Early allogeneic immune modulation after establishment of donor hematopoietic cell-induced mixed chimerism in a nonhuman primate kidney transplant model.

Background: Mixed lymphohematopoietic chimerism is a proven strategy for achieving operational transplant tolerance, though the underlying immunologic mechanisms are incompletely understood. Methods: A post-transplant, non-myeloablative, tomotherapy-based total lymphoid (TLI) irradiation protocol combined with anti-thymocyte globulin and T cell co-stimulatory blockade (belatacept) induction was applied to a 3-5 MHC antigen mismatched rhesus macaque kidney and hematopoietic cell transplant model. Mechanistic investigations of early (60 days post-transplant) allogeneic immune modulation induced by mixed chimerism were conducted. Results: Chimeric animals demonstrated expansion of circulating and graft-infiltrating CD4+CD25+Foxp3+ regulatory T cells (Tregs), as well as increased differentiation of allo-protective CD8+ T cell phenotypes compared to naïve and non-chimeric animals. In vitro mixed lymphocyte reaction (MLR) responses and donor-specific antibody production were suppressed in animals with mixed chimerism. PD-1 upregulation was observed among CD8+ T effector memory (CD28-CD95+) subsets in chimeric hosts only. PD-1 blockade in donor-specific functional assays augmented MLR and cytotoxic responses and was associated with increased intracellular granzyme B and extracellular IFN-γ production. Conclusions: These studies demonstrated that donor immune cell engraftment was associated with early immunomodulation via mechanisms of homeostatic expansion of Tregs and early PD-1 upregulation among CD8+ T effector memory cells. These responses may contribute to TLI-based mixed chimerism-induced allogenic tolerance.

Strategies to Improve the Utilization and Function of DCD Livers.

Despite the increased usage of livers from donation after circulatory death (DCD) donors in the last decade, many patients remaining on the waitlist who need a liver transplant. Recent efforts have focused on maximizing the utilization and outcomes of these allografts using advances in machine perfusion technology and other perioperative strategies such as normothermic regional perfusion (NRP). In addition to the standard donor and recipient matching that is required with DCD donation, new data regarding the impact of graft steatosis, extensive European experience with NRP, and the increasing use of normothermic and hypothermic machine perfusion have shown immense potential in increasing DCD organ overall utilization and improved outcomes. These techniques, along with viability testing of extended criteria donors, have generated early promising data to consider the use of higher-risk donor organs and more widespread adoption of these techniques in the United States. This review explores the most recent international literature regarding strategies to optimize the utilization and outcomes of DCD liver allografts, including donor-recipient matching, perioperative strategies including NRP versus rapid controlled DCD recovery, viability assessment of discarded livers, and postoperative strategies including machine perfusion versus pharmacologic interventions.

CD28-Selective Inhibition Prolongs Non-Human Primate Kidney Transplant Survival.

Costimulation blockade using belatacept results in improved renal function after kidney transplant as well as decreased likelihood of death/graft loss and reduced cardiovascular risk; however, higher rates and grades of acute rejection have prevented its widespread clinical adoption. Treatment with belatacept blocks both positive (CD28) and negative (CTLA-4) T cell signaling. CD28-selective therapies may offer improved potency by blocking CD28-mediated costimulation while leaving CTLA-4 mediated coinhibitory signals intact. Here we test a novel domain antibody directed at CD28 (anti-CD28 dAb (BMS-931699)) in a non-human primate kidney transplant model. Sixteen macaques underwent native nephrectomy and received life-sustaining renal allotransplantation from an MHC-mismatched donor. Animals were treated with belatacept alone, anti-CD28 dAb alone, or anti-CD28 dAb plus clinically relevant maintenance (MMF, Steroids) and induction therapy with either anti-IL-2R or T cell depletion. Treatment with anti-CD28 dAb extended survival compared to belatacept monotherapy (MST 187 vs. 29 days, p=0.07). The combination of anti-CD28 dAb and conventional immunosuppression further prolonged survival to MST ∼270 days. Animals maintained protective immunity with no significant infectious issues. These data demonstrate CD28-directed therapy is a safe and effective next-generation costimulatory blockade strategy with a demonstrated survival benefit and presumed advantage over belatacept by maintaining intact CTLA-4 coinhibitory signaling.

Liver Transplantation as a New Standard of Care in Patients With Perihilar Cholangiocarcinoma? Results From an International Benchmark Study.

OBJECTIVE: To define benchmark values for liver transplantation (LT) in patients with perihilar cholangiocarcinoma (PHC) enabling unbiased comparisons. BACKGROUND: Transplantation for PHC is used with reluctance in many centers and even contraindicated in several countries. Although benchmark values for LT are available, there is a lack of specific data on LT performed for PHC. METHODS: PHC patients considered for LT after Mayo-like protocol were analyzed in 17 reference centers in 2 continents over the recent 5-year period (2014-2018). The minimum follow-up was 1 year. Benchmark patients were defined as operated at high-volume centers (≥50 overall LT/year) after neoadjuvant chemoradiotherapy, with a tumor diameter <3 cm, negative lymph nodes, and with the absence of relevant comorbidities. Benchmark cutoff values were derived from the 75th to 25th percentiles of the median values of all benchmark centers. RESULTS: One hundred thirty-four consecutive patients underwent LT after completion of the neoadjuvant treatment. Of those, 89.6% qualified as benchmark cases. Benchmark cutoffs were 90-day mortality ≤5.2%; comprehensive complication index at 1 year of ≤33.7; grade ≥3 complication rates ≤66.7%. These values were better than benchmark values for other indications of LT. Five-year disease-free survival was largely superior compared with a matched group of nodal negative patients undergoing curative liver resection (n=106) (62% vs 32%, P <0.001). CONCLUSION: This multicenter benchmark study demonstrates that LT offers excellent outcomes with superior oncological results in early stage PHC patients, even in candidates for surgery. This provocative observation should lead to a change in available therapeutic algorithms for PHC.

Progress in kidney transplantation: The role for systems immunology.

The development of systems biology represents an immense breakthrough in our ability to perform translational research and deliver personalized and precision medicine. A multidisciplinary approach in combination with use of novel techniques allows for the extraction and analysis of vast quantities of data even from the volume and source limited samples that can be obtained from human subjects. Continued advances in microfluidics, scalability and affordability of sequencing technologies, and development of data analysis tools have made the application of a multi-omics, or systems, approach more accessible for use outside of specialized centers. The study of alloimmune and protective immune responses after solid organ transplant offers innumerable opportunities for a multi-omics approach, however, transplant immunology labs are only just beginning to adopt the systems methodology. In this review, we focus on advances in biological techniques and how they are improving our understanding of the immune system and its interactions, highlighting potential applications in transplant immunology. First, we describe the techniques that are available, with emphasis on major advances that allow for increased scalability. Then, we review initial applications in the field of transplantation with a focus on topics that are nearing clinical integration. Finally, we examine major barriers to adapting these methods and discuss potential future developments.

Optimal Timing of Administration of Direct-acting Antivirals for Patients With Hepatitis C-associated Hepatocellular Carcinoma Undergoing Liver Transplantation.

OBJECTIVE: To investigate the optimal timing of direct acting antiviral (DAA) administration in patients with hepatitis C-associated hepatocellular carcinoma (HCC) undergoing liver transplantation (LT). SUMMARY OF BACKGROUND DATA: In patients with hepatitis C (HCV) associated HCC undergoing LT, the optimal timing of direct-acting antivirals (DAA) administration to achieve sustained virologic response (SVR) and improved oncologic outcomes remains a topic of much debate. METHODS: The United States HCC LT Consortium (2015-2019) was reviewed for patients with primary HCV-associated HCC who underwent LT and received DAA therapy at 20 institutions. Primary outcomes were SVR and HCC recurrence-free survival (RFS). RESULTS: Of 857 patients, 725 were within Milan criteria. SVR was associated with improved 5-year RFS (92% vs 77%, P < 0.01). Patients who received DAAs pre-LT, 0-3 months post-LT, and ≥3 months post-LT had SVR rates of 91%, 92%, and 82%, and 5-year RFS of 93%, 94%, and 87%, respectively. Among 427 HCV treatment-naïve patients (no previous interferon therapy), patients who achieved SVR with DAAs had improved 5-year RFS (93% vs 76%, P < 0.01). Patients who received DAAs pre-LT, 0-3 months post-LT, and ≥3 months post-LT had SVR rates of 91%, 93%, and 78% (P < 0.01) and 5-year RFS of 93%, 100%, and 83% (P = 0.01). CONCLUSIONS: The optimal timing of DAA therapy appears to be 0 to 3 months after LT for HCV-associated HCC, given increased rates of SVR and improved RFS. Delayed administration after transplant should be avoided. A prospective randomized controlled trial is warranted to validate these results.

A longitudinal cadaver-based simulation curriculum creates sustainable increases in resident confidence and exposure to fundamental techniques: Results of a 5-year program evaluation.

INTRODUCTION: For the past five years, our surgical residency program has led a cadaver-based simulation course focused on fundamental surgical maneuvers. This study aimed to quantify the impact of this course on resident exposure to surgical skills and longitudinal impact on resident education. METHODS: General surgery residents participated in an annual cadaver-based simulation curriculum. Participants completed surveys regarding improvements in knowledge and confidence; these results were stratified between course iterations (P1: 2 years, 2014-15; P2: 3 years, 2016-2018). RESULTS: Residents reported a sustained increase in knowledge of anatomy and technical dissection, confidence in performing operative skills independently, and exposure to operative skills that were otherwise not encountered in clinical rotations. Junior residents demonstrated an increase in gaining skills they would otherwise not achieve (87% vs. 98%, p = 0.028) and confidence to safely perform these procedures in the clinical setting (94% vs. 100%, p = 0.077). CONCLUSION: This annual, longitudinal cadaver-based skills course focused on fundamental maneuvers demonstrates a sustained impact in resident and faculty surgical confidence in resident's operative skills as a component of a longitudinal simulation curriculum to enhance competency-based promotion.

Donor-specific antibodies in liver transplantation: challenges in diagnosis and determining clinical impact.

PURPOSE OF REVIEW: Our understanding of the clinical impact of donor-specific antibodies in liver transplant recipients has evolved in recent years as outcomes for liver allografts have improved and advances in diagnostic testing have made recognition of antibody mediated rejection in transplant patients more sensitive. RECENT FINDINGS: Two main types of donor-specific antibodies - preformed and de novo - have been reported in the literature to have a negative impact on graft survival, and researchers have been able to further identify subclasses of class II donor-specific antibodies as being the most clinically impactful. Furthermore, there is evidence that donor-specific antibody formation can augment cellular rejection in liver grafts and lead to worsened clinical outcomes. Recent data have shown a higher prevalence of donor-specific antibody formation than previously reported. SUMMARY: This review explores the most recent literature regarding the clinical impact of both preformed and de-novo donor-specific antibodies and potential management guidelines for patients undergoing liver transplantation. The best practice guidelines for undergoing monitoring for donor-specific antibody formation and protocol biopsies in sensitized patients will depend on further multiinstitutional studies.

"The Living Monument": The Desegregation of Grady Memorial Hospital and the Changing South.

Grady Memorial Hospital is a pillar of public medical and surgical care in the Southeast. The evolution of this institution, both in its physical structure as well as its approach to patient care, mirrors the cultural and social changes that have occurred in the American South. Grady Memorial Hospital opened its doors in 1892 built in the heart of Atlanta's black community. With its separate and unequal facilities and services for black and white patients, the concept of "the Gradies" was born. Virtually, every aspect of care at Grady continued to be segregated by race until the mid-20th century. In 1958, the opening of the "New Grady" further cemented this legacy of the separate "Gradies," with patients segregated by hospital wing. By the 1960s, civil rights activists brought change to Atlanta. The Atlanta Student Movement, with the support of Dr. Martin Luther King Jr., led protests outside of Grady, and a series of judicial and legislative rulings integrated medical boards and public hospitals. Eventually, the desegregation of Grady occurred with a quiet memo that belied years of struggle: on June 1, 1965, a memo from hospital superintendent Bill Pinkston read "All phases of the hospital are on a non-racial basis, effective today." The future of Grady is deeply rooted in its past, and Grady's mission is unchanged from its inception in 1892: "It will nurse the poor and rich alike and will be an asylum for black and white."

Can a Structured, Video-Based Cadaver Curriculum Demonstrating Proficiency Enhance Resident Operative Autonomy?

INTRODUCTION: Autonomy is of foremost concern in the current era of surgical residency, and it is especially important to trainees when considering their surgical education. Factors impacting trainee independence include the restriction of clinical work hours and the development of advanced minimally invasive techniques such as robotics, which requires separate technical education outside of conventional surgical education. Moreover, when residents are left to learn fundamental exposures via their clinical experience alone, they run the risk of not being exposed to some fundamental skills based on case volume and type. The Department of Surgery at Emory University developed a cadaver-based simulation curriculum to standardize exposure to fundamental operative skills and enhance proficiency outside the operating room, with the larger aim of improving resident autonomy. METHODS: Residents were assigned to small groups led by a chief resident with an even distribution of postgraduate year (PGY) levels. Each group participated in core surgical exposures and fundamental maneuvers on a cadaver over a 6-hour session. Residents were tested on skills according to their PGY level about 1 month after the course. Testing included recitation of the skill in an oral boards format, highlighting major steps, followed by performance of the skill. All steps were video-recorded with no resident identifiers. These were reviewed by 2 independent, blinded faculty examiners who assigned proficiency grades to each resident video. RESULTS: Three hundred and thirty-three individual procedure evaluations were done over the 5-year period. Senior residents (PGY3-5) had 86% pass rate while junior residents (PGY1-2) had 70% pass rate. Overall, 21% of residents failed to achieve competence in their assigned skills. Junior residents were less likely to achieve competence compared to senior residents. Faculty graders had improved congruence in grading as the course progressed through the 5 years. The most recent 2 years had >80% congruence in faculty grading compared to less than 50% congruence in the first 2 years. 81% of attendings agreed this course positively influenced the granting of autonomy in the operating room. CONCLUSIONS: A cadaveric skills course focused on fundamental maneuvers with objective confirmation of achieving competency is a viable adjunct to clinical operative experience. Video-recorded evaluation, of these fundamental skills improved both resident and attending confidence in trainee operative skill.

Visceral Kaposi Sarcoma Presenting as Small Bowel Intussusception: A Rare Presentation and Call to Action.

Surgical emergencies related to visceral involvement of Kaposi sarcoma (KS) are rare complications of the disease. In this report, we describe a case of visceral KS causing small bowel intussusception in a young, previously undiagnosed human immunodeficiency virus (HIV)-positive patient. Southern surgeons should be particularly attentive to HIV/AIDS-related disease as a cause of surgical pathology, particularly in the southeast, and can play a significant advocacy role for improved access to HIV/AIDS diagnostic and treatment services.

Long-term survival of pig-to-rhesus macaque renal xenografts is dependent on CD4 T cell depletion.

The shortage of available organs remains the greatest barrier to expanding access to transplant. Despite advances in genetic editing and immunosuppression, survival in experimental models of kidney xenotransplant has generally been limited to <100 days. We found that pretransplant selection of recipients with low titers of anti-pig antibodies significantly improved survival in a pig-to-rhesus macaque kidney transplant model (6 days vs median survival time 235 days). Immunosuppression included transient pan-T cell depletion and an anti-CD154-based maintenance regimen. Selective depletion of CD4+ T cells but not CD8+ T cells resulted in long-term survival (median survival time >400 days vs 6 days). These studies suggested that CD4+ T cells may have a more prominent role in xenograft rejection compared with CD8+ T cells. Although animals that received selective depletion of CD8+ T cells showed signs of early cellular rejection (marked CD4+ infiltrates), animals receiving selective CD4+ depletion exhibited normal biopsy results until late, when signs of chronic antibody rejection were present. In vitro study results suggested that rhesus CD4+ T cells required the presence of SLA class II to mount an effective proliferative response. The combination of low pretransplant anti-pig antibody and CD4 depletion resulted in consistent, long-term xenograft survival.

CD122 signaling in CD8+ memory T cells drives costimulation-independent rejection.

Interrupting T cell costimulatory signals as a strategy to control undesired immune responses, such as occur in autoimmunity or transplantation, has the potential to alleviate many of the unwanted side effects associated with current immunosuppressive therapies. Belatacept, a high-affinity version of CTLA4-Ig that blocks ligand ligation to CD28, has been approved for use in kidney transplant recipients. Despite the long-term benefits associated with its use, such as improved renal function and lower cardiovascular risk, a subset of patients treated with belatacept experience elevated rates of acute T cell-mediated rejection, tempering enthusiasm for its use. Here we demonstrate that costimulation-independent T cell alloreactivity relies on signaling through CD122, the shared IL-2 and IL-15 receptor ß-chain. Combined costimulatory and CD122 blockade improved survival of transplanted tissue in mice and nonhuman primates by controlling proliferation and effector function of CD8+ T cells. The high-affinity IL-2 receptor was dispensable for memory CD8+ T cell responses, whereas signaling through CD122 as a component of the high-affinity IL-15 receptor was critical for costimulation-independent memory CD8+ T cell recall, distinguishing specific roles for IL-2 and IL-15 in T cell activation. These studies outline a novel approach for clinical optimization of costimulatory blockade strategies in transplantation by targeting CD122.

Xenoantigen Deletion and Chemical Immunosuppression Can Prolong Renal Xenograft Survival.

OBJECTIVE: Xenotransplantation using pig organs could end the donor organ shortage for transplantation, but humans have xenoreactive antibodies that cause early graft rejection. Genome editing can eliminate xenoantigens in donor pigs to minimize the impact of these xenoantibodies. Here we determine whether an improved cross-match and chemical immunosuppression could result in prolonged kidney xenograft survival in a pig-to-rhesus preclinical model. METHODS: Double xenoantigen (Gal and Sda) knockout (DKO) pigs were created using CRISPR/Cas. Serum from rhesus monkeys (n = 43) was cross-matched with cells from the DKO pigs. Kidneys from the DKO pigs were transplanted into rhesus monkeys (n = 6) that had the least reactive cross-matches. The rhesus recipients were immunosuppressed with anti-CD4 and anti-CD8 T-cell depletion, anti-CD154, mycophenolic acid, and steroids. RESULTS: Rhesus antibody binding to DKO cells is reduced, but all still have positive CDC and flow cross-match. Three grafts were rejected early at 5, 6, and 6 days. Longer survival was achieved in recipients with survival to 35, 100, and 435 days. Each of the 3 early graft losses was secondary to IgM antibody-mediated rejection. The 435-day graft loss occurred secondary to IgG antibody-mediated rejection. CONCLUSIONS: Reducing xenoantigens in donor pigs and chemical immunosuppression can be used to achieve prolonged renal xenograft survival in a preclinical model, suggesting that if a negative cross-match can be obtained for humans then prolonged survival could be achieved.

Alloimmunity But Not Viral Immunity Promotes Allograft Loss in a Mouse Model of Polyomavirus-Associated Allograft Injury.

BACKGROUND: The interplay between viral infection and alloimmunity is known to influence the fate of transplanted organs. Clarifying how local virus-associated inflammation/injury and antiviral immunity can alter host alloimmune responses in transplantation remains a critical question. METHODS: We used a mouse model of polyomavirus (PyV) infection and kidney transplantation to investigate the roles of direct viral pathology, the antiviral immune response, and alloimmunity in the pathogenesis of PyV-associated allograft injury. We have previously shown that an effective primary T cell response is required in PyV-associated graft injury. RESULTS: Here we show that the transfer of primed antidonor, but not antiviral, T cells results in PyV-associated allograft injury. In further studies, we use a surrogate minor antigen model (ovalbumin) and show that only antidonor specific T cells and not antiviral specific T cells are sufficient to mediate injury. Lastly, we demonstrate that local but not systemic virus-mediated inflammation and injury within the graft itself are required. CONCLUSIONS: These data suggest that in this mouse model, the predominant mechanism of allograft injury in PyV-associated injury is due to an augmented alloimmune T cell response driven by virus-induced inflammation/injury within the graft. These studies highlight the important interplay between viral infection and alloimmunity in a model system.

Cadaver-Based Simulation Increases Resident Confidence, Initial Exposure to Fundamental Techniques, and May Augment Operative Autonomy.

OBJECTIVE: Surgical simulation is an important adjunct in surgical education. The majority of operative procedures can be simplified to core components. This study aimed to quantify a cadaver-based simulation course utility in improving exposure to fundamental maneuvers, resident and attending confidence in trainee capability, and if this led to earlier operative independence. DESIGN: A list of fundamental surgical procedures was established by a faculty panel. Residents were assigned to a group led by a chief resident. Residents performed skills on cadavers appropriate for PGY level. A video-recorded examination where they narrated and demonstrated a task independently was then graded by attendings using standardized rubrics. Participants completed surveys regarding improvements in knowledge and confidence. SETTING: The course was conducted at the Emory University School of Medicine and the T3 Laboratories in Atlanta, GA. PARTICIPANTS: A total of 133 residents and 41 attendings participated in the course. 133 (100%) participating residents and 32 (78%) attendings completed surveys. RESULTS: Resident confidence in completing the assigned skill independently increased from 3 (2-3) to 4 (3-4), p < 0.01. Residents stated that a median of 40% (interquartile range: 20%-60%) of procedures were performed for the first time in the course, and the same number had been performed only in the course. The percentage of skills attendings believed residents could perform independently increased from 40% (40%-60%) to 60% (60%->80%), p < 0.04. Attendings were more likely to grant autonomy in the operating room after this exercise (4 [3-5]). CONCLUSIONS: A cadaveric skills course focused on fundamental maneuvers with objective confirmation of success is a viable adjunct to clinical operative experience. Residents were formally exposed to fundamental surgical maneuvers earlier as a result of this course. This activity improved both resident and attending confidence in trainee operative skill, resulting in increased attending willingness to grant a higher level of autonomy in the operating room.

Urine proteomics in kidney transplantation.

The transplanted kidney, through its urinary output, provides a medium through which the molecular constitution can provide insight into either the healthy function or developing dysfunction of a newly transplanted organ. An assay that would detect the aberration of early biomarkers of allograft injury using only urine samples from patients would provide many advantages over the current use of creatinine and tissue biopsies, as these means are either relatively non-specific or very invasive. Several urine biomarkers have been correlated with allograft injury, including CXCL9, CXCL10, CCL2, NGAL, IL-18, cystatin C, KIM-1 and Tim-3. The recent results of the CTOT-01 trial serve to validate the predictive value of the CXCL9 biomarker as a non-invasive biomarker for rejection and a prognostic indicator of graft function. There is now a preponderance of evidence showing a value of urinary monitoring of CXCL9 and CXCL10 with respect to detection of acute kidney allograft rejection. The value of the assay has been validated as a means of reducing the need for kidney transplant biopsy and applying biopsy in a more targeted manner. Additional goals for non-invasive monitoring would include predictive value prior to creatinine elevation that in turn would permit earlier, preemptive treatment of rejection.