Systemic Inflammation Differences in Brain-vs. Circulatory-Dead Donors: Impact on Lung Transplant Recipients.

Brain death triggers a systemic inflammatory response. Whether systemic inflammation is different in lung donors after brain- (DBD) or circulatory-death (DCD) is unknown, but this may potentially increase the incidence of primary graft dysfunction (PGD) after lung transplantation. We compared the plasma levels of interleukin (IL)-6, IL-8, IL-10 and TNF-α in BDB and DCD and their respective recipients, as well as their relationship with PGD and mortality after LT. A prospective, observational, multicenter, comparative, cohort-nested study that included 40 DBD and 40 DCD lung donors matched and their respective recipients. Relevant clinical information and blood samples were collected before/during lung retrieval in donors and before/during/after (24, 48 and 72 h) LT in recipients. Incidence of PGD and short-term mortality after LT was recorded. Plasma levels of all determined cytokines were numerically higher in DBD than in DCD donors and reached statistical significance for IL-6, IL-10 and IL-8. In recipients with PGD the donor's plasma levels of TNF-α were higher. The post-operative mortality rate was very low and similar in both groups. DBD is associated with higher systemic inflammation than DCD donors, and higher TNF-α plasma levels in donors are associated with a higher incidence of PGD.

Early postoperative hemodynamic instability after heart transplantation - incidence and metabolic indicators.

BACKGROUND: Primary graft dysfunction (PGD) is the main cause of death in the first 30 days after heart transplantation (HTX), accounting for approximately 40% of mortality. The study's primary aim was to assess the incidence of PGD, following the International Society for Heart and Lung Transplantation consensus, and to compare it with the incidence of significant postoperative hypotension despite administration of high-dose inotropes and vasoconstrictors. The secondary aim of the study was to determine changes in biochemical markers that accompany the phenomenon. METHODS: Forty-five patients who underwent HTX between 2010 and 2015 were enrolled in this study, and detailed hemodynamic and metabolic data from the first 48 postoperative hours were collected and analyzed. Hemodynamic instability was defined as significant postoperative hypotension (mean arterial pressure (MAP) < 60 mmHg) combined with a high inotrope score (> 10). Data for long-term mortality were obtained from the population registration office. RESULTS: PGD incidence was relatively low (17.8%); however, hemodynamic instability was common (40%). Among unstable patients, MAP was insufficient for end-organ perfusion (51.4 ± 9.5 mmHg) but no decrease in left ventricular function was observed (cardiac index, 2.65 ± 0.6 l/min/m2; left ventricular ejection fraction, 52.9 ± 15.5%). Within this group, mean systemic vascular resistance index (961 ± 288 dyn*s*m2/cm5) was low despite receiving high doses of vasoactive agent (norepinephrine 0.21 (0.06-0.27) µg/kg/min during first 24 h postoperatively and 0.21 (0.01-0.27) µg/kg/min during next 24 h postoperatively). After HTX, serum lactate levels were initially significantly higher in patients with hemodynamic instability (p = 0.002); however, impaired lactate clearance was not observed (p = 0.366), and lactate levels normalized within the first 24 h postoperatively. Postoperative hemodynamic instability altered the long-term outcome and increased 5-year mortality after HTX (p = 0.034). CONCLUSIONS: Hemodynamic instability is a more common phenomenon than PGD. Only early postoperative serum lactate levels correspond with hemodynamic instability following HTX. Postoperative hemodynamic instability is associated with poor long-term survival among HTX recipients.

Proteomic profiling identifies CLEC4C expression as a novel biomarker of primary graft dysfunction after heart transplantation.

PURPOSE: Clinical models to identify patients at high risk of primary graft dysfunction (PGD) after heart transplantation (HT) are limited, and the underlying pathophysiology of this common post-transplant complication remains poorly understood. We sought to identify whether pre-transplant levels of circulating proteins reporting on immune activation and inflammation are associated with incident PGD. METHODS: The study population consisted of 219 adult heart transplant recipients identified between 2016 and 2020 at Duke University Medical Center, randomly divided into derivation (n = 131) and validation (n = 88) sets. PGD was defined using modified ISHLT criteria. Proteomic profiling was performed using Olink panels (n = 354 proteins) with serum samples collected immediately prior to transplantation. Association between normalized relative protein expression and PGD was tested using univariate and multivariable (recipient age, creatinine, mechanical circulatory support, and sex; donor age; ischemic time) models. Significant proteins identified in the derivation set (p < 0.05 in univariate models), were then tested in the validation set. Pathway enrichment analysis was used to test candidate biological processes. The predictive performance of proteins was compared to that of the RADIAL score. RESULTS: Nine proteins were associated with PGD in univariate models in the derivation set. Of these, only CLEC4C remained associated with PGD in the validation set after Bonferroni correction (OR [95% CI] = 3.04 [1.74,5.82], p = 2.8 × 10-4). Patterns of association were consistent for CLEC4C in analyses stratified by biventricular/left ventricular and isolated right ventricular PGD. Pathway analysis identified interferon-alpha response and C-type lectin signaling as significantly enriched biologic processes. The RADIAL score was a poor predictor of PGD (AUC = 0.55). CLEC4C alone (AUC = 0.66, p = 0.048) and in combination with the clinical covariates from the multivariable model (AUC = 0.69, p = 0.018) improved discrimination for the primary outcome. CONCLUSIONS: Pre-transplantation circulating levels of CLEC4C, a protein marker of plasmacytoid dendritic cells (pDCs), may identify HT recipients at risk for PGD. Further studies are needed to better understand the potential role pDCs and the innate immune response in PGD.

Plasma kallikrein predicts primary graft dysfunction after heart transplant.

BACKGROUND: Primary graft dysfunction (PGD) is the leading cause of early mortality after heart transplant. Pre-transplant predictors of PGD remain elusive and its etiology remains unclear. METHODS: Microvesicles were isolated from 88 pre-transplant serum samples and underwent proteomic evaluation using TMT mass spectrometry. Monte Carlo cross validation (MCCV) was used to predict the occurrence of severe PGD after transplant using recipient pre-transplant clinical characteristics and serum microvesicle proteomic data. Putative biological functions and pathways were assessed using gene set enrichment analysis (GSEA) within the MCCV prediction methodology. RESULTS: Using our MCCV prediction methodology, decreased levels of plasma kallikrein (KLKB1), a critical regulator of the kinin-kallikrein system, was the most predictive factor identified for PGD (AUROC 0.6444 [0.6293, 0.6655]; odds 0.1959 [0.0592, 0.3663]. Furthermore, a predictive panel combining KLKB1 with inotrope therapy achieved peak performance (AUROC 0.7181 [0.7020, 0.7372]) across and within (AUROCs of 0.66-0.78) each cohort. A classifier utilizing KLKB1 and inotrope therapy outperforms existing composite scores by more than 50 percent. The diagnostic utility of the classifier was validated on 65 consecutive transplant patients, resulting in an AUROC of 0.71 and a negative predictive value of 0.92-0.96. Differential expression analysis revealed a enrichment in inflammatory and immune pathways prior to PGD. CONCLUSIONS: Pre-transplant level of KLKB1 is a robust predictor of post-transplant PGD. The combination with pre-transplant inotrope therapy enhances the prediction of PGD compared to pre-transplant KLKB1 levels alone and the resulting classifier equation validates within a prospective validation cohort. Inflammation and immune pathway enrichment characterize the pre-transplant proteomic signature predictive of PGD.

The Role of Galectins in Chronic Lung Allograft Dysfunction.

BACKGROUND: Galectins are proteins that bind ß-galactosides such as N-acetyllactosamine present in N-linked and O-linked glycoproteins and that seem to be implicated in inflammatory and immune responses as well as fibrotic mechanisms. This preliminary study investigated serum galectins as clinical biomarkers in lung transplant patients with chronic lung allograft dysfunction (CLAD), phenotype bronchiolitis obliterans syndrome (BOS). MATERIALS AND METHODS: Nineteen lung transplant patients [median age (IQR), 55 (45-62) years; 53% males] were enrolled in the study. Peripheral blood concentrations of galectins-1, 3 and 9 were determined with commercial ELISA kits. RESULTS: Galectin-1 concentrations were higher in BOS than in stable LTX patients (p = 0.0394). In logistic regression analysis, testing BOS group as dependent variable with Gal-1 and 3 as independent variables, area under the receiver operating characteristics (AUROC) curve was 98.9% (NPV 90% and PPV 88.9%, p = 0.0003). With the stable LTX group as dependent variable and Gal-1, 3 and 9 as independent variables, AUROC was 92.6% (NPV 100% and PPV 90%, p = 0.0023). In stable patients were observed an inverse correlation of Gal-3 with DLCO% and KCO%, and between Gal-9 and KCO%. CONCLUSION: Galectins-1, 3 and 9 are possible clinical biomarkers in lung transplant patients with diagnostic and prognostic meaning. These molecules may be directly implicated in the pathological mechanisms of BOS. The hypothesis that they could be new therapeutic targets in BOS patients is intriguing and also worth exploring.

Use of donor-derived-cell-free DNA as a marker of early allograft injury in primary graft dysfunction (PGD) to predict the risk of chronic lung allograft dysfunction (CLAD).

BACKGROUND: Primary graft dysfunction (PGD) is a risk factor for chronic lung allograft dysfunction (CLAD). However, the association between PGD and degree of allograft injury remains poorly defined. In this study, we leverage a novel biomarker for allograft injury, percentage donor-derived cell-free DNA (%ddcfDNA), to study the association between PGD, degree of allograft injury, and the development of CLAD. METHODS: This prospective cohort study recruited 99 lung transplant recipients and collected plasma samples on days 1, 3, and 7 for %ddcfDNA measurements. Clinical data on day 3 was used to adjudicate for PGD. %ddcfDNA levels were compared between PGD grades. In PGD patients, %ddcfDNA was compared between those who developed CLAD and those who did not. RESULTS: On posttransplant day 3, %ddcfDNA was higher in PGD than in non-PGD patients (median [IQR]: 12.2% [8.2, 22.0] vs 8.5% [5.6, 13.2] p = 0.01). %ddcfDNA correlated with the severity grade of PGD (r = 0.24, p = 0.02). Within the PGD group, higher levels of %ddcfDNA correlated with increased risk of developing CLAD (log OR(SE) 1.38 (0.53), p = 0.009). PGD patients who developed CLAD showed ∼2-times higher %ddcfDNA levels than patients who did not develop CLAD (median [IQR]: 22.4% [11.8, 27.6] vs 9.9% [6.7, 14.9], p = 0.007). CONCLUSION: PGD patients demonstrated increased early posttransplant allograft injury, as measured by %ddcfDNA, in comparison to non-PGD patients, and these high %ddcfDNA levels were associated with subsequent development of CLAD. This study suggests that %ddcfDNA identifies PGD patients at greater risk of CLAD than PGD alone.

Circulating exosomes with lung self-antigens as a biomarker for chronic lung allograft dysfunction: A retrospective analysis.

BACKGROUND: Exosomes isolated from plasma of lung transplant recipients (LTxRs) with bronchiolitis obliterans syndrome (BOS) contain human leukocyte antigens and lung self-antigens (SAgs), K-alpha 1 tubulin (Kα1T) and collagen type V (Col-V). The aim was to determine the use of circulating exosomes with lung SAgs as a biomarker for BOS. METHODS: Circulating exosomes were isolated retrospectively from plasma from LTxRs at diagnosis of BOS and at 6 and 12 months before the diagnosis (n = 41) and from stable time-matched controls (n = 30) at 2 transplant centers by ultracentrifugation. Exosomes were validated using Nanosight, and lung SAgs (Kα1T and Col-V) were detected by immunoblot and semiquantitated using ImageJ software. RESULTS: Circulating exosomes from BOS and stable LTxRs demonstrated 61- to 181-nm vesicles with markers Alix and CD9. Exosomes from LTxRs with BOS (n = 21) showed increased levels of lung SAgs compared with stable (n = 10). A validation study using 2 separate cohorts of LTxRs with BOS and stable time-matched controls from 2 centers also demonstrated significantly increased lung SAgs-containing exosomes at 6 and 12 months before BOS. CONCLUSIONS: Circulating exosomes isolated from LTxRs with BOS demonstrated increased levels of lung SAgs (Kα1T and Col-V) 12 months before the diagnosis (100% specificity and 90% sensitivity), indicating that circulating exosomes with lung SAgs can be used as a non-invasive biomarker for identifying LTxRs at risk for BOS.

Preoperative Neutrophil-to-Lymphocyte Ratio as an Independent Predictor of 1-Year Graft Loss and Mortality After Orthotopic Liver Transplantation.

INTRODUCTION: Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are low-cost and readily available inflammation markers. Previously, we revealed that the high preoperative neutrophil level is a recipient-related risk factor for the primary liver graft dysfunction (PGD), associated with a higher risk of early retransplantation or death. Here we aimed to evaluate the prognostic significance of preoperative neutrophil level, as well as the NLR and PLR in predicting a 1-year outcome of the orthotopic liver transplantation (OLTx). MATERIALS AND METHODS: One hundred and thirty-four patients who underwent the OLTx between 2012 and 2017 were enrolled. Analysis included, inter alia, etiology of liver failure and preoperative blood morphology. In the statistical analysis, the logistic regression model and receiver operator characteristic analysis were applied. RESULTS: In 1-year follow-up, 11% of patients died and 5% were retransplanted. Acute liver failure (ALF; odds ratio [OR] = 8.62, P = .007), autoimmune hepatitis (AIH; R = 5.25, P = .006), neutrophil level (OR = 1.23, P = .0003), MELD (OR = 1.05, P = .038), and the NLR (OR = 1.16, P = .001) were significant predictors of these detrimental outcomes. The multivariate analysis revealed etiology (AIH, P < .001 or ALF, P = .006) and NLR (P = .008) as the only independent predictors of 1-year graft loss or patient's death. Receiver operator characteristic analysis pointed at the NLR above 5.48 as their highly sensitive and specific risk factor. The PLR was not a prognostic biomarker. CONCLUSION: Achieved results call for further studies on the influence of the preoperative balance between systemic inflammation and immunity, expressed with the NLR on the long-term liver graft function.

C-reactive protein at ICU admission as a marker of early graft dysfunction after liver transplant. A prospective, single-center cohort study.

OBJECTIVE: To explore the behavior of C-reactive protein (CRP) after orthotopic liver transplantation (OLT) during the first postoperative days, and its usefulness as a marker of severe early allograft dysfunction (EAD). DESIGN: A prospective, single-center cohort study was carried out. SETTING: The Intensive Care Unit (ICU) of a regional hospital with a liver transplant program since 1997. PATIENTS: The study comprised a total of 183 patients admitted to our ICU immediately after liver transplantation between 2009 and 2015. VARIABLES OF INTEREST: C-reactive protein levels upon ICU admission and after 24 and 48h, severe EAD and hospital mortality. RESULTS: The CRP levels after OLT were: upon ICU admission 57.5 (51.6-63.3)mg/L, after 24h 80.1 (72.9-87.3)mg/L and after 48h 69.9 (62.5-77.4)mg/L. Severe EAD patients (14.2%) had higher mortality (23.1 vs 2.5; OR 11.48: 2.98-44.19) and lower CRP upon ICU admission (39.3 [29.8-48.7]mg/L) than the patients without EAD (0.5 [53.9-67.0]; p<0.05] - the best cut-off point being 68mg/L (sensitivity 92.3%; specificity 40.1%; Youden index 0.33). Lower CRP upon ICU admission was correlated to higher mortality (24.5 [9.2-39.7] vs 59.4 [53.4-65.4]; p<0.01, AUC 0.79 [0.65-0.92]). CONCLUSION: Liver transplant is a strong inflammatory stimulus accompanied by high levels of C-reactive protein. A blunted rise in CRP on the first postoperative day after OLT may be a marker of poor allograft function and is related to hospital mortality.

A novel mechanism for immune regulation after human lung transplantation.

OBJECTIVE: Lung transplantation is therapeutic for end-stage lung disease, but survival is limited due to bronchiolitis obliterans syndrome and restrictive chronic lung allograft dysfunction. We sought a common denominator in lung transplant recipients, analyzing risk factors that trigger immune responses that lead to bronchiolitis obliterans syndrome. METHODS: We collected blood from patients who underwent lung transplant at our institution. Exosomes were isolated from the sera of recipients with risk factors for chronic rejection and from stable recipients. Exosomes were analyzed with western blot, using antibodies to lung self-antigens K alpha 1 tubulin and collagen-V, costimulatory molecules (costimulatory molecule 80, costimulatory molecule 86), transcription factors (nuclear factor kappa-light-chain-enhancer of activated B cells, hypoxia-inducible factor 1α, Class II Major Histocompatibility Complex Transactivator), and 20S proteasome. RESULTS: Of the 90 patients included, we identified 5 with grade 3 primary graft dysfunction, 5 without, 15 with respiratory viral infection, 10 with acute rejection, 10 with donor-specific antibodies (DSA), 5 without DSA, and 10 who were stable for exosome isolation. Recipients with grade 3 primary graft dysfunction, respiratory viral infection, acute rejection, and DSA had exosomes containing self-antigens; exosomes from stable recipients did not. Exosomes from recipients with grade 3 primary graft dysfunction, acute rejection, and DSA also demonstrated costimulatory molecule 80, costimulatory molecule 86, major histocompatibility complex class II, transcription factor, and 20S proteasome. CONCLUSIONS: Transplanted lungs with grade 3 primary graft dysfunction, symptomatic respiratory viral infection, acute rejection, and immune responses induce exosomes that contain self-antigens, costimulatory molecules, major histocompatibility complex class II, transcription factors, and 20S proteasome. Release of circulating exosomes post-transplant from the aforementioned stress-inducing insults augment immunity and may play an important role in the pathogenesis of bronchiolitis obliterans syndrome.

Postreperfusion plasma endothelial activation markers are associated with acute kidney injury after lung transplantation.

Acute kidney injury (AKI) is common after lung transplantation, but molecular markers remain poorly studied. The endothelial activation markers soluble thrombomodulin (sTM), protein C, and plasminogen activator inhibitor-1 (PAI-1) are implicated in kidney microcirculatory injury in animal models of AKI. We tested the association of 6-hour postreperfusion plasma levels of these markers with posttransplant AKI severity in patients enrolled in the Lung Transplant Outcomes Group prospective cohort study at the University of Pennsylvania during two eras: 2004-06 (n = 61) and 2013-15 (n = 67). We defined AKI stage through postoperative day 5 using Kidney Disease Improving Global Outcomes creatinine criteria. We used multivariable ordinal logistic regression to determine the association of each biomarker with AKI, adjusted for primary graft dysfunction and extracorporeal life support. AKI occurred in 57 (45%) patients across both eras: 28 (22%) stage 1, 29 (23%) stage 2-3. Higher sTM and lower protein C plasma levels were associated with AKI stage in each era and remained so in multivariable models utilizing both eras (sTM: OR 1.76 [95% CI 1.19-2.60] per standard deviation, P = .005; protein C: OR 0.54 [1.19-2.60], P = .003). We conclude that 6-hour postreperfusion plasma sTM and protein C levels are associated with early postlung transplant AKI severity.

Arterial Lactate Concentration at the End of Liver Transplantation Is an Early Predictor of Primary Graft Dysfunction.

BACKGROUND: Although many prognostic factors of primary graft dysfunction after liver transplantation (LT) are available, it remains difficult to predict failure in a given recipient. OBJECTIVE: We aimed to determine whether the intraoperative assay of arterial lactate concentration at the end of LT (LCEOT) might constitute a reliable biological test to predict early outcomes [primary nonfunction (PNF), early graft dysfunction (EAD)]. METHODS: We reviewed data from a prospective database in a single center concerning patients transplanted between January 2015 and December 2016 (n = 296). RESULTS: There was no statistical imbalance between the training (year 2015) and validation groups (year 2016) for epidemiological and perioperative feature. Ten patients (3.4%) presented with PNF, and EAD occurred in 62 patients (20.9%); 9 patients died before postoperative day (POD) 90. LCEOT ≥5 mmol/L was the best cut-off point to predict PNF (Se=83.3%, SP=74.3%, positive likelihood ratio (LR+)=3.65, negative likelihood ratio (LR-)=0.25, diagnostic odds ratio (DOR)=14.44) and was predictive of PNF (P = 0.02), EAD (P = 0.05), and death ≤ POD90 (P = 0.06). Added to the validated BAR-score, LCEOT improved its predictive value regarding POD 90 survival with a better AUC (0.87) than BAR score (0.74). The predictive value of LCEOT was confirmed in the validation cohort. CONCLUSION: As a reflection of both hypoperfusion and tissue damage, the assay of arterial LCEOT ≥5 mmol/L appears to be a strong predictor of early graft outcomes and may be used as an endpoint in studies assessing the impact of perioperative management. Its accessibility and low cost could impose it as a reliable parameter to anticipate postoperative management and help clinicians for decision-making in the first PODs.

Endothelial Glycocalyx Shedding Predicts Donor Organ Acceptability and Is Associated With Primary Graft Dysfunction in Lung Transplant Recipients.

BACKGROUND: The endothelial glycocalyx, a sieve-like structure located on the luminal surface of all blood vessels, has been found to be integral to regulation of capillary permeability and mechanotransduction. Given this, we investigated the role of endothelial glycocalyx breakdown products in organ donors and recipients in terms of acceptability for transplant and risk of primary graft dysfunction (PGD). METHODS: Endothelial glycocalyx breakdown products were measured in the peripheral blood of 135 intended and actual organ donors. Breakdown product levels were tested for association with donor demographic and clinical data, organ acceptability for transplant along with lung recipient outcomes (n = 35). Liquid chromatography mass spectrometry analysis was performed to confirm glycosaminoglycan levels and sulfation patterns on donor samples (n = 15). In transplant recipients (n = 50), levels were measured pretransplant and daily for 4 days posttransplant. Levels were correlated with PGD severity and intubation time. RESULTS: Decreased hyaluronan levels in peripheral blood independently predicted organ acceptability in intended and actual donors (odds ratio, 0.96; [95% confidence interval, 0.93-0.99] P = 0.026). Furthermore, high donor syndecan-1 levels were associated with PGD in recipients (3142 [1575-4829] versus 6229 [4009-8093] pg/mL; P = 0.045). In recipient blood, levels of syndecan-1 were correlated with severe (grades 2-3) PGD at 72 hours posttransplant (5982 [3016-17191] versus 3060 [2005-4824] pg/mL; P = 0.01). CONCLUSIONS: Endothelial glycocalyx breakdown occurs in lung transplant donors and recipients and predicts organ acceptability and development of PGD. Glycocalyx breakdown products may be useful biomarkers in transplantation, and interventions to protect the glycocalyx could improve transplant outcomes.

Serum Factor V Is a Continuous Biomarker of Graft Dysfunction and a Predictor of Graft Loss After Liver Transplantation.

BACKGROUND: Factor V has never been compared to a validated early allograft dysfunction (EAD) definition. We aimed to assess factor V as a biomarker of EAD and a predictor of graft loss after liver transplantation (LT). METHODS: We retrospectively assessed the serum factor V levels on postoperative day 1 after LT. Patients were divided according to their factor V levels into the ≤36.1 U/mL and > 36.1 U/mL groups. The primary outcome was graft loss within 1, 3, and 6 months. The secondary outcome was EAD, as defined by Olthoff et al. Predictors of outcomes were identified by multivariable logistic regression. RESULTS: Two hundred twenty-seven patients were included in the study: 74 with factor V of 36.1 U/mL or less and 153 with factor V >36.1 U/mL. EAD was diagnosed in 41 (55.4%) of 74 patients with factor V of 36.1 U/mL or less and in 20/153 (13.1%) patients with factor V >36.1 U/mL (P < 0.001). According to the multivariable regression model, factor V was a continuous marker of EAD (odds ratio [OR], 0.96; 95% confidence interval [CI], 0.94-0.98 per U/mL). Among the study groups, the 1-, 3-, and 6-month graft survival rates were 82%, 74%, and 74%, respectively, for patients with factor V of 36.1 U/mL or less and 98%, 95%, and 95%, respectively, for patients with factor V >36.1 U/mL (P = 0.001). Factor V was a continuous predictor for 3- and 6-month graft losses (OR, 0.96; 95% CI, 0.94-0.99 and OR, 0.97; 95% CI, 0.94-0.99 per U/mL), whereas EAD was not significant when adjusted for factor V. CONCLUSION: Factor V is an early marker for EAD and is a continuous predictor of short-term graft loss after LT.

Donor-derived cell-free DNA is associated with acute rejection and decreased oxygenation in primary graft dysfunction after living donor-lobar lung transplantation.

Donor-derived cell-free DNA (dd-cf-DNA) has been shown to be an informative biomarker of rejection after lung transplantation (LT) from deceased donors. However, in living-donor lobar LT, because small grafts from blood relatives are implanted with short ischemic times, the detection of dd-cf-DNA might be challenging. Our study was aimed at examining the role of dd-cf-DNA measurement in the diagnosis of primary graft dysfunction and acute rejection early after living-donor lobar LT. Immediately after LT, marked increase of the plasma dd-cf-DNA levels was noted, with the levels subsequently reaching a plateau with the resolution of primary graft dysfunction. Increased plasma levels of dd-cf-DNA were significantly correlated with decreased oxygenation immediately (p = 0.022) and at 72 hours (p = 0.046) after LT. Significantly higher plasma dd-cf-DNA levels were observed in patients with acute rejection (median, 12.0%) than in those with infection (median, 4.2%) (p = 0.028) or in a stable condition (median, 1.1%) (p = 0.001). Thus, measurement of the plasma levels of dd-cf-DNA might be useful to monitor the severity of primary graft dysfunction, and plasma dd-cf-DNA could be a potential biomarker for the diagnosis of acute rejection after LT.

Usefulness of Asymmetric Dimethylarginine to Predict Outcomes After Heart Transplantation.

Asymmetric dimethylarginine (ADMA) is a key mediator of vascular homeostasis and an independent predictor of the development of accelerated cardiac allograft vasculopathy after heart transplantation. However, its association with clinical outcomes in heart transplant recipients has not been described. Plasma levels of ADMA were assayed within 8 weeks following transplantation (baseline) using a competitive enzyme-linked immunosorbent assay. The primary end point was the composite of nonfatal myocardial infarction, percutaneous coronary intervention, retransplantation, or death at 5-year follow-up. Kaplan-Meier curves were generated to assess the association between baseline ADMA levels (stratified at 0.70 µM, a previously established cutoff) and cumulative event-free survival. Multivariate Cox regression was performed to adjust for other candidate predictors. In 69 heart transplant recipients at Stanford, the primary end point occurred in 11 patients (16%)-4 percutaneous coronary intervention, 1 retransplant, and 6 deaths-during 5-years follow-up. Patients with baseline ADMA ≥0.70 µM had lower cumulative 5-year event-free survival (77% vs 93%, p = 0.059). In multivariate Cox analysis, baseline ADMA was the only significant predictor of the primary end point (hazard ratio 1.33, 95% confidence interval 1.03 to 1.72 per 0.1 µM; p = 0.031). This association remained significant even after restricting the end point to death or retransplantation (hazard ratio 1.48, 95% confidence interval 1.12 to 1.97 per 0.1 µM; p = 0.006). In conclusion, elevated baseline plasma levels of ADMA independently predicted 5-year clinical outcomes after heart transplantation, suggesting that ADMA has potential prognostic value in the heart transplant arena.

Serum 25-Hydroxyvitamin D Level Is Not Related to Regulatory T Cells or Kidney Function in Renal Transplant Recipients.

BACKGROUND: Vitamin D and regulatory T cells (Tregs) are both involved in promoting peripheral tolerance and limiting chronic inflammatory diseases. Renal transplant recipients (RTRs) are likely to have low vitamin D levels, which may influence their immune status. AIM: The aim of our study was to assess the usefulness of serum 25-hydroxyvitamin D (25(OH)D) and Tregs in estimation of the protolerogenic milieu in RTRs within 1 year after kidney transplantation. METHODS: 26 RTRs (15M/11F, aged 49.1 ± 15.4 years) 3 to 13 months after kidney transplantation and 24 healthy volunteers were enrolled for the study. The serum level of 25(OH)D was measured with ELISA and peripheral blood immune cell populations (T lymphocytes, helper T lymphocytes, and Tregs) were assessed by flow cytometry. RESULTS: Severe 25(OH)D deficiency (<10 ng/mL) was found in one RTR (3%) and moderate deficiency (<20 ng/mL) in 12 (46%), while vitamin D sufficiency was found in 6 patients (23%). The RTRs did not differ from the control group in observed 25(OH)D levels. None of the cell populations were related to the level of 25(OH)D in the control group. In RTRs, there was a negative association between 25(OH)D and total T lymphocyte count (rs = -0.45, P = .023), but 25(OH)D was not related to any other cell population or kidney function. CONCLUSION: The results of our study suggest that serum 25(OH)D is not sufficiently reflective of vitamin D status to apply this measure in assessment of protolerogenic milieu in RTRs.

Elevated donor plasminogen activator inhibitor-1 levels and the risk of primary graft dysfunction.

Primary graft dysfunction (PGD) following lung transplantation is associated with elevated recipient plasma levels of plasminogen activator inhibitor-1 (PAI-1) and the receptor for advanced glycation end products (RAGE). However, the significance of these biomarkers in the donor plasma is uncertain. We hypothesized that elevated donor plasma levels of PAI-1 and RAGE would be associated with recipient PGD. We carried out a prospective unmatched case-control study of double-lung transplant recipients between May 2014 and September 2015. We compared donor plasma levels of PAI-1 and RAGE using rank-sum tests and t tests, in 12 recipients who developed PGD grade 2 or 3 within 72 hours postoperatively with 13 recipients who did not. Recipients who developed PGD had higher donor plasma levels of PAI-1 than recipients who did not (median 2.7 ng/mL vs 1.4; P = .03). Recipients with PGD also had numerically higher donor plasma levels of RAGE than recipients without PGD, although this difference did not achieve statistical significance (median 1061 pg/mL vs 679; P = .12). Systemic inflammatory responses in the donor, as reflected by elevated plasma levels of PAI-1, may contribute to the risk of developing PGD. Rapid biomarker assessment of easily available plasma samples may assist in donor lung selection and risk stratification.

Glycome Patterns of Perfusate in Livers Before Transplantation Associate With Primary Nonfunction.

BACKGROUND & AIMS: Primary nonfunction (PNF) is a rare complication after liver transplantation that requires urgent retransplantation. PNF is associated with livers from extended criteria donors. Clinical and biochemical factors have not been identified that reliably associate with graft function after liver transplantation. Serum patterns of N-glycans associate with changes in the liver. We analyzed perfusate from grafted liver to identify protein glycosylation patterns associated with PNF. METHODS: We performed a prospective study of consecutive patients who underwent liver transplantation (66 patients, from 1 center, in the derivation set, and 56 patients, from 2 centers, in the validation set) in Belgium, from October 1, 2011, through April 30, 2017. All donor grafts were transported using cold static storage, and perfusate samples were collected from the livers by flushing of hepatic veins before transplantation. Protein-linked N-glycans were isolated from perfusate samples and analyzed with a multicapillary electrophoresis-based ABI3130 sequencer. We compared glycan patterns between patients with vs without PNF of transplanted livers. PNF was defined as the need for urgent retransplantation when a graft had no evidence of function, after exclusion of other causes, such as hepatic artery thrombosis or acute cellular rejection. RESULTS: The relative abundance of a single glycan, agalacto core-alpha-1,6-fucosylated biantennary glycan (NGA2F) was significantly increased in perfusate of livers given to 4 patients who developed PNF after liver transplantation compared with livers given to patients who did not develop PNF. Level of NGA2F identified patients with PNF with 100% accuracy. This glycomarker was the only factor associated with PNF in multivariate analysis in the derivation and the validation sets (P < .0001). CONCLUSIONS: In an analysis of patients who underwent liver transplantation, we associated graft perfusate level of glycan NGA2F present on perfusate proteins with development of PNF with 100% accuracy, and validated this finding in a separate cohort of patients. This biomarker might be used to assess grafts before transplantation, especially when high-risk organs are under consideration.

Cell-free hemoglobin promotes primary graft dysfunction through oxidative lung endothelial injury.

Primary graft dysfunction (PGD) is acute lung injury within 72 hours of lung transplantation. We hypothesized that cell-free hemoglobin (CFH) contributes to PGD by increasing lung microvascular permeability and tested this in patients, ex vivo human lungs, and cultured human lung microvascular endothelial cells. In a nested case control study of 40 patients with severe PGD at 72 hours and 80 matched controls without PGD, elevated preoperative CFH was independently associated with increased PGD risk (odds ratio [OR] 2.75, 95%CI, 1.23-6.16, P = 0.014). The effect of CFH on PGD was magnified by reperfusion fraction of inspired oxygen (FiO2) ≥ 0.40 (OR 3.41, P = 0.031). Isolated perfused human lungs exposed to intravascular CFH (100 mg/dl) developed increased vascular permeability as measured by lung weight (CFH 14.4% vs. control 0.65%, P = 0.047) and extravasation of Evans blue-labeled albumin dye (EBD) into the airspace (P = 0.027). CFH (1 mg/dl) also increased paracellular permeability of human pulmonary microvascular endothelial cell monolayers (hPMVECs). Hyperoxia (FiO2 = 0.95) increased human lung and hPMVEC permeability compared with normoxia (FiO2 = 0.21). Treatment with acetaminophen (15 µg/ml), a specific hemoprotein reductant, prevented CFH-dependent permeability in human lungs (P = 0.046) and hPMVECs (P = 0.037). In summary, CFH may mediate PGD through oxidative effects on microvascular permeability, which are augmented by hyperoxia and abrogated by acetaminophen.