Acoustofluidic-based therapeutic apheresis system.

Therapeutic apheresis aims to selectively remove pathogenic substances, such as antibodies that trigger various symptoms and diseases. Unfortunately, current apheresis devices cannot handle small blood volumes in infants or small animals, hindering the testing of animal model advancements. This limitation restricts our ability to provide treatment options for particularly susceptible infants and children with limited therapeutic alternatives. Here, we report our solution to these challenges through an acoustofluidic-based therapeutic apheresis system designed for processing small blood volumes. Our design integrates an acoustofluidic device with a fluidic stabilizer array on a chip, separating blood components from minimal extracorporeal volumes. We carried out plasma apheresis in mouse models, each with a blood volume of just 280 µL. Additionally, we achieved successful plasmapheresis in a sensitized mouse, significantly lowering preformed donor-specific antibodies and enabling desensitization in a transplantation model. Our system offers a new solution for small-sized subjects, filling a critical gap in existing technologies and providing potential benefits for a wide range of patients.

Allosensitisation in NHP results in cross-reactive anti-SLA antibodies not detected by a lymphocyte-based flow cytometry crossmatch.

Xenotransplantation is a potential option for individuals for whom an acceptable human allograft is unavailable. Individuals with broadly reactive HLA antibodies due to prior exposure to foreign HLA are potential candidates for a clinical xenotransplant trial. It remains controversial if allosensitisation results in the development of cross-reactive antibodies against SLA. This may require increased histocompatibility scrutiny for highly sensitised individuals prior to enrollment in a clinical trial. Serum samples were obtained from non-human primates sensitised via serial skin transplantation from maximally MHC-mismatched donor, as reported. Sera from pre- and post-allosensitisation timepoints were assessed in a flow crossmatch (FXM) for IgM and IgG binding to pig splenocytes with or without red blood cell adsorption. Xenoreactive antibodies were eluted from pig splenocytes and screened on a single antigen HLA bead assay. A MHC Matchmaker algorithm was developed to predict potential conserved amino acid motifs among the pig, NHP, and human. Our sensitised NHP model was used to demonstrate that allosensitisation does not result in an appreciable difference in xenoreactive antibody binding in a cell-based FXM. However, antibody elution and screening on single antigen HLA beads suggest the existence of potential cross-reactive antibodies against SLA. The cross-reactive IgG after allosensitisation were predicted by comparing the recipient Mamu alleles against its previous allograft donor Mamu alleles and the donor pig SLA alleles. Our study suggests that allosensitisation could elevate cross-reactive antibodies, but a more sensitive assay than a cell-based FXM is required to detect them. The MHC Matchmaker algorithm was developed as a potential tool to help determine amino acid motif conservation and reactivity pattern.

Subnormothermic Oxygenated Machine Perfusion (24 h) in DCD Kidney Transplantation.

Background: Ex vivo kidney perfusion is an evolving platform that demonstrates promise in preserving and rehabilitating the kidney grafts. Despite this, there is little consensus on the optimal perfusion conditions. Hypothermic perfusion offers limited functional assessment, whereas normothermic perfusion requires a more complex mechanical system and perfusate. Subnormothermic machine perfusion (SNMP) has the potential to combine the advantages of both approaches but has undergone limited investigation. Therefore, the present study sought to determine the suitability of SNMP for extended kidney preservation. Methods: SNMP at 22-25 °C was performed on a portable device for 24 h with porcine kidneys. Graft assessment included measurement of mechanical parameters and biochemical analysis of the perfusate using point-of-care tests. To investigate the viability of kidneys preserved by SNMP, porcine kidney autotransplants were performed in a donation after circulatory death (DCD) model. SNMP was also compared with static cold storage (SCS). Finally, follow-up experiments were conducted in a subset of human kidneys to test the translational significance of findings in porcine kidneys. Results: In the perfusion-only cohort, porcine kidneys all displayed successful perfusion for 24 h by SNMP, evidenced by stable mechanical parameters and biological markers of graft function. Furthermore, in the transplant cohort, DCD grafts with 30 min of warm ischemic injury demonstrated superior posttransplant graft function when preserved by SNMP in comparison with SCS. Finally, human kidneys that underwent 24-h perfusion exhibited stable functional and biological parameters consistent with observations in porcine organs. Conclusions: These observations demonstrate the suitability and cross-species generalizability of subnormothermic machine perfusion to maintain stable kidney perfusion and provide foundational evidence for improved posttransplant graft function of DCD kidneys after SNMP compared with SCS.

Contemporary trends and outcomes after liver transplantation and resection for intrahepatic cholangiocarcinoma.

BACKGROUND: Liver transplantation (LT) has been shown to be superior to resection in highly selected patients with perihilar cholangiocarcinoma (CCA), yet has traditionally been contraindicated for intrahepatic CCA (iCCA). Herein, we aimed to examine contemporary trends and outcomes for surgical resection and LT for iCCA. METHODS: The National Cancer Database was queried for patients presenting with stage I-III iCCA between 2010 and 2018 who underwent resection or LT. Overall survival (OS) was compared with Kaplan-Meier and multivariable Cox proportional hazards methods stratified by management. Secondary analysis of patients undergoing transplant for CCA was performed with the United Network for Organ Sharing database. RESULTS: Of 2565 patients, 2412 (94.0%) underwent resection and 153 (5.96%) LT of whom 84 (54.9%) received neoadjuvant therapy. Utilization of LT remained between 3.9% and 7.8% annually. Unadjusted 5-year OS was higher for LT than resection (59.8% vs 39.9%, P = .0067), yet adjusted analysis revealed no significant difference in mortality (hazard ratio, 0.91; 95% CI, 0.66-1.27; P = .58). On secondary analysis including 437 patients with all subtypes of CCA, unadjusted 5-year OS was higher for non-CCA indications (79% vs 52%-54%, P < .001). CONCLUSION: Utilization of LT for iCCA remains low and many cases are likely incidental. Although partial hepatectomy remains the standard of care for patients with resectable disease, our findings suggest that highly selected patients with unresectable iCCA may achieve favorable outcomes after LT. Granular, prospective data are needed to identify patients most likely to benefit from transplant and allocate scarce liver grafts.

The impact of IdeS (imlifidase) on allo-specific, xeno-reactive, and protective antibodies in a sensitized rhesus macaque model.

BACKGROUND: Highly sensitized patients face many barriers to kidney transplantation, including higher rates of antibody-mediated rejection after HLA-incompatible transplant. IdeS, an endopeptidase that cleaves IgG nonspecifically, has been trialed as desensitization prior to kidney transplant, and successfully cleaves donor-specific antibody (DSA), albeit with rebound. METHODS: IdeS was generated and tested (2 mg/kg, IV) in two naïve and four allosensitized nonhuman primates (NHP). Peripheral blood samples were collected at regular intervals following IdeS administration. Total IgG, total IgM, and anti-CMV antibodies were quantified with ELISA, and donor-specific antibody (DSA) and anti-pig antibodies were evaluated using flow cytometric crossmatch. B cell populations were assessed using flow cytometry. RESULTS: IdeS successfully cleaved rhesus IgG in vitro. In allosensitized NHP, robust reduction of total, DSA, anti-pig, and anti-CMV IgG was observed within one day following IdeS administration. Rapid rebound of all IgG antibody populations was observed, with antibody levels returning to baseline around day 14 post-infusion. Total IgM level was not affected by IdeS. Interestingly, a comparable reduction in antibody populations was observed after the second dose of IdeS. However, we have not observed any significant modulation of B cell subpopulations after IdeS. CONCLUSIONS: This study evaluated efficacy of IdeS in the allosensitized NHP in IgG with various specificities, mirroring antibody kinetics in human patients. The efficacy of IdeS on preexisting anti-pig antibodies may be useful in clinical xenotransplantation. However, given the limitation of IdeS on its durability as a monotherapy, optimization of IdeS with other agents targeting the humoral response is further needed.

Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

The anti-CD40L monoclonal antibody AT-1501 promotes islet and kidney allograft survival and function in nonhuman primates.

Prior studies of anti-CD40 ligand (CD40L)-based immunosuppression demonstrated effective prevention of islet and kidney allograft rejection in nonhuman primate models; however, clinical development was halted because of thromboembolic complications. An anti-CD40L-specific monoclonal antibody, AT-1501 (Tegoprubart), was engineered to minimize risk of thromboembolic complications by reducing binding to Fcγ receptors expressed on platelets while preserving binding to CD40L. AT-1501 was tested in both a cynomolgus macaque model of intrahepatic islet allotransplantation and a rhesus macaque model of kidney allotransplantation. AT-1501 monotherapy led to long-term graft survival in both islet and kidney transplant models, confirming its immunosuppressive potential. Furthermore, AT-1501-based regimens after islet transplant resulted in higher C-peptide, greater appetite leading to weight gain, and reduced occurrence of cytomegalovirus reactivation compared with conventional immunosuppression. These data support AT-1501 as a safe and effective agent to promote both islet and kidney allograft survival and function in nonhuman primate models, warranting further testing in clinical trials.

Revisiting Medical Student Expectations on the Surgery Clerkship.

OBJECTIVE: Medical students frequently report ambiguity of expectations in their feedback of the surgery clerkship. Herein, we aimed to gauge general surgery resident and attending expectations of students on their clerkship. DESIGN: Residents and attending surgeons were surveyed on medical student expectations for rounding and floor duties, the operating room, clinic, and professionalism. RESULTS: There were slight differences in expectations between residents and attendings, which were then utilized to facilitate a discussion to create consensus expectations for students. Early outcomes demonstrate improved understanding of expectations by medical students. CONCLUSION: Identifying differences in resident and attending expectations of medical students on their surgery clerkship can help improve the alignment of such expectations. We hope that longterm, this intervention can facilitate a better learning environment for medical students on their surgery clerkship.

Desensitization and belatacept-based maintenance therapy in pregnancy-sensitized monkeys receiving a kidney transplant.

Among sensitized patients awaiting a transplant, females are disproportionately represented, partly because of pregnancy-induced sensitization. Using female NHPs sensitized by pregnancy alone, we examined the efficacy of costimulation blockade and proteasome inhibition for desensitization. Three animals received no desensitization (control), and seven animals received weekly carfilzomib (27 mg/m2) and belatacept (20 mg/kg) before kidney transplantation. All animals received renal allografts from crossmatch-positive/maximally MHC-mismatched donors. Controls and three desensitized animals received tacrolimus-based immunosuppression. Four desensitized animals received additional belatacept with tacrolimus-based immunosuppression. Multiparous females had less circulating donor-specific antibody when compared to skin-sensitized males before transplantation. While females receiving desensitization showed only a marginal survival benefit over control females (MST = 11 days versus 63 days), additional belatacept to posttransplant maintenance significantly prolonged graft survival (MST > 164 days) and suppressed posttransplant DSA and circulating follicular helper T-like cells. This combination of therapies demonstrates great potential to reduce antibody-mediated rejection in sensitized recipients.

Xenorecognition and costimulation of porcine endothelium-derived extracellular vesicles in initiating human porcine-specific T cell immune responses.

Porcine vascular endothelial cells (PECs) form a mechanistic centerpiece of xenograft rejection. Here, we determined that resting PECs release swine leukocyte antigen class I (SLA-I) but not swine leukocyte antigen class-II DR (SLA-DR) expressing extracellular vesicles (EVs) and investigated whether these EVs proficiently initiate xenoreactive T cell responses via direct xenorecognition and costimulation. Human T cells acquired SLA-I+ EVs with or without direct contact to PECs, and these EVs colocalized with T cell receptors. Although interferon gamma-activated PECs released SLA-DR+ EVs, the binding of SLA-DR+ EVs to T cells was sparse. Human T cells demonstrated low levels of proliferation without direct contact to PECs, but marked T cell proliferation was induced following exposure to EVs. EV-induced proliferation proceeded independent of monocytes/macrophages, suggesting that EVs delivered both a T cell receptor signal and costimulation. Costimulation blockade targeting B7, CD40L, or CD11a significantly reduced T cell proliferation to PEC-derived EVs. These findings indicate that endothelial-derived EVs can directly initiate T cell-mediated immune responses, and suggest that inhibiting the release of SLA-I EVs from organ xenografts has the potential to modify the xenograft rejection. We propose a secondary-direct pathway for T cell activation via xenoantigen recognition/costimulation by endothelial-derived EVs.

Attitudes of patients with renal disease on xenotransplantation: A systematic review.

BACKGROUND: Recent years have seen major advancements in xenotransplantation: the first pig-to-human heart transplant, the development of a brain-dead recipient model for kidney xenotransplantation, and the registration of the first xenokidney clinical trial. The attitudes of patients with kidney disease or transplants on xenotransplantation and an assessment of their reservations and considerations regarding the technology are crucial to successful clinical translation and eventual widespread implementation. METHODS: This systematic review was registered through PROSPERO (CRD42022344581) prior to initiation of the study and reported using the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines. We included studies that evaluated attitudes towards and willingness to undergo xenotransplantation in patients with end-stage renal disease (ESRD), including those who had already undergone transplantation. MEDLINE (via Ovid), Embase (via Elsevier), and Web of Science (via Clarivate) were searched from database inception to July 15, 2022 by an experienced medical librarian for studies on xenotransplantation and attitudes. Abstracts and full text were screened using Covidence software and data items regarding study methodology, patient demographics, and attitudes regarding xenotransplantation were extracted using Microsoft Excel. Risk of bias assessments were performed using the Critical Appraisal Skills Programmed and National Institute of Health study quality assessment tools. RESULTS: Of 1992 studies identified, 14 studies met the inclusion criteria. These studies were conducted across eight countries, four in the United States, for a total of 3114 patients on the kidney waitlist or with a kidney transplant. All patients were over 17 years old and 58% were male. Acceptance of a xenotransplant was assessed using surveys in 12 studies. Sixty-three percent (n = 1354) of kidney patients reported that they would accept a xenotransplant with function comparable to that of an allotransplant. Acceptance of xenografts with inferior function to allografts (15%) or as bridge organs (35%) to allotransplantation was lower. Specific concerns expressed by patients included graft function, infection, social stigma, and animal rights. Subgroup analyses showed higher acceptance in already transplanted compared to waitlist patients and white compared to Black Americans. CONCLUSION: An understanding of patient attitudes and reservations is key to the successful execution of the first xenotransplantation clinical trials. This study compiles important factors to consider, such as patient concerns, attitudes regarding practical clinical scenarios for the use of xenotransplantation, and the impact of demographic factors on acceptance of this emerging technology.

Incidence of Postreperfusion Hyperfibrinolysis in Liver Transplantation by Donor Type and Observed Treatment Strategies.

BACKGROUND: Hyperfibrinolysis is a possible complication during liver transplantation, particularly immediately after reperfusion. METHODS: We performed a retrospective study to examine the incidence, treatment, and resolution of postreperfusion hyperfibrinolysis in patients undergoing liver transplantation at Duke University Hospital from 2015 to 2020. RESULTS: Out of 535 patients undergoing liver transplantation, 21 or 3.9%, 95% CI (2.5-5.9), had hyperfibrinolysis after reperfusion. Hyperfibrinolysis occurred in 16 of 511 (3.1%) patients receiving livers from DBD donors, 5 of 18 (27.8%) patients receiving livers from donation after circulatory death (DCD) donors, and 0 of 6 (0.0%) patients receiving livers from living donors. Fibrinolysis was treated with cryoprecipitate (12/21), a combination of cryoprecipitate and tranexamic acid (3/21), or neither (6/21) and resolved within several hours in all cases. CONCLUSIONS: Anesthesiologists should be aware of the possibility of postreperfusion hyperfibrinolysis in liver transplantation, particularly with DCD donors, and may consider treatment with cryoprecipitate or tranexamic acid. Further work is needed to identify any potential differences, such as faster resolution of fibrinolysis, between different treatment modalities.

AAV9-mediated gene delivery to liver grafts during static cold storage in a rat liver transplant model.

Introduction: Recombinant adeno-associated virus (rAAV) is a novel strategy used clinically for gene delivery, but has not been characterized in the context of organ transplantation. We sought to determine the efficacy of rAAV-mediated gene delivery during static cold storage (SCS) prior to liver transplantation. Methods: A triple-plasmid transfection protocol was used to produce rAAV subtype-9 vectors containing firefly luciferase genomes in HEK293 cells. Lewis rat liver grafts were flushed and stored in cold HTK solution. Three experimental groups received rAAV at different doses, administered via the portal vein as a bolus during SCS. A control group did not receive rAAV (N = 2). Recipients then underwent syngeneic liver transplantation. Bioluminescence imaging to quantify in vivo luciferase expression was performed on post-operative days 7, 14, 28, and 56. Results: Control animals demonstrated no bioluminescent activity, while animals receiving rAAV-treated livers had increasing bioluminescence, peaking at four weeks but sustained to the eight-week endpoint. This result was confirmed by experimental endpoint tissue luciferase activity assay. Discussion: rAAV mediates gene transduction in liver grafts when administered during SCS and has potential for gene therapy applications in solid organ transplantation.

Complement-targeting therapeutics for ischemia-reperfusion injury in transplantation and the potential for ex vivo delivery.

Organ shortages and an expanding waitlist have led to increased utilization of marginal organs. All donor organs are subject to varying degrees of IRI during the transplant process. Extended criteria organs, including those from older donors and organs donated after circulatory death are especially vulnerable to ischemia-reperfusion injury (IRI). Involvement of the complement cascade in mediating IRI has been studied extensively. Complement plays a vital role in the propagation of IRI and subsequent recruitment of the adaptive immune elements. Complement inhibition at various points of the pathway has been shown to mitigate IRI and minimize future immune-mediated injury in preclinical models. The recent introduction of ex vivo machine perfusion platforms provides an ideal window for therapeutic interventions. Here we review the role of complement in IRI by organ system and highlight potential therapeutic targets for intervention during ex vivo machine preservation of donor organs.

Addition of interleukin-6 receptor blockade to carfilzomib-based desensitization in a highly sensitized nonhuman primate model.

Sensitized patients, those who had prior exposure to foreign human leukocyte antigens, are transplanted at lower rates due to challenges in finding suitable organs. Desensitization strategies have permitted highly sensitized patients to undergo kidney transplantation, albeit with higher rates of rejection. This study assesses targeting plasma cell and interleukin (IL)-6 receptor for desensitization in a sensitized nonhuman primate kidney transplantation model. All animals were sensitized using two sequential skin transplants from maximally major histocompatibility complex-mismatched donors. Carfilzomib (CFZ)/tocilizumab (TCZ) desensitization (N = 6) successfully decreased donor-specific antibody (DSA) titers and prevented the expansion of B cells compared to CFZ monotherapy (N = 3). Dual desensitization further delayed, but did not prevent humoral rebound, as evidenced by a delayed increase in post-kidney transplant DSA titers. Accordingly, CFZ/TCZ desensitization conferred a significant survival advantage over CFZ monotherapy. A trend toward increased T follicular helper cells was also observed in the dual therapy group along the same timeline as an increase in DSA and subsequent graft loss. Cytomegalovirus reactivation also occurred in the CFZ/TCZ group but was prevented with ganciclovir prophylaxis. In accordance with prior studies of CFZ-based dual desensitization strategies, the addition of IL-6 receptor blockade resulted in desensitization with further suppression of posttransplant humoral response compared to CFZ monotherapy.

Complement-targeted therapies in kidney transplantation-insights from preclinical studies.

Aberrant activation of the complement system contributes to solid-organ graft dysfunction and failure. In kidney transplantation, the complement system is implicated in the pathogenesis of antibody- and cell-mediated rejection, ischemia-reperfusion injury, and vascular injury. This has led to the evaluation of select complement inhibitors (e.g., C1 and C5 inhibitors) in clinical trials with mixed results. However, the complement system is highly complex: it is composed of more than 50 fluid-phase and surface-bound elements, including several complement-activated receptors-all potential therapeutic targets in kidney transplantation. Generation of targeted pharmaceuticals and use of gene editing tools have led to an improved understanding of the intricacies of the complement system in allo- and xeno-transplantation. This review summarizes our current knowledge of the role of the complement system as it relates to rejection in kidney transplantation, specifically reviewing evidence gained from pre-clinical models (rodent and nonhuman primate) that may potentially be translated to clinical trials.

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.

Gene Therapy: Will the Promise of Optimizing Lung Allografts Become Reality?

Lung transplantation is the definitive therapy for patients living with end-stage lung disease. Despite significant progress made in the field, graft survival remains the lowest of all solid organ transplants. Additionally, the lung has among the lowest of organ utilization rates-among eligible donors, only 22% of lungs from multi-organ donors were transplanted in 2019. Novel strategies are needed to rehabilitate marginal organs and improve graft survival. Gene therapy is one promising strategy in optimizing donor allografts. Over-expression or inhibition of specific genes can be achieved to target various pathways of graft injury, including ischemic-reperfusion injuries, humoral or cellular rejection, and chronic lung allograft dysfunction. Experiments in animal models have historically utilized adenovirus-based vectors and the majority of literature in lung transplantation has focused on overexpression of IL-10. Although several strategies were shown to prevent rejection and prolong graft survival in preclinical models, none have led to clinical translation. The past decade has seen a renaissance in the field of gene therapy and two AAV-based in vivo gene therapies are now FDA-approved for clinical use. Concurrently, normothermic ex vivo machine perfusion technology has emerged as an alternative to traditional static cold storage. This preservation method keeps organs physiologically active during storage and thus potentially offers a platform for gene therapy. This review will explore the advantages and disadvantages of various gene therapy modalities, review various candidate genes implicated in various stages of allograft injury and summarize the recent efforts in optimizing donor lungs using gene therapy.