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.

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.

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.

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.