Donor extracellular vesicle trafficking via the pleural space represents a novel pathway for allorecognition after lung transplantation.

Restoration of lymphatic drainage across the bronchial anastomosis after lung transplantation requires several weeks. As donor antigen and antigen presenting cell trafficking via lymphatics into graft-draining lymph nodes is an important component of the alloresponse, alternative pathways must exist that account for rapid rejection after pulmonary transplantation. Here, we describe a novel allorecognition pathway mediated through donor extracellular vesicle (EV) trafficking to mediastinal lymph nodes via the pleural space. Pleural fluid collected early after lung transplantation in rats and humans contains donor-specific EVs. In a fully MHC mismatched rat model of lung transplantation, we demonstrate EVs carrying donor antigen preferentially accumulate in mediastinal lymph nodes and colocalize with MHC II expressing cells within 4 h of engraftment. Injection of allogeneic EVs into pleural space of syngeneic lung transplant recipients confirmed their selective trafficking to mediastinal lymph nodes and resulted in activation of T cells in mediastinal, but not peripheral lymph nodes. Thus, we have uncovered an alternative pathway of donor antigen trafficking where pulmonary EVs released into the pleural space traffic to locoregional lymph nodes via pleural lymphatics. This pathway obviates the need for restoration of lymphatics across the bronchial anastomosis for trafficking of donor antigen to draining lymph nodes.

Direct relationship between transvalvular velocity and cardiac dysfunction, morbidity, and mortality in patients with aortic stenosis.

OBJECTIVE: Current guidelines recommend intervention in subjects with severe symptomatic aortic stenosis (AS), even though any degree of AS is associated with a higher risk of mortality. We investigated the association between the degree of AS, delineated by transvalvular flow velocity, and patient morbidity and mortality. METHODS: Medically managed patients aged 40-95 years with maximum flow velocity (Vmax ) by echocardiography between 2013 and 2018 were stratified into five groups (A-E) based on the 75th, 90th, 97.5th, and the 99th percentiles of Vmax distribution. Patient characteristics, cardiac structural changes, and end-organ disease were compared using Kruskal-Wallis and Cochran-Armitage tests. Mortality over a median of 2.8 (1.52-4.8) years was compared using Kaplan-Meier curves and risk estimates were derived from the Cox model. RESULTS: The Vmax was reported in 37,131 patients. There was a steady increase (from Group A towards E) in age, Caucasian race, structural cardiac changes, end-organ morbidities, and all-cause mortality. In reference to Group A, there as an increased risk of mortality in Groups B (hazard ratio [HR] = 1.3; confidence interval [CI]: 1.2-1.35; p < .0001), C (HR = 1.5; CI: 1.4-1.6; p < .0001), and D (HR = 1.8; CI: 1.6-2; p < .0001), with an exponential increase in Group E (HR = 2.5; CI: 2.2-2.8; p < .0001). CONCLUSIONS: A direct, strong correlation exists between the degree of AS and cardiac structural changes and mortality. Patients with Vmax ≥ 97.5th percentile (≥3.2 m/s) might benefit from early intervention.

Noninvasive diagnosis of recurrent autoimmune type 1 diabetes after islet cell transplantation.

Islet cell transplantation is curative therapy for patients with complicated autoimmune type 1 diabetes (T1D). We report the diagnostic potential of circulating transplant islet-specific exosomes to noninvasively distinguish pancreatic ß cell injury secondary to recurrent autoimmunity vs immunologic rejection. A T1D patient with hypoglycemic unawareness underwent islet transplantation and maintained normoglycemia until posttransplant day 1098 before requiring exogenous insulin. Plasma analysis showed decreased donor islet exosome quantities on day 1001, before hyperglycemia onset. This drop in islet exosome quantity signified islet injury, but did not distinguish injury type. However, analysis of purified transplant islet exosome cargoes showed decrease in insulin-containing exosomes, but not glucagon-containing exosomes, indicating selective destruction of transplanted ß cells secondary to recurrent T1D autoimmunity. Furthermore, donor islet exosome cargo analysis showed time-specific increase in islet autoantigen, glutamic acid decarboxylase 65 (GAD65), implicated in T1D autoimmunity. Time-matched analysis of plasma transplant islet exosomes in 3 control subjects undergoing islet cell transplantation failed to show changes in islet exosome quantities or intraexosomal cargo expression of insulin, glucagon, and GAD65. This is the first report of noninvasive diagnosis of recurrent autoimmunity after islet cell transplantation, suggesting that transplant tissue exosome platform may serve as a biomarker in islet transplant diagnostics.

Framework for pediatric robotic surgery program development.

Pediatric robotic surgery has seen increasing implementation for its many benefits over the past two decades. As more pediatric surgeons gain exposure to robotic surgery, the interest in utilizing this technology is growing. However, there are no guidelines or existing framework for developing pediatric general surgery robotic programs. Programmatic development can be challenging, requiring institutional support, a minimum 12-month multistep process in partnership with the robot manufacturer, and organization of a local dedicated team. A cornerstone to all program building is collaboration and communication with key stakeholders who are committed to establishing a robotic surgery program. In this manuscript, we detail numerous best practices for implementation, followed by three variations of programmatic development, each drawing lessons from one of three practice settings: (i) A children's hospital in a large medical center associated with an adult hospital, (ii) a free-standing children's hospital, and (iii) a community-based practice. We aim for this article to provide a framework that can serve as a guide for those beginning this process, consolidating the key resources and strategies used to develop a robust pediatric robotic surgery program.

Living in disadvantaged neighborhoods linked to less intervention for severe aortic stenosis.

To investigate the association between area deprivation index (ADI) and aortic valve replacement (AVR) in patients with severe aortic stenosis (AS). Patients aged 40-95 years with severe AS confirmed by echocardiography were included. The 9-digit zip code of patient residence address was used to identify the ADI ranking, based on which patients were divided into 5 groups (with Group E being most deprived). The rates of AV intervention were compared among 5 groups using competing risks analysis, with death as a competing event. We included 1751 patients with severe AS from 2013 to 2018 followed for a median 2.8 (interquartile range, 1.5-4.8) years. The more distressed ADI groups tended to be younger (P = 0.002), female (P < 0.001), and of African American race (P < 0.001), have higher presentation of sepsis (P = 0.031), arrhythmia (P = 0.022), less likely to have previous diagnosis of AS (P < 0.001); and were less likely to undergo AVR (52.5% vs 46.9% vs 46.1% vs 48.9% vs 39.7%, P = 0.023). Using competing risk analysis, the highest ADI group (E) were the least and the lowest ADI group (A) the most likely to undergo AVR (Gray's test, P = 0.025). The association between ADI ranking and AVR rates was influenced by sex and race. Within group analysis, there was significant association between race and AVR (Gray's test, P < 0.001), and between sex and AVR (Gray's test, P < 0.001). Patients with severe AS living in more deprived neighborhoods were less likely to undergo aortic valve interventions, which was influenced by female gender, and African American race.

One and Five-Year Mortality Risk Prediction in Patients with Moderate and Severe Aortic Stenosis.

(1) Background: Our goal was to develop a risk prediction model for mortality in patients with moderate and severe aortic stenosis (AS). (2) Methods: All patients aged 40−95 years, with echocardiographic evidence of moderate and severe AS at a single institution, were studied over a median of 2.8 (1.5−4.8) years, between 2013−2018. Patient characteristics and mortality were compared using Chi-squares, t-tests, and Kaplan−Meier (KM) curves, as appropriate. The risk calculation for mortality was derived using the Cox proportional hazards model. A risk score was calculated for each parameter, and the total sum of scores predicted the individualized risks of 1-and 5-year mortality. (3) Results: A total of 1991 patients with severe and 2212 with moderate AS were included. Severe AS patients were older, had a lower ejection fraction %, were more likely to be Caucasian, and had lower rates of obesity and smoking, but had higher rates of cardiac comorbidities and AVR (49.3% vs. 2.8%, p < 0.0001). The unadjusted overall mortality was 41.7% vs. 41%, p = 0.6530, and was not different using KM curves (log rank, p = 0.0853). The models included only patients with complete follow-up (3966 in the 1-year, and 816 in the 5-year model) and included 13 variables related to patient characteristics, degree of AS, and AVR. The C-statistic was 0.75 and 0.72 for the 1-year and the 5-year models, respectively. (4) Conclusions: Patients with moderate and severe AS experience high morbidity and mortality. The usage of a risk prediction model may provide guidance for clinical decision making in complex patients.

Screening Tool to Identify Patients with Advanced Aortic Valve Stenosis.

(1) Background: The clinical burden of aortic stenosis (AS) remains high in Western countries. Yet, there are no screening algorithms for this condition. We developed a risk prediction model to guide targeted screening for patients with AS. (2) Methods: We performed a cross-sectional analysis of all echocardiographic studies performed between 2013 and 2018 at a tertiary academic care center. We included reports of unique patients aged from 40 to 95 years. A logistic regression model was fitted for the risk of moderate and severe AS, with readily available demographics and comorbidity variables. Model performance was assessed by the C-index, and its calibration was judged by a calibration plot. (3) Results: Among the 38,788 reports yielded by inclusion criteria, there were 4200 (10.8%) patients with ≥moderate AS. The multivariable model demonstrated multiple variables to be associated with AS, including age, male gender, Caucasian race, Body Mass Index ≥ 30, and cardiovascular comorbidities and medications. C-statistics of the model was 0.77 and was well calibrated according to the calibration plot. An integer point system was developed to calculate the predicted risk of ≥moderate AS, which ranged from 0.0002 to 0.7711. The lower 20% of risk was approximately 0.15 (corresponds to a score of 252), while the upper 20% of risk was about 0.60 (corresponds to a score of 332 points). (4) Conclusions: We developed a risk prediction model to predict patients' risk of having ≥moderate AS based on demographic and clinical variables from a large population cohort. This tool may guide targeted screening for patients with advanced AS in the general population.

Long-term outcomes of aortic root procedures for heterogenous ascending aneurysm disease in bicuspid aortic valve syndrome.

OBJECTIVE: Surgery for ascending aneurysms in bicuspid aortic valve syndrome primarily includes Bentall root replacement, aortic valve replacement with supracoronary ascending aorta replacement (AVRSCAAR), and valve-sparing root reimplantation (VSRR). Comparative analysis of long-term clinical and functional outcomes of these procedures is detailed. METHODS: From 1997 to 2017, 635 patients with bicuspid aortic valve undergoing root complex-focused procedures electively were stratified by valvulopathy (ie, aortic stenosis vs aortic insufficiency) and substratified into ascending or root aneurysm phenotype. Inverse probability weights were calculated to adjust for baseline differences. RESULTS: Kaplan-Meier curves for all-cause mortality demonstrated no difference between Bentall versus AVRSCAAR for aortic stenosis and aortic insufficiency presentations (log-rank P > .05). In patients with aortic stenosis, multivariable Cox regression showed significantly decreased risk of stroke for biologic AVRSCAAR (hazard ratio, 0.04; P = .013). Aortic reoperation rates were similar for biologic versus mechanical valves (P = .353). In patients with aortic insufficiency, similar long-term mortality (hazard ratio, 0.95; P = .93), but lower stroke risk in biologic AVRSCAAR group by Cox regression, and lower aortic reoperation rate was noted (coefficient < 0.01; P < .001). Comparing Bentall to VSRR, mortality (hazard ratio, 0.12; P = .022) was significantly improved in patients undergoing VSRR, but recurrence of moderate or greater aortic insufficiency was higher in VSRR by multistate model (beta coefficient 2.63; P < .001). CONCLUSIONS: A tailored approach to heterogeneous ascending aneurysm pathologies in bicuspid aortic valve syndrome utilizing Bentall, AVRSCAAR, and VSRR procedures renders excellent long-term clinical and functional outcomes, with biologic conduits showing equivalent to improved clinical outcomes.

Circulating Donor Lung-specific Exosome Profiles Enable Noninvasive Monitoring of Acute Rejection in a Rodent Orthotopic Lung Transplantation Model.

BACKGROUND: There is a critical need for development of biomarkers to noninvasively monitor for lung transplant rejection. We investigated the potential of circulating donor lung-specific exosome profiles for time-sensitive diagnosis of acute rejection in a rat orthotopic lung transplant model. METHODS: Left lungs from Wistar transgenic rats expressing human CD63-GFP, an exosome marker, were transplanted into fully MHC-mismatched Lewis recipients or syngeneic controls. Recipient blood was collected between 4 h and 10 d after transplantation, and plasma was processed for exosome isolation by size exclusion column chromatography and ultracentrifugation. Circulating donor exosomes were profiled using antihuman CD63 antibody quantum dot on the nanoparticle detector and via GFP trigger on the nanoparticle flow cytometer. RESULTS: In syngeneic controls, steady-state levels of circulating donor exosomes were detected at all posttransplant time points. Allogeneic grafts lost perfusion by day 8, consistent with acute rejection. Levels of circulating donor exosomes peaked on day 1, decreased significantly by day 2, and then reached baseline levels by day 3. Notably, decrease in peripheral donor exosome levels occurred before grafts had histological evidence of acute rejection. CONCLUSIONS: Circulating donor lung-specific exosome profiles enable an early detection of acute rejection before histologic manifestation of injury to the pulmonary allograft. As acute rejection episodes are a major risk factor for the development of chronic lung allograft dysfunction, this biomarker may provide a novel noninvasive diagnostic platform that can translate into earlier therapeutic intervention for lung transplant patients.

Syncytiotrophoblast extracellular microvesicle profiles in maternal circulation for noninvasive diagnosis of preeclampsia.

Preeclampsia is the most common placental pathology in pregnant females, with increased morbidity and mortality incurred on the mother and the fetus. There is a need for improved biomarkers for diagnosis and monitoring of this condition. Placental syncytiotrophoblasts at the maternal-fetal interface release nanoparticles, including extracellular microvesicles, into the maternal blood during pregnancy. Syncytiotrophoblast extracellular microvesicles (STEVs) are being studied for their diagnostic potential and for their potential physiologic role in preeclampsia. We hypothesized that STEV profiles in maternal circulation would be altered under conditions of preeclampsia compared to normal pregnancy. Extracellular vesicles (EVs) released by BeWo cells in vitro showed high expression of syncytin-1, but no plac1 expression, demonstrating that trophoblast cell EVs express syncytin-1 on their surface. Placental alkaline phosphatase also showed high expression on BeWo EVs, but due to concern for cross reactivity to highly prevalent isoforms of intestinal and bone alkaline phosphatase, we utilized syncytin-1 as a marker for STEVs. In vivo, syncytin-1 protein expression was confirmed in maternal plasma EVs from Control and Preeclampsia subjects by Western blot, and overall, lower expression was noted in samples from patients with preeclampsia (n = 8). By nanoparticle analysis, EV profiles from Control and Preeclampsia groups showed similar total plasma EV quantities (p = 0.313) and size distribution (p = 0.415), but STEV quantitative signal, marked by syncytin-1 specific EVs, was significantly decreased in the Preeclampsia group (p = 2.8 × 10-11). Receiver operating characteristic curve demonstrated that STEV signal threshold cut-off of <0.316 was 95.2% sensitive and 95.6% specific for diagnosis of preeclampsia in this cohort (area under curve = 0.975 ± 0.020). In conclusion, we report that the syncytin-1 expressing EV profiles in maternal plasma might serve as a placental tissue specific biomarker for preeclampsia.

Predictors of Recurrent Aortic Insufficiency in Type I Bicuspid Aortic Valve Repair.

BACKGROUND: Annular stabilization techniques in bicuspid aortic valve (BAV) repair include valve-sparing root reimplantation (VSRR), external subannular aortic ring (ESAR), and subcommissural annuloplasty (SCA). Unlike VSRR that offers neoroot creation, ESAR and SCA offer annular reduction only. We compared long-term functional outcomes to understand BAV repair durability. METHODS: From 2004 to 2017, 137 patients underwent Sievers type I BAV repair (VSRR, n = 54; ESAR, n = 22; SCA, n = 51). Prospectively maintained BAV repair database was queried for clinical and functional outcomes. Data were analyzed by logistic regression, threshold regression, multistate survival, and transition models for BAV repair durability. RESULTS: VSRR patients had larger preoperative sinus dimensions (p < 0.001), but mean preoperative annulus size was similar for VSRR, ESAR, and SCA (29.3 ± 3.7 mm, 29.8 ± 3.8 mm, and 29.7 ± 3.8mm, respectively; p = 0.807). Degree of annular reduction (p = 0.280) was comparable between the groups. Intraoperative postrepair freedom from aortic insufficiency (AI) 1+ or greater was 100% across the entire cohort. By logistic regression, important predictors of recurrent AI (1+ and ≥2+) were preoperative annulus of 30 mm or more for SCA. Threshold regression confirmed annulus of 30 mm or more as risk factor for recurrent AI of 1+ or greater for SCA. Risk to relapse from no AI to AI 1+ was equal between the groups; however, once AI 1+ was reached, there was a 2.5-fold increased risk for patients with annulus of 30 mm or more who underwent SCA to progress to recurrent AI of 2+ or greater. CONCLUSIONS: VSRR is associated with improved longitudinal BAV durability compared with SCA. Preoperative annulus diameter of 30 mm or more is associated with increased recurrent AI, especially for SCA patients. For annular indications, ESAR might offer comparable functional outcomes with VSRR; however, further follow-up is critical.