Heart Transplant Rejection Research Articles

Mechanism Exploration on the Immunoregulation of Allogeneic Heart Transplantation Rejection in Rats With Exosome miRNA and Proteins From Overexpressed IDO1 BMSCs.

Immunoregulation and indoleamine 2,3-dioxygenase 1 (IDO1) play pivotal roles in the rejection of allogeneic organ transplantation. This study aims to elucidate the immune-related functional mechanisms of exosomes (Exos) derived from bone marrow-derived mesenchymal stem cells (BMSCs) overexpressing IDO1 in the context of allogeneic heart transplantation (HTx) rejection. A rat model of allogeneic HTx was established. Exos were extracted after transfection with oe-IDO1 and oe-NC from rat BMSCs. Exos were administered via the caudal vein for treatment. The survival of rats was analyzed, and reverse transcription qualitative PCR (RT-qPCR) and immunohistochemistry (IHC) were employed to detect the expression of related genes. Histopathological examination was conducted using hematoxylin and eosin (HE) staining, and flow cytometry was utilized to analyze T-cell apoptosis. Proteomics and RNA-seq analyses were performed on Exos. The data were subjected to functional enrichment analysis using the R language. A protein interaction network was constructed using the STRING database, and miRWalk, TargetScan, and miRDB databases predicted the target genes, differentially expressed miRNAs, and transcription factors (TFs). Exos from BMSCs overexpressing IDO1 prolonged the survival time of rats undergoing allogeneic HTx. These Exos reduced inflammatory cell infiltration, mitigated myocardial damage, induced CD4 T-cell apoptosis, and alleviated transplantation rejection. The correlation between Exos from BMSCs overexpressing IDO1 and immune regulation was profound. Notably, 13 immune-related differential proteins (Anxa1, Anxa2, C3, Ctsb, Hp, Il1rap, Ntn1, Ptx3, Thbs1, Hspa1b, Vegfc, Dcn, and Ptpn11) and 10 significantly different miRNAs were identified. Finally, six key immune proteins related to IDO1 were identified through common enrichment pathways, including Thbs1, Dcn, Ptpn11, Hspa1b, Il1rap, and Vegfc. Thirteen TFs of IDO1-related key miRNAs were obtained, and a TF-miRNA-mRNA-proteins regulatory network was constructed. Exosome miRNA derived from BMSCs overexpressing IDO1 may influence T-cell activation and regulate HTx rejection by interacting with mRNA.

ВОПРОСЫ ДОГОСПИТАЛЬНОЙ ДИАГНОСТИКИ ОСТРОЙ РЕАКЦИИ ОТТОРЖЕНИЯ У ПАЦИЕНТА, ПЕРЕНЕСШЕГО ОРТОТОПИЧЕСКУЮ ТРАНСПЛАНТАЦИЮ СЕРДЦА

An acute cardiac transplant rejection is a reaction of the recipient’s immune system, which includes the cellular and/or humoral pathway of the immune response. This reaction is directed against the tissues of the “alien” donor organ. The alertness of clinicians regarding the possible development of an acute rejection reaction during the first year after heart transplantation is due to the extremely high risk of developing life-threatening conditions mediated by graft dysfunction. These include various cardiac arrhythmias and a progressive decrease in myocardial contractility. In case of untimely diagnosis of this serious complication, the risk of mortality of the patient who underwent orthotopic heart transplantation is extremely high. What changes in clinical, laboratory and instrumental examination parameters at the outpatient stage can help a practicing cardiologist to suspect the presence of an acute rejection reaction of a heart transplant in a patient who has undergone orthotopic heart transplantation? At present, there is no unambiguous answer to this question, and the topic of early detection and prediction of the development of an acute heart transplant rejection reaction is extremely relevant for modern scientific research and is of great practical interest in the field of transplantology and cardiology, in particular.

Cardiac magnetic resonance assessment of acute rejection and cardiac allograft vasculopathy in pediatric heart transplant

In pediatric heart transplant (PHT), cardiac catheterization with endomyocardial biopsy (EMB) is standard for diagnosing acute rejection (AR) and cardiac allograft vasculopathy (CAV) but is costly and invasive. To evaluate the ability of cardiac magnetic resonance (CMR) to noninvasively identify differences in PHT patients with AR and CAV. Patients were enrolled at three children's hospitals. Data were collected from surveillance EMB or EMB for-cause AR. Patients were excluded if they had concurrent diagnoses of AR and CAV, CMR obtained >7days from AR diagnosis, they had EMB negative AR, or could not undergo contrasted, unsedated CMR. Kruskal-Wallis test was used to compare groups: (1) No AR or CAV (Healthy), (2) AR, (3) CAV. Wilcoxon rank-sum test was used for pairwise comparisons. Fifty-nine patients met inclusion criteria (median age 17years [IQR 15-19]) 10 (17%) with AR, and 11 (19%) with CAV. AR subjects had worse left ventricular ejection fraction compared to Healthy patients (p=0.001). Global circumferential strain (GCS) was worse in AR (p=0.054) and CAV (p=0.019), compared to Healthy patients. ECV, native T1, and T2 z-scores were elevated in patients with AR. CMR was able to identify differences between CAV and AR. CAV subjects had normal global function but abnormal GCS which may suggest subclinical dysfunction. AR patients have abnormal function and tissue characteristics consistent with edema (elevated ECV, native T1 and T2 z-scores). Characterization of CMR patterns is critical for the development of noninvasive biomarkers for PHT and may decrease dependence on EMB.

Serial electrocardiograms at follow-up for early detection of transplanted heart rejection: A viewpoint

This viewpoint proposed that serial electrocardiograms (ECG) could be used to monitor for heart transplantation (HT) rejection, based on the expected attenuation of the amplitude of ECG QRS complexes (attQRS) engendered by the rejection-induced decrease in electrical resistance due to the underlying myocardial edema (ME). Previous work in humans has shown attQRS in the setting of a diverse array of edematous states, affecting the myocardium (i.e, ME) and the body volume conductor “enveloping” the heart. Also, animal and human experience has revealed low electrical resistance during mild/moderate HT rejection. Studies with serial correlations of endomyocardial biopsy (EMB), echocardiography, cardiac magnetic resonance imaging, and ECG are recommended, which will merely require recording of an ECG, when EMB and imaging studies are carried out for monitoring of post-HT rejection.

Incidence of Acute Rejection Compared With Endomyocardial Biopsy Complications for Heart Transplant Patients in the Contemporary Era.

The reference standard of detecting acute rejection (AR) in adult heart transplant (HTx) patients is an endomyocardial biopsy (EMB). The majority of EMBs are performed in asymptomatic patients. However, the incidence of treated AR compared with EMB complications has not been compared in the contemporary era (2010-current). The authors retrospectively analyzed 2769 EMBs obtained in 326 consecutive HTx patients between August 2019 and August 2022. Variables included surveillance versus for-cause indication, recipient and donor characteristics, EMB procedural data and pathological grades, treatment for AR, and clinical outcomes. The overall EMB complications rate was 1.6%. EMBs performed within 1 mo after HTx compared with after 1 mo from HTx showed significantly increased complications (OR, 12.74, P < 0.001). The treated AR rate was 14.2% in the for-cause EMBs and 1.2% in the surveillance EMBs. We found the incidence of AR versus EMB complications was significantly lower in the surveillance compared with the for-cause EMB group (OR, 0.05, P < 0.001). We also found the incidence of EMB complications was higher than treated AR in surveillance EMBs. The yield of surveillance EMBs has declined in the contemporary era, with a higher incidence of EMB complications compared with detected AR. The risk of EMB complications was highest within 1 mo after HTx. Surveillance EMB protocols in the contemporary era may need to be reevaluated.

Novel Diagnostics for Pediatric Heart Transplant Rejection

Novel Diagnostics for Pediatric Heart Transplant Rejection

Heart transplantation : Current situation

Heart transplantation is the gold-standard in the treatment of terminal heart failure. The shortage of donor hearts represents the major obstacle in patient care and necessitates the creation of waiting lists and allocation algorithms. The Transplantation Act regulates donor heart allocation according to the urgency and the prospects of success. Donor hearts can be implanted following the classical biatrial or the modern bicaval valve implantation technique with a slightly lower spectrum of complications. Modern mechanical perfusion systems enable extended transport times. After heart transplantation rejection reactions must be controlled by an individually adjusted immunosuppression to guarantee long-term survival with as few complications as possible.

Role of NTproBNP and donor-derived cell-free DNA in the diagnosis of acute rejection in heart transplant recipients: a prospective study

Abstract Background Donor-derived cell-free DNA (dd-cfDNA) has shown a good ability to rule out rejection in heart transplant (HT) recipients, but there is scarce information on the utility of NTproBNP (and the combination of both biomarkers) in this setting. Methods We prospectively obtained levels of both biomarkers in all patients included in the FreeDNA-CAR study (NCT 04973943), just before performing surveillance endomyocardial biopsies (EMB) at 0.5, 1, 2, 3, 4, 6 and 12 months post-HT. Primary end-point was the correlation between dd-cfDNA and NTproBNP levels. Results A total of 206 HT patients from 12 different centers were included (mean age 54±12 years, 74% male), with a median of 5.6 EBM per patient. After excluding visits with missing data, 883 pairs of EBM-both biomarkers were available for analysis. ACR, defined as grade ≥2R, was present in 37 EMB (4.2%), and AMR ≥1 in 16 EMB (1.8%). Median (IQR) NTproBNP values for each ACR group were: 0R 1067 pg/ml (454-2563), 1R 1300 pg/ml (562-3298) and 2R-3R 1780 pg/ml (943-4880) and median dd-cfDNA values: 0R 0.08% (0.038-0.178), 1R 0.105% (0.04-0.34), 2R-3R 0.22% (0.058-0.7). Regarding AMR, median NTproBNP in pAMR0 was 1200 pg/ml (504-2840) and in pAMR≥1 2230 pg/ml (806-4256) (Figures 1A and 1B). Using a GEE (generalized estimating equation) approach to account for repeated measures in each individual, a statistically significant correlation between both biomarkers was found, with a coefficient of 0.001 (CI95% 0.0006-0.002), meaning that for every increase of 100 units of NTproBNP, %dd-cfDNA increased in 0.001, p &amp;lt;0.001 (Figure 2). Moreover, in a multivariable analysis, both biomarkers were associated with the risk of acute rejection, with an OR 1.48 (CI95% 1.04-2.11), p=0.03 for dd-cfDNA, and OR 1.007 (IC95% 1.001-1-013) p=0.017 for each additional 100 pg/ml of NTproBNP. Conclusion In a cohort of HT patients in their first post-transplant year, dd-cfDNA and NT-proBNP levels were associated with acute rejection and showed a good correlation between them. Further studies are needed in order to determine if the combination of both biomarkers could improve individual performance and increase the number of EMB that could be safely eliminated.Median (IQR) NTproBNP levels (pg/ml)Linear prediction of dd-cfDNA

Donor-derived cell-free DNA kinetics in the first year post-transplant in patients without acute cellular rejection

Abstract Background Donor-derived cell-free DNA (dd-cfDNA) appears to be an excellent biomarker for acute cellular rejection (ACR) in heart transplant (HT) recipients. However, a significant rise in the immediate post-transplant phase probably related to direct myocardial damage during harvesting-implantation process has been described, and could potentially affect the biomarker’s performance in the first post-HT month. We sought to describe the biomarker’s kinetics in the first year post-HT in patients without ACR. Methods Prospective, multicentre study performed between 2019 and 2022. All patients underwent surveillance endomyocardial biopsy (EMB) at 7 pre-specified time points in the first post-HT year (0.5, 1, 2, 3, 4, 6 and 12 months). Dd-cfDNA levels were determined prior to each EMB, using Next Generation Sequencing technology. Results A total of 1006 pairs of EMB-dd-cfDNA from 206 patients were available for analysis (mean age 54 ±12 years, 74% male). Of these, 44 (4.3%) had clinically relevant rejection, defined as ACR grade ≥2, and 962 (95.7%) did not (562 0R and 400 1R). 15 samples that had pAMR≥1 were excluded from this analysis. We divided the 947 samples with ACR 0-1 into three subgroups, according to the time of blood sampling: at 15 days (time point 1), at 30 days (time point 2) and 2 to 12 months post-HT (time point 3). Samples with 0R that were drawn at 15 and 30 days showed significantly higher levels of dd-cfDNA than those drawn between the second month and first year post-transplant (p &amp;lt;0.001) (Figure 1A). When we included patients with 1R, a similar trend was observed (p &amp;lt;0.001) (Figure 1B). Median dd-cfDNA levels for 0R and for 0-1R at the three time points are shown in Figure 2. Conclusions Dd-cfDNA levels in patients without clinically relevant rejection are significantly higher in the first month post-transplant. Further studies are needed in order to determine if a different cut-off point for ACR during the first post-HT month is useful for ACR diagnosis.Donor-derived cell-free DNA kineticsDd-cfDNA values at three time points

Abstract 15922: Integrative Analysis of Extracellular Vesicles in Heart Transplant Rejection: From Biomarker Discovery to Novel Immune Mechanisms

Introduction: At the forefront of post-heart transplant care is early detection of transplant rejection. However, the current gold standard for detecting acute cellular transplant rejection is recurrent invasive endomyocardial biopsies (EMB). We aimed at developing a non-invasive biomarker for acute cellular heart transplant rejection (ACR) using a plasma extracellular vesicle (EV) platform. Methods: Peripheral and coronary sinus plasma samples were prospectively collected from 183 (31.7% female) patients from 2020 to 2023 during routine surveillance EMB. A discovery cohort comprised five patients who displayed ISHLT 0-1R biopsy followed by an ISHLT 2R grade rejection event. Plasma EVs were isolated from the baseline (0-1R) and rejection (2R) time points. Subsequently, integrative analysis of plasma EV proteomic, miRNomic, and metabolomics was performed pairwise with the samples. Results: The study collected 943 sample time points from 183 patients. Of these sample collections, 43 (4.5%) cases were 2R rejections. Our proteomic analysis of the plasma EVs identified 28 proteins found explicitly during a grade 2R rejection and 87 differentially expressed proteins during a 2R rejection event. An Ingenuity Pathway Analysis (IPA) analysis of the upregulated proteins highlighted EVs mediated mTOR signaling and antigen presentation as central to rejection events. Although all patients in the discovery cohort were negative for antibody-mediated rejection (AMR), the proteomic findings also revealed increased B-cell receptor signaling, implying a mechanistic intersection of ACR and AMR. Conclusions: A distinct set of proteins were found in plasma EVs during ACR events, providing potentially specific and sensitive biomarkers to reduce the need for EMB that could be exploited for ACR diagnosis. The proteins identified formed an immune signaling network that suggests a novel role for EVs in ACR pathogenesis.

Therapeutic potential role of vitamin C in prevention and control of heart transplant rejection and cardiac allograft vasculopathy. A need for consideration.

The burden of cardiovascular diseases is rising rapidly globally. Heart transplant is one of the most last resort medical option for patients with heart failure. Unfortunately, this surgical intervention is associated with several serious complications including heart transplant rejection (HTR) and Cardiac Allograft Vasculopathy (CAV) which can manifest just within few years'posttransplant. These complications affect significantly the prognosis and quality of life among postheart transplant patients. Several medications including immunosuppressant, antibiotics, antihypertensive,and statins have been used during posttransplant care so as to address such complications. Unfortunately, most of those drugs are expensive and pose a number of serious side effects to the patients enough to compromise patients'quality of life too.Several studies on Vitamin C are therapeutically suggestive that it can be used during postheart transplant care with more cost-effective benefits with less and minimized side effects compared to the current drugs in place. It should be considered pharmacologically that Vitamin C has a great potential role clinically in prevention and control of HTR and CAV development. On the light of such findings as described above, we recommend more studies especially clinical trials and molecular studies to determine whether Vitamin C can be repositioned to replace or to be used along the current drug regimens used in postheart transplant care for prevention and control of HTR and CAV.

Accuracy of free-breathing multi-parametric SASHA in identifying T1 and T2 elevations in pediatric orthotopic heart transplant patients.

T1/T2 parametric mapping may reveal patterns of elevation ("hotspots") in myocardial diseases, such as rejection in orthotopic heart transplant (OHT) patients. This study aimed to evaluate the diagnostic accuracy of free-breathing (FB) multi-parametric SAturation recovery single-SHot Acquisition (mSASHA) T1/T2 mapping in identifying hotspots present on conventional Breath-held Modified Look-Locker Inversion recovery (BH MOLLI) T1 and T2-prepared balanced steady-state free-precession (BH T2p-bSSFP) maps in pediatric OHT patients. Pediatric OHT patients underwent noncontrast 1.5T CMR with BH MOLLI T1 and T2p-bSSFP and prototype FB mSASHA T1/T2 mapping in 8 short-axis slices. FB and BH T1/T2 hotspots were segmented using semi-automated thresholding (ITK-SNAP) and their 3D coordinate locations were collected (3-Matic, Materialise, Leuven, Belgium). Receiver operator characteristic curve analysis and measures of central tendency were utilized. 40 imaging datasets from 23 pediatric OHT patients were obtained. FB mSASHA yielded a sensitivity of 82.8% for T1 and 80% for T2 maps when compared to the standard BH MOLLI, as well as 100% specificity for both T1 and T2 maps. When identified on both FB and BH maps, hotspots overlapped in all cases, with an average long axis offset between FB and BH hotspot centers of 5.8mm (IQR 3.5-8.2) on T1 and 5.9mm (IQR 3.5-8.2) on T2 maps. FB mSASHA T1/T2 maps can identify hotspots present on conventional BH T1/T2 maps in pediatric patients with OHT, with high sensitivity, specificity, and overlap in 3D space. Free-breathing mapping may improve patient comfort and facilitate OHT assessment in younger patient populations.

Microneedle-mediated transdermal delivery of FK506 for treatment of acute cardiac allograft rejection

FK506 has been widely used to prevent acute rejection in heart transplantation (HT). However, its strong hydrophobicity, poor bioavailability, high pharmacokinetic variability, and narrow therapeutic window are obstacles to its application in HT. It has been demonstrated that localized and sustained immunosuppressant delivery may be a suitable route for FK506 administration. Hence, the objective of this study was to evaluate the efficacy of a FK506-loaded microneedle (FK506 MN) in the treatment of acute rejection in HT. The physicochemical properties of FK506 MN were evaluated, and it exhibited constant release over the course of at least 9 days with a 94.6 % cumulative release rate. Also, the FK506 MN treatment could successfully alleviate acute rejection and prolong allograft survival compared with the untreated group (mean survival time, 12.33 versus 7.25 days). Furthermore, T cell and macrophage infiltration as well as secretion of IL-2, IL-6 and IFN-γ were dramatically reduced in the FK506 MN group. As expected, no hepatoxicity and nephrotoxicity were observed after the application of FK506 MN. Taken together, FK506 MN based drug delivery system provides a novelty strategy to deliver FK506 in heart transplantation, which may be also suitable for other organ transplantations in the future.

Use of microaxial flow pumps in adolescents.

The Impella 5.5 has been successfully used in the adult population; however, safety and efficacy data in patients aged less than 18 years are limited. Six pediatric patients, aged 13 to 16years and weighing 45 to 113kg, underwent axillary artery graft placement and attempted placement of the Impella 5.5 device at our institution between August 2020 and March 2023. Indications for implantation were heart failure secondary to myocarditis (2), rejection of prior orthotopic heart transplant, idiopathic dilated cardiomyopathy (2), and heart failure after transposition of the great arteries repair. Placement was unsuccessful in a 13.8-year-old female patient due to prohibitively acute angulation of the right subclavian artery, and venoarterial extracorporeal membrane oxygenation cannulation was performed via the axillary graft. In 5 patients with successful Impella 5.5 placement, median duration of support was 13.5days (range, 7-42days). One experienced cardiac arrest secondary to coagulation-associated device failure, requiring temporary HeartMate3 implantation. Four patients were bridged to transplant; 3 patients received a transplant directly from Impella 5.5, and 1 patient received a transplant after HeartMate3. The final patient received the HeartMate3 on Impella day 42 and is awaiting transplant. Although exact size cutoffs and anatomy are still being determined, our experience provides a framework for use of the Impella 5.5 in adolescents.

Plasma Levels of High-sensitivity Cardiac Troponin I and Histopathological Findings in Heart Transplant Patients: A Single-center Cross-sectional Study

Abstract Introduction: This study aimed to evaluate the association between plasma levels of high-sensitivity cardiac troponin I (cTnI) in heart transplant patients and the grading of graft rejection. Methods: In a cross-sectional study, 92 biopsy samples from the patients who had undergone heart transplantation during 2022, at Rajaie Cardiovascular Medical and Research Center, the largest referral center in Tehran, Iran, were evaluated for heart transplant rejection. Transplant rejection grades, high-sensitivity troponin I plasma levels, and the relationship between them were assessed statistically. Results: Forty-two patients (mean age 37 ± 9.6 years, 26 (62%) male) were assessed. The mean transplant age was 3 ± 1.8 years. In terms of graft rejection, 30 (33%) biopsies showed Grade 1R and 4 (4%) showed Grade 2R. Quilty effect was observed in 43 (47%). The median (Q1–Q3) of the cTnI was 0.02 (0.02–0.75) and 43 (47%) had high troponin level (&gt;0.03 ng/mL). A positive association was observed between high cTnI and rejection (P = 0.025). Conclusion: The association between the troponin level and rejection grade suggested the considerable myocardial injury during the rejection process.

Total Lymphoid Irradiation in the Treatment of Heart Transplant Rejection: Single Institution Long-Term Results

Acute cardiac allograft rejection is a major cause of morbidity and mortality post-transplant. Total lymphoid irradiation (TLI) has been shown to cause long-lasting alterations in lymphocyte sub-sets, and thereby induces a state of partial response that is useful to prevent organ transplant rejection. In our previous report, treatment with TLI resulted in decreased episodes of acute rejection and steroid dosage requirements for up to 9.1 years post cardiac transplantation. In this study, we report an update on the short- and long-term effects of TLI in the treatment of cardiac transplant rejection. We performed a retrospective analysis of patients with intractable cardiac allograft transplantation evaluated in the department of radiation oncology at our institution over 23 years (1998-2021). Cardiac allograft rejection was defined as multiple or persistent episodes of rejection despite aggressive immunosuppressive therapy. The prescribed dose was 8 Gy in 10 fractions to be delivered over 5 weeks. All episodes of pathologic acute cardiac rejection, duration of immunosuppression, and episodes of acute infection requiring treatment before and after TLI were documented. Overall survival (OS) was analyzed using Kaplan-Meier method. The median time to first cardiac allograft rejection and the median time to first acute rejection was monitored. Of the 29 patients included, 21 (72.4%) had ISHLT grade 1R, 5 (17.2%), grade 2R, and 2(6.9%) grade 3R rejection. Rejection grade was unknown for 1 patient. The mean number of acute rejections after transplant was 14.4 episodes per patient (range 1-44) with median time to first rejection of 0.63 months. The median radiation dose was 8 Gy (range 7.2-8 Gy) delivered over a mean of 31 days. Treatment was stopped early in 2 (6.5%) patients (after 7.2 Gy and 7.9 Gy) for severe bone marrow suppression and drop in CD4 counts, respectively. The number of rejections dropped from 14.2 to 3.97 per patient over a median time of 29.0 months vs 28.1 months pre and post TLI (p = 0.01). The frequency of ISHLT acute grade 3R rejections decreased from 0.58 to 0.06 episodes per patient pre and post TLI (p = 0.02). Immunosuppressant requirements decreased from 2.83 before TLI to 2.69 after TLI (p = 0.71). Infection rates per patient were 0.71 pre TLI and remained at 0.63 post TLI. Treatment was well tolerated with 4 (13.8%) grade 2 or higher radiation toxicity. Median follow up was 76.3 months. Median and 3-year OS were 53.4 months and 60.9%, respectively. TLI is a safe and effective treatment for control of intractable cardiac rejection. The episodes of acute rejection and immunosuppressant requirements are decreased, but with long-term follow up, these patients continue to experience rejection. However, TLI lowers the frequency of ISHLT moderate to severe rejection episodes for up to 6.4 years. There was no increased risk of severe infections and there were only minimal short- and long-term grade 2 or higher radiation toxicities.

Relationship between blood and tissue-based rejection-related transcripts in heart transplantation

PurposeThe purpose of the study is to investigate the relationship between blood and tissue-derived rejection-related transcripts from blood gene expression profiling (GEP) and molecular microscope in the setting of allograft rejection in heart transplant. MethodsAll heart transplant patients from August 2021 to May 2022 with both circulating blood GEP (AlloMap; CareDx) and endomyocardial biopsy with molecular microscope diagnostic system (MMDx, One Lambda) within 4 weeks were included (N=173 samples). We obtained individual blood GEP-based mRNA transcript expression levels of the 11 target genes from CareDx. Student’s t-test was performed to compare blood GEP transcript expression levels between no rejection and rejection as assessed by MMDx. A Scatter plot with Spearman correlation analysis was performed to compare the relationship between transcript expression levels from AlloMap and MMDx, with and without allograft rejection. ResultsThere were 52 samples (30.1%) with antibody-mediated rejection (ABMR) and 15 samples (8.7%) with T-cell-mediated rejection (TCMR), as assessed by MMDx. Expression of one of the blood ITGA4 (Integrin alpha 4) expression level was elevated in ABMR, compared to no ABMR (4607.5 vs 4217.5; p=0.019). Most tissue RAT expression levels were elevated in ABMR, and tissue ROBO4 expression correlated with the blood ITGA4 expression with moderate or greater effect size in all samples (Spearman’s R 0.31; p<0.001). There was also a positive correlation between blood ITGA4 and tissue ROBO4 expression in samples without ABMR (Spearman’s R 0.33; p<0.001), but no correlation between blood ITGA4 and tissue ROBO4 expression in samples with ABMR (Spearman’s R=0.009; p=0.513). DiscussionCirculating blood ITGA4 expression is elevated in AMR and correlate with myocardial expression of ROBO4. The knowledge of individual transcript expression levels in blood and in tissue may provide insights into various disease processes in heart transplant patients. Taken together, the results of our study reveal an overlap between two objective post-heart transplant rejection surveillance methods, identify potential novel markers for ABMR, and reveal the need for a deeper understanding of molecular mechanisms underlying allograft rejection.

Reduction of HLA donor specific antibodies in heart transplant patients treated with proteasome inhibitors for antibody mediated rejection.

In this project, we describe proteasome inhibitor (PI) treatment of antibody-mediated rejection (AMR) in heart transplantation (HTX). From January 2018 to September 2021, 10 patients were treated with PI for AMR: carfilzomib (CFZ) n=8; bortezomib (BTZ) n=2. Patients received 1-3 cycles of PI. All patients had ≥1 strong donor-specific antibody (DSA) (mean fluorescence intensity [MFI]>8000) in undiluted serum. Most DSAs (20/21) had HLA class II specificity. The MFI of strong DSAs had a median reduction of 56% (IQR=13%-89%) in undiluted serum and 92% (IQR=53%-95%) at 1:16 dilution. Seventeen DSAs in seven patients were reduced>50% at 1:16 dilution after treatment. Four DSAs from three patients did not respond. DSA with MFI>8000 at 1:16 dilution was less responsive totreatment. 60% (6/10) patients presented with graft dysfunction; 4/6 recovered ejection fraction>40% after treatment. Pathologic AMR was resolved in 5/7 (71.4%) of patients within 1year after treatment. 9/10 (90%) patients survived to 1year after AMR diagnosis. Using PI in AMR resulted in significant DSA reduction with some resolution of graft dysfunction. Larger studies are needed to evaluate PI for AMR.

Single-cell analysis of graft-infiltrating host cells identifies caspase-1 as a potential therapeutic target for heart transplant rejection.

Understanding the cellular mechanisms underlying early allograft rejection is crucial for the development of effective immunosuppressant strategies. This study aims to investigate the cellular composition of graft-infiltrating cells during the early rejection stage at a single-cell level and identify potential therapeutic targets. A heterotopic heart transplant model was established using enhanced green fluorescent protein (eGFP)-expressing mice as recipients of allogeneic or syngeneic grafts. At 3 days post-transplant, eGFP-positive cells infiltrating the grafts were sorted and subjected to single-cell RNA-seq analysis. Potential molecular targets were evaluated by assessing graft survival and functions following administration of various pharmacological inhibitors. A total of 27,053 cells recovered from syngrafts and allografts were classified into 20 clusters based on expression profiles and annotated with a reference dataset. Innate immune cells, including monocytes, macrophages, neutrophils, and dendritic cells, constituted the major infiltrating cell types (>90%) in the grafts. Lymphocytes, fibroblasts, and endothelial cells represented a smaller population. Allografts exhibited significantly increased proportions of monocyte-derived cells involved in antigen processing and presentation, as well as activated lymphocytes, as compared to syngrafts. Differential expression analysis revealed upregulation of interferon activation-related genes in the innate immune cells infiltrating allografts. Pro-inflammatory polarization gene signatures were also enriched in these infiltrating cells of allografts. Gene profiling and intercellular communication analysis identified natural killer cells as the primary source of interferon-γ signaling, activating inflammatory monocytes that displayed strong signals of major histocompatibility complexes and co-stimulatory molecules. The inflammatory response was also associated with promoted T cell proliferation and activation in allografts during the early transplant stages. Notably, caspase-1 exhibited specific upregulation in inflammatory monocytes in response to interferon signaling. The regulon analysis also revealed a significant enrichment of interferon-related motifs within the transcriptional regulatory network of downstream inflammatory genes including caspase-1. Remarkably, pharmacological inhibition of caspase-1 was shown to reduce immune infiltration, prevent acute graft rejection, and improve cardiac contractile function. The single-cell transcriptional profile highlighted the crucial role of caspase-1 in interferon-mediated inflammatory monocytes infiltrating heart transplants, suggesting its potential as a therapeutic target for attenuating rejection.

Digital Pathology for Analyzing Endomyocardial Biopsy Specimens

Introduction: Visual assessment of endomyocardial biopsy (EMB) specimens may result in errors due to the human factor and inability to obtain quantitative data on the intensity of immunohistochemical (IHC) reactions and severity of pathological changes in the heart transplant. Detailed digital characterization of EMB specimens based on automated or semi-automated computer-assisted morphometry improves the diagnostic accuracy of EMB. Objective: To develop a method of and algorithms for digital computer-assisted analysis of EMB specimens to quantify pathomorphological and immunophenotypic changes in the myocardium of patients with various types and grades of heart transplant rejection. Materials and methods: We studied 257 EMB specimens from 56 heart transplant recipients at Scientific Research Institute – Ochapovsky Regional Clinical Hospital No. 1 (Krasnodar, Russian Federation). Sections were stained with hematoxylin and eosin. We used the streptavidin-biotin method to determine the CD68 expression and computer-assisted morphometry of digital images to measure the area of pathological changes and CD68 expression. Results: When developing a method for computer-assisted analysis of EMB specimens, we proposed a new evaluation criterion: staining area coefficient (%) that is a ratio of the total stained area to the total section area. We created and field-tested algorithms for digital morphometric analysis to assess the intensity of IHC reactions and to identify the relative area of pathological changes in the EBM specimens. Conclusions: New digital criteria for diagnosing heart transplant rejections are to improve the accuracy of EMB results interpretation, but can also be used for analyzing other biopsy specimens.