineteen in the control group completed the study. The mean estimated glomerular filtration rate after KT did not differ between the groups. Graft biopsy was performed in two patients (12.5%) and nine (47.4%) in the biomarker and control groups, respectively, with the proportion being significantly lower in the biomarker group (p = 0.027). One patient (6.3%) in the biomarker group and two (10.5%) in the control group were diagnosed with AR, and the AR incidence did not differ between the groups. The tacrolimus trough level was significantly lower in the biomarker group than in the control group at 1 year after KT (p = 0.006). Integrated omics biomarker monitoring may help prevent unnecessary or high-complication-risk biopsy and enables tailored immunosuppression by predicting the risk of AR in KTRs.

BK polyomavirus DNAemia, allograft rejection, and de novo donor-specific antibodies after lowering target tacrolimus levels in pediatric kidney transplant recipients.

BACKGROUND: BK polyomavirus (BKV) DNAemia is a challenging infectious complication after kidney transplant (KT). Reduction of immunosuppression is the mainstay of management, and tacrolimus is often the first immunosuppressive medication adjusted upon the diagnosis of BKV DNAemia. This study aimed to evaluate the impact of a new institutional protocol with lower target tacrolimus levels on BKV DNAemia, allograft rejection, and de novo donor-specific antibodies (dnDSA) among pediatric KT recipients. METHODS: We conducted a retrospective chart review of all KT episodes between January 2013 and December 2018. The new protocol with lower target tacrolimus levels was implemented in March 2015. One hundred twenty-seven patients were included in primary analysis. All patients received induction with basiliximab and methylprednisolone and were maintained on a steroid-based immunosuppressive regimen. RESULTS: In the post-intervention cohort, cumulative incidence of BKV DNAemia at 100 days (13.4% vs. 17.8%, p = .605) and 18 months post-KT (34.1% vs. 26.7%, p = .504) was not significantly different from the pre-intervention cohort. Biopsy-proven rejection rate did not change. However, we observed a trend toward earlier development of dnDSA in the post-intervention cohort using the Kaplan-Meier survival analysis (log-rank p = .06). Younger recipient age at the time of transplant was found to slightly increase the risk of BKV DNAemia (OR: 1.09, 95% CI [1.01, 1.16], p = .024). There was an association between BKV DNAemia and biopsy-proven rejection of any type (adjustedOR: 2.77, 95% CI [1.26, 6.23], p = .012), especially acute T-cell-mediated rejection grade 1A and above (adjustedOR: 2.95, 95% CI [1.06, 8.30], p = .037), after adjusted for recipient age at the time of transplant. CONCLUSIONS: Targeting lower tacrolimus levels did not decrease the incidence of BKV DNAemia within 100 days or 18 months post-KT, nor did it increase the risk of biopsy-proven rejection among pediatric KT recipients in our center. However, there was a trend toward earlier development of dnDSA, which may portend worse long-term graft outcome post-KT. Our findings highlight the need for individualized immunosuppressive regimens based on immunologic and infectious risk factors and the importance of implementing innovative biomarkers to guide therapy and improve outcomes.

Prevalence of iron deficiency and anemia in pediatric heart transplant recipients.

INTRODUCTION: The prevalence of iron deficiency and anemia in the setting of modern-day maintenance immunosuppression in pediatric heart transplant (HTx) recipients is unclear. The primary aim was to determine the prevalence of iron deficiency (serum ferritin < 30 ng/mL ± transferrin saturation < 20%) and anemia per World Health Organization diagnostic criteria and associated risk factors. METHODS: Single-center, cross-sectional analysis of 200 consecutive pediatric HTx recipients (<21 years old) from 2005 to 2021. Data were collected at 1-year post-HTx at the time of annual protocol biopsy. RESULTS: Median age at transplant was 3 years (IQR .5-12.2). The median ferritin level was 32 ng/mL with 46% having ferritin < 30 ng/mL. Median transferrin saturation (TSAT) was 22% with 47% having TSAT < 20%. Median hemoglobin was 11 g/dL with 54% having anemia. Multivariable analysis revealed lower absolute lymphocyte count, TSAT < 20%, and estimated glomerular filtration rate <75 mL/min/1.73 m2 were independently associated with anemia. Ferritin < 30 ng/mL in isolation was not associated with anemia. Ferritin < 30 ng/mL may aid in detecting absolute iron deficiency while TSAT < 20% may be useful in identifying patients with functional iron deficiency ± anemia in pediatric HTx recipients. CONCLUSION: Iron deficiency and anemia are highly prevalent in pediatric HTx recipients. Future studies are needed to assess the impact of iron deficiency, whether with or without anemia, on clinical outcomes in pediatric HTx recipients.

Sex-specific patterns of donor-derived cell-free DNA in heart transplant rejection: An analysis from the Genomic Research Alliance for Transplantation (GRAfT).

BACKGROUND: Noninvasive methods for surveillance of acute rejection are increasingly used in heart transplantation (HT), including donor-derived cell-free DNA (dd-cfDNA). As other cardiac biomarkers differ by sex, we hypothesized that there may be sex-specific differences in the performance of dd-cfDNA for the detection of acute rejection. The purpose of the current study was to examine patterns of dd-cfDNA seen in quiescence and acute rejection in male and female transplant recipients. METHODS: Patients enrolled in the Genomic Research Alliance for Transplantation who were ≥18 years at the time of HT were included. Rejection was defined by endomyocardial biopsy with acute cellular rejection (ACR) grade ≥2R and/or antibody-mediated rejection ≥ pAMR 1. dd-cfDNA was quantitated using shotgun sequencing. Median dd-cfDNA levels were compared between sexes during quiescence and rejection. The performance of dd-cfDNA by sex was assessed using area under the receiver operator characteristic (AUROC) curve. Allograft injury was defined as dd-cfDNA ≥0.25%. RESULTS: One hundred fifty-one unique patients (49 female, 32%) were included in the analysis with 1,119 available dd-cfDNA measurements. Baseline characteristics including demographics and comorbidities were not significantly different between sexes. During quiescence, there were no significant sex differences in median dd-cfDNA level (0.04% [IQR 0.00, 0.16] in females vs 0.03% [IQR 0.00, 0.12] in males, p = 0.22). There were no significant sex differences in median dd-cfDNA for ACR (0.33% [0.21, 0.36] in females vs 0.32% [0.21, 1.10] in males, p = 0.57). Overall, median dd-cfDNA levels were higher in antibody-mediated rejection (AMR) than ACR but did not significantly differ by sex (0.50% [IQR 0.18, 0.82] in females vs 0.63% [IQR 0.32, 1.95] in males, p = 0.51). Elevated dd-cfDNA detected ACR/AMR with an AUROC of 0.83 in females and 0.89 in males, p-value for comparison = 0.16. CONCLUSIONS: There were no significant sex differences in dd-cfDNA levels during quiescence and rejection. Performance characteristics were similar, suggesting similar diagnostic thresholds can be used in men and women for rejection surveillance.

Bioinformatics analysis characterizes immune infiltration landscape and identifies potential blood biomarkers for heart transplantation.

BACKGROUND: Cardiac allograft rejection (AR) remains a significant complication following heart transplantation. The primary objective of our study is to gain a comprehensive understanding of the fundamental mechanisms involved in AR and identify possible therapeutic targets. METHODS: We acquired the GSE87301 dataset from the Gene Expression Omnibus database. In GSE87301, a comparison was conducted on blood samples from patients with and without cardiac allograft rejection (AR and NAR) to detect differentially expressed genes (DEGs). Enrichment analysis was conducted to identify the pathways that show significant enrichment during AR. Machine learning techniques, including the least absolute shrinkage and selection operator regression (LASSO) and random forest (RF) algorithms, were employed to identify potential genes for the diagnosis of AR. The diagnostic value was evaluated using a nomogram and receiver operating characteristic (ROC) curve. Additionally, immune cell infiltration was analyzed to explore any dysregulation of immune cells in AR. RESULTS: A total of 114 DEGs were identified from the GSE87301 dataset. These DEGs were mainly found to be enriched in pathways related to the immune system. To identify the signature genes, the LASSO and RF algorithms were used, and four genes, namely ALAS2, HBD, EPB42, and FECH, were identified. The performance of these signature genes was evaluated using the receiver operating characteristic curve (ROC) analysis, which showed that the area under the curve (AUC) values for ALAS2, HBD, EPB42, and FECH were 0.906, 0.881, 0.900, and 0.856, respectively. These findings were further confirmed in the independent datasets and clinical samples. The selection of these specific genes was made to construct a nomogram, which demonstrated excellent diagnostic ability. Additionally, the results of the single-sample gene set enrichment analysis (ssGSEA) revealed that these genes may be involved in immune cell infiltration. CONCLUSION: We identified four signature genes (ALAS2, HBD, EPB42, and FECH) as potential peripheral blood diagnostic candidates for AR diagnosis. Additionally, a nomogram was constructed to aid in the diagnosis of heart transplantation. This study offers valuable insights into the identification of candidate genes for heart transplantation using peripheral blood samples.

Serum IL-6 predicts risk of kidney transplant failure independently of immunological risk.

Interleukin-6 (IL-6) is an important immune mediator and a target for novel antibody therapies. In this study, we aimed to determine whether serum IL-6 levels are associated with immunological risk, allograft rejection and outcomes in kidney transplant (Ktx) patients. We retrospectively analyzed the data of 104 patients who underwent Ktx at our center between 2011 and 2015. The patients were divided into high- and low-risk groups (n = 52 per group) based on panel reactive antibody (PRA) percentage ≥ 35%, the existence of pre-Ktx donor-specific antibodies (DSA), or a previous transplant. IL-6 concentrations were measured before and at 3 months, 12 months, and 3 years after Ktx. Serum IL-6 levels tended to be higher in high-risk patients than in low-risk patients prior to Ktx and at 12 months after Ktx; however, the difference did not reach statistical significance (pre-Ktx, high-risk: 1.995 ± 2.79 pg/ml vs. low-risk: 1.43 ± 1.76 pg/ml, p = 0.051; 12 mo. high-risk: 1.16 ± 1.87 pg/ml vs. low-risk: 0.78 ± 1.13 pg/ml, p = 0.067). IL-6 levels were correlated with the types (no rejection, T cell mediated rejection (TCMR), antibody-mediated rejection (ABMR), or both) and time (<1 year vs. >1 year after Ktx) of rejection, as well as patient and graft survival. Patients with both TCMR and ABMR had significantly higher IL-6 levels at 3 months (14.1 ± 25.2 pg/ml) than patients with ABMR (3.4 ± 4.8 pg/ml, p = 0.017), with TCMR (1.7 ± 1.3 pg/ml, p < 0.001), and without rejection (1.7 ± 1.4 pg/ml, p < 0.001). Three years after Ktx, patients with AMBR had significantly higher IL-6 levels (5.30 ± 7.66 pg/ml) than patients with TCMR (1.81 ± 1.61 pg/ml, p = 0.009) and patients without rejection (1.19 ± 0.95 pg/ml; p = 0.001). Moreover, three years after Ktx IL-6 levels were significantly higher in patients with late rejections (3.5 ± 5.4 pg/ml) than those without rejections (1.2 ± 1.0 pg/ml) (p = 0.006). The risk of death-censored graft failure was significantly increased in patients with elevated IL-6 levels at 12 months (IL-6 level > 1.396 pg/ml, HR 4.61, p = 0.007) and 3 years (IL-6 level > 1.976 pg/ml, HR 6.75, p = 0.003), but elevated IL-6 levels were not associated with a higher risk of death. Overall, our study highlights IL-6 as a risk factor for allograft failure and confirms that IL-6 levels are higher in patients developing ABMR compared to TCMR alone or no rejection.

Serum Extracellular Vesicle Protein Profiling for Prediction of Corneal Transplant Rejection.

BACKGROUND: Corneal transplantation is the most common transplant procedure worldwide. Despite immune and angiogenic privilege of the cornea, 50% to 70% of corneal transplants fail in high-risk recipients, primarily because of immune rejection. Therefore, it is crucial to identify predictive biomarkers of rejection to improve transplant survival. METHODS: In search for predictive biomarkers, we performed proteomics analysis of serum extracellular vesicles (EVs) in a fully major histocompatibility complex-mismatched (C57BL/6-to-BALB/c) murine corneal transplantation model, wherein 50% of transplants undergo rejection by day 28 following transplantation. RESULTS: Our time course study revealed a decrease in the number of serum EVs on day 1, followed by a gradual increase by day 7. A comparative analysis of proteomics profiles of EVs from transplant recipients with rejection (rejectors) and without rejection (nonrejectors) found a distinct enrichment of histocompatibility 2, Q region locus 2, which is a part of major histocompatibility complex-class I of donor C57BL/6 mice, in day 7 EVs of rejectors, compared with nonrejectors, syngeneic controls, or naïve mice. In contrast, serum amyloid A2, a protein induced in response to injury, was increased in day 7 EVs of nonrejectors. CONCLUSIONS: Our findings offer noninvasive EV-based potential biomarkers for predicting corneal allograft rejection or tolerance.

Donor-derived cell-free DNA as a diagnostic marker for kidney-allograft rejection: A systematic review and meta-analysis.

Donor-derived cell-free DNA (dd-cfDNA) has emerged as a promising biomarker for detecting graft rejection. This study aimed to evaluate the diagnostic accuracy and clinical value of applying it to kidney transplant rejection. Relevant literature on dd-cfDNA diagnostics in kidney transplant rejection was reviewed from PubMed, Embase, Cochrane Library, and Web of Science databases up to 2023. Data and study characteristics were extracted independently by two researchers, and disagreements were resolved through discussion. Diagnostic accuracy data for any rejection (AR) and antibody-mediated rejection (ABMR) were analyzed separately. Potential heterogeneity was analyzed by subgroup analysis or meta-regression. Funnel plots were used to clarify the presence or absence of publication bias. Nine publications provided data on dd-cfDNA accuracy in diagnosing patients with AR. The pooled sensitivity, specificity, and the area under the receiver operating characteristic (AUROC) curve with 95% confidence intervals (CI) were 0.59 (95% CI, 0.48-0.69), 0.83 (95% CI, 0.76-0.88), and 0.80 (95% CI, 0.76-0.83), respectively. Additionally, 12 studies focused on the diagnostic accuracy of dd-cfDNA for ABMR, showing pooled sensitivity, specificity, and the AUROC curve with 95% CI of 0.81 (95% CI, 0.72-0.88), 0.80 (95% CI, 0.73-0.86), and 0.87 (95% CI, 0.84-0.90), respectively. Study type, age group, and sample size contributed to heterogeneity. In summary, our findings indicate that while plasma dd-cfDNA accuracy in diagnosing patients with AR is limited by significant heterogeneity, it is a valuable biomarker for diagnosing ABMR.

Association of Serum Osteoprotegerin With Vascular Calcification, and Cardiovascular and Graft Outcomes in Kidney Transplant Patients: Results From the KNOW-KT.

BACKGROUND: Vascular calcification and stiffness contribute to increased cardiovascular morbidity in patients with chronic kidney disease. This study investigated associations between serum osteoprotegerin (OPG) levels and vascular calcification or stiffness to assess cardiovascular and graft outcomes in kidney transplant patients. METHODS: The KoreaN cohort study for Outcome in patients With Kidney Transplantation was a prospective multicenter cohort study. Serum OPG levels were measured at baseline and 3 y after transplantation in 1018 patients. Patients were classified into high and low OPG groups according to median serum OPG levels. The median follow-up duration was 93.5 mo. RESULTS: The mean age was 45.8 ±â€…11.7 y and 62.9% were men. Patients with high OPG had significantly higher coronary artery calcium scores, abdominal aortic calcification scores, and brachial-ankle pulse wave velocities than those with lower OPG; these parameters remained significant for 5 y after transplantation. The 3-y OPG levels were lower than baseline values ( P < 0.001) and were positively correlated ( r = 0.42, P < 0.001). Multivariate Cox regression analysis showed that high OPG levels were significantly associated with posttransplant cardiovascular events ( P = 0.008) and death-censored graft loss ( P = 0.004). Similar findings regarding posttransplant cardiovascular events ( P = 0.012) and death-censored graft loss ( P = 0.037) were noted in patients with high OPG at the 3-y follow-up. Mediation analyses revealed that coronary artery calcium scores, abdominal aortic calcification scores, and brachial-ankle pulse wave velocities could act as mediators between serum OPG levels and posttransplant cardiovascular events. CONCLUSIONS: Serum OPG concentration is associated with vascular calcification and stiffness and could be a significant risk factor for cardiovascular outcomes and graft loss in patients undergoing kidney transplantation.

Screening of novel biomarkers for acute kidney transplant rejection using DIA-MS based proteomics.

BACKGROUND: Kidney transplantation is the preferred treatment for patients with end-stage renal disease. However, acute rejection poses a threat to the graft long-term survival. The aim of this study was to identify novel biomarkers to detect acute kidney transplant rejection. METHODS: The serum proteomic profiling of kidney transplant patients with T cell-mediated acute rejection (TCMR) and stable allograft function (STA) was analyzed using data-independent acquisition mass spectrometry (DIA-MS). The differentially expressed proteins (DEPs) of interest were further verified by enzyme-linked immunosorbent assay (ELISA). RESULTS: A total of 131 DEPs were identified between STA and TCMR patients, 114 DEPs were identified between mild and severe TCMR patients. The verification results showed that remarkable higher concentrations of serum amyloid A protein 1 (SAA1) and insulin like growth factor binding protein 2 (IGFBP2), and lower fetuin-A (AHSG) concentration were found in TCMR patients when compared with STA patients. We also found higher SAA1 concentration in severe TCMR group when compared with mild TCMR group. The receiver operating characteristics (ROC) analysis further confirmed that combination of SAA1, AHSG, and IGFBP2 had excellent performance in the acute rejection diagnosis. CONCLUSIONS: Our data demonstrated that serum SAA1, AHSG, and IGFBP2 could be effective biomarkers for diagnosing acute rejection after kidney transplantation. DIA-MS has great potential in biomarker screening of kidney transplantation.

Pre-transplant angiotensin II receptor type I antibodies in pediatric renal transplant recipients: An observational cohort study.

BACKGROUND: The role of angiotensin II type 1 receptor antibodies (AT1R-Ab) in pediatric renal transplantation is unclear. Here, we evaluated pre-transplant AT1R-Ab on transplant outcomes in the first 5 years. Secondary analysis compared pre-transplant AT1R-Ab levels by age. METHODS: Thirty-six patients, 2-20 years old, were divided into two groups: pre-transplant AT1R-Ab- (<17 U/ml; n = 18) and pre-transplant AT1R-Ab+ (≥17 U/ml; n = 18). eGFR was determined at 6-month, 1-, 2-, and 4-year post-transplant. Allograft biopsies were performed in the setting of strong HLA-DSA (MFI > 10 000), AT1R-Ab ≥17 U/ml, and/or elevated creatinine. RESULTS: Mean age in pre-transplant AT1R-Ab- was 13.3 years vs. 11.0 in pre-transplant AT1R-Ab+ (p = 0.16). At 6 months, mean eGFR was 111.3 ml/min/1.73 m2 in pre-transplant AT1R-Ab- vs. 100.2 in pre-transplant AT1R-Ab + at 1 year, 103.6 ml/min/1.73 m2 vs. 100.5; at 2 years, 98.9 ml/min/1.73 m2 vs. and 93.7; at 4 years, 72.6 ml/min/1.73 m2 vs. 80.9. 11/36 patients had acute rejection (6 in pre-transplant AT1R-Ab-, 5 in pre-transplant AT1R-Ab + ). There was no difference in rejection rates. All 6 subjects with de novo HLA-DSA and AT1R-Ab ≥17 U/ml at the time of biopsy experienced rejection. Mean age in those with the AT1R-Ab ≥40 U/ml was 10.0 years vs. 13.2 in those <40 U/ml (p = 0.07). CONCLUSION: In our small cohort, pre-transplant AT1R-Ab ≥17 U/ml was not associated with reduced graft function or rejection. The pathogenicity of pre-transplant AT1R-Ab in pediatric kidney transplantation requires further investigation.

Cytosporone B (Csn-B), an NR4A1 agonist, attenuates acute cardiac allograft rejection by inducing differential apoptosis of CD4+T cells.

CD4+T cell-mediated acute rejection remains a major factor that affects the early survival of transplanted organs post-transplantation. Here, we reveal that nuclear receptor subfamily 4 Group A member 1 (Nr4A1) was upregulated during cardiac allograft rejection and that the increased Nr4A1 was primarily localized in intragraft-infiltrating CD4+T cells. Nr4A1 acts as a transcription factor with an important role in CD4+T cell apoptosis, differentiation and T cell dysfunction, which indicates that Nr4A1 may play a critical role in transplant rejection. Cytosporone B (Csn-B) is a naturally occurring agonist of Nr4A1, and the role of Csn-B in the physiological process of cardiac rejection is poorly defined. This study constructed an acute rejection model of abdominal heterotopic cardiac transplantation in mice and investigated whether Csn-B could attenuate acute transplant rejection by modulating the CD4+T lymphocyte response. The results showed that Csn-B prolonged murine cardiac allograft survival and reduced inflammation in allografts. Subsequently, it was confirmed that Csn-B functions by inducing non-Treg apoptosis and promoting Treg cell differentiation. Finally, we also confirmed that Csn-B attenuates acute rejection by directly targeting Nr4A1 in CD4+T cells. Our data suggest that Csn-B is a promising novel therapeutic approach for acute cardiac allograft rejection.