Honoring the gift: The transformative potential of transplant-declined human organs.

For decades, transplantation has been a life-saving treatment for those fortunate enough to gain access. Nevertheless, many patients die waiting for an organ and countless more never make it onto the waitlist because of a shortage of donor organs. Concurrently, thousands of donated organs are declined for transplant each year because of concerns about poor outcomes post-transplant. The decline of any donated organ-even if medically justified-is tragic for both the donor family and potential recipients. In this Personal Viewpoint, we discuss the need for a new mindset in how we honor the gift of organ donation. We believe that the use of transplant-declined human organs in translational research has the potential to hasten breakthrough discoveries in a multitude of scientific and medical areas. More importantly, such breakthroughs will allow us to properly value every donated organ. We further discuss the many practical challenges that such research presents and offer some possible solutions based on experiences in our own research laboratories. Finally, we share our perspective on what we believe are the necessary next steps to ensure a future where every donated organ realizes its full potential to impact the lives of current and future patients.

An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

The liver lymphatic system is essential for maintaining tissue fluid balance and immune function. The detailed structure of lymphatic vessels (LVs) in the liver remains to be fully demonstrated. The aim of this study is to reveal LV structures in normal and diseased livers by developing a tissue-clearing and coimmunolabeling protocol optimized for the tissue size and the processing time for three-dimensional (3-D) visualization and quantification of LVs in the liver. We showed that our optimized protocol enables in-depth exploration of lymphatic networks in the liver, consisting of LVs along the portal tract (deep lymphatic system) and within the collagenous Glisson's capsule (superficial lymphatic system) in different species. With this protocol, we have shown 3-D LVs configurations in relation to blood vessels and bile ducts in cholestatic mouse livers, in which LVs were highly dilated and predominantly found around highly proliferating bile ducts and peribiliary vascular plexuses in the portal tract. We also established a quantification method using a 3-D volume-rendering approach. We observed a 1.6-fold (P < 0.05) increase in the average diameter of LVs and a 2.4-fold increase (P < 0.05) in the average branch number of LVs in cholestatic/fibrotic livers compared with control livers. Furthermore, cholestatic/fibrotic livers showed a 4.3-fold increase (P < 0.05) in total volume of LVs compared with control livers. Our optimized protocol and quantification method demonstrate an efficient and simple liver tissue-clearing procedure that allows the comprehensive analysis of liver lymphatic system.NEW & NOTEWORTHY This article showed a comprehensive 3-D-structural analysis of liver lymphatic vessel (LV) in normal and diseased livers in relation to blood vessels and bile ducts. In addition to the LVs highly localized at the portal tract, we revealed capsular LVs in mouse, rat, and human livers. In cholestatic livers, LVs are significantly increased and dilated compared with normal livers. Our optimized protocol provides detailed spatial information for LVs remodeling in normal and pathological conditions.

Lysis of cold-storage-induced microvascular obstructions for ex vivo revitalization of marginal human kidneys.

Thousands of kidneys from higher-risk donors are discarded annually because of the increased likelihood of complications posttransplant. Given the severe organ shortage, there is a critical need to improve utilization of these organs. To this end, normothermic machine perfusion (NMP) has emerged as a platform for ex vivo assessment and potential repair of marginal organs. In a recent study of 8 transplant-declined human kidneys on NMP, we discovered microvascular obstructions that impaired microvascular blood flow. However, the nature and physiologic impact of these lesions were unknown. Here, in a study of 39 human kidneys, we have identified that prolonged cold storage of human kidneys induces accumulation of fibrinogen within tubular epithelium. Restoration of normoxic conditions-either ex vivo during NMP or in vivo following transplant-triggered intravascular release of fibrinogen correlating with red blood cell aggregation and microvascular plugging. Combined delivery of plasminogen and tissue plasminogen activator during NMP lysed the plugs leading to a significant reduction in markers of renal injury, improvement in indicators of renal function, and improved delivery of vascular-targeted nanoparticles. Our study suggests a new mechanism of cold storage injury in marginal organs and provides a simple treatment with immediate translational potential.

The future of marginal kidney repair in the context of normothermic machine perfusion.

Normothermic machine perfusion (NMP) is a technique that utilizes extracorporeal membrane oxygenation to recondition and repair kidneys at near body temperature prior to transplantation. The application of this new technology has been fueled by a significant increase in the use of the kidneys that were donated after cardiac death, which are more susceptible to ischemic injury. Preliminary results indicate that NMP itself may be able to repair marginal organs prior to transplantation. In addition, NMP serves as a platform for delivery of therapeutics. The isolated setting of NMP obviates problems of targeting a particular therapy to an intended organ and has the potential to reduce the harmful effects of systemic drug delivery. There are a number of emerging therapies that have shown promise in this platform. Nutrients, therapeutic gases, mesenchymal stromal cells, gene therapies, and nanoparticles, a newly explored modality, have been successfully delivered during NMP. These technologies may be effective at blocking multiple mechanisms of ischemia- reperfusion injury (IRI) and improving renal transplant outcomes. This review addresses the mechanisms of renal IRI, examines the potential for NMP as a platform for pretransplant allograft modulation, and discusses the introduction of various therapies in this setting.

Quantitative microscopy-based measurements of circulating nanoparticle concentration using microliter blood volumes.

Nanoparticles (NPs) are potential drug delivery vehicles for treatment of a broad range of diseases. Intravenous (IV) administration, the most common form of delivery, is relatively non-invasive and provides (in theory) access throughout the circulatory system. However, in practice, many IV injected NPs are quickly eliminated by specialized phagocytes in the liver and spleen. Consequently, new materials have been developed with the capacity to significantly extend the circulating half-life of IV administered NPs. Unfortunately, current procedures for measuring circulation half-lives are often expensive, time consuming, and can require large blood volumes that are not compatible with mouse models of disease. Here we describe a simple and reliable procedure for measuring circulation half-life utilizing quantitative microscopy. This method requires only 2µL of blood and minimal sample preparation, yet provides robust quantitative results.

Revisiting the Principles of Preservation in an Era of Pandemic Obesity.

The current obesity epidemic has caused a significant decline in the health of our donor population. Organs from obese deceased donors are more prone to ischemia reperfusion injury resulting from organ preservation. As a consequence, these donors are more likely to be discarded under the assumption that nothing can be done to make them viable for transplant. Our current methods of organ preservation-which remain relatively unchanged over the last ~40 years-were originally adopted in the context of a much healthier donor population. But methods that are suitable for healthier deceased donors are likely not optimal for organs from obese donors. Naturally occurring models of acute obesity and fasting in hibernating mammals demonstrate that obesity and resilience to cold preservation-like conditions are not mutually exclusive. Moreover, recent advances in our understanding of the metabolic dysfunction that underlies obesity suggest that it may be possible to improve the resilience of organs from obese deceased donors. In this mini-review, we explore how we might adapt our current practice of organ preservation to better suit the current reality of our deceased donor population.

Surface conjugation of antibodies improves nanoparticle uptake in bronchial epithelial cells.

BACKGROUND: Advances in Molecular Therapy have made gene editing through systemic or topical administration of reagents a feasible strategy to treat genetic diseases in a rational manner. Encapsulation of therapeutic agents in nanoparticles can improve intracellular delivery of therapeutic agents, provided that the nanoparticles are efficiently taken up within the target cells. In prior work we had established proof-of-principle that nanoparticles carrying gene editing reagents can mediate site-specific gene editing in fetal and adult animals in vivo that results in functional disease improvement in rodent models of ß-thalassemia and cystic fibrosis. Modification of the surface of nanoparticles to include targeting molecules (e.g. antibodies) holds the promise of improving cellular uptake and specific cellular binding. METHODS AND FINDINGS: To improve particle uptake for diseases of the airway, like cystic fibrosis, our group tested the impact of nanoparticle surface modification with cell surface marker antibodies on uptake in human bronchial epithelial cells in vitro. Binding kinetics of antibodies (Podoplanin, Muc 1, Surfactant Protein C, and Intracellular Adhesion Molecule-1 (ICAM)) were determined to select appropriate antibodies for cellular targeting. The best target-specific antibody among those screened was ICAM antibody. Surface conjugation of nanoparticles with antibodies against ICAM improved cellular uptake in bronchial epithelial cells up to 24-fold. CONCLUSIONS: This is a first demonstration of improved nanoparticle uptake in epithelial cells using conjugation of target specific antibodies. Improved binding, uptake or specificity of particles delivered systemically or to the luminal surface of the airway would potentially improve efficacy, reduce the necessary dose and thus safety of administered therapeutic agents. Incremental improvement in the efficacy and safety of particle-based therapeutic strategies may allow genetic diseases such as cystic fibrosis to be cured on a fundamental genetic level before birth or shortly after birth.

A digital pathology tool for quantification of color features in histologic specimens.

In preclinical research, histological analysis of tissue samples is often limited to qualitative or semiquantitative scoring assessments. The reliability of this analysis can be impaired by the subjectivity of these approaches, even when read by experienced pathologists. Furthermore, the laborious nature of manual image assessments often leads to the analysis being restricted to a relatively small number of images that may not accurately represent the whole sample. Thus, there is a clear need for automated image analysis tools that can provide robust and rapid quantification of histologic samples from paraffin-embedded or cryopreserved tissues. To address this need, we have developed a color image analysis algorithm (DigiPath) to quantify distinct color features in histologic sections. We demonstrate the utility of this tool across multiple types of tissue samples and pathologic features, and compare results from our program to other quantitative approaches such as color thresholding and hand tracing. We believe this tool will enable more thorough and reliable characterization of histological samples to facilitate better rigor and reproducibility in tissue-based analyses.

Poly(amine-co-ester) nanoparticles for effective Nogo-B knockdown in the liver.

Degradable poly(amine-co-ester) (PACE) terpolymers hold tremendous promise for siRNA delivery because these materials can be formulated into delivery vehicles with highly efficient siRNA encapsulation, providing effective knockdown with low toxicity. Here, we demonstrate that PACE nanoparticles (NPs) provide substantial protein knockdown in human embryonic kidney cells (HEK293) and hard-to-transfect primary human umbilical vein endothelial cells (HUVECs). After intravenous administration, NPs of solid PACE (sPACE)-synthesized with high monomer content of a hydrophobic lactone-accumulated in the liver and, to a lesser extent, in other tissues. Within the liver, a substantial fraction of sPACE NPs were phagocytosed by liver macrophages, while a smaller fraction of NPs accumulated in hepatic stellate cells and liver sinusoidal endothelial cells, suggesting that sPACE NPs could deliver siRNA to diverse cell populations within the liver. To test this hypothesis, we loaded sPACE NPs with siRNA designed to knockdown Nogo-B, a protein that has been implicated in the progression of alcoholic liver disease and liver fibrosis. These sPACE:siRNA NPs produced up to 60% Nogo-B protein suppression in the liver after systemic administration. We demonstrate that sPACE NPs can effectively deliver siRNA therapeutics to the liver to mediate protein knockdown in vivo.

Nanoparticle targeting to the endothelium during normothermic machine perfusion of human kidneys.

Ex vivo normothermic machine perfusion (NMP) is a new clinical strategy to assess and resuscitate organs likely to be declined for transplantation, thereby increasing the number of viable organs available. Short periods of NMP provide a window of opportunity to deliver therapeutics directly to the organ and, in particular, to the vascular endothelial cells (ECs) that constitute the first point of contact with the recipient's immune system. ECs are the primary targets of both ischemia-reperfusion injury and damage from preformed antidonor antibodies, and reduction of perioperative EC injury could have long-term benefits by reducing the intensity of the host's alloimmune response. Using NMP to administer therapeutics directly to the graft avoids many of the limitations associated with systemic drug delivery. We have previously shown that polymeric nanoparticles (NPs) can serve as depots for long-term drug release, but ensuring robust NP accumulation within a target cell type (graft ECs in this case) remains a fundamental challenge of nanomedicine. We show that surface conjugation of an anti-CD31 antibody enhances targeting of NPs to graft ECs of human kidneys undergoing NMP. Using a two-color quantitative microscopy approach, we demonstrate that targeting can enhance EC accumulation by about 5- to 10-fold or higher in discrete regions of the renal vasculature. In addition, our studies reveal that NPs can also nonspecifically accumulate within obstructed regions of the vasculature that are poorly perfused. These quantitative preclinical human studies demonstrate the therapeutic potential for targeted nanomedicines delivered during ex vivo NMP.