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Background: Identification of heart transplant (HTx) rejection currently relies on immunohistology and immunohistochemistry. We aimed to identify specific sets of microRNAs (miRNAs) to characterize acute cellular rejection (ACR), antibody-mediated rejection (pAMR), and mixed rejection (MR) in monitoring formalin-fixed paraffin-embedded (FFPE) endomyocardial biopsies (EMBs) in HTx patients., Methods: In this study we selected 33 adult HTx patients. For each, we chose the first positive EMB for study of each type of rejection. The next-generation sequencing (NGS) IonProton technique and reverse transcript quantitative polymerase chain reaction (RT-qPCR) analysis were performed on FFPE EMBs. Using logistic regression analysis we created unique miRNA signatures as predictive models of each rejection. In situ PCR was carried out on the same EMBs., Results: We obtained >2,257 mature miRNAs from all the EMBs. The 3 types of rejection showed a different miRNA profile for each group. The logistic regression model formed by miRNAs 208a, 126-5p, and 135a-5p identified MR; that formed by miRNAs 27b-3p, 29b-3p, and 199a-3p identified ACR; and that formed by miRNAs 208a, 29b-3p, 135a-5p, and 144-3p identified pAMR. The expression of miRNAs on tissue, through in situ PCR, showed different expressions of the same miRNA in different rejections. miRNA 126-5p was expressed in endothelial cells in ACR but in cardiomyocytes in pAMR. In ACR, miRNA 29b-3p was significantly overexpressed and detected in fibroblasts, whereas in pAMR it was underexpressed and detected only in cardiomyocytes., Conclusions: miRNA profiling on FFPE EMBs differentiates the 3 types of rejection. Localization of expression of miRNAs on tissue showed different expression of the same miRNA for different cells, suggesting different roles of the same miRNA in different rejections., (Copyright © 2018 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

This multicenter case-controlled pilot study evaluated myocardial inflammatory burden (IB) and phenotype in endomyocardial biopsies (EMBs) with and without pathologic antibody-mediated rejection (pAMR). Sixty-five EMBs from five European heart transplant centers were centrally reviewed as positive (grade 2, n = 28), suspicious (grade 1, n = 7) or negative (n = 30) for pAMR. Absolute counts of total, intravascular (IV) and extravascular (EV) immunophenotyped mononuclear cells were correlated with pAMR grade, capillary C4d deposition, donor specific antibody (DSA) status and acute cellular rejection (ACR). In pAMR+ biopsies, equivalent number of IV CD3+ T lymphocytes (23 ± 4/0.225 mm(2) ) and CD68+ macrophages (21 ± 4/0.225 mm(2) ) were seen. IB and cell phenotype correlated with pAMR grade, C4d positivity and DSA positivity (p < 0.0001). High numbers of IV T lymphocytes were associated with low grade ACR (p = 0.002). In late-occurring AMR EV plasma cells occurring in 34% of pAMR+ EMBs were associated with higher IB. The IB in AMR correlated with pAMR+, C4d positivity and DSA positivity. In pAMR+ equivalent numbers of IV T lymphocytes and macrophages were found. The presence of plasma cells was associated with a higher IB and occurrence of pAMR late after transplantation., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Among the various complications of heart transplantation (HTx), the vasculopathy of the allograft (CAV), a phenomenon of chronic rejection, is still a serious problem. Recently, the literature has shown that low testosterone levels in men are associated with cardiovascular disease. In this study, we evaluated the influence of testosterone plasma levels on CAV development., Methods: We studied, with a prospective observational study, all consecutive male HTx patients evaluated from May 2010 to June 2011 at our center. All subjects underwent accurate medical history collection, physical examination, biochemical blood tests, hormone levels, transthoracic Doppler echocardiography, coronary flow velocity reserve assessment, and coronary angiogram., Results: HTx subjects with CAV had significant lower total testosterone plasma levels (12.9±3.9 vs. 15.8±5.8 nmol/L), free testosterone (0.26±0.07 vs. 0.31±0.08 nmol/L), and coronary flow velocity reserve (2.35±0.60 vs. 2.81±0.78 s) with respect to No-CAV patients. Considering the patients as a whole group, a significant negative relation was found between free and total testosterone plasma levels and some cardiovascular risk factors (cholesterol and fasting blood glucose). A significant linear inverse relation was found between total and free testosterone plasma levels and CAV grading. Only free testosterone plasma levels were independent predictors for CAV., Conclusions: We showed for the first time the influence of testosterone plasma levels on CAV development: indirectly increasing traditional risk factors and directly with a probable influence on alloimmune response.

Background: The aim of our study was to evaluate the role of intravascular macrophages in the diagnosis of early and late antibody-mediated rejection (AMR) on endomyocardial biopsies (EMBs)., Methods: We reviewed 1,420 consecutive EMBs from 131 patients and selected 75 C4d+ EMBs. The C4d+ group was compared with a control group (66 patients) matched for age, gender, date of transplantation, follow-up, immunosuppressive regimen and primary heart disease. A total of 141 EMBs were evaluated. Immunoperoxidase staining for C4d and CD68 were performed. Post-transplant IgG anti-HLA reactivity was investigated by Luminex technology. Clinical data were also collected. Fourteen EMBs were available from 11 symptomatic AMR patients., Results: Of the 141 EMBs evaluated, 53 were positive for intravascular macrophages (CD68); among them, 32 were also positive for C4d (32 of 53, 60.4%). Of the 88 CD68- EMBs, 43 were also C4d+ (43 of 88, 48.9%). Of the 53 CD68+ EMBs, 30 EMBs were within the first year since transplantation (30 of 53, 57.8%), and among these 21 were also positive for C4d (21 of 30, 70.0%). In the late period, among the 23 CD68+ EMBs (23 of 53, 42.2%) 11 were also positive for C4d (11 of 23, 47.8%). In the early period, intravascular macrophages were more common in symptomatic (3 of 3, 100%) than asymptomatic (3 of 11, 27.3%) patients. Sensitivity and specificity of intravascular macrophages in predicting donor-specific antibodies (DSA) within the first year were 50.0% and 100.0%, respectively., Conclusions: Intravascular macrophages predict C4d, DSA and symptoms early after transplantation; however, in the late period, they are unable to identify patients with circulating DSA, C4d and/or symptoms., (Copyright © 2013 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

We report the case of a 68-year-old woman who underwent heart transplantation for hypertrophic cardiomyopathy. Two months after the transplant she developed mild fever and dyspnea with a marked drop in left ventricle ejection fraction of 31%. Coronary angiography was negative for cardiac allograft vasculopathy. Endomyocardial biopsy revealed ischemic damage with no evidence of acute cellular rejection, antibody-mediated rejection or viral myocarditis. A neoplastic process was suspected even though full-body computerized tomography was negative for malignancy. The patient died 4 months after transplantation. The autopsy showed acute antero-septal myocardial infarction due to a nodular epicardial EBV-related posttransplant lymphoproliferative disorder (PTLD) infiltrating the left anterior descending coronary artery with occlusive neoplastic thrombosis. We highlight two major aspects of this case: (1) the unusual occurrence of early PTLD involving the cardiac allograft and causing a fatal outcome, (2) the application of an immunological technique for HLA-DRB1 typing to posttransplant paraffin-embedded autopsy material to identify the recipient origin of this early malignancy, thus excluding a possible donor-transmitted neoplasm., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: The present study evaluated if morphologic parameters detect signs of early sub-clinical or latent stages of antibody-mediated rejection (AMR) and their correlation with C4d staining in cardiac transplants recipients., Methods: The study reviewed 1,270 endomyocardial biopsies (EMB) from 131 patients. Of these, 61 stained positive for C4d in the absence of acute cellular rejection >2R. Sixty-six EMB specimens negative for C4d were matched for pre-transplant diagnosis, time after transplantation, age, and acute cellular rejection (ACR) grading. Histopathologic evaluation and C4d staining were performed on formalin-fixed, paraffin-embedded sections using the C4d polyclonal antibody., Results: Of the 8 histologic characteristics evaluated, only endothelial swelling (78.7% sensitivity, 28.8% specificity; positive likelihood ratio, 1.10) and interstitial edema (77% sensitivity, 31.8% specificity; positive likelihood ratio, 1.13) could be considered fair predictors of C4d capillary positivity. The presence of mononuclear cells in capillaries in relation to C4d positivity showed 39.3% sensitivity and 68.2% specificity. Combining the parameters endothelial swelling and mononuclear cells in capillaries, sensitivity was 31.1% (95% confidence interval [CI] 19.9-44.3) and specificity was 71.2% (95 CI, 58.8-81.7), with a positive likelihood ratio of 1.08 (95% CI, 0.68-1.84)., Conclusions: Our results showed that histologic parameters did not always detect signs of early sub-clinical or latent stages of AMR. Combining the parameters of endothelial swelling and intracapillary mononuclear cells did not significantly improve the sensitivity or specificity. Screening recommendations should, therefore, be modified to include more sensitive tests such as C4d staining in the routine protocol to improve patient risk stratification., (Copyright © 2011 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Humoral rejection in heart transplantation is associated with graft dysfunction, circulating anti-donor antibodies and C4d deposits in endomyocardial biopsies. Detecting C4d capillary positivity is of diagnostic and prognostic value. C4d positivity can be found in solid-organ transplants in cases of "accommodation," a form of humoral rejection without graft dysfunction. Accommodation might reflect a change in antibodies or in the antigen, or the graft acquiring a resistance to injury by antibodies and complement. We present a case of accommodation in the setting of adult ABO-compatible orthotopic heart transplantation, which was diagnosed according to the recently introduced ISHLT criteria for humoral rejection: despite this immunologic profile, the patient never showed signs of graft dysfunction. Physicians should be aware of the accommodation phenomenon so they can identify this subset of patients and assess its long-term effects on chronic rejection and outcome in transplanted patients.

Due to the shortage of organ donors, heart transplantation cannot be offered to many infants with end-stage heart failure; this issue leads to mortality rates of 30-50% in patients in the paediatric age group awaiting operation. ABO-incompatible heart transplantation has been performed safely with no particular or invasive preparatory procedures other than plasma exchange during cardiopulmonary bypass for removing preformed antibodies, with no reports of hyperacute rejection. We report our first clinical experience of heart transplantation on a 2-month-old-infant (blood group O), diagnosed with intracardiac tumour, in which the donor was a 19-day-old newborn of blood group A. Sharing the know-how about ABO-incompatible heart transplantation in newborns and infants awaiting transplantation will help in decreasing mortality among this group of patients.

Abnormally high myocardial performance index (MPI) is a Doppler-derived marker of combined systolic and diastolic left ventricular (LV) dysfunction. To identify early stage allograft dysfunction by MPI, we studied 154 long-term heart transplantation (HT) recipients (131 male, aged 51 +/- 13 years at HT, mean follow up 8.4 +/- 3.5 years), with normal left ventricular ejection fraction (LVEF) and free from acute rejection (AR), and 25 normals (13 male, aged 39 +/- 16 years). Rejection score (RS) on endomyocardial biopsy was calculated in the first year. MPI was prolonged (0.45 +/- 0.18 vs. 0.28 +/- 0.10, P = 0.0001) in patients and directly related with mean time from HT (P = 0.001), higher cumulative dosages of cyclosporine at 3 months (P = 0.01), 6 months (P = 0.03), 1 year (P = 0.02), 3 years (P = 0.04) and with cumulative dosage of methylprednisolone at 1 year (P = 0.002). The index was inversely related with mean age at HT (P = 0.002) and tended to be directly related with RS at 1 year (P = 0.05). Thus, MPI is abnormal in long-term HT recipients with normal LVEF. Its direct relation with time from HT as well as immunosuppressive load suggests an early stage of graft dysfunction because of chronic rejection. Extended prospective studies are warranted to clarify its potential role as a negative prognostic marker in HT.

We studied 361 patients, to evaluate risk factors for cardiac allograft vasculopathy (CAV) onset and severity/diffusion in heart transplantation (HT). Rejection scores (RS) on endomyocardial biopsy were calculated (first year and whole follow-up). CAV onset was defined as any lesion seen at yearly angiography. A CAV severity/diffusion index was calculated for each patient summing up the scores of all lesions. Cox multivariate analysis included: donor age, sex, and weight; recipient sex, age, pre-HT diagnosis, hypertension, diabetes and hyperlipidemia post-HT; number of treated rejections and RS; and immunosuppressive dosage at 3, 6, and 12 months. CAV frequency was 2% at 1 year, 22% at 5 and 39% at 10 years. Risk factors for CAV onset were older donor age [p < 0.0001, relative risk (RR) = 9.9], male donor (p < 0.001, RR = 3.2), high RS for severe (> or = 3A) grades (p < 0.02, RR = 2.01), high cyclosporine at 3 months (p < 0.02, RR = 1.9). Risk factors for CAV severity/diffusion were higher donor weight (p < 0.01, RR = 7.5), high prednisone dosage at 1 year (p < 0.0001, RR = 21.1), and coronary disease pre-HT (p < 0.002, RR = 9.7). High RS was an independent predictor for CAV onset, not severity/diffusion. This suggests an immune basis for CAV onset and nonimmune modulation for progression. High RS for severe grades may provide a predictor for patients at risk.

Unlabelled: Photopheresis (ECP) is a new immunomodulatory therapy in which recipient lymphocytes are treated extracorporeally with 8-methoxypsoralen and ultraviolet light. The treatment seems to induce an inhibition of both humoral and cellular rejection after transplantation., Objective: Since recurrent rejection (RR) continues to be a severe complication after heart transplantation (HTx) and the immunosuppressive regimes used for the treatment are often associated with increased morbidity and mortality, we investigated whether ECP could have a beneficial effect on the number and severity of rejection episodes., Methods: Eleven HTX recipients (5 M and 6 F, mean age 48.5 yrs) with RR were enrolled in the study. ECP was performed at weekly intervals during the 1st month, at 2 week intervals during the 2nd and 3rd month, and then monthly for another 3 months., Results: The fraction of biopsies (EMB) with a grade 0/1A rejection increased during ECP from 46% to 72% while the EMB showing a 3A/3B rejection decreased from 42% to 18%. It is also noteworthy that out of the 78 EMB performed during ECP only one showed a 3B rejection in comparison with 13 out of 110 EMB in the pre-ECP period. Six rejection relapses were observed in a total follow-up of 60 months, two of them occurring during the tapering of oral steroid. Four relapses were reversed by ECP, one by i.v. steroids and the last by methotrexate after the failure of both i.v. steroids and ECP. The mean doses of immunosuppressive drugs resulted lower after 6 months of ECP: steroids were reduced from 13 to 8.25 mg/day, cyclosporine from 375 to 285 mg/day, azathioprine from 55 to 35 mg/day., Conclusions: ECP is a well tolerated treatment. Its administration allows better RR control and significant reduction in immunosuppressive therapy.