RNA expression profiling of nonhuman primate renal allograft rejection identifies tolerance.

Tolerance induction to prevent allograft rejection is a long-standing clinical goal. However, convincing and dependable tolerance identification remains elusive. Hypothesizing that intragraft RNA expression is informative in both rejection and tolerance, we profile intrarenal allograft RNA expression in a mixed chimerism renal allograft model of cynomolgus monkeys and identify biologically significant tolerance. Analysis of 67 genes identified 3 dominant factors, each with a different pattern of gene expressions, relating to T cell-mediated rejection (TCMR), chronic antibody-mediated rejection (CAMR), or Tolerance. Clustering these 3 factors created 9 groups. One of the 9 clustered groups, the Tolerance cluster, showed the lowest probability of terminal rejection, the longest duration of allograft survival, and the lowest relative risk of terminal rejection. The Tolerance factor consists of a novel set of gene expressions including cytokine and immunoregulatory genes adding mechanistic insights into tolerance. The Tolerance factor could not be identified within current pathologic diagnostic categories. The TCMR and CAMR factors are dominant to the Tolerance factor, causing rejection even if the Tolerance factor is present. These 3 factors determine the probability of terminal rejection or tolerance. This novel a posteriori approach permits identification of pathways of rejection, including tolerance.

RNA expression profiling of renal allografts in a nonhuman primate identifies variation in NK and endothelial gene expression.

RNA transcript expression estimates are a promising method to study the mechanisms and classification of renal allograft rejections. Here we use the Nanostring platform to profile RNA expression in renal allografts in a nonhuman primate (NHP), the Cynomolgus monkey. We analyzed protocol and indication 278 archival renal allograft samples, both protocol and indication from 76 animals with diagnoses of chronic antibody-mediated rejection (CAMR), acute cellular rejection (TCMR), and MIXED (both CAMR and TCMR), plus normals and samples with no pathological rejection using a Cynomolgus-specific probe set of 67 genes. Analysis identified RNA expression heterogeneity of endothelial and NK genes within CAMR and TCMR, including the stages of CAMR. Three factors were partitioned into additional groups. One group with the longest allograft survival time is pure CAMR without NK or CD3. Three mixed groups show variation in NK and CD3. TCMR was split into 2 groups with variation in NK genes. Additional validation of the complete gene-set correlated many of the genes with diagnoses of CAMR, MIXED, and TCMR rejections and with Banff histologic criteria defined in human subjects. These NHP data demonstrate the utility of RNA expression profiling to identify additional heterogeneity of endothelial and NK RNA gene expressions.

Chronic Antibody-Mediated Rejection in Nonhuman Primate Renal Allografts: Validation of Human Histological and Molecular Phenotypes.

Molecular testing represents a promising adjunct for the diagnosis of antibody-mediated rejection (AMR). Here, we apply a novel gene expression platform in sequential formalin-fixed paraffin-embedded samples from nonhuman primate (NHP) renal transplants. We analyzed 34 previously described gene transcripts related to AMR in humans in 197 archival NHP samples, including 102 from recipients that developed chronic AMR, 80 from recipients without AMR, and 15 normal native nephrectomies. Three endothelial genes (VWF, DARC, and CAV1), derived from 10-fold cross-validation receiver operating characteristic curve analysis, demonstrated excellent discrimination between AMR and non-AMR samples (area under the curve = 0.92). This three-gene set correlated with classic features of AMR, including glomerulitis, capillaritis, glomerulopathy, C4d deposition, and DSAs (r = 0.39-0.63, p < 0.001). Principal component analysis confirmed the association between three-gene set expression and AMR and highlighted the ambiguity of v lesions and ptc lesions between AMR and T cell-mediated rejection (TCMR). Elevated three-gene set expression corresponded with the development of immunopathological evidence of rejection and often preceded it. Many recipients demonstrated mixed AMR and TCMR, suggesting that this represents the natural pattern of rejection. These data provide NHP animal model validation of recent updates to the Banff classification including the assessment of molecular markers for diagnosing AMR.

Immunosuppression With CD40 Costimulatory Blockade Plus Rapamycin for Simultaneous Islet-Kidney Transplantation in Nonhuman Primates.

The lack of a reliable immunosuppressive regimen that effectively suppresses both renal and islet allograft rejection without islet toxicity hampers a wider clinical application of simultaneous islet-kidney transplantation (SIK). Seven MHC-mismatched SIKs were performed in diabetic cynomolgus monkeys. Two recipients received rabbit antithymocyte globulin (ATG) induction followed by daily tacrolimus and rapamycin (ATG/Tac/Rapa), and five recipients were treated with anti-CD40 monoclonal antibody (mAb) and rapamycin (aCD40/Rapa). Anti-inflammatory therapy, including anti-interleukin-6 receptor mAb and anti-tumor necrosis factor-α mAb, was given in both groups. The ATG/Tac/Rapa recipients failed to achieve long-term islet allograft survival (19 and 26 days) due to poor islet engraftment and cytomegalovirus pneumonia. In contrast, the aCD40/Rapa regimen provided long-term islet and kidney allograft survival (90, 94, >120, >120, and >120 days), with only one recipient developing evidence of allograft rejection. The aCD40/Rapa regimen was also tested in four kidney-alone transplant recipients. All four recipients achieved long-term renal allograft survival (100% at day 120), which was superior to renal allograft survival (62.9% at day 120) with triple immunosuppressive regimen (tacrolimus, mycophenolate mofetil, and steroids). The combination of anti-CD40 mAb and rapamycin is an effective and nontoxic immunosuppressive regimen that uses only clinically available agents for kidney and islet recipients.

The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions.

The 13th Banff Conference on Allograft Pathology was held in Vancouver, British Columbia, Canada from October 5 to 10, 2015. The cardiac session was devoted to current diagnostic issues in heart transplantation with a focus on antibody-mediated rejection (AMR) and small vessel arteriopathy. Specific topics included the strengths and limitations of the current rejection grading system, the central role of microvascular injury in AMR and approaches to semiquantitative assessment of histopathologic and immunophenotypic indicators, the role of AMR in the development of cardiac allograft vasculopathy, the important role of serologic antibody detection in the management of transplant recipients, and the potential application of new molecular approaches to the elucidation of the pathophysiology of AMR and potential for improving the current diagnostic system. Herein we summarize the key points from the presentations, the comprehensive, open and wide-ranging multidisciplinary discussion that was generated, and considerations for future endeavors.

Testing the Efficacy of Contrast-Enhanced Ultrasound in Detecting Transplant Rejection Using a Murine Model of Heart Transplantation.

One of the key unmet needs to improve long-term outcomes of heart transplantation is to develop accurate, noninvasive, and practical diagnostic tools to detect transplant rejection. Early intragraft inflammation and endothelial cell injuries occur prior to advanced transplant rejection. We developed a novel diagnostic imaging platform to detect early declines in microvascular perfusion (MP) of cardiac transplants using contrast-enhanced ultrasonography (CEUS). The efficacy of CEUS in detecting transplant rejection was tested in a murine model of heart transplants, a standard preclinical model of solid organ transplant. As compared to the syngeneic groups, a progressive decline in MP was demonstrated in the allografts undergoing acute transplant rejection (40%, 64%, and 92% on days 4, 6, and 8 posttransplantation, respectively) and chronic rejection (33%, 33%, and 92% on days 5, 14, and 30 posttransplantation, respectively). Our perfusion studies showed restoration of MP following antirejection therapy, highlighting its potential to help monitor efficacy of antirejection therapy. Our data suggest that early endothelial cell injury and platelet aggregation contributed to the early MP decline observed in the allografts. High-resolution MP mapping may allow for noninvasive detection of heart transplant rejection. The data presented have the potential to help in the development of next-generation imaging approaches to diagnose transplant rejection.

Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

Long-term lung transplantation in nonhuman primates.

Despite advances in surgical technique and clinical care, lung transplantation still remains a short-term solution for the treatment of end-stage lung disease. To date, there has been limited experience in experimental lung transplantation using nonhuman primate models. Therefore, we have endeavored to develop a long-term, nonhuman primate model of orthotopic lung transplantation for the ultimate purpose of designing protocols to induce tolerance of lung grafts. Here, we report our initial results in developing this model and our observation that the nonhuman primate lung is particularly prone to rejection. This propensity toward rejection may be a consequence of 1) upregulated nonspecific inflammation, and 2) a larger number of pre-existing alloreactive memory T cells, leading to augmented deleterious immune responses. Our data show that triple-drug immunosuppression mimicking clinical practice is not sufficient to prevent acute rejection in nonhuman primate lung transplantation. The addition of horse-derived anti-thymocyte globulin and a monoclonal antibody to the IL-6 receptor allowed six out of six lung recipients to be free of rejection for over 120 days.

Tolerance of Lung Allografts Achieved in Nonhuman Primates via Mixed Hematopoietic Chimerism.

While the induction of transient mixed chimerism has tolerized MHC-mismatched renal grafts in nonhuman primates and patients, this approach has not been successful for more immunogenic organs. Here, we describe a modified delayed-tolerance-induction protocol resulting in three out of four monkeys achieving long-term lung allograft survival without ongoing immunosuppression. Two of the tolerant monkeys displayed stable mixed lymphoid chimerism, and the other showed transient chimerism. Serial biopsies and post-mortem specimens from the tolerant monkeys revealed no signs of chronic rejection. The tolerant recipients also exhibited T cell unresponsiveness and a lack of alloantibody. This is the first report of durable mixed chimerism and successful tolerance induction of MHC-mismatched lungs in primates.

Pilot Study Evaluating Regulatory T Cell-Promoting Immunosuppression and Nonimmunogenic Donor Antigen Delivery in a Nonhuman Primate Islet Allotransplantation Model.

The full potential of islet transplantation will only be realized through the development of tolerogenic regimens that obviate the need for maintenance immunosuppression. Here, we report an immunotherapy regimen that combines 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI)-treated donor lymphoid cell infusion (ECDI-DLI) with thymoglobulin, anti-interleukin-6 receptor antibody and rapamycin to achieve prolonged allogeneic islet graft survival in a nonhuman primate (NHP) model. Prolonged graft survival is associated with Treg expansion, donor-specific T cell hyporesponsiveness and a transient absence of donor-specific alloantibody production during the period of graft survival. This regimen shows promise for clinical translation.

Use of CTLA4Ig for induction of mixed chimerism and renal allograft tolerance in nonhuman primates.

We have previously reported successful induction of renal allograft tolerance via a mixed chimerism approach in nonhuman primates. In those studies, we found that costimulatory blockade with anti-CD154 mAb was an effective adjunctive therapy for induction of renal allograft tolerance. However, since anti-CD154 mAb is not clinically available, we have evaluated CTLA4Ig as an alternative agent for effecting costimulation blockade in this treatment protocol. Two CTLA4Igs, abatacept and belatacept, were substituted for anti-CD154 mAb in the conditioning regimen (low dose total body irradiation, thymic irradiation, anti-thymocyte globulin and a 1-month posttransplant course of cyclosporine [CyA]). Three recipients treated with the abatacept regimen failed to develop comparable lymphoid chimerism to that achieved with anti-CD154 mAb treatment and these recipients rejected their kidney allografts early. With the belatacept regimen, four of five recipients developed chimerism and three of these achieved long-term renal allograft survival (>861, >796 and >378 days) without maintenance immunosuppression. Neither chimerism nor long-term allograft survival were achieved in two recipients treated with the belatacept regimen but with a lower, subtherapeutic dose of CyA. This study indicates that CD28/B7 blockade with belatacept can provide a clinically applicable alternative to anti-CD154 mAb for promoting chimerism and renal allograft tolerance.

Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation.

To reduce widespread shortages, attempts are made to use more marginal livers for transplantation. Many of these grafts are discarded for fear of inferior survival rates or biliary complications. Recent advances in organ preservation have shown that ex vivo subnormothermic machine perfusion has the potential to improve preservation and recover marginal livers pretransplantation. To determine the feasibility in human livers, we assessed the effect of 3 h of oxygenated subnormothermic machine perfusion (21°C) on seven livers discarded for transplantation. Biochemical and microscopic assessment revealed minimal injury sustained during perfusion. Improved oxygen uptake (1.30 [1.11-1.94] to 6.74 [4.15-8.16] mL O2 /min kg liver), lactate levels (4.04 [3.70-5.99] to 2.29 [1.20-3.43] mmol/L) and adenosine triphosphate content (45.0 [70.6-87.5] pmol/mg preperfusion to 167.5 [151.5-237.2] pmol/mg after perfusion) were observed. Liver function, reflected by urea, albumin and bile production, was seen during perfusion. Bile production increased and the composition of bile (bile salts/phospholipid ratio, pH and bicarbonate concentration) became more favorable. In conclusion, ex vivo subnormothermic machine perfusion effectively maintains liver function with minimal injury and sustains or improves various hepatobiliary parameters postischemia.

Long-term results in recipients of combined HLA-mismatched kidney and bone marrow transplantation without maintenance immunosuppression.

We report here the long-term results of HLA-mismatched kidney transplantation without maintenance immunosuppression (IS) in 10 subjects following combined kidney and bone marrow transplantation. All subjects were treated with nonmyeloablative conditioning and an 8- to 14-month course of calcineurin inhibitor with or without rituximab. All 10 subjects developed transient chimerism, and in seven of these, IS was successfully discontinued for 4 or more years. Currently, four subjects remain IS free for periods of 4.5-11.4 years, while three required reinstitution of IS after 5-8 years due to recurrence of original disease or chronic antibody-mediated rejection. Of the 10 renal allografts, three failed due to thrombotic microangiopathy or rejection. When compared with 21 immunologically similar living donor kidney recipients treated with conventional IS, the long-term IS-free survivors developed significantly fewer posttransplant complications. Although most recipients treated with none or two doses of rituximab developed donor-specific antibody (DSA), no DSA was detected in recipients treated with four doses of rituximab. Although further revisions of the current conditioning regimen are planned in order to improve consistency of the results, this study shows that long-term stable kidney allograft survival without maintenance IS can be achieved following transient mixed chimerism induction.

Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

Polyreactive antibodies developing amidst humoral rejection of human kidney grafts bind apoptotic cells and activate complement.

Antibody mediated rejection (AMR) is associated with a variety of graft-reactive antibodies following kidney transplant. To characterize these antibodies, we immortalized 107 B cell clones from a patient with AMR. In a previous study, we showed that six clones were reacting to multiple self-antigens as well as to HLA and MICA for two of them, thus displaying a pattern of polyreactivity. We show here that all six polyreactive clones also reacted to apoptotic but not viable cells. More generally we observed a nearly perfect overlap between polyreactivity and reactivity to apoptotic cells. Functionally, polyreactive antibodies can activate complement, resulting in the deposition of C3d and C4d at the surface of target cells. Testing the serum of 88 kidney transplant recipients revealed a significantly higher IgG reactivity to apoptotic cells in AMR patients than in patients with stable graft function. Moreover, total IgG purified from AMR patients had increased complement activating properties compared to IgG from non-AMR patients. Overall, our studies show the development of polyreactive antibodies cross-reactive to apoptotic cells during AMR. Further studies are now warranted to determine their contribution to the detection of C4d in graft biopsies as well as their role in the pathophysiology of AMR.

Alefacept promotes immunosuppression-free renal allograft survival in nonhuman primates via depletion of recipient memory T cells.

Renal allograft tolerance has been achieved in MHC-mismatched primates via nonmyeloablative conditioning beginning 6 days prior to planned kidney and donor bone marrow transplantation (DBMT). To extend the applicability of this approach to deceased donor transplantation, we recently developed a novel-conditioning regimen, the "delayed protocol" in which donor bone marrow (DBM) is transplanted several months after kidney transplantation. However, activation/expansion of donor-reactive CD8(+) memory T cells (TMEM) occurring during the interval between kidney and DBM transplantation impaired tolerance induction using this strategy. In the current study, we tested whether, Alefacept, a fusion protein which targets LFA-3/CD2 interactions and selectively depletes CD2(high) CD8(+) effector memory T cells (TEM) could similarly induce long-term immunosuppression-free renal allograft survival but avoid the deleterious effects of anti-CD8 mAb treatment. We found that Alefacept significantly delayed the expansion of CD2(high) cells including CD8(+) TEM while sparing naïve CD8(+) T and NK cells and achieved mixed chimerism and long-term immunosuppression-free renal allograft survival. In conclusion, elimination of CD2(high) T cells represents a promising approach to prevent electively the expansion/activation of donor-reactive TEM and promotes tolerance induction via the delayed protocol mixed chimerism approach.

Psychological Impacts of Retained Bullets From the Perspective of Survivors.

INTRODUCTION: Despite a high prevalence of retained bullet fragments (RBFs) after firearm related injury (FRI) there is limited data on the full spectrum of their consequences, particularly the psychological impacts on those injured. Further, the experiences of FRI survivors with RBFs are missing from existing literature. The objective of this study was to explore the psychological impacts of RBFs on individuals who have experienced recent FRI. METHODS: Adult (18-65 years) survivors of FRI with radiographically confirmed RBFs were purposively selected from an urban Level 1 trauma center in Atlanta, Georgia, to participate in an in-depth interview. Interviews were conducted between March 2019 and February 2020. Thematic analysis was used to identify a range of psychological effects from RBFs. RESULTS: Interviews from 24 FRI survivors were analyzed: the majority of participants were Black males (N = 22, 92%) with a mean age of 32 years whose FRI occurred ∼8.6 months prior to data collection. The psychological effects of RBFs were grouped into four categories: physical health (eg, pain, limited mobility), emotional well-being (eg, anger, fear), social isolation, and occupational welfare (eg, disability leading to inability to work). A range of coping mechanisms were also identified. CONCLUSION: Survivors of FRI with RBFs experience a range of psychological impacts that are far-reaching and affect daily activities, mobility, pain and emotional wellbeing. Study results indicate a need for enhanced resources to support those with RBFs. Further, changes to clinical protocols are warranted on removal of RBFs and communication about the effects of leaving RBFs in situ.

Characterizing injury patterns and outcomes in hospitalized trauma patients with non-English Language Preferences.

INTRODUCTION: Patients with Non-English Language Preferences (NELP) experience challenges navigating the US healthcare system which can lead to disparate outcomes. This study sought to investigate injury patterns and outcomes in hospitalized trauma patients with NELP. METHODS: A retrospective review was performed at a trauma center from January 2019-December 2020. An institutional database of all emergency department video consultations for interpreter services was cross-referenced with the trauma registry and comparisons were made between NELP and English-preferred (EP) speaking patients. RESULTS: During the study, 257 NELP patients were hospitalized after traumatic injury. Twenty-two percent had work related injuries compared to only 3.0% in the EP cohort (p < 0.001). When propensity score matched, there were no significant differences in ICU and hospital length of stay or mortality between NELP and EP patients. DISCUSSION: Trauma patients are linguistically diverse and understanding their injury patterns and outcomes is crucial for guiding culturally and linguistically appropriate injury prevention.

Development and validation of a brief screener for posttraumatic stress disorder risk in emergency medical settings.

OBJECTIVE: Predicting risk of posttraumatic stress disorder (PTSD) in the acute care setting is challenging given the pace and acute care demands in the emergency department (ED) and the infeasibility of using time-consuming assessments. Currently, no accurate brief screening for long-term PTSD risk is routinely used in the ED. One instrument widely used in the ED is the 27-item Immediate Stress Reaction Checklist (ISRC). The aim of this study was to develop a short screener using a machine learning approach and to investigate whether accurate PTSD prediction in the ED can be achieved with substantially fewer items than the IRSC. METHOD: This prospective longitudinal cohort study examined the development and validation of a brief screening instrument in two independent samples, a model development sample (N = 253) and an external validation sample (N = 93). We used a feature selection algorithm to identify a minimal subset of features of the ISRC and tested this subset in a predictive model to investigate if we can accurately predict long-term PTSD outcomes. RESULTS: We were able to identify a reduced subset of 5 highly predictive features of the ISRC in the model development sample (AUC = 0.80), and we were able to validate those findings in the external validation sample (AUC = 0.84) to discriminate non-remitting vs. resilient trajectories. CONCLUSION: This study developed and validated a brief 5-item screener in the ED setting, which may help to improve the diagnostic process of PTSD in the acute care setting and help ED clinicians plan follow-up care when patients are still in contact with the healthcare system. This could reduce the burden on patients and decrease the risk of chronic PTSD.

Overcoming memory T-cell responses for induction of delayed tolerance in nonhuman primates.

The presence of alloreactive memory T cells is a major barrier for induction of tolerance in primates. In theory, delaying conditioning for tolerance induction until after organ transplantation could further decrease the efficacy of the regimen, since preexisting alloreactive memory T cells might be stimulated by the transplanted organ. Here, we show that such "delayed tolerance" can be induced in nonhuman primates through the mixed chimerism approach, if specific modifications to overcome/avoid donor-specific memory T-cell responses are provided. These modifications include adequate depletion of CD8+ memory T cells and timing of donor bone marrow administration to minimize levels of proinflammatory cytokines. Using this modified approach, mixed chimerism was induced successfully in 11 of 13 recipients of previously placed renal allografts and long-term survival without immunosuppression could be achieved in at least 6 of these 11 animals.