Subnormothermic and Normothermic Ex Vivo Liver Perfusion as a Novel Preservation Technique

Due to the worldwide organ shortage, interest in the use of marginal liver allografts has increased. More widespread use of marginal grafts is limited by graft injury from cold storage and the risk of poor outcomes after transplantation. Warm (subnormothermic and normothermic) ex vivo liver perfusion has emerged as a novel preservation strategy to recover marginal organs and potentially increase the organ pool. Over the last decade, advances in the field have taken warm ex vivo liver perfusion from the laboratory to clinical trials. While most investigation thus far has focused on the rescue of marginal grafts for expansion of the donor pool, warm perfusion (WP) preservation also has great potential to facilitate novel graft interventions prior to transplantation

Need for Pretransplant Midodrine Does Not Negatively Impact Simultaneous Liver-kidney Transplant Outcomes.

BACKGROUND: Midodrine is often needed pretransplant to improve hemodynamics in simultaneous liver-kidney transplant candidates. Previous research has shown that patients requiring midodrine before kidney transplant alone have increased posttransplant risk for delayed allograft function, graft failure, and death. However, the impact of pretransplant midodrine use on outcomes after simultaneous liver-kidney transplant is unknown. METHODS: We performed a retrospective study of all adult (age ≥18 y) simultaneous liver-kidney transplant recipients from a single academic transplant center from February 1, 2002, to June 30, 2019. RESULTS: Sixty-four simultaneous liver-kidney transplants were performed in our institution during this time period, of which, 43 were not on midodrine before transplant, 17 were on midodrine alone, and 4 were on intravenous (IV) vasopressor therapy. Despite the midodrine group having a higher MELD-Na at listing, higher MELD-Na at transplant, and being older, there were no significant differences in key outcomes including delayed renal allograft function, estimated glomerular filtration rate at transplant discharge, and estimated glomerular filtration rate at 1 y after transplant compared with the nonmidodrine group. There was no significant difference in graft failure or survival at last follow-up. CONCLUSIONS: Our study suggests that need for pretransplant midodrine should not be a barrier to simultaneous liver-kidney transplant

Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs

Successful Heart-Liver Transplant Using Dual-organ Normothermic Perfusion in a Patient With Fontan Failure

Advances in surgical technique and multidisciplinary management have improved long-term survival for patients born with single ventricle physiology. However, patients who have undergone Fontan completion remain at risk for long-term comorbidities associated with the complex hemodynamic changes following the procedure, including Fontan failure and Fontan-associated liver disease.1 Combined heart-liver transplantation (CHLT) is a rare but lifesaving procedure that has been described in the setting of heart and liver failure secondary to Fontan failure.2 As long-term survival continues to improve for Fontan patients, the incidence of Fontan-associated liver disease will increase. Thus, improving CHLT outcomes and access to both organs is a strong priority. Here, we describe a successful CHLT for a patient with chronic ventricular dysfunction and Fontan-associated liver disease. The key innovation in this case was the use of normothermic machine perfusion (NMP) to preserve both the heart and liver grafts. This approach extended the preservation time for the liver while also mitigating the risk of ischemic injury and reducing the time pressure constraints on the heart transplant team. Notably, this stands in contrast to traditional static cold storage (SCS), where metabolic activity is reduced through hypothermia, but the extended cold ischemic time can lead to increased vulnerability to reperfusion injury

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

Education Theory Made Practical - Volume 4

This project is made possible with funding by the Government of Ontario and through eCampusOntario’s support of the Virtual Learning Strategy.1. Cognitive Load Theory2. Epstein’s Mindful Practitioner3. Joplin’s Five Stage Model of Experiential Learning4. Kolb’s Experiential Learning Theory5. Maslow’s Hierarchy of Needs6. Miller’s Pyramid7. Multiple Resource Theory8. Prototype Theory9. Self-regulated Learning Theory10. Siu & Reiter’s TAU ApproachThis is the fourth volume of the popular series Education Theory Made Practical which has been brought to you as a joint effort between the Academic Life in Emergency Medicine (ALiEM) Faculty Incubator program and the International Clinician Educator Blog. This project is made possible in part with funding from the Government of Ontario and through eCampusOntario's support of the Virtual Learning Strategy. To learn more about the Virtual Learning Strategy visit https://vls.ecampusontario.c

Characteristics of liver transplant candidates delisted following recompensation and predictors of such delisting in alcohol-related liver disease: a case-control study

Whether and when recovery beyond the need for transplant may occur in patients listed for decompensation remains unclear. This study aimed to investigate the characteristics of patients delisted following recompensation. Seventy-seven patients who were listed between 2005 and 2015 for decompensation, but later delisted following recompensation were included. Alcohol-related liver disease (ALD) was the underlying etiology in the majority (n=47, 61%). Listing characteristics of these patients were compared with those of decompensated ALD patients who either underwent deceased donor liver transplantation or died on the waiting list. The model for end-stage liver disease (MELD) score <20 and serum albumin ≥32g/l at listing were the only independent predictors of recompensation/delisting in ALD. The probability of recompensation was 70% when both factors were present at listing. Interestingly, about a tenth of decompensated ALD patients who died on the waiting list (median duration on waiting list 11 months) and a quarter of decompensated ALD patients who underwent living donor liver transplantation (median duration on waiting list 2 months) also had both factors at listing. In conclusion, ALD seems to be the most favorable etiology for recompensation beyond the need for transplantation. Both MELD and serum albumin at listing independently predict recompensation/delisting in ALD. It seems advisable to implement a period of observation for ALD patients with both favorable factors, before embarking on living donor liver transplantation

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

IntroductionRecombinant 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.MethodsA 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.ResultsControl 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.DiscussionrAAV mediates gene transduction in liver grafts when administered during SCS and has potential for gene therapy applications in solid organ transplantation

Antibody binding loop insertions as diversity elements

In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments

Hippocampal pyramidal cells: the reemergence of cortical lamination

The increasing resolution of tract-tracing studies has led to the definition of segments along the transverse axis of the hippocampal pyramidal cell layer, which may represent functionally defined elements. This review will summarize evidence for a morphological and functional differentiation of pyramidal cells along the radial (deep to superficial) axis of the cell layer. In many species, deep and superficial sublayers can be identified histologically throughout large parts of the septotemporal extent of the hippocampus. Neurons in these sublayers are generated during different periods of development. During development, deep and superficial cells express genes (Sox5, SatB2) that also specify the phenotypes of superficial and deep cells in the neocortex. Deep and superficial cells differ neurochemically (e.g. calbindin and zinc) and in their adult gene expression patterns. These markers also distinguish sublayers in the septal hippocampus, where they are not readily apparent histologically in rat or mouse. Deep and superficial pyramidal cells differ in septal, striatal, and neocortical efferent connections. Distributions of deep and superficial pyramidal cell dendrites and studies in reeler or sparsely GFP-expressing mice indicate that this also applies to afferent pathways. Histological, neurochemical, and connective differences between deep and superficial neurons may correlate with (patho-) physiological phenomena specific to pyramidal cells at different radial locations. We feel that an appreciation of radial subdivisions in the pyramidal cell layer reminiscent of lamination in other cortical areas may be critical in the interpretation of studies of hippocampal anatomy and function