Introducing thymus for promoting transplantation tolerance.

Establishing tolerance remains a central, if elusive, goal of transplantation. In solid-organ transplantation, one strategy for inducing tolerance has been cotransplantation of various forms of thymic tissue along with another organ. As one of the biological foundations of central tolerance, thymic tissue carries with it the ability to induce tolerance to any other organ or tissue from the same donor (or another donor tissue-matched to the thymic tissue) if successfully transplanted. In this review, we outline the history of this approach as well as work to date on its application in organ transplantation, concluding with future directions. We also review our experience with allogeneic processed thymus tissue for the treatment of congenital athymia, encompassing complete DiGeorge syndrome and other rare genetic disorders, and consider whether allogeneic processed thymic tissue implantation may offer a novel method for future experimentation with tolerance induction in organ transplantation.

Preoperative carfilzomib and lulizumab based desensitization prolongs graft survival in a sensitized non-human primate model.

Sensitized patients are difficult to transplant due to pre-formed anti-donor immunity. We have previously reported successful desensitization using carfilzomib and belatacept in a non-human primate (NHP) model. Here we evaluated selective blockade of the co-stimulatory signal (CD28-B7) with Lulizumab, which preserves the co-inhibitory signal (CTLA4-B7). Five maximally MHC-mismatched pairs of NHPs were sensitized to each other with two sequential skin transplants. Individuals from each pair were randomized to either desensitization with once-weekly Carfilzomib (27mg/m2 IV) and Lulizumab (12.5mg/kg SC) over four weeks, or no desensitization (Control). NHPs then underwent life-sustaining kidney transplantation from their previous skin donor. Rhesus-specific anti-thymocyte globulin was used as induction therapy and immunosuppression maintained with tacrolimus, mycophenolate, and methylprednisolone. Desensitized subjects demonstrated a significant reduction in donor-specific antibody, follicular helper T cells (CD4+PD-1+ICOS+), and proliferating B cells (CD20+Ki67+) in the lymph nodes. Interestingly, regulatory T cell (CD4+CD25+CD127lo) frequency was maintained after desensitization in addition to increased frequency of naïve CD4 T cells (CCR7+CD45RA+) and naïve B cells (IgD+CD27-CD20+) in circulation. This was associated with significant prolongation in graft survival (MST = 5.8 ± 4.0 vs. 64.8 ± 36.3; p<0.05) and lower antibody-mediated rejection scores compared to control animals. However, all desensitized animals eventually developed AMR and graft failure. Desensitization with CFZ and Lulizumab improves allograft survival in allosensitized NHPs, by transient control of the germinal center and shifting of the immune system to a more naive phenotype. This regimen may translate into clinical practice to improve outcomes of highly sensitized transplant patients.

Expanding donor availability in lung transplantation: A case report of 5000 miles traveled.

We present the case of a 41-year-old female who underwent bilateral lung transplantation after the donor lungs were placed on a normothermic ex vivo lung perfusion and ventilation device and flown nearly 5000 miles from Honolulu, Hawaii to Durham, North Carolina. The patient experienced no primary graft dysfunction. One year after transplantation she has remained rejection-free and exhibits excellent pulmonary function. This case highlights the challenge that active organ preservation systems pose to questions of organ allocation and geographic sharing.

Coagulation, inflammation, and CD46 transgene expression in neonatal porcine islet xenotransplantation.

BACKGROUND: Thrombosis is a known consequence of intraportal islet transplantation, particularly for xenogeneic islets. To define the origins of thrombosis after islet xenotransplantation and relate it to early inflammation, we examined porcine islets transplanted into non-human primates using a dual-transplant model to directly compare islet characteristics. METHODS: α1,3-Galactosyltransferase gene-knockout (GTKO) islets with and without expression of the human complement regulatory transgene CD46 (hCD46) were studied. Biologically inert polyethylene microspheres were used to examine the generic pro-thrombotic effects of particle embolization. Immunohistochemistry was performed 1 and 24 hours after transplantation. RESULTS: Xeno-islet transplantation activated both extrinsic and intrinsic coagulation pathways. The intrinsic pathway was also initiated by microsphere embolization, while extrinsic pathway tissue factor (TF) and platelet aggregation were more specific to engrafted islets. hCD46 expression significantly reduced TF, platelet, fibrin, and factor XIIIa accumulation in and around islets but did not alter intrinsic factor activation. Layers of TF+ cells emerged around islets within 24 hours, particularly co-localized with vimentin, and identified as CD3+ and CD68+ cells inflammatory cells. CONCLUSIONS: These findings detail the origins of thrombosis following islet xenotransplantation, relate it to early immune activation, and suggest a role for transgenic hCD46 expression in its mitigation. Layers of TF-positive inflammatory cells and fibroblasts around islets at 24 hours may have important roles in the progressive events of thrombosis, inflammatory cell recruitment, rejection, and the ultimate outcome of transplanted grafts. These suggest that the strategies targeting these elements could yield more progress toward successful xenogeneic islet engraftment and survival.

Kidney transplantation using alemtuzumab, belatacept, and sirolimus: Five-year follow-up.

Kidney transplant outcomes are limited by toxicities associated with calcineurin inhibitors and steroids. This trial was conducted to determine whether a costimulation blockade (CoB)-based regimen could achieve acceptable long-term outcomes and graft survival could be maintained solely with CoB. Forty patients underwent alemtuzumab induction followed by belatacept and sirolimus maintenance therapy. Patients were offered weaning to belatacept monotherapy after 1 year and followed for 5 years. Five-year patient and graft survival rates were 100% and 95%, respectively. Graft function remained stable with a mean estimated glomerular filtration rates of 67 ± 21 and 71 ± 19 at 36 and 60 months, respectively. There was no clinical rejection in the first year; subclinical rejection was detected by protocol biopsy in 4 patients. Twelve patients were successfully weaned to belatacept monotherapy. Cytomegalovirus and Epstein-Barr virus reactivations were well controlled, but 9 patients experienced transient BK viremia during the first year. Alemtuzumab produced profound lymphopenia followed by gradual T cell and more rapid B cell reconstitution to a repertoire deviated toward naïve cells with increased regulatory T cells. This regimen effectively prevents allograft rejection without using steroids or calcineurin inhibitors, enriches for naïve cells susceptible to control with CoB, and permits control of rejection with belatacept monotherapy in selected patients.

B cells in transplant tolerance and rejection: friends or foes?

Our understanding of the role of B cells in organ transplantation remains incomplete and continues to grow. The majority of research has focused on the detrimental role of antibodies that drive the development of pathogenesis of the transplanted organ. However, it has been shown that not all donor-specific antibodies are harmful and in some circumstances can even promote tolerance through the mechanism of accommodation. Furthermore, B cells can have effects on transplanted organs through their interaction with T cells, namely antigen presentation, cytokine production, and costimulation. More recently, the role and importance of Bregs was introduced to the field of transplantation. Due to this functional and ontogenetic heterogeneity, targeting B cells in transplantation may bring undesired immunologic side effects including increased rejection. Therefore, the selective control of B cells that contribute to the humoral response against donor antigens will continue to be an important and challenging area of research and potentially lead to improved long-term transplant outcomes.

The role of human CD46 in early xenoislet engraftment in a dual transplant model.

BACKGROUND: Membrane cofactor protein CD46 attenuates the complement cascade by facilitating cleavage of C3b and C4b. In solid organ xenotransplantation, organs expressing CD46 have been shown to resist hyperacute rejection. However, the incremental value of human CD46 expression for islet xenotransplantation remains poorly defined. METHODS: This study attempted to delineate the role of CD46 in early neonatal porcine islet engraftment by comparing Gal-knocked out (GKO) and hCD46-transgenic (GKO/CD46) islets in a dual transplant model. Seven rhesus macaques underwent dual transplant and were sacrificed at 1 hour (n = 4) or 24 hours (n = 3). Both hemilivers were recovered and fixed for immunohistochemistry (CD46, insulin, neutrophil elastase, platelet, IgM, IgG, C3d, C4d, CD68, Caspase 3). Quantitative immunohistochemical analysis was performed using the Aperio Imagescope. RESULTS: Within 1 hour of intraportal infusion of xenografts, no differences were observed between the two types of islets in terms of platelet, antibody, or complement deposition. Cellular infiltration and islet apoptotic activity were also similar at 1 hour. At 24 hours, GKO/CD46 islets demonstrated significantly less platelet deposition (P = 0.01) and neutrophil infiltration (P = 0.01) compared to GKO islets. In contrast, C3d (P = 0.38) and C4d (P = 0.45) deposition was equal between the two genotypes. CONCLUSIONS: Our findings suggest that expression of hCD46 on NPIs potentially provides a measurable incremental survival advantage in vivo by reducing early thrombo-inflammatory events associated with instant blood-mediated inflammatory reaction (IBMIR) following intraportal islet infusion.

The past, present, and future of costimulation blockade in organ transplantation.

PURPOSE OF REVIEW: Manipulating costimulatory signals has been shown to alter T cell responses and prolong graft survival in solid organ transplantation. Our understanding of and ability to target various costimulation pathways continues to evolve. RECENT FINDINGS: Since the approval of belatacept in kidney transplantation, many additional biologics have been developed targeting clinically relevant costimulation signaling axes including CD40-CD40L, inducible costimulator-inducible costimulator ligand (ICOS-ICOSL), and OX40-OX40L. Currently, the effects of costimulation blockade on posttransplant humoral responses, tolerance induction, and xenotransplantation are under active investigation. Here, we will discuss these pathways as well as preclinical and clinical outcomes of biologics targeting these pathways in organ transplantation. SUMMARY: Targeting costimultion is a promising approach for not only controlling T cell but also B cell responses. Consequently, costimulation blockade shows considerable potential for improving outcomes in antibody-mediated rejection and xenotransplantation.

Incidence, risk, and prevention of ventilator-associated pneumonia in adult cardiac surgical patients: a systematic review.

Ventilator-associated pneumonia remains a major cause of morbidity and mortality in postoperative heart surgery patients. We present a systematic review of the literature on the incidence, risk factors, and prevention of this condition in a population at heightened risk.

A novel method for in vitro culture and expansion of nonhuman primate B cells.

BACKGROUND: Given the role of B cells in sensitization and antibody-mediated rejection pathogenesis, the ability to identify, isolate, and study B cells in vitro is critical for understanding these processes and developing novel therapeutics. While in vivo nonhuman primate models have been used to this end, an in vitro nonhuman primate model of B cell activation and proliferation has not been developed. METHODS: CD20+ B cells and CD3+ T cells were isolated using magnetic bead separation from the peripheral blood of naive and skin allograft sensitized nonhuman primates. Allogeneic B and T cells were co-cultured in plates pre-coated with murine stromal cells engineered to express human CD40L and stimulated with cytokines. Cells and supernatants were harvested every 2 days for immune phenotyping and donor specific antibody quantification by flow cytometry. RESULTS: The optimized culture system consisted of MS40L cells co-cultured with B and allogenic T cells and stimulated with cytokines. This culture system resulted in increased memory cells and plasmablasts over time compared to other culture systems. Comparison of culture of naïve and sensitized nonhuman primate samples revealed faster B cell exhaustion and marginally increased plasmablast differentiation in sensitized culture. Donor-specific antibody production was not observed in either culture group. CONCLUSIONS: This study describes the first in vitro nonhuman primate model of B cell activation and proliferation using both naïve and allosensitized samples. This model provides an opportunity for exploration of B cell mechanisms and novel therapeutics and is a preliminary step in the development of an in vitro germinal center model.

Belatacept-Based Maintenance Immunosuppression Controls the Post-Transplant Humoral Immune Response in Highly Sensitized Nonhuman Primates.

Preexisting donor-specific antibodies (DSA) to MHC antigens increase the risk of antibody-mediated rejection (AMR) in sensitized transplant recipients and reduces graft survival. Pretransplant desensitization with costimulation blockade and proteasome inhibition has facilitated transplantation in our preclinical nonhuman primate (NHP) model. However, long-term graft survival is limited by rebound of DSA after transplantation. In this study, we performed kidney transplants between highly sensitized, maximally MHC-mismatched NHPs (n=14). At kidney transplantation, primates received T cell depletion with rhesus-specific anti-thymocyte globulin (rhATG; n=10) or monoclonal anti-CD4 and anti-CD8 antibodies (n=4). Maintenance immunosuppression consisted of belatacept and tacrolimus (n=5) or belatacept and rapamycin (n=9) with steroids. Rebound of DSA post-kidney transplantation was significantly reduced compared with maintenance immunosuppression with tacrolimus, mycophenolate, and steroids. Protocol lymph node biopsy specimens showed a decrease in germinal center activity, with low frequencies of T follicular helper cells and class-switched B cells after kidney transplantation. Combined belatacept and rapamycin was superior in controlling viral reactivation, enabling weaning of ganciclovir prophylaxis. Tacrolimus was associated with increased morbidity that included cytomegalovirus and parvovirus viremia and post-transplant lymphoproliferative disorder. All primates in the tacrolimus/belatacept group failed discontinuation of antiviral therapy. Overall, belatacept-based immunosuppression increased AMR-free graft survival by controlling post-transplant humoral responses in highly sensitized NHP recipients and should be further investigated in a human clinical trial.

A simple scoring system to estimate perioperative mortality following liver resection for primary liver malignancy-the Hepatectomy Risk Score (HeRS).

BACKGROUND: Selection of the optimal treatment modality for primary liver cancers remains complex, balancing patient condition, liver function, and extent of disease. In individuals with preserved liver function, liver resection remains the primary approach for treatment with curative intent but may be associated with significant mortality. The purpose of this study was to establish a simple scoring system based on Model for End-stage Liver Disease (MELD) and extent of resection to guide risk assessment for liver resections. METHODS: The 2005-2015 NSQIP database was queried for patients undergoing liver resection for primary liver malignancy. We first developed a model that incorporated the extent of resection (1 point for major hepatectomy) and a MELD-Na score category of low (MELD-Na =6, 1 point), medium (MELD-Na =7-10, 2 points) or high (MELD-Na >10, 3 points) with a score range of 1-4, called the Hepatic Resection Risk Score (HeRS). We tested the predictive value of this model on the dataset using logistic regression. We next developed an optimal multivariable model using backwards sequential selection of variables under logistic regression. We performed K-fold cross validation on both models. Receiver operating characteristics were plotted and the optimal sensitivity and specificity for each model were calculated to obtain positive and negative predictive values. RESULTS: A total of 4,510 patients were included. HeRS was associated with increased odds of 30-day mortality [HeRS =2: OR =3.23 (1.16-8.99), P=0.025; HeRS =3: OR =6.54 (2.39-17.90), P<0.001; HeRS =4: OR =13.69 (4.90-38.22), P<0.001]. The AUC for this model was 0.66. The AUC for the optimal multivariable model was higher at 0.76. Under K-fold cross validation, the positive predictive value (PPV) and negative predictive value (NPV) of these two models were similar at PPV =6.4% and NPV =97.7% for the HeRS only model and PPV =8.4% and NPV =98.1% for the optimal multivariable model. CONCLUSIONS: The HeRS offers a simple heuristic for estimating 30-day mortality after resection of primary liver malignancy. More complicated models offer better performance but at the expense of being more difficult to integrate into clinical practice.

C3 complement inhibition prevents antibody-mediated rejection and prolongs renal allograft survival in sensitized non-human primates.

Sensitized kidney transplant recipients experience high rates of antibody-mediated rejection due to the presence of donor-specific antibodies and immunologic memory. Here we show that transient peri-transplant treatment with the central complement component C3 inhibitor Cp40 significantly prolongs median allograft survival in a sensitized nonhuman primate model. Despite donor-specific antibody levels remaining high, fifty percent of Cp40-treated primates maintain normal kidney function beyond the last day of treatment. Interestingly, presence of antibodies of the IgM class associates with reduced median graft survival (8 vs. 40 days; p = 0.02). Cp40 does not alter lymphocyte depletion by rhesus-specific anti-thymocyte globulin, but inhibits lymphocyte activation and proliferation, resulting in reduced antibody-mediated injury and complement deposition. In summary, Cp40 prevents acute antibody-mediated rejection and prolongs graft survival in primates, and inhibits T and B cell activation and proliferation, suggesting an immunomodulatory effect beyond its direct impact on antibody-mediated injury.

Point-of-Care Assessment of DCD Livers During Normothermic Machine Perfusion in a Nonhuman Primate Model.

Normothermic machine perfusion (NMP) provides clinicians an opportunity to assess marginal livers before transplantation. However, objective criteria and point-of-care (POC) biomarkers to predict risk and guide decision making are lacking. In this investigation, we characterized trends in POC biomarkers during NMP and compared primate donation after circulatory death (DCD) livers with short and prolonged warm ischemic injury. Following asystole, livers were subjected to either 5 minutes (DCD-5min, n = 4) or 45 minutes (DCD-45min, n = 4) of warm ischemia time. Livers were flushed with heparinized UW solution, and preserved in cold storage before NMP. During flow-controlled NMP, circulating perfusate and tissue biopsies were collected at 0, 2, 4, 6, and 8 hours for analysis. DCD-45min livers had greater terminal portal vein pressure (8.5 vs. 13.3 mm Hg, P = 0.027) and terminal portal vein resistance (16.3 vs. 32.4 Wood units, P = 0.005). During perfusion, DCD-45min livers had equivalent terminal lactate clearance (93% vs. 96%, P = 0.344), greater terminal alanine aminotransferase (163 vs. 883 U/L, P = 0.002), and greater terminal perfusate gamma glutamyltransferase (GGT) (5.0 vs. 31.7 U/L, P = 0.002). DCD-45min livers had higher circulating levels of flavin mononucleotide (FMN) at hours 2 and 4 of perfusion (136 vs. 250 ng/mL, P = 0.029; and 158 vs. 293 ng/mL, P = 0.003; respectively). DCD-5min livers produced more bile and demonstrated progressive decline in bile lactate dehydrogenase, whereas DCD-45min livers did not. On blinded histologic evaluation, DCD-45min livers demonstrated greater injury and necrosis at late stages of perfusion, indicative of nonviability. Conclusion: Objective criteria are needed to define graft viability during NMP. Perfusate lactate clearance does not discriminate between viable and nonviable livers during NMP. Perfusate GGT and FMN may represent POC biomarkers predictive of liver injury during NMP.

Minimally Invasive Pulmonary Fibroelastoma Resection.

Pulmonary fibroelastomas are a rare primary cardiac tumor with less than 50 cases reported in the literature to date. We performed a minimally invasive valve-sparing tumor resection through a left anterior mini-incision (LAMI). The procedure was performed without cardiac arrest or aortic cross clamp, expediting postoperative recovery and allowing for an uncomplicated discharge on postoperative day 5. LAMI is a safe and reliable alternative to median sternotomy for patients requiring interventions on the right ventricular outflow tract and main pulmonary artery, including pulmonary fibroelastoma resection and pulmonary valve replacement when needed.

A Minimally Invasive Approach for Placing Sew-On Epicardial Leads in the Child.

Traditionally, pacing leads are placed transvenously, although smaller pediatric patients who require permanent pacemakers may benefit from delaying tranvenous lead placement until they are larger. Alternative, minimally invasive atrioventricular pacing options have not previously existed for this patient population, leaving many of these children with large sternotomies or thoracotomies. Using three port sites and an adjustable shaft dual-needle suturing device, we placed a steroid-eluting, sew-on epicardial lead on the right atrium of a 9-year-old patient. This is one of the earliest reported cases of a minimally invasive technique for sew-on epicardial lead placement on the atrium of a child. Although based on a single case, we believe that this approach is safe, reliable, and reproducible and that it can be used to place leads on any aspect of the heart. Adoption of this technique will allow for earlier atrioventricular pacing, which may decrease the incidence of pacemaker syndrome, and thus improve overall outcomes in this patient population.

Anatomic thoracoscopic segmentectomy for early-stage lung cancer.

Over the past 20 years, there have been significant advancements in thoracoscopic surgical techniques as well as in lung cancer screening protocols, which have identified greater numbers of smaller lung tumors (<2 cm) that are more frequently operable and curable. These advancements have led to new interest in the thoracoscopic (VATS) approach to segmentectomy. This article will discuss the outcomes and technical considerations associated with VATS segmentectomy.

Design of interventional studies in thoracic surgery.

Interventional studies differ from observational studies in that one or more specific interventions are evaluated. Randomized controlled trials remain the gold standard for interventional studies and can take different forms. In surgical studies, the three types of randomized controlled trials most commonly encountered are: (I) trials that compare two different medical treatments for patients undergoing surgery; (II) trials that evaluate two different surgical techniques and (III) studies that compare surgery vs. non-operative management. When an intervention is to be evaluated but a randomized controlled trial is not feasible, alternative interventional study designs may be considered.

The Role of Extent of Surgical Resection and Lymph Node Assessment for Clinical Stage I Pulmonary Lepidic Adenocarcinoma: An Analysis of 1991 Patients.

BACKGROUND: This study examined the association of extent of lung resection, pathologic nodal evaluation, and survival for patients with clinical stage I (cT1-2N0M0) adenocarcinoma with lepidic histologic features in the National Cancer Data Base. METHODS: The association between extent of surgical resection and long-term survival for patients in the National Cancer Data Base with clinical stage I lepidic adenocarcinoma who underwent lobectomy or sublobar resection was evaluated using Kaplan-Meier and Cox proportional hazards regression analyses. RESULTS: Of the 1991 patients with cT1-2N0M0 lepidic adenocarcinoma who met the study criteria, 1544 underwent lobectomy and 447 underwent sublobar resection. Patients treated with sublobar resection were older, more likely to be female, and had higher Charlson/Deyo comorbidity scores, but they had smaller tumors and lower T status. Of the patients treated with lobectomy, 6% (n = 92) were upstaged because of positive nodal disease, with a median of seven lymph nodes sampled (interquartile range 4-10). In an analysis of the entire cohort, lobectomy was associated with a significant survival advantage over sublobar resection in univariate analysis (median survival 9.2 versus 7.5 years, p = 0.022, 5-year survival 70.5% versus 67.8%) and after multivariable adjustment (hazard ratio = 0.81, 95% confidence interval: 0.68-0.95, p = 0.011). However, lobectomy was no longer independently associated with improved survival when compared with sublobar resection (hazard ratio = 0.99, 95% confidence interval: 0.77-1.27, p = 0.905) in a multivariable analysis of a subset of patients in which only those patients who had undergone a sublobar resection including lymph node sampling were compared with patients treated with lobectomy. CONCLUSIONS: Surgeons treating patients with stage I lung adenocarcinoma with lepidic features should cautiously utilize sublobar resection rather than lobectomy, and they must always perform adequate pathologic lymph node evaluation.

Hospital Bed Type, the Electronic Medical Record, and Safe Bed Elevation in the Intensive Care Setting.

In mechanically ventilated patients, head of bed (HOB) elevation above 30° decreases the risk of ventilator-associated pneumonia. The research team studied (a) compliance with proper HOB elevation in their cardiac surgical intensive care unit, (b) the accuracy of HOB angles recorded in the electronic medical record (EMR), and (c) the effect of bed type on (a) and (b). Nurses were polled to discover how HOB angles were measured in practice. HOB angles were compliant in 80% of observations. Compliance was more frequent in beds with side-of-bed angle indicators (SBI) than beds with under-bed angle indicators (UBI; 88% vs 77%, P = .04). Charting in the EMR was accurate in 50% of SBI bed observations but only 20% of UBI bed observations (P < .0001). Sixty-seven percent of nurses used the SBI; 27% used the UBI; 6% used estimation alone. Though compliance was suboptimal, compliance and EMR accuracy were significantly associated with bed type. Bedside indicators are underutilized.