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Post-transplant lymphoproliferative disorder (PTLD) comprises a broad spectrum of diseases and is a rare but serious complication of solid organ transplantation. We report the case of a 45-year-old simultaneous pancreas and kidney (SPK) transplant recipient with diffuse, early-onset PTLD, manifesting as jejunal perforation at 6 months after transplantation. The patient underwent urgent small bowel resection of the affected portion of jejunum. The surgical pathology report was significant for diffuse large B-cell lymphoma. Subsequently, the patient underwent a full workup, including upper and lower endoscopy and whole-body positron emission tomography that revealed involvement of the axial skeleton and multiple abdominal organs with sparing of the grafts. He was treated with rituximab and intrathecal methotrexate for central nervous system prophylaxis. The patient experienced complete resolution of disease by positron emission tomography 8 months after initial presentation. We found no previous report in the literature of intestinal perforation as the initial presentation of PTLD in SPK transplant recipients.

Background Microvascular endothelial cells (ECs) are central to an allograft's immunogenicity. Cold ischemia and reperfusion injury associated with static cold storage and warm reperfusion activates ECs and increases the immunogenicity of the allograft. After reperfusion, mitochondrial permeability transition pore (mPTP) opening contributes to mitochondrial dysfunction in the allograft, which correlates to alloimmune rejection. Current understanding of this relationship, however, centers on the whole allograft instead of ECs. This study aimed to elucidate the relationship between EC mPTP opening and their immunophenotype. Methods Mitochondrial metabolic fitness and glycolysis in ECs were assessed in parallel with metabolic gene microarray postreperfusion. NIM811 was used to inhibit mPTP opening to rescue mitochondrial fitness. The immunogenicity of NIM811-treated ECs was determined via levels of EC's proinflammatory cytokines and allogeneic CD8 T cell cocultures. Finally, EC surface expression of adhesion, costimulatory, coinhibitory, MHC-I molecules, and MHC-I machinery protein levels were characterized. Results Genes for glycolysis, tricarboxylic acid cycle, fatty acid synthesis, gluconeogenesis were upregulated at 6 hours postreperfusion but either normalized or downregulated at 24 hours postreperfusion. As mitochondrial fitness was reduced, glycolysis increased during the first 6 hours postreperfusion. Endothelial cell treatment with NIM811 during the early postreperfusion period rescued mitochondrial fitness and reduced EC immunogenicity by decreasing CCL2, KC release, and VCAM-1, MHC-I, TAP1 expression. Conclusions Static cold storage and warm reperfusion leads to a reduction in mitochondrial fitness in microvascular ECs due to mPTP opening. Further, mPTP opening promotes increased EC immunogenicity that can be prevented by NIM811 treatment.

AIMS: Following an acute myocardial infarction (MI) the extracellular matrix (ECM) undergoes remodeling in order to prevent dilation of the infarct area and maintain cardiac output. Excessive and prolonged inflammation following an MI exacerbates adverse ventricular remodeling. Macrophages are an integral part of the inflammatory response that contribute to this remodeling. Treatment with histone deacetylase (HDAC) inhibitors preserves LV function and myocardial remodeling in the post-MI heart. This study tested whether inhibition of HDAC activity resulted in preserving post-MI LV function through the regulation of macrophage phenotype and early resolution of inflammation. METHODS AND RESULTS: HDAC inhibition does not affect the recruitment of CD45(+) leukocytes, CD45(+)/CD11b(+) inflammatory monocytes or CD45(+)/CD11b(+)CD86(+) inflammatory macrophages for the first 3 days following infarct. Further, HDAC inhibition does not change the high expression level of the inflammatory cytokines in the first days following MI. However, by day 7, there was a significant reduction in the levels of CD45(+)/Cd11b(+) and CD45(+)/CD11b(+)/CD86(+) cells with HDAC inhibition. Remarkably, HDAC inhibition resulted in the dramatic increase in the recruitment of CD45(+)/CD11b(+)/CD206(+) alternatively activated macrophages as early as 1 Day which remained significantly elevated until 5 days post-MI. qRT-PCR revealed that HDAC inhibitor treatment shifts the cytokine and chemokine environment towards an M2 phenotype with upregulation of M2 markers at 1 and 5 days post-MI. Importantly, HDAC inhibition correlates with significant preservation of both LV ejection fraction and end-diastolic volume and is associated with a significant increase in micro-vessel density in the border zone at 14 days post-MI. CONCLUSION: Inhibition of HDAC activity result in the early recruitment of reparative CD45(+)/CD11b(+)/CD206(+) macrophages in the post-MI heart and correlates with improved ventricular function and remodeling. This work identifies a very promising therapeutic opportunity to manage macrophage phenotype and enhance resolution of inflammation in the post-MI heart.

(a) Rapamycin nanotherapeutic pre-treatment improves tracheal allograft outcome after transplantation. (b) Nanotherapy reduces aortic allograft vasculopathy. (c) Dose dependency of the nanotherapy in aortic interposition allografts., Hypothermic preservation is the standard of care for storing organs prior to transplantation. Endothelial and epithelial injury associated with hypothermic storage causes downstream graft injury and, as such, the choice of an ideal donor organ preservation solution remains controversial. Cold storage solutions, by design, minimize cellular necrosis and optimize cellular osmotic potential, but do little to assuage immunological cell activation or immune cell priming post transplantation. Thus, here we explore the efficacy of our previously described novel Targeted Rapamycin Micelles (TRaM) as an additive to standard-of-care University of Wisconsin preservation solution as a means to alter the immunological microenvironment post transplantation using in vivo models of tracheal and aortic allograft transplantation. In all models of transplantation, grafts pre-treated with 100 ng mL–1 of TRaM augmented preservation solution ex vivo showed a significant inhibition of chronic rejection post-transplantation, as compared to UW augmented with free rapamycin at a ten-fold higher dose. Here, for the first time, we present a novel method of organ pretreatment using a nanotherapeutic-based cellular targeted delivery system that enables donor administration of rapamycin, at a ten-fold decreased dose during cold storage. Clinically, these pretreatment strategies may positively impact post-transplant outcomes and can be readily translated to clinical scenarios.

Objective Determine the impact of cytolytic versus IL-2 receptor antibody (IL-2RA) induction on acute rejection, graft loss and death in African-American (AA) kidney transplant (KTX) recipients. Background AAs are underrepresented in clinical trials in transplantation; thus, there is controversy regarding the optimal choice of perioperative antibody induction in KTX to improve outcomes. Methods National cohort study using US transplant registry data from January 1, 2000 to December 31, 2009 in adult solitary AA KTX recipients, with at least 5 years of follow-up. Multivariable logistic and Cox regression were utilized to assess the outcomes of acute rejection, graft loss, and mortality, with interaction terms to assess effect modification. Results Twenty-five thousand eighty-four adult AAs receiving solitary KTX were included, 16,927 (67.5%) received cytolytic induction and 8157 (32.5%) received IL-2RA induction. After adjustment for recipient sociodemographics, donor, and transplant characteristics, the use of cytolytic induction therapy reduced the risk of acute rejection by 32% (OR 0.68, 0.62-0.75), graft loss by 9% (HR 0.91, 0.86-0.97), and death by 12% (HR 0.88, 0.83-0.94). There were a number of significant effect modifiers, including public insurance, panel reactive antibody, delayed graft function, and steroid withdrawal; in these groups, cytolytic induction substantially improved clinical outcomes. Conclusions These data demonstrate that cytolytic induction therapy, as compared with IL-2RA, reduces the risk of rejection, graft loss, and death in adult AA KTX recipients, particularly in those who are sensitized, receive public insurance, develop delayed graft function, or undergo steroid withdrawal.

Arterial conduits are a well-recognized technique used in liver transplantation to achieve allograft arterial inflow when conventional hepatic arterial inflow is compromised. Indications for ectopic inflow include native arterial disease at the time of initial transplantation, as well as reconstruction in the setting of thrombotic complications. Although supraceliac or infrarenal aortic reconstructions are preferred approaches, the right common iliac artery represents a viable alternative. We present the case of a morbidly obese patient with occlusive atheromatous plaque at the celiac origin not amenable to preoperative angioplasty who underwent reconstruction with a donor iliac artery conduit to the recipient right common iliac artery. His hepatic arterial inflow remained patent postoperatively with no thrombotic or hemorrhagic complications.

Medication non-adherence is common after transplant and a major contributor to graft loss. The objective of this pilot study was to obtain preliminary safety and adherence data of a complete once-daily immunosuppression regimen of Extended-release-tacrolimus (LCP-tac), everolimus, and prednisone vs LCP-tac, mycophenolate Twice daily (BID), and prednisone through a randomized controlled pilot study of 40 patients (20 in each arm). At 3 ± 2 months post-transplant, patients were randomized to receive LCP-tac and everolimus once daily or LCP-tac and mycophenolate BID (control arm) for 6 months; 80 met eligibility, and 40 were randomized. Mean age was 51 ± 14 years, 33% were female, 45% African American, and 55% had a Calculated panel reactive antibody (cPRA) >20%. Both regimens were well-tolerated, and medication side effect burden tended to be less severe in the intervention group. Self-reported high medication adherence decreased in the control arm from baseline to 6 months (80%-59%), while remaining the same in the intervention arm throughout the study (45%-47%). For safety assessment, there was no rejection, graft loss, or death in either arm. These results provide preliminary evidence of the safety of a novel once-daily immunosuppression regimen. The impact of this once-daily regimen on medication adherence requires a larger long-term study.

A lack of research exploring post-transplant process optimization to reduce readmissions and increasing readmission rates at our center from 2009 to 2013 led to this study, aimed at assessing the effect of patient and process factors on 30-d readmission rates after kidney transplantation. This was a retrospective case-control study in adult kidney transplant recipients. Univariate and multivariate analyses were utilized to assess patient and process determinants of 30-d readmissions. 384 patients were included; 30-d readmissions were significantly associated with graft loss and death (p = 0.001). Diabetes (p = 0.049), pharmacist identification of poor understanding or adherence, and prolonged time on hemodialysis prior to transplant were associated with an increased risk of 30-d readmissions. After controlling for risk factors, readmission rates were only independently predicted by pharmacist identification of patient lack of understanding or adherence regarding post-transplant medications and dialysis exposure for more than three yr (OR 2.3, 95% CI 1.10-4.71, p = 0.026 and OR 2.1, 95% CI 1.22, 3.70, respectively), both of which were significantly modified by history of diabetes. Thirty-d readmissions are attributable to both patient and process-level factors. These data suggest that a lack of post-transplant medication knowledge in high-risk patients drives early hospital readmission.

Background Diabetes mellitus (DM) is associated with increased post-operative complications in various surgeries. Little data exist regarding the impact of long-standing DM (>25 years) on outcomes in pancreas transplantation (PTX). The objectives of our study were to determine if long-standing pre-transplant DM (>25 years) was associated with inferior outcomes following PTX. Methods Using a 13-year (April, 2000-May, 2012) retrospective analysis, we examined demographic and transplant factors, complications, and outcomes in patients without (Group A) and with (Group B) long-standing (>25 years) pre-PTX DM. Results Mean follow-up was 4.2 years. Of 214 consecutive PTX performed, 137 (105 simultaneous PTX (SPK), 25 PTX after kidney (PAK), 7 PTX alone (PTA)) had pre-PTX duration of DM recorded, including 65 in Group A and 72 in Group B. There were no differences between cohorts with respect to demographics. There were no differences in post-PTX surgical/medical complications. There were no differences in outcomes between cohorts (ie, rejection, graft loss or death). Conclusions This large-scale analysis demonstrated that PTX can be performed in patients with long-standing DM with excellent patient and graft outcomes. Long-standing DM did not lead to an increased post-PTX infections or complications. Our study suggests that duration of DM should not impact PTX candidacy.

Purpose Microvascular endothelial cells (mECs) are central to promoting recipient T-cell responses by acting as semi-professional antigen-presenting cells. Alterations to mECs as a consequence of brain death, organ preservation, and ischemia reperfusion injury act to further increase mEC immunogenicity. Recent studies have demonstrated that altering mitochondrial fusion/fission can change the phenotype of T cells and dendritic cells. However, whether this has an impact on mECs has not been described. Therefore, in the present study, we sought to modulate mitochondrial fusion/fission in donor mECs prior to transplantation and assessed the in-vitro and in-vivo impacts on mEC-T cell interaction. Methods The functional consequence of mEC mitochondrial fusion on T-cell responses was investigated in a co-culture system, whereby mECs were pre-treated with M1, a fusion promoter, and Mdivi1, a fission inhibitor, to promote mitochondrial fusion prior to being co-cultured with allogeneic CD8+ T-cells for 7 days. T-cell granzyme B, interferon gamma, and mEC surface proteins were assessed after co-culturing. In addition, Balb/c brain-dead donor hearts were pre-treated with M1/Mdivi1 and were then heterotopically transplanted into C57BL/6 recipients. In-vivo donor mEC mitochondrial fusion was validated by confocal microscopy at the end of donor pre-treatment, and survival rates of the transplanted hearts were determined. Results In vitro, pre-treating mECs with M1/Mdivi1 significantly decreased granzyme B and interferon gamma release from co-cultured CD8+ T-cells. Treatment with M1/Mdivi1 led to decreases in VCAM-1 and ICAM-1 and an increase in PD-L1 EC surface expression. Pre-treating donor hearts with M1/Mdivi1 elongated mEC mitochondria in vivo and resulted in a significant improvement in cardiac allograft survival post transplantation (with a median of 9.5 days in M1/Mdivi1-pretreated group vs 6 days in control group, n=4, p=0.0091). Conclusion Promoting mitochondrial fusion/inhibiting fission in donor mEC induces immunoprotective effects both in vitro and in vivo in cardiac transplantation.

Background Allograft arteriopathy is still a leading cause of late organ failure. The aortic allograft model in mice has been used to study chronic rejection and has given useful information in the development of graft arteriosclerosis. However, the technical difficulties of small vessel anastomoses still continue to limit its widespread use. We introduce a new simple method for aortic transplantation in mice. Methods The descending aorta or infrarenal aorta from the donor mouse was anastomosed to the infrarenal aorta using a cuff technique. Aortic transplantation was performed in 30 mice, 10 isografts and 20 allografts. No immunosuppression was administered, and the recipients were sacrificed at day 28. The grafts were histologically analyzed. Results Implantation of grafts could be completed in an average of 23 min. There was no technical failure in all 60 anastomoses. The overall survival rate was 93.3%. Histology of aortas revealed typical aspects of chronic rejection in the allografts at day 28. No significant lesion was observed in isografts. Conclusions We have developed an innovative, stable, and simple aortic transplantation model in mice, which is useful for vascular research in transplantation and beyond.

Over the past four decades, advances in the technology supporting solid organ transplantation have been remarkable. Transplantation is now entering a new era where multidisciplinary approaches, including the use of bioengineering, are being utilized in the pursuit of perfection for the field. In this review article, we will introduce and recap two broad categories that are on the verge of revolutionizing the utilization of donor organs and delivery of immunosuppression, metabolic additives, and gene therapies. Machine perfusion techniques and nanotechnology are areas of significant interest in the transplantation community and make up the next generation of bench to bedside research endeavors. In this review, we will summarize the progress made in the field of machine perfusion and nanotechnology as it applies to liver transplantation.

Undifferentiated embryonal sarcoma of the liver (UESL) is a rare but aggressive pediatric tumor. Historically, treatment for UESL has consisted of combination therapy with surgical resection and chemotherapy. In some cases, surgical resection is not appropriate due to limiting factors such as tumor size, location, and lack of response to treatment. Orthotopic liver transplant (OLT) has been suggested as a promising alternative in these cases. We present a case of an 8 year-old boy with a large undifferentiated embryonal sarcoma of the liver who experienced disease progression after initial neoadjuvant chemotherapy. His tumor was considered unresectable after internal tumor board review due to its size and significant vascularity, ultimately making OLT the only curative option for this patient. Keywords: Undifferentiated embryonal sarcoma of the liver, Pediatric liver tumors, Orthotopic liver transplant, Pediatric liver transplantation

Purpose Donor Brain death (BD) provokes activation of inflammatory pathways resulting in acute lung injury (BD-ALI), characterized by the breakdown of endothelial and epithelial barrier function. These injuries are further propagated in the early transplant period by ischemia reperfusion injury (IRI) which together adversely affect post-transplant outcomes. We have developed and characterized a novel small molecule gap/tight junction stabilizer, aCT1, which we have shown to improve cellular communication and integrity. Herein we explored the use of aCT1 as a pretreatment therapeutic to protect donor lungs from the effects of BD-ALI. Methods A mouse model of BD and orthotopic left lung transplant (LTx) were utilized to examine the effects of aCT1 on barrier function stabilization in BD and subsequent impact on LTx outcomes. Following BD induction donors were nebulized with aCT1 or vehicle and ventilated for a further 3 hours. Lungs were then removed to be flushed with and stored in Perfadex solution at 4°C. After 18 hours of cold storage the right lung was processed for histological analysis and the left lung transplanted into CD45.1 B6 mice. Transplanted lungs were then harvested 6 hours post-Tx for analysis of graft injury via histopathology and protein quantification. For controls a standard living donor (LD) model was used for comparison. Results After 3 hrs of BD induction and 18 hours cold ischemia, lungs had BD-ALI features which included significant increases in immune cell infiltration, edema, and endothelial activation. Treatment with aCT1 had no impact on BD-ALI features. 6 hrs post-Tx lungs from BD donors had significantly increased IRI as compared to LD controls, as determined by increased immune cell infiltration and pathological assessment of injury. Treatment of BD donors with aCT1 significantly reduced this BD injury profile, with pathological injury not dissimilar to that seen in LD controls (BD+aCT1 3 vs. BD 8.25, BD+aCT1 3 vs. LD 3, respectively. p Conclusion These data support gap junction stabilization as a viable option for protecting donor lungs against BD-ALI exacerbated ischemia reperfusion injury.

Purpose Lung transplant (LTx) recipients with significant cigarette smoke (CS) exposure histories continue to have poorer long-term outcomes. We utilized a systems biology approach to analyze a novel murine model of allogeneic LTx into CS exposed recipients. Through in silico modeling, we sought to identify therapeutic targets that could modulate post-Tx outcomes in the CS exposed patient population. Methods C57Bl/6 mice were either chronically exposed to CS or room air (NS) prior to receiving an orthotopic left LTx from non-smoke Balb/c age matched controls. Lungs were harvested at 7 days post-LTx, after which histological, nanostring mRNA immune profiling, and high dimensional mass cytometry (CyTOF) assays were performed. Results We demonstrate that acute rejection is exacerbated in CS recipients as compared to NS controls. Principle component analysis of nanostring data revealed significant differences IL17A, CTLA4, IL6, and IL4 pathways in CS vs NS. In silico predictive modeling suggested this phenotype could be reversed by the PI3K pathway inhibitor LY294002 (LY). Therapy of CS exposed mice with LY restored the CS recipient to a rejection profile in keeping with that seen in NS recipients. CyTOF and flow cytometry revealed significant alterations in splenic CD8+ central memory (CM) versus effector memory (EM) ratios, with EM predominating in CS mice pre-LTx, as compared to NS. Treatment with LY differentially restored the CM:EM ratio, in vitro and in vivo, in CS samples, and was associated with reversing the rejection phenotype in LY-treated CS recipients to that seen in NS controls. Finally, reconstitution of ex vivo-treated and -untreated CD8+ cells into CD8 KO mice prior to LTx replicated our observed rejection patterns, further implicating the CD8+ population as a key regulator of CS related alloimmunity. Conclusion Pre-transplant CS exposure, particularly in COPD and IPF patients, predisposes to poorer post-LTx outcomes. Using a clinically relevant LTx system and in silico modeling we have identified PI3K inhibition as a novel therapeutic approach predicted to be efficacious in CS exposed LTx recipients.

Acute liver failure (ALF) is a rare clinical syndrome associated with a high case fatality rate. Asymptomatic primary infection with Epstein-Barr virus (EBV) is common in the general population while acute hepatitis and jaundice are much less common and ALF has been rarely reported. We reviewed the presenting features as well as clinical outcomes amongst consecutive adults with EBV-related ALF.Amongst the 1,887 adult ALF patients enrolled into the US ALF Study Group from January 1998 to February 2012, there were four patients (0.21 %) with EBV-related ALF. Diagnostic criteria for acute EBV infection included compatible serologies and/or the detection of EBV-encoded RNA (EBER) in liver tissue.Median patient age was 30 years (range 18-44); 75 % were male, and only 25 % were immunosuppressed. The median presenting ALT was 504 IU/mL (range 156-4,920), median Alk P was 431 (range 136-1,009), and median bilirubin was 17 mg/dL (range 13-22.1). Liver biopsy findings ranged from cholestasis to submassive necrosis with EBER + staining in two of the three samples tested. Although all of the patients were treated with an antiviral agent, two died of ALF, one underwent liver transplantation (LT) and one survived with supportive care and is well at 5 years. A review of the literature identified four additional LT recipients with favorable long-term outcomes.Primary EBV infection accounts for1 % of consecutive adult ALF cases but is associated with a high case fatality rate. LT is associated with favorable short- and long-term outcomes.

Background Human regulatory T cells (Treg) offer an attractive adjunctive therapy to reduce current reliance on lifelong, nonspecific immunosuppression after transplantation. Here, we evaluated the ability of ex vivo expanded human Treg to prevent the rejection of islets of Langerhans in a humanized mouse model and examined the mechanisms involved. Methods We engrafted human pancreatic islets of Langerhans into the renal subcapsular space of immunodeficient BALB/c.rag2−/−.cγ−/− mice, previously rendered diabetic via injection of the β-cell toxin streptozocin. After the establishment of stable euglycemia, mice were reconstituted with allogeneic human peripheral blood mononuclear cells (PBMC) and the resultant alloreactive response studied. Ex vivo expanded CD25highCD4+ human Treg, which expressed FoxP3, CTLA-4, and CD62L and remained CD127low, were then cotransferred together with human PBMC and islet allografts and monitored for evidence of rejection. Results Human islets transplanted into diabetic immunodeficient mice reversed diabetes but were rejected rapidly after the mice were reconstituted with allogeneic human PBMC. Cotransfer of purified, ex vivo expanded human Treg prolonged islet allograft survival resulting in the accumulation of Treg in the peripheral lymphoid tissue and suppression of proliferation and interferon-γ production by T cells. In vitro, Treg suppressed activation of signal transducers and activators of transcription and inhibited the effector differentiation of responder T cells. Conclusions Ex vivo expanded Treg retain regulatory activity in vivo, can protect a human islet allograft from rejection by suppressing signal transducers and activators of transcription activation and inhibiting T-cell differentiation, and have clinical potential as an adjunctive cellular therapy., Supplemental digital content is available in the article.

ObjectivesSteroids are a mainstay of treatment in orthotopic liver transplantation (OLT) and are associated with significant morbidity. This trial was conducted to assess the efficacy of steroids avoidance.MethodsPatients undergoing OLT between June 2002 and April 2005 were entered into a prospective, randomized trial of complete steroids avoidance and followed until November 2011. Recipients received either standard therapy (n = 50) or complete steroids avoidance (n = 50). Analyses were performed on an intention‐to‐treat basis. The mean follow‐up of all recipients was 2095 ± 117 days. Sixteen (32%) recipients randomized to the steroids avoidance group ultimately received steroids for clinical indications.ResultsIncidences of diabetes and hypertension prior to or after OLT were similar in both groups, as was the incidence of rejection. Patient and graft survival rates at 1, 3 and 5 years were lower in the steroids avoidance group than in the standard therapy group (patient survival: 1‐year, 80% versus 86%; 3‐year, 68% versus 76%; 5‐year, 60% versus 72%; graft survival: 1‐year, 76% versus 76%; 3‐year, 64% versus 74%; 5‐year, 56% versus 72%), but the differences were not statistically different.conclusionsComplete steroids avoidance provides liver transplant recipients with minimal benefit and appears to result in a concerning trend towards decreased graft and recipient survival. The present data support the use of at least a short course of steroids after liver transplantation.

BACKGROUND: Composite tissue transplantation effectively reconstructs the most complex defects, but its use is limited because of harmful immunosuppression and the high susceptibility of skin to rejection. Development of tolerance is an ideal solution, and protocols using regulatory T cells (Tregs) to achieve this have been promising in experimental animal models. The aim of this study was to investigate the ability of human Tregs to regulate immune responses to a human skin allograft in vivo. METHODS: We isolated and expanded naturally occurring CD127loCD25+CD4+ human Tregs from peripheral blood mononuclear cells (PBMCs) and examined their phenotype and suppressive activity in vitro. Using a clinically relevant chimeric humanized mouse system, we transplanted mice with human skin grafts followed by allogeneic populations of PBMCs with or without Tregs derived from the same PBMC donor. RESULTS: Ex vivo-expanded Tregs maintain the appropriate Treg markers and retain suppressive activity against allostimulated and polyclonally stimulated autologous PBMCs in vitro. Mice receiving allogeneic PBMCs alone consistently reject human skin grafts, whereas those also receiving Tregs display stable long-term human skin transplant survival along with a reduction in the CD8+ human cellular graft infiltrate. CONCLUSIONS: We show for the first time the unique ability of human Tregs to prevent the rejection of a skin allograft in vivo, highlighting the therapeutic potential of these cells clinically.

In this study, we developed a stable, nontoxic novel micelle nanoparticle to attenuate responses of endothelial cell (EC) inflammation when subjected to oxidative stress, such as observed in organ transplantation. Targeted Rapamycin Micelles (TRaM) were synthesized using PEG-PE-amine and N-palmitoyl homocysteine (PHC) with further tailoring of the micelle using targeting peptides (cRGD) and labeling with far-red fluorescent dye for tracking during cellular uptake studies. Our results revealed that the TRaM was approximately 10 nm in diameter and underwent successful internalization in Human Umbilical Vein EC (HUVEC) lines. Uptake efficiency of TRaM nanoparticles was improved with the addition of a targeting moiety. In addition, our TRaM therapy was able to downregulate both mouse cardiac endothelial cell (MCEC) and HUVEC production and release of the pro-inflammatory cytokines, IL-6 and IL-8 in normal oxygen tension and hypoxic conditions. We were also able to demonstrate a dose-dependent uptake of TRaM therapy into biologic tissues ex vivo. Taken together, these data demonstrate the feasibility of targeted drug delivery in transplantation, which has the potential for conferring local immunosuppressive effects without systemic consequences while also dampening endothelial cell injury responses.

The number of patients requiring organ transplants still outpaces the number of available transplantable organs. During the process of orthotopic liver transplantation (OLTx), donor organs undergo significant stress resulting from ischemia and reperfusion. Healthy organs respond to this stressful environment with compensatory mechanisms that ideally allow for complete recovery. However, "marginal" organs do not compensate as well. Hepatic steatosis typically renders an organ nontransplantable; a liver with 30% or more fat has a 25% chance of primary nonfunction (PNF) or graft failure after a technically sound operation. In this study, we report on the significant markers of cellular ultrastructural change in steatotic livers. These include glycogen content, mitochondrial swelling, and hepatocellular blebbing. The data disclosed here argue that further investigation of these factors in marginal organs subjected to I/R may better facilitate our understanding of PNF.

Regulatory T cells (T(regs)) manipulated ex vivo have potential as cellular therapeutics in autoimmunity and transplantation. Although it is possible to expand naturally occurring T(regs), an attractive alternative possibility, particularly suited to solid organ and bone marrow transplantation, is the stimulation of total T cell populations with defined allogeneic antigen-presenting cells (APCs) under conditions that lead to the generation or expansion of donor-reactive, adaptive T(regs). Here we demonstrate that stimulation of mouse CD4(+) T cells by immature allogeneic dendritic cells combined with pharmacological inhibition of phosphodiesterase 3 (PDE) resulted in a functional enrichment of Foxp3(+) T cells. Without further manipulation or selection, the resultant population delayed skin allograft rejection mediated by polyclonal CD4(+) effectors or donor-reactive CD8(+) T cell receptor transgenic T cells and inhibited both effector cell proliferation and T cell priming for interferon-γ production. Notably, PDE inhibition also enhanced the enrichment of human Foxp3(+) CD4(+) T cells driven by allogeneic APCs. These cells inhibited T cell proliferation in a standard in vitro mixed lymphocyte assay and, moreover, attenuated the development of vasculopathy mediated by autologous peripheral blood mononuclear cells in a functionally relevant humanized mouse transplant model. These data establish a method for the ex vivo generation of graft-reactive, functional mouse and human T(regs) that uses a clinically approved agent, making pharmacological PDE inhibition a potential strategy for T(reg)-based therapies.