Cardiac Rejection Research Articles

Feasibility of Interleukin-6 Receptor Blockade in Cardiac Antibody-mediated Rejection.

Antibody-mediated rejection (AMR) remains a significant cause of heart transplant mortality with few effective therapies. This study aimed to describe initial experience of using interleukin-6 receptor blockade with tocilizumab in the treatment of acute cardiac AMR at Barnes-Jewish Hospital/Washington University Transplant Center from July 2017 to May 2021 (n = 7). Clinical, echocardiographic, and serum alloantibody data were analyzed before and after treatment. All participants demonstrated marked improvement in functional status. Echocardiographic data following 4-6 mo of tocilizumab revealed significant improvements in biventricular systolic function for all participants. Consistent reductions in donor-specific HLA or angiotensin type I receptor antibodies were not observed, suggesting that tocilizumab may act downstream of antibody production. No patient experienced drug-related complications that necessitated discontinuation of therapy. These findings provide initial insights into the safety and efficacy of interleukin-6 receptor blockade in the treatment of cardiac AMR and support the design of larger prospective studies.

Combining Serum miR-144-3p and miR-652-3p as Potential Biomarkers for the Early Diagnosis and Stratification of Acute Cellular Rejection in Heart Transplantation Patients.

There is a dire need for specific, noninvasive biomarkers that can accurately detect cardiac acute cellular rejection (ACR) early. Previously, we described miR-144-3p as an excellent candidate for detecting grade ≥2R ACR. Now, we investigated the combination of miR-144-3p with miR-652-3p, other differentially expressed serum miRNA we previously described, to improve diagnostic accuracy mainly in mild rejection to avoid reaching severe stages. We selected miR-652-3p from a preliminary RNA-seq study to be validated by reverse transcription-quantitative polymerase chain reaction on 212 consecutive serum samples from transplantation recipients undergoing routine endomyocardial biopsies to subsequently combine them with miR-144-3p results and investigate their diagnostic capability. We confirmed the miR-652-3p overexpression (P < 0.0001) and its capability to discriminate between patients with and without ACR of any grade (P < 0.0001). The combined serum levels of miR-144-3p and miR-652-3p were significantly higher in patients with rejection regardless of posttransplantation time (P < 0.0001). This combination resulted in a diagnostic efficacy for 1R (area under the curve = 0.794) and ≥2R (area under the curve = 0.892; P < 0.0001) that was superior to each biomarker alone. Furthermore, it was a strong independent predictor of ACR for 1R (odds ratio of 10.950; P < 0.0001) and ≥2R (odds ratio of 14.289; P < 0.01). We demonstrated that an appropriate combination of blood-based biomarkers could exhibit greater efficiency for cardiac rejection diagnosis. The combined detection of abnormal expression of miR-144-3p and miR-652-3p in the serum of ACR patients can improve the diagnostic sensitivity of rejection at an early stage and contribute to increasing the diagnostic accuracy, mainly in the lower rejection grades.

Validation of the clinical utility of microRNA as noninvasive biomarkers of cardiac allograft rejection: A prospective longitudinal multicenter study

While studies have shown an association between microRNAs and cardiac rejection, the clinical relevance of a pre-identified miRNA signature as a non-invasive biomarker has never been assessed in prospective multicentric unselected cohorts. To address this unmet need, we designed a prospective study (NCT02672683) including recipients from 11 centers between 08/2016 to 03/2018. The objective was to validate the association between three previously identified circulating microRNA (10a, 92a, 155) and the histopathological diagnosis of rejection. Both relative and absolute (sensitivity analysis) quantifications of microRNAs were performed. Overall, 461 patients were included (831 biopsies, 79 rejections). A per-protocol interim analysis (258 biopsies, 49 rejections) did not find any association between microRNA and rejection (microRNA10-a: OR=1.05, 95%CI=0.87-1.27, p=0.61; 92-a: OR=0.98, 95%CI=0.87-1.10, p=0.68; 155: OR=0.91, 95%CI=0.76-1.10, p=0.33). These results were confirmed in the sensitivity analysis. The analysis of the remaining sera was stopped for futility. This study shows no clinical utility of circulating microRNAs 10a, 92a and 155 monitoring in heart allograft recipients.

Matrix Metalloproteinases and Heart Transplantation-A Pathophysiological and Clinical View.

Heart transplantation is undergoing a continuous development, with rates of success increasing substantially due to advances in immunosuppressive therapy and surgical techniques. The most worrying complication occurring after cardiac transplantation is graft rejection, a phenomenon that is much affected by matrix metalloproteinases (MMPs), with the role of these proteases in the cardiac remodeling process being well established in the literature. A detailed investigation of the association between MMPs and cardiac rejection is necessary for the future development of more targeted therapies in transplanted patients, and to discover prognostic serum and immunohistochemical markers that will lead to more organized therapeutic management in these patients. The aim of this review is therefore to highlight the main MMPs relevant to cardiovascular pathology, with particular emphasis on those involved in complications related to heart transplantation, including cardiac graft rejection.

The results of orthotopic heart transplantation using the bicaval technique

Circulatory failure, which develops in the final stages of the course of the most heart diseases, is a link in the progressive process. This process is accompanied by a significant decrease in the quality of life, as well as high disability and mortality.&#x0D; Heart transplantation increases the survival of patients with chronic heart failure, increases tolerance to physical exertion, improves the quality of life of patients and allows most of them to return to vigorous activity.&#x0D; Aim. The work is devoted to the analysis of the results of orthotopic heart transplantation in patients with a progressive course of chronic heart failure that cannot be treated conservatively.&#x0D; Materials and methods. We analyzed the clinical course of heart transplants in 55 patients. There were 46 (83.6 %) men and 9 (16.4 %) women among the recipients. The average age was 46.0 ± 10.0 years.&#x0D; Results. The result of the conducted retrospective study revealed that in-hospital (early) and one-year (longterm) survival among patients in our study was 92.7 % and 81.8 %, respectively.&#x0D; Conclusions. Heart transplantation is the most effective method of treatment for terminal heart failure. The quality of life of heart transplant patients improves significantly after surgery and is the main factor in the development of heart transplantation nowadays.&#x0D; The main causes of death were acute graft dysfunction, rejection reactions, and coronary heart disease of the transplanted heart. Problems related to cardiac graft dysfunction and rejection in the early and long-term period still need to be definitively resolved.

Reduced incidence of cardiac rejection in multi-organ transplants: A propensity matched study.

Rejection remains a primary cause of graft loss after heart transplant (HT). Recognizing the immunomodulation of multi-organ transplant can enhance our understanding of the mechanisms of cardiac rejection. This retrospective cohort study identified patients from the UNOS database with isolated heart (H, N=37 433), heart-kidney (HKi, N=1516), heart-liver (HLi, N=286), and heart-lung (HLu, N=408) transplants from 2004 to 2019. Propensity score matching reduced baseline differences between groups. Outcomes included risk of rejection prior to transplant hospital discharge and within 1 year, and mortality within 1 year of transplant. In the propensity score matched data, the relative risk of being treated for rejection prior to transplant hospital discharge was 61% lower for HKi (RR .39, 95% CI .29, .53) and 87% lower for HLi (RR .13, 95% CI .05, .37) compared to H. Similarly, the probability of being treated for rejection in the first year after transplant remained lower in HKi (RR .45, 95% CI .35, .57) and HLi (RR .13, 95% CI .06, .28) compared to H. The 1-year survival analysis revealed an equivalent risk of death in HKi (HR .84, 95% CI .68, 1.03) and HLi (HR 1.41, 95% CI .83, 2.41) compared to H, while HLu had a higher risk of death in the first year after transplant (HR 1.65, 95% CI 1.17, 2.33). Recipients of HKi and HLi experience a reduced risk of rejection when compared to H, but an equivalent risk of 1yr mortality. These findings have important implications for the future of HT medicine.

Small-molecule BCL6 inhibitor protects chronic cardiac transplant rejection and inhibits T follicular helper cell expansion and humoral response.

Background: B cell lymphoma 6 (BCL6) is an important transcription factor of T follicular helper (Tfh) cells, which regulate the humoral response by supporting the maturation of germinal center B cells and plasma cells. The aim of this study is to investigate the expansion of T follicular helper cells and the effect of the BCL6 inhibitor FX1 in acute and chronic cardiac transplant rejection models. Methods: A mouse model of acute and chronic cardiac transplant rejection was established. Splenocytes were collected at different time points after transplantation for CXCR5+PD-1+ and CXCR5+BCL6+ Tfh cells detection by flow cytometry (FCM). Next, we treated the cardiac transplant with BCL6 inhibitor FX1 and the survival of grafts was recorded. The hematoxylin and eosin, Elastica van Gieson, and Masson staining of cardiac grafts was performed for the pathological analysis. Furthermore, the proportion and number of CD4+ T cells, effector CD4+ T cells (CD44+CD62L-), proliferating CD4+ T cells (Ki67+), and Tfh cells in the spleen were detected by FCM. The cells related to humoral response (plasma cells, germinal center B cells, IgG1+ B cells) and donor-specific antibody were also detected. Results: We found that the Tfh cells were significantly increased in the recipient mice on day 14 post transplantation. During the acute cardiac transplant rejection, even the BCL6 inhibitor FX1 did not prolong the survival or attenuate the immune response of cardiac graft, the expansion of Tfh cell expansion inhibit. During the chronic cardiac transplant rejection, FX1 prolonged survival of cardiac graft, and prevented occlusion and fibrosis of vascular in cardiac grafts. FX1 also decreased the proportion and number of splenic CD4+ T cells, effector CD4+ T cells, proliferating CD4+ T cells, and Tfh cells in mice with chronic rejection. Moreover, FX1 also inhibited the proportion and number of splenic plasma cells, germinal center B cells, IgG1+ B cells, and the donor-specific antibody in recipient mice. Conclusion: We found BCL6 inhibitor FX1 protects chronic cardiac transplant rejection and inhibits the expansion of Tfh cells and the humoral response, which suggest that BCL6 is a potential therapeutic target of the treatment for chronic cardiac transplant rejection.

Cardiomyocytic cyclic GMP-AMP synthase is critical for the induction of experimental cardiac graft rejection

ObjectiveDuring cardiac transplantation, cellular injury and DNA damage can result in the accumulation of cytosolic double-stranded DNA (dsDNA), which can activate the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) signaling pathway and thus induce multiple proinflammatory responses. However, the role of the cGAS-STING pathway in cardiac transplantation remains unclear. This study explored the role of cardiomyocytic cGAS in mouse heart transplantation during the ischemia/reperfusion and rejection processes. Methods and ResultsCytosolic dsDNA accumulation and cGAS-STING signaling pathway component upregulation were observed in the grafts posttransplantation. The use of cGAS-deficient donor tissues led to significantly prolonged graft survival. The underlying mechanisms involved decreased expression and phosphorylation of downstream proteins, including TANK binding kinase 1 and nuclear factor κB. In parallel, notably diminished expression levels of various proinflammatory cytokines were observed. Accordingly, substantially decreased proportions of macrophages (CD11b+F4/80+) and CD8+ T cells were observed in the spleen. The activation of CD8+ T cells (CD8+CD69+) within the graft and the proportion of effector memory (CD44highCD62Llow) lymphocytes in the spleen were notably decreased. Treatment with the cGAS inhibitor Ru.521 led to significantly prolonged graft survival. ConclusionsCardiomyocytic cGAS plays a critical role by sensing cytosolic dsDNA during cardiac transplantation and could serve as a potential therapeutic target to prevent graft rejection.

The dynamic cellular landscape of grafts with acute rejection after heart transplantation.

Acute cellular rejection (ACR) is a major barrier to the long-term survival of cardiac allografts. Although immune cells are well known to play critical roles in ACR, the dynamic cellular landscape of allografts with ACR remains obscure. Single-cell RNA sequencing (scRNA-seq) was carried out for mouse cardiac allografts with ACR. Bioinformatic analysis was performed, and subsequent transplant experiments were conducted to validate the findings. Despite an overall large depletion of cardiac fibroblasts (CFBs), highly expanded cytotoxic T lymphocytes and a CXCL10+Gbp2+ subcluster of CFBs were enriched within grafts at the late stage. CXCL10+Gbp2+ CFBs featured strong interferon responsiveness and high expression of chemokines and major histocompatibility complex molecules, implying their involvement in the recruitment and activation of immune cells. Cell‒cell communication analysis revealed that CXCL9/CXCL10-CXCR3 might contribute to regulating CXCL10+Gbp2+ CFB-induced chemotaxis and immune cell recruitment. In vivo transplant studies revealed the therapeutic potential of CXCR3 antagonism in transplant rejection. The findings of our study unveiled a novel CFB subcluster that might mediate acute cardiac rejection. Targeting CXCR3 could prolong allograft survival.

Beyond the AJR: Cardiac MRI Accurately Detects Acute Cardiac Transplant Rejection.

Beyond the AJR: Cardiac MRI Accurately Detects Acute Cardiac Transplant RejectionChristopher J. François, MD1Audio Available | Share

Inhibiting NFAT5 With KRN2 Mitigates Acute Allograft Rejection in a Murine Heart Transplantation Model.

Despite advancements in immunosuppressive therapy, acute allograft rejection remains an important challenge for heart transplantation patients. Nuclear factor of activated T-cells 5 (NFAT5), a member of the family of Rel homology domain-containing factors that plays an important role in regulating immune responses of T lymphocytes, may be closely associated with cardiac rejection. KRN2, as a specific inhibitor of NFAT5, is injected intraperitoneally daily starting from day 0 after murine heart transplantation. When compared with saline treatment, KRN2 treatment can improve allograft survival. Histologic examination revealed that the KRN2 treatment group experienced less-severe rejection, and enzyme-linked immunosorbent assay revealed lower levels of inflammatory cytokines in circulating serum. The proportion and number of T-cell subpopulations in the spleens were analyzed by flow cytometry. We found that KRN2 treatment reduced the proportions of CD4 + IFN-γ + , CD4 + IL-17A + , and CD4 + IL-4 + Th cells, whereas increasing CD4 + Foxp3 + Treg cells compared with the control group. These findings suggest that KRN2 attenuates acute allograft rejection by regulating CD4 + T lymphocyte responses. NFAT5 could be a promising therapeutic target for preventing acute allograft rejection.

Abstract 14413: Possible Antibody Mediated Cardiac Rejection After Combined Heart-Kidney Transplant in a Sensitized Patient Associated to COVID-19 Pneumonia

Background: Antibody mediated rejection (AMR) starts with the appearance of donor-specific antibodies (DSA), followed by graft injury that may be subclinical and can progress to cardiac allograft vasculopathy (CAV) and chronic graft dysfunction. Case report A 48-year-old man with a past medical history of right nephrectomy due to congenital renal atrophy and dilated cardiomyopathy required heart transplantation at age 31, presented an episode of AMR grade 2 ten years post-transplant developing CAV with graft loss and end-stage renal disease. He was listed for a combined heart-kidney transplant. His panel reactive antibody (PRA) level was 40%, requiring desensitization therapy (plasmapheresis, intravenous gamma globulin and rituximab). At age 44, he received a heart-kidney transplant. Methylprednisolone and rabbit antithymocyte globulin were used for induction and desensitization therapy with plasmapheresis and intravenous gamma globulin at months 0-3-6-12 was continued. Maintenance immunosuppression therapy continued with tacrolimus, mycophenolic acid and prednisone. During follow-up persistent circulating DSA anti-class II HLA were detected and PRA reached 80%. He was admitted with a multifocal COVID-19 pneumonia. He had received 4 doses of the messenger RNA vaccine BNT162b2. Dexamethasone was started and mycophenolic acid was discontinued for 14 days. He did not need mechanical ventilation. Echocardiogram revealed marked concentric LVH, LVEF = 60%, grade II diastolic dysfunction and reduced global longitudinal strain (-7,6%). Pro-BNP was 962 pg/ml, and Troponin T 100 pg/ml. Endomyocardial biopsy showed no signs of cellular rejection nor capillary injury. Immunopathologic findings were negative. Coronary angiogram showed no significant lesions. AMR was suspected, the patient was treated with methylprednisolone, plasmapheresis and intravenous gamma globulin. Clinical response was satisfactory, and he was discharged in functional class I (NYHA). Conclusions Although both histological and immunopathologic studies were negative, clinical suspicion for AMR in a highly sensitized patient prevailed in the context of a recent infectious intercurrence. Other diagnosis cannot be completely ruled out in this clinical scenario.

Screening for cardiac allograft rejection among heart transplant recipients using an electrocardiogram-based deep learning model

Abstract Background Current approaches utilizing non-invasive methods to screen for cardiac allograft rejection (gene expression profiling and cell free DNA) have yet to be broadly integrated into heart transplant management and have shown limited discrimination (AUCs of 0.68 and 0.77, respectively). Changes in the electrocardiogram (ECG) have been reported at the time of severe cardiac rejection, including low voltages and conduction abnormalities. It remains unknown if subtle ECG changes correlating with cardiac allograft rejection can be detected earlier using machine learning methods. Purpose We sought to develop an artificial intelligence (AI) model to detect cardiac allograft rejection based on the 12 lead ECG. Methods We identified all patients who underwent a heart transplant at 3 hospital sites within a single health system from Jan 1998 through Apr 2021 and extracted digital 12-lead ECG data as well as endomyocardial biopsy pathology results from the electronic medical record. We partitioned our data into a training (80%), validation (10%), and test set (10%) based on a group stratification sampling. Each patient was present in only one set and each set had a positivity rate of 2.6% with 6,074/758/758 ECGs belonging to 1,146/140/141 unique patients in each set respectively. Cardiac allograft rejection was defined as moderate or severe acute cellular rejection based on International Society for Heart and Lung Transplantation (ISHLT) guidelines. A convolutional neural network, using the 12-lead ECG data as input, was trained with hyperparameter optimization for regularization, learning rate adjustments, and class weights. Model performance metrics were based on the test data and estimated using the final model architecture. Results 1,587 heart transplant recipients who had at least one endomyocardial biopsy were evaluated for inclusion. We limited our sample to ECGs performed within 30 days of the biopsy date (7,590 ECGs, representing 1,425 unique patients). Our study population had a median age of 55.8 years and 28.7% were female. The median number of ECG-biopsy pairs per patient was 5. The majority of endomyocardial biopsy results were classified as none or mild rejection (97.1%), and 2.9% had moderate/severe rejection. The ECG-based AI model detected cardiac allograft rejection with an area under the receiver operative curve (AUC) of 0.84 in the test set. The sensitivity, specificity, positive and negative predictive values were 95%, 52.6%. 5.2% and 99.7% respectively. Conclusions An AI-ECG model appears to outperform novel non-invasive laboratory tests (gene expression profiling and cell free DNA) for detecting cardiac allograft rejection and does not require a blood draw or the additional complexities surrounding sample processing. This model relies on a readily available and relatively inexpensive test, the ECG. In addition, AI predictions can be made available within a few minutes following ECG acquisition. Funding Acknowledgement Type of funding sources: Private hospital(s). Main funding source(s): Mayo Clinic

Implication of long non-coding RNAs in the pathophysiology of cardiac rejection and its accuracy in diagnosis

Abstract Background The complementation of the endomyocardial biopsy or even its replacement by the use of a non-invasive method is one of the great challenges in the investigation of cardiac rejection. However, the limitations of the serum markers identified so far limit their use in clinical practice. In recent years, many long non-coding RNAs (lncRNAs) have been identified along with their role in different biological functions. However, the utility of lncRNAs in cardiac rejection has not been explored. Purpose We aimed to determine the expression of lncRNAs in serum of patient with cardiac rejection and evaluate the diagnostic capacity as potential biomarkers for the non-invasive detection of mild (grade 1R) or moderate/severe (grade ≥2R) cellular rejection. Methods RNA sequencing analysis (Illumina HiSeq 2500) was performed on forty serum samples from transplant recipients undergoing routine endomyocardial biopsies. Twenty eight samples were diagnosed with RCA (RCA Grade 1R, n=16; and RCA Grade ≥2R, n=12) and 12 samples had no diagnosed rejection. Results Of the 10,144 lncRNAs detected in serum, 5.1% presented differential expression in patients with cardiac rejection of Grade ≥2R. Fifty-six molecules showed excellent diagnostic ability in moderate-severe rejection (AUC &amp;gt;0.900) and fifty-one molecule were even able to detect mild rejection with good diagnostic ability for moderate-severe rejection (AUC &amp;gt;0.800). Three lncRNAs stand out: EGFR-AS1 (≥2R, AUC=0.871, p&amp;lt;0.01; 1R, AUC=0.921, p&amp;lt;0.0001), AC008649.2 (≥2R, AUC=0.867, p&amp;lt;0.01; 1R, AUC=0.807, p&amp;lt;0.01) and AP001652.1 (≥2R, AUC=0.848, p&amp;lt;0.01; 1R, AUC=0.855, p&amp;lt;0.01). Conclusions A large number of lncRNAs with excellent diagnostic capacity for acute cellular rejection have been identified. In addition, we highlight 3 long non-coding (EGFR-AS1, AC008649.2 and AP001652.1) that maintain a good diagnostic capacity even in the mildest degrees. Therefore, we propose these non-coding RNAs as potential biomarkers of cardiac rejection. Although the function of these non-coding RNAs in acute cellular rejection is unknown, it is important to take into account in future studies the pathways in which they participate in order to elucidate the mechanisms involved in the development of this pathology. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)

Non-invasive diagnosis of cardiac rejection using small non-coding RNAs

Abstract Background Endomyocardial biopsy is the standard technique for rejection surveillance in heart transplantation recipients and is an invasive method with important limitations and clearly associated with both a risk of procedural complications and serious long-term sequelae when performed repeatedly. All of these considerations highlight the need to identify non-invasive alternative methods capable of discriminating between the different grades of rejection and reducing the need for endomyocardial biopsy. Purpose We conducted a non-targeted transcriptomic study focused on identifying serum small non-coding RNAs, mainly aimed on the less studied types, to evaluate their diagnostic accuracy for detecting rejection episodes. Methods We included consecutive serum samples from transplant recipients undergoing routine endomyocardial biopsies. Non-coding RNA sequencing analysis (Illumina HiSeq 2500) was performed on 40 samples, 28 diagnosed with RCA (RCA Grade 1R, n=16; and RCA Grade ≥2R, n=12) and 12 samples without cardiac rejection. Results 636 non-coding RNAs were detected among the scaRNAs, snoRNAs, snRNAs and miscRNAs biotypes. But, neither piRNA nor siRNA biotypes were detected. Of all of them, 41 were altered in patients with rejection Grade ≥2R, finding no alteration in the scaRNAs biotype. Three molecules of miscRNA biotype showed the best results with excellent diagnostic capacity in moderate/severe rejection: RN7SL840P (AUC=0.938, p&amp;lt;0.01), Y_RNA [ENSG00000223302] (AUC=0.931, p&amp;lt;0.0001) and Y_RNA [ENSG00000202273] (AUC=0.903, p&amp;lt;0.001). In addition, RN7SKP5 showed a good capacity for the diagnosis of Grade ≥2R (AUC=0.806, p&amp;lt;0.05) and preserving the accuracy at Grade 1R (AUC=0.781, p&amp;lt;0.05). Conclusions The 6.5% of the small non-coding RNAs detected have differential expression in patients with acute cellular rejection. The non-detection of the piRNAs and siRNAs biotypes stands out, as well as the absence of alteration in the scaRNAs biotype. We propose serum miscRNA levels as potential biomarkers of cardiac rejection. We highlight the role of RN7SL840P due to its excellent diagnostic capacity for RCA, and RN7SKP5 since it also allows the detection of mild rejection. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)

Combining serum miR-144-3p and miR-652-3p as potential biomarkers for the early diagnosis and stratification of acute cellular rejection in heart transplantation patients

Abstract Background There is a dire need for specific, noninvasive biomarkers that can accurately and early detect cardiac acute cellular rejection (ACR). Previously, we described miR-144-3p as an excellent candidate to detect Grade ≥2R ACR. Purpose Now, we investigated the combination of miR-144-3p with other differentially expressed serum miRNA that we previously described to improve diagnostic accuracy, mainly in mild rejection to avoid reaching severe stages. Methods We selected miR-652-3p, miR-6747-3p and miR-221-3p from a preliminary RNA-seq study to be validated by RT-qPCR on 212 consecutive serum samples from transplantation recipients undergoing routine endomyocardial biopsies to subsequently combine them with miR-144-3p results and investigate their diagnostic capability. Results We confirmed the miR-652-3p overexpression (p&amp;lt;0.0001) and its capability to discriminate between patients with and without ACR of any grade (p&amp;lt;0.0001), but not miR-6747-3p or miR-221-3p. The combined serum levels of miR-144-3p and miR-652-3p were significantly higher in patients with rejection regardless of posttransplantation time (p&amp;lt;0.0001). This combination resulted in a diagnostic efficacy for 1R (AUC=0.794) and ≥2R (AUC=0.892) (p&amp;lt;0.0001) that was superior to each biomarker alone. Furthermore, it was a strong independent predictor of ACR for 1R (odds ratio of 10.950, p&amp;lt;0.0001) and ≥2R (odds ratio of 14.289, p&amp;lt;0.01). Conclusions We demonstrated that an appropriate combination of blood-based biomarkers can exhibit greater efficiency for cardiac rejection diagnosis. The combined detection of abnormal expression of miR-144-3p and miR-652-3p in the serum of ACR patients can improve the diagnostic sensitivity of rejection at the early stage and contribute to increasing the diagnostic accuracy, mainly in the lower rejection grades. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)

Insights from DOCK2 in cell function and pathophysiology.

Dedicator of cytokinesis 2 (DOCK2) can activate the downstream small G protein Rac and regulate cytoskeletal reorganization. DOCK2 is essential for critical physiological processes such as migration, activation, proliferation, and effects of immune cells, including lymphocytes, neutrophils, macrophages, and dendritic cells. For example, DOCK2 is involved in the development and activation of T and B lymphocytes by affecting synapse formation and inhibiting the development of the Th2 lineage by downregulating IL-4Rα surface expression. Not only that, DOCK2 may be a molecular target for controlling cardiac transplant rejection and Alzheimer’s disease (AD). Patients with defects in the DOCK2 gene also exhibit a variety of impaired cellular functions, such as chemotactic responses of lymphocytes and reactive oxygen species (ROS) production by neutrophils. To date, DOCK2 has been shown to be involved in the development of various diseases, including AD, pneumonia, myocarditis, colitis, tumors, etc. DOCK2 plays different roles in these diseases and the degree of inflammatory response has a different impact on the progression of disease. In this paper, we present a review of recent advances in the function of DOCK2 in various immune cells and its role in various diseases.

A Review of Biomarkers of Cardiac Allograft Rejection: Toward an Integrated Diagnosis of Rejection.

Despite major advances in immunosuppression, allograft rejection remains an important complication after heart transplantation, and it is associated with increased morbidity and mortality. The gold standard invasive strategy to monitor and diagnose cardiac allograft rejection, based on the pathologic evaluation of endomyocardial biopsies, suffers from many limitations including the low prevalence of rejection, sample bias, high inter-observer variability, and international working formulations based on arbitrary cut-offs that simplify the landscape of rejection. The development of innovative diagnostic and prognostic strategies—integrating conventional histology, molecular profiling of allograft biopsy, and the discovery of new tissue or circulating biomarkers—is one of the major challenges of translational medicine in solid organ transplantation, and particularly in heart transplantation. Major advances in the field of biomarkers of rejection have paved the way for a paradigm shift in the monitoring and diagnosis of cardiac allograft rejection. We review the recent developments in the field, including non-invasive biomarkers to minimize the number of protocol endomyocardial biopsies and tissue biomarkers as companion tools of pathology to refine the diagnosis of cardiac rejection. Finally, we discuss the potential role of these biomarkers to provide an integrated bio-histomolecular diagnosis of cardiac allograft rejection.

Effect of cardiac graft rejection on semilunar valve function: implications for heart valve transplantation.

The treatment of neonates with unrepairable heart valve dysfunction remains an unsolved problem because there are no growing heart valve replacements. Heart valve transplantation is a potential approach to deliver growing heart valve replacements. Therefore, we retrospectively analysed the semilunar valve function of orthotopic heart transplants during rejection episodes. We included children who underwent orthotopic heart transplantation at our institution and experienced at least one episode of rejection between 1/1/2010 and 1/1/2020. Semilunar valve function was analysed using echocardiography at baseline, during rejection and approximately 3 months after rejection. Included were a total of 31 episodes of rejection. All patients had either no (27) or trivial (4) aortic insufficiency prior to rejection. One patient developed mild aortic insufficiency during a rejection episode (P = 0.73), and all patients had either no (21) or trivial (7) aortic insufficiency at follow-up (P = 0.40). All patients had mild or less pulmonary insufficiency prior to rejection, which did not significantly change during (P = 0.40) or following rejection (P = 0.35). Similarly, compared to maximum pressure gradients across the valves at baseline, which were trivial, there was no appreciable change in the gradient across the aortic valve during (P = 0.50) or following rejection (P = 0.42), nor was there any meaningful change in the gradient across the pulmonary valve during (P = 0.55) or following rejection (P = 0.91). This study demonstrated that there was no echocardiographic evidence of change in semilunar valve function during episodes of rejection in patient with heart transplants. These findings indicate that heart valve transplants require lower levels of immune suppression than orthotopic heart transplants and provide partial foundational evidence to justify future research that will determine whether heart valve transplantation may deliver growing heart valve replacements for children.

ZBTB20 regulates cardiac allograft rejection through NFкB-mediated inflammation in mouse heart transplantation

Allograft rejection is a major obstacle for the long-term survival of heart transplantation (Htx) patients. The cardiac allograft rejection requires the activation of macrophages and effector T cells. In this study, we explored the role of zinc-finger and BTB domain containing protein 20 (ZBTB20) in the regulation of heart allograft rejection. Flow cytometry analysis of the spleen cells from mice undergoing an acute cardiac rejection revealed that the ZBTB20 protein expression was upregulated in both T and B cells(n = 4，P < 0.01). In addition, ZBTB20 gene knockdown significantly prolonged the survival of heart allografts in mice(n = 4，P < 0.01). Lack of ZBTB20 increased the expression of Foxp3 and limited the response of T helper 1 (Th1) cells(n = 4，P < 0.01). The ZBTB20-related regulation occurred through the activation of the NFкB pathway. In conclusion, our data suggest that ZBTB20 is involved in the regulation of T cells involved in acute heart allograft rejection. Hence, downregulation of ZBTB20 expression may inhibit T cells to prolong heart transplant survival.