Validation of a multiomic model of plasma extracellular vesicle PD-L1 and radiomics for prediction of response to immunotherapy in NSCLC.

BACKGROUND: Immune-checkpoint inhibitors (ICIs) have showed unprecedent efficacy in the treatment of patients with advanced non-small cell lung cancer (NSCLC). However, not all patients manifest clinical benefit due to the lack of reliable predictive biomarkers. We showed preliminary data on the predictive role of the combination of radiomics and plasma extracellular vesicle (EV) PD-L1 to predict durable response to ICIs. MAIN BODY: Here, we validated this model in a prospective cohort of patients receiving ICIs plus chemotherapy and compared it with patients undergoing chemotherapy alone. This multiparametric model showed high sensitivity and specificity at identifying non-responders to ICIs and outperformed tissue PD-L1, being directly correlated with tumor change. SHORT CONCLUSION: These findings indicate that the combination of radiomics and EV PD-L1 dynamics is a minimally invasive and promising biomarker for the stratification of patients to receive ICIs.

Baseline extracellular vesicle miRNA-30c and autophagic CTCs predict chemoradiotherapy resistance and outcomes in patients with lung cancer.

Concurrent chemoradiotherapy (cCRT) is the mainstay of treatment for patients diagnosed with locally advanced non-small cell lung cancer (NSCLC). One significant challenge in the effectiveness of this therapy is the potential development of resistance mechanisms, where autophagy up-regulation has been proposed as a key contributing factor. However, there is a lack of reliable biomarkers to predict outcomes on these patients. Interestingly, for addressing this gap, extracellular vesicles (EVs) and circulating tumor cells (CTCs) have emerged as potential sources of such biomarkers. In this study, we investigated EV-associated miRNAs and presence of autophagic CTCs in prospectively collected serial samples from 38 patients with stage III NSCLC undergoing cCRT. Our findings revealed that non-responders exhibited low levels of baseline EV miR-375, miR-200c, and miR-30c. In particular, EV miR-30c showed high predictive value with an area under the curve of 87.2%. Low EV miR-30c and the presence of autophagic-activated CTCs emerged as independent predictive biomarkers for shorter relapse-free survival and overall survival. Furthermore, in experimental models simulating the effects of chemo- and radiotherapy, the administration of miR-30c, either through direct transfection or encapsulation into human EVs, led to the inhibition of autophagy in these cells. This is the first report demonstrating that EV miR-30c inhibits tumor autophagy and its quantification, together with autophagic-activated CTCs, could be used as biomarkers for the stratification and monitoring of patients with NSCLC undergoing cCRT, and they may hold promising potential for guiding subsequent consolidation treatment with immunotherapy or other novel therapies based on autophagy inhibitors.

Augmentation of Histone Deacetylase 6 Activity Impairs Mitochondrial Respiratory Complex I in Ischemic/Reperfused Diabetic Hearts.

BACKGROUND: Diabetes augments activity of histone deacetylase 6 (HDAC6) and generation of tumor necrosis factor α (TNFα) and impairs the physiological function of mitochondrial complex I (mCI) which oxidizes reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide to sustain the tricarboxylic acid cycle and ß-oxidation. Here we examined how HDAC6 regulates TNFα production, mCI activity, mitochondrial morphology and NADH levels, and cardiac function in ischemic/reperfused diabetic hearts. METHODS: HDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent myocardial ischemia/reperfusion injury in vivo or ex vivo in a Langendorff-perfused system. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. We compared the activities of HDAC6 and mCI, TNFα and mitochondrial NADH levels, mitochondrial morphology, myocardial infarct size, and cardiac function between groups. RESULTS: Myocardial ischemia/reperfusion injury and diabetes synergistically augmented myocardial HDCA6 activity, myocardial TNFα levels, and mitochondrial fission and inhibited mCI activity. Interestingly, neutralization of TNFα with an anti-TNFα monoclonal antibody augmented myocardial mCI activity. Importantly, genetic disruption or inhibition of HDAC6 with tubastatin A decreased TNFα levels, mitochondrial fission, and myocardial mitochondrial NADH levels in ischemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and ameliorated cardiac dysfunction. In H9c2 cardiomyocytes cultured in high glucose, hypoxia/reoxygenation augmented HDAC6 activity and TNFα levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown. CONCLUSIONS: Augmenting HDAC6 activity inhibits mCI activity by increasing TNFα levels in ischemic/reperfused diabetic hearts. The HDAC6 inhibitor, tubastatin A, has high therapeutic potential for acute myocardial infarction in diabetes.

Exosomes mediated fibrogenesis in dilated cardiomyopathy through a MicroRNA pathway.

Cardiac fibrosis is a hallmark in late-stage familial dilated cardiomyopathy (DCM) patients, although the underlying mechanism remains elusive. Cardiac exosomes (Exos) have been reported relating to fibrosis in ischemic cardiomyopathy. Thus, we investigated whether Exos secreted from the familial DCM cardiomyocytes could promote fibrogenesis. Using human iPSCs differentiated cardiomyocytes we isolated Exos of angiotensin II stimulation conditioned media from either DCM or control (CTL) cardiomyocytes. Of interest, cultured cardiac fibroblasts had increased fibrogenesis following exposure to DCM-Exos rather than CTL-Exos. Meanwhile, injecting DCM-Exos into mouse hearts enhanced cardiac fibrosis and impaired cardiac function. Mechanistically, we identified the upregulation of miRNA-218-5p in the DCM-Exos as a critical contributor to fibrogenesis. MiRNA-218-5p activated TGF-ß signaling via suppression of TNFAIP3, a master inflammation inhibitor. In conclusion, our results illustrate a profibrotic effect of cardiomyocytes-derived Exos that highlights an additional pathogenesis pathway for cardiac fibrosis in DCM.

Baseline extracellular vesicle TGF-ß is a predictive biomarker for response to immune checkpoint inhibitors and survival in non-small cell lung cancer.

BACKGROUND: Immune-checkpoint inhibitors (ICIs) are an effective therapeutic strategy, improving the survival of patients with lung cancer compared with conventional treatments. However, novel predictive biomarkers are needed to stratify which patients derive clinical benefit because the currently used and highly heterogenic histological PD-L1 has shown low accuracy. Liquid biopsy is the analysis of biomarkers in body fluids and represents a minimally invasive tool that can be used to monitor tumor evolution and treatment effects, potentially reducing biases associated with tumor heterogeneity associated with tissue biopsies. In this context, cytokines, such as transforming growth factor-ß (TGF-ß), can be found free in circulation in the blood and packaged into extracellular vesicles (EVs), which have a specific delivery tropism and can affect in tumor/immune system interaction. TGF-ß is an immunosuppressive cytokine that plays a crucial role in tumor immune escape, treatment resistance, and metastasis. Thus, we aimed to evaluate the predictive value of circulating and EV TGF-ß in patients with non-small-cell lung cancer receiving ICIs. METHODS: Plasma samples were collected in 33 patients with advanced non-small-cell lung cancer before and during treatment with ICIs. EV were isolated from plasma by serial ultracentrifugation methods and circulating and EV TGF-ß expression levels were evaluated by enzyme-linked immunosorbent assay. RESULTS: Baseline high expression of TGF-ß in EVs was associated with nonresponse to ICIs as well as shorter progression-free survival and overall survival, outperforming circulating TGF-ß levels and tissue PD-L1 as a predictive biomarker. CONCLUSION: If validated, EV TGF-ß could be used to improve patient stratification, increasing the effectiveness of treatment with ICIs and potentially informing combinatory treatments with TGF-ß blockade. PLAIN LANGUAGE SUMMARY: Treatment with immune-checkpoint inhibitors (ICIs) has improved the survival of some patients with lung cancer. However, the majority of patients do not benefit from this treatment, making it essential to develop more reliable biomarkers to identify patients most likely to benefit. In this pilot study, the expression of transforming growth factor-ß (TGF-ß) in blood circulation and in extracellular vesicles was analyzed. The levels of extracellular vesicle TGF-ß before treatment were able to determine which patients would benefit from treatment with ICIs and have a longer survival with higher accuracy than circulating TGF-ß and tissue PD-L1, which is the currently used biomarker in clinical practice.

Comparative efficacy and mechanism of action of cardiac progenitor cells after cardiac injury.

Successful cell therapy requires cells to resist the hostile ischemic myocardium, be retained to continue secreting cardioprotective growth factors/exosomes, and resist immunological host responses. Clinically relevant stem/progenitor cells in a rodent model of acute myocardial infarction (MI) demonstrated that neonatal cardiac mesenchymal stromal cells (nMSCs) provide the most robust cardiac functional recovery. Transplanted nMSCs significantly increased the number of tissue reparative macrophages and regulatory T-cells and decreased monocyte-derived inflammatory macrophages and neutrophils in the host myocardium. mRNA microarray and single-cell analyses combined with targeted depletion studies established CD47 in nMSCs as a key molecule responsible for cell retention in the myocardium through an antiphagocytic mechanism regulated by miR34a-5p. Gain and loss-of-function studies demonstrated that miR34a-5p also regulated the production of exosomes and cardioprotective paracrine factors in the nMSC secretome. In conclusion, miR34a-5p and CD47 play an important role in determining the composition of nMSCs' secretome and immune evasion, respectively.

Transplanted allogeneic cardiac progenitor cells secrete GDF-15 and stimulate an active immune remodeling process in the ischemic myocardium.

BACKGROUND: Despite promising results in clinical studies, the mechanism for the beneficial effects of allogenic cell-based therapies remains unclear. Macrophages are not only critical mediators of inflammation but also critical players in cardiac remodeling. We hypothesized that transplanted allogenic rat cardiac progenitor cells (rCPCs) augment T-regulatory cells which ultimately promote proliferation of M2 like macrophages by an as-yet undefined mechanism. METHODS AND RESULTS: To test this hypothesis, we used crossover rat strains for exploring the mechanism of myocardial repair by allogenic CPCs. Human CPCs (hCPCs) were isolated from adult patients undergoing coronary artery bypass grafting, and rat CPCs (rCPCs) were isolated from male Wistar-Kyoto (WKY) rat hearts. Allogenic rCPCs suppressed the proliferation of T-cells observed in mixed lymphocyte reactions in vitro. Transplanted syngeneic or allogeneic rCPCs significantly increased cardiac function in a rat myocardial infarct (MI) model, whereas xenogeneic CPCs did not. Allogeneic rCPCs stimulated immunomodulatory responses by specifically increasing T-regulatory cells and M2 polarization, while maintaining their cardiac recovery potential and safety profile. Mechanistically, we confirmed the inactivation of NF-kB in Treg cells and increased M2 macrophages in the myocardium after MI by transplanted CPCs derived GDF15 and it's uptake by CD48 receptor on immune cells. CONCLUSION: Collectively, these findings strongly support the active immunomodulatory properties and robust therapeutic potential of allogenic CPCs in post-MI cardiac dysfunction.

Extracellular vesicle PD-L1 dynamics predict durable response to immune-checkpoint inhibitors and survival in patients with non-small cell lung cancer.

BACKGROUND: Immune-checkpoint inhibitors (ICIs) changed the therapeutic landscape of patients with lung cancer. However, only a subset of them derived clinical benefit and evidenced the need to identify reliable predictive biomarkers. Liquid biopsy is the non-invasive and repeatable analysis of biological material in body fluids and a promising tool for cancer biomarkers discovery. In particular, there is growing evidence that extracellular vesicles (EVs) play an important role in tumor progression and in tumor-immune interactions. Thus, we evaluated whether extracellular vesicle PD-L1 expression could be used as a biomarker for prediction of durable treatment response and survival in patients with non-small cell lung cancer (NSCLC) undergoing treatment with ICIs. METHODS: Dynamic changes in EV PD-L1 were analyzed in plasma samples collected before and at 9 ± 1 weeks during treatment in a retrospective and a prospective independent cohorts of 33 and 39 patients, respectively. RESULTS: As a result, an increase in EV PD-L1 was observed in non-responders in comparison to responders and was an independent biomarker for shorter progression-free survival and overall survival. To the contrary, tissue PD-L1 expression, the commonly used biomarker, was not predictive neither for durable response nor survival. CONCLUSION: These findings indicate that EV PD-L1 dynamics could be used to stratify patients with advanced NSCLC who would experience durable benefit from ICIs.

Safety and efficacy of transcoronary transfer of human neonatal stem cells to ischemic myocardium using a novel cell-delivery system (CIRCULATE catheter) in swine model of acute myocardial infarction.

Introduction: Stem cell-based therapies have shown promise in adults with ischemic cardiomyopathy and children with congenital heart diseases, especially those without available therapeutic options. Human neonatal mesenchymal stem cells (nMSCs) have greater regenerative potential than adult stem cells. Aim: To describe our experience with a novel catheter system for transcoronary delivery of cell-based therapies (CIRCULATE catheter) in the intra-coronary delivery of nMSCs in a swine acute myocardial infarct model. Material and methods: A newly developed catheter system (CIRCULATE catheter) with several unique features, including an expandable intra-coronary reservoir with spirally placed side holes of varying diameter, was used. nMSCs together with their secretome were used for the treatment. Pigs underwent myocardial infarction by inflating a 2.5 mm angioplasty balloon in the left anterior descending artery for 60 min. After reperfusion, stem cell therapy or placebo was administered via the novel catheter. TTE was performed at baseline, 1 h after the procedure, and before the euthanasia. Troponin blood concertation was evaluated at baseline, and after 48 h. The heart was harvested, sliced, and stained with triphenyl tetrazolium chloride (TTC). Infarct size to area-at-risk ratio was calculated. Troponin was assessed at baseline and after 48 h. Results: Thirty-nine pigs were operated with the mortality rate of 5.13% (exclusively malignant arrhythmia). Infarct size to area-at-risk ratio was significantly lower in the treatment group. Treated animals had higher ejection fraction than controls. Conclusions: Intra-coronary delivery of neonatal mesenchymal stem cells reduces the infarct size and restores myocardial function in a swine model. The novel catheter system (CIRCULATE catheter) tested in this study was safe and effective in transcoronary cell delivery of human neonatal mesenchymal stem cells.

Stem Cell Therapy in Single-Ventricle Physiology: Recent Progress and Future Directions.

Current surgical and medical treatment options for single ventricle physiology conditions remain palliative. On the long term, despite treatment, the systemic ventricle has a significant risk of developing failure. There are unmet needs to develop novel treatment modalities to help ameliorate the ventricular dysfunction. Advances in the field of stem cell therapy have been promising for the treatment of heart failure. Numerous stem cell populations have been identified. Preclinical studies in small and large animal models provide evidence for effectiveness of this treatment modality and reveal several mechanisms of action by which stem cells exert their effect. Many clinical trials have been designed to further investigate the therapeutic potential that stem cell therapy may hold for pediatric populations with single ventricle physiology. In this review, we discuss the stem cell types used in these populations, some preclinical studies, and the clinical trials of stem cell therapy in single ventricle patients.

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.

Improving extracellular vesicles visualization: From static to motion.

In the last decade, extracellular vesicles (EVs) have become a hot topic. The findings on EVs content and effects have made them a major field of interest in cancer research. EVs, are able to be internalized through integrins expressed in parental cells, in a tissue specific manner, as a key step of cancer progression and pre-metastatic niche formation. However, this specificity might lead to new opportunities in cancer treatment by using EVs as devices for drug delivery. For future applications of EVs in cancer, improved protocols and methods for EVs isolation and visualization are required. Our group has put efforts on developing a protocol able to track the EVs for in vivo internalization analysis. We showed, for the first time, the videos of labeled EVs uptake by living lung cancer cells.

Respiratory viral infection in lung transplantation induces exosomes that trigger chronic rejection.

BACKGROUND: Respiratory viral infections can increase the risk of chronic lung allograft dysfunction after lung transplantation, but the mechanisms are unknown. In this study, we determined whether symptomatic respiratory viral infections after lung transplantation induce circulating exosomes that contain lung-associated self-antigens and assessed whether these exosomes activate immune responses to self-antigens. METHODS: Serum samples were collected from lung transplant recipients with symptomatic lower- and upper-tract respiratory viral infections and from non-symptomatic stable recipients. Exosomes were isolated via ultracentrifugation; purity was determined using sucrose cushion; and presence of lung self-antigens, 20S proteasome, and viral antigens for rhinovirus, coronavirus, and respiratory syncytial virus were determined using immunoblot. Mice were immunized with circulating exosomes from each group and resulting differential immune responses and lung histology were analyzed. RESULTS: Exosomes containing self-antigens, 20S proteasome, and viral antigens were detected at significantly higher levels (p < 0.05) in serum of recipients with symptomatic respiratory viral infections (n = 35) as compared with stable controls (n = 32). Mice immunized with exosomes from recipients with respiratory viral infections developed immune responses to self-antigens, fibrosis, small airway occlusion, and significant cellular infiltration; mice immunized with exosomes from controls did not (p < 0.05). CONCLUSIONS: Circulating exosomes isolated from lung transplant recipients diagnosed with respiratory viral infections contained lung self-antigens, viral antigens, and 20S proteasome and elicited immune responses to lung self-antigens that resulted in development of chronic lung allograft dysfunction in immunized mice.

Liquid biopsy tracking of lung tumor evolutions over time.

Introduction: The rise of the personalized era in lung cancer prompted the evaluation of novel diagnostic tools to overcome some of the limits of traditional tumor genotyping. Liquid biopsy refers to a multitude of minimally invasive techniques that can allow a real-time biomolecular characterization of the tumor through the analysis of human body fluids.Areas covered: Herein we provide a comprehensive overview of the role of liquid biopsy in lung cancer, mainly focusing on the most studied members of the liquid biopsy family, cell-free DNA (cfDNA) and circulating tumor cells (CTCs).Expert opinion: Among the different components of the large liquid biopsy family, cfDNA is the most studied and widely adopted source for tumor genotyping in lung cancer, already entered clinical practice for detection of both sensitizing and resistance EGFR mutations. However, the impressive technological advances made in the last few years are expanding its potential applications, allowing a more comprehensive plasma genotyping through next-generation sequencing and moving from advanced/metastatic disease to novel frontiers, such as early detection and minimal residual disease evaluation.

Circulating exosomes derived from transplanted progenitor cells aid the functional recovery of ischemic myocardium.

The stem cell field is hindered by its inability to noninvasively monitor transplanted cells within the target organ in a repeatable, time-sensitive, and condition-specific manner. We hypothesized that quantifying and characterizing transplanted cell-derived exosomes in the recipient plasma would enable reliable, noninvasive surveillance of the conditional activity of the transplanted cells. To test this hypothesis, we used a human-into-rat xenogeneic myocardial infarction model comparing two well-studied progenitor cell types: cardiosphere-derived cells (CDCs) and c-kit+ cardiac progenitor cells (CPCs), both derived from the right atrial appendage of adults undergoing cardiopulmonary bypass. CPCs outperformed the CDCs in cell-based and in vivo regenerative assays. To noninvasively monitor the activity of transplanted CDCs or CPCs in vivo, we purified progenitor cell-specific exosomes from recipient total plasma exosomes. Seven days after transplantation, the concentration of plasma CPC-specific exosomes increased about twofold compared to CDC-specific exosomes. Computational pathway analysis failed to link CPC or CDC cellular messenger RNA (mRNA) with observed myocardial recovery, although recovery was linked to the microRNA (miRNA) cargo of CPC exosomes purified from recipient plasma. We further identified mechanistic pathways governing specific outcomes related to myocardial recovery associated with transplanted CPCs. Collectively, these findings demonstrate the potential of circulating progenitor cell-specific exosomes as a liquid biopsy that provides a noninvasive window into the conditional state of the transplanted cells. These data implicate the surveillance potential of cell-specific exosomes for allogeneic cell therapies.

Stem Cell Therapy for Hypoplastic Left Heart Syndrome: Mechanism, Clinical Application, and Future Directions.

Hypoplastic left heart syndrome is a type of congenital heart disease characterized by underdevelopment of the left ventricle, outflow tract, and aorta. The condition is fatal if aggressive palliative operations are not undertaken, but even after the complete 3-staged surgical palliation, there is significant morbidity because of progressive and ultimately intractable right ventricular failure. For this reason, there is interest in developing novel therapies for the management of right ventricular dysfunction in patients with hypoplastic left heart syndrome. Stem cell therapy may represent one such innovative approach. The field has identified numerous stem cell populations from different tissues (cardiac or bone marrow or umbilical cord blood), different age groups (adult versus neonate-derived), and different donors (autologous versus allogeneic), with preclinical and clinical experience demonstrating the potential utility of each cell type. Preclinical trials in small and large animal models have elucidated several mechanisms by which stem cells affect the injured myocardium. Our current understanding of stem cell activity is undergoing a shift from a paradigm based on cellular engraftment and differentiation to one recognizing a primarily paracrine effect. Recent studies have comprehensively evaluated the individual components of the stem cells' secretomes, shedding new light on the intracellular and extracellular pathways at the center of their therapeutic effects. This research has laid the groundwork for clinical application, and there are now several trials of stem cell therapies in pediatric populations that will provide important insights into the value of this therapeutic strategy in the management of hypoplastic left heart syndrome and other forms of congenital heart disease. This article reviews the many stem cell types applied to congenital heart disease, their preclinical investigation and the mechanisms by which they might affect right ventricular dysfunction in patients with hypoplastic left heart syndrome, and finally, the completed and ongoing clinical trials of stem cell therapy in patients with congenital heart disease.

Regenerative medicine therapy for single ventricle congenital heart disease.

One of the most complex forms of congenital heart disease (CHD) involving single ventricle physiology is hypoplastic left heart syndrome (HLHS), characterized by underdevelopment of the left ventricle (LV), mitral and aortic valves, and narrowing of the ascending aorta. The underdeveloped LV is incapable of providing long-term systemic flow, and if left untreated, the condition is fatal. Current treatment for this condition consists of three consecutive staged palliative operations: the first is conducted within the first few weeks of birth, the second between 4 to 6 months, and the third and final surgery within the first 4 years. At the conclusion of the third surgery, systemic perfusion is provided by the right ventricle (RV), and deoxygenated blood flows passively to the pulmonary vasculature. Despite these palliative interventions, the RV, which is ill suited to provide long-term systemic perfusion, is prone to eventual failure. In the absence of satisfying curative treatments, stem cell therapy may represent one innovative approach to the management of RV dysfunction in HLHS patients. Several stem cell populations from different tissues (cardiac and non-cardiac), different age groups (adult- vs. neonate-derived), and different donors (autologous vs. allogeneic), are under active investigation. Preclinical trials in small and large animal models have elucidated several mechanisms by which these stem cells affect the injured myocardium, and are driving the shift from a paradigm based upon cellular engraftment and differentiation to one based primarily on paracrine effects. Recent studies have comprehensively evaluated the individual components of the stem cells' secretomes, shedding new light on the intracellular and extracellular pathways at the center of their therapeutic effects. This research has laid the groundwork for clinical application, and there are now several trials of stem cell therapies in pediatric populations that will provide important insights into the value of this therapeutic strategy in the management of HLHS and other forms of CHD. This article reviews the many stem cell types applied to CHD, their preclinical investigation and the mechanisms by which they might affect RV dysfunction in HLHS patients, and finally, the completed and ongoing clinical trials of stem cell therapy in patients with CHD.

Circulating Exosomes with Distinct Properties during Chronic Lung Allograft Rejection.

Circulating exosomes containing donor HLA and lung-associated self-antigens (SAg) are thought to play an important role in allograft rejection after human lung transplantation. We characterized exosomes isolated from serum of 10 lung transplant recipients (LTxR) diagnosed with bronchiolitis obliterans syndrome (BOS) and compared them with exosomes isolated from serum of 10 stable LTxR. Lung-associated SAg (K-α-1-tubulin [Kα1T] and collagen V [Col-V]), MHC class II molecules, costimulatory molecules CD40, CD80, and CD86, and transcription factors class II MHC trans-activator, NF-κB, hypoxia-inducible factor 1-α, IL-1R-associated kinase 1, MyD88, and 20S proteasome were detected in exosomes from BOS, but not stable LTxR. In contrast, adhesion molecules were present in both groups. C57BL/6 mice immunized with exosomes from BOS but not stable LTxR demonstrated Ab to SAg (Col-V, 33.5 ± 15.7 versus 10.4 ± 6.4, p = 0.021; Kα1T, 925 ± 403 versus 317 ± 285, p = 0.044) and HLA (mean fluorescence intensity: BOS, 8450; stable, 632; p < 0.05). Furthermore, splenic lymphocytes demonstrated increased frequency of lung SAg-specific IL-17 (Col-V, 128 ± 46 versus 31 ± 21, p = 0.013; Kα1T, 194 ± 47 versus 67 ± 43, p = 0.014) and IFN-γ (Col-V, 165 ± 79 versus 38 ± 40, p = 0.042; Kα1T, 232 ± 64 versus 118 ± 39, p = 0.012). Reduced levels of IL-10-producing cells were seen in BOS exosome immunized mice compared with mice immunized with stable exosomes (Col-V, 59 ± 23 versus 211 ± 85, p = 0.016; Kα1T, 78 ± 49 versus 295 ± 104, p = 0.017). Owing to the unique immune-stimulating properties of exosomes induced during rejection, we propose that they play an important role in eliciting both alloantigen- and SAg-specific immunity, leading to chronic rejection after lung transplantation.

Exosomes expressing the self-antigens myosin and vimentin play an important role in syngeneic cardiac transplant rejection induced by antibodies to cardiac myosin.

Long-term success of heart transplantation is hindered by humoral and cell-mediated immune responses. We studied preexisting antibodies to cardiac self-antigens, myosin and vimentin, and exosomes induced by antibodies to self-antigens in eliciting immune responses to cardiac grafts. After syngeneic heterotopic murine heart transplantation, rabbit anti-myosin or normal rabbit immunoglobulin was administered at day 0 or 7. Sera were collected after heartbeat cessation, cellular infiltration was analyzed, and exosomes were isolated from sera. Histopathologic examination of the controls' transplanted hearts demonstrated normal architecture, and their sera demonstrated neither antibodies to self-antigens nor exosomes expressing self-antigens. Administration of antibodies to cardiac myosin immediately posttransplantation (day 0) but not on day 7 triggered graft failure on day 7, and histopathologic examination revealed marked cellular infiltration with neutrophils and lymphocytes. Histopathologic examination of rejected hearts also demonstrated myocyte damage as sera had increased antibodies to myosin and vimentin and development of exosomes expressing self-antigens. Administration of exosomes isolated from failed grafts containing self-antigens induced graft dysfunction; exosomes isolated from stable mice did not induce graft failure. Antibodies to self-antigens can induce exosomes containing self-antigens, initiating an immune response and causing graft failure after cardiac transplantation.