heterotopic-auxiliary-whole-liver-rat-transplant-model-utilizing

Heterotopic Auxiliary Whole Liver Rat Transplant Model Utilizing a Hepaticoureterostomy for Allograft Rejection Studies

Small animal transplant models are indispensable for organ tolerance studies investigating feasible therapeutic interventions in preclinical studies. Rat ...

University of Wisconsin-Madison

Donor brain death (BD) triggers a systemic inflammatory response that reduces organ quality and increases immunogenicity of the graft. We characterized the early innate immune response induced by BD in the liver and peripheral blood of hemodinamically stable non-human primates (NHP).

Early activation of the inflammatory response in the liver of brain-dead non-human primates.

Most pancreas and islet grafts are recovered from brain-dead (BD) donors. In this study we characterized the early inflammatory response induced by brain death in pancreata and islets from nonhuman primate donors and evaluated the effect of targeted anti-inflammatory intervention in the protection of pancreatic islets prior to transplantation. BD donors were monitored for 6 h and assigned to three experimental groups: group 1: BD-untreated donors (BD-UT) (n = 7), group 2: BD + donor pretreatment with IL-1ra (n = 6), and group 3: non-BD animals serving as controls (n = 7). We observed an IL-1ra-dependent reduction in the mobilization and activation of neutrophils from bone marrow and a significantly reduced accumulation of CD68(+) leukocytes in the pancreas and islets after brain death induction. Donor treatment with IL-1ra significantly decreased chemokine mRNA expression (MCP-1, IL-8, and MIP-1a) and attenuated the activation of circulating neutrophils and intraislet macrophages as demonstrated by a reduction in intracellular IL-1β, IL-6, MCP-1, and MIP-1α expression. As a result, IL-1ra dramatically improved viability, mitochondrial membrane polarity, and islet engraftment in mice transplanted using a minimal islet mass. These results suggest that early immunomodulation targeting inflammation in the BD donor may represent an effective therapeutic strategy to improve islet quality and function prior to transplantation.

Donor Pretreatment With IL-1 Receptor Antagonist Attenuates Inflammation and Improves Functional Potency in Islets From Brain-Dead Nonhuman Primates.

The complement system is an essential component of innate immunity and plays a major role in the pathogenesis of ischemia-reperfusion injury (IRI). In this study, we investigated the impact of human C1-inhibitor (C1INH) on the early inflammatory response to IRI and the subsequent progression to fibrosis in mice. We evaluated structural damage, renal function, acute inflammatory response, progression to fibrosis and overall survival at 90-days post-injury. Animals receiving C1INH prior to reperfusion had a significant improvement in survival rate along with superior renal function when compared to vehicle (PBS) treated counterparts. Pre-treatment with C1INH also prevented acute IL-6, CXCL1 and MCP-1 up-regulation, C5a release, C3b deposition and infiltration by neutrophils and macrophages into renal tissue. This anti-inflammatory effect correlated with a significant reduction in the expression of markers of fibrosis alpha smooth muscle actin, desmin and picrosirius red at 30 and 90 days post-IRI and reduced renal levels of TGF-β1 when compared to untreated controls. Our findings indicate that intravenous delivery of C1INH prior to ischemic injury protects kidneys from inflammatory injury and subsequent progression to fibrosis. We conclude that early complement blockade in the context of IRI constitutes an effective strategy in the prevention of fibrosis after ischemic acute kidney injury.

Complement inhibition attenuates acute kidney injury after ischemia-reperfusion and limits progression to renal fibrosis in mice.

Renal transplant outcomes result from a combination of factors. Traditionally, donor factors were summarized by classifying kidneys as extended criteria or standard criteria. In 2014, the nomenclature changed to describe donor factors with the kidney donor profile index (KDPI). We aim to evaluate the relationship between KDPI and delayed graft function (DGF), and the impact KDPI on transplant outcomes for both donor after cardiac death (DCD) and donor after brain death (DBD).

The impact of kidney donor profile index on delayed graft function and transplant outcomes: A single-center analysis.

Long-term graft survival is an ongoing challenge in the field of islet transplantation. With the growing demand for transplantable organs, therapies to improve organ quality and reduce the incidence of graft dysfunction are of paramount importance. We evaluated the protective role of a recombinant DNA repair protein targeted to mitochondria (Exscien I-III), as a therapeutic agent using a rodent model of pancreatic islet transplantation. We first investigated the effect of therapy on isolated rat islets cultured with pro-inflammatory cytokines (interleukin-1 β, interferon γ, and tumor necrosis factor α) for 48 h and documented a significant reduction in apoptosis by flow cytometry, improved viability by immunofluorescence, and conserved functional potency in vitro and in vivo in Exscien I-III-treated islets. We then tested the effect of therapy in systemic inflammation using a rat model of donor brain death (BD) sustained for a 6-h period. Donor rats were allocated to 4 groups: (non-BD + vehicle, non-BD + Exscien I-III, BD + vehicle, and BD + Exscien I-III) and treated with Exscien I-III (4 mg/kg) or vehicle 30 min after BD induction. Sham (non-BD)-operated animals receiving either Exscien I-III or vehicle served as controls. Islets purified from BD + Exscien I-III-treated donors showed a significant increase in glucose-stimulated insulin release in vitro when compared to islets from vehicle-treated counterparts. In addition, donor treatment with Exscien I-III attenuated the effects of BD and significantly improved the functional potency of transplanted islets in vivo. Our data indicate that mitochondrially targeted antioxidant therapy is a novel strategy to protect pancreas and islet quality from the deleterious effects of cytokines in culture and during the inflammatory response associated with donation after BD. The potential for rapid translation into clinical practice makes Exscien I-III an attractive therapeutic option for the management of brain-dead donors or as an additive to islets in culture after isolation setting.

Novel Fusion Protein Targeting Mitochondrial DNA Improves Pancreatic Islet Functional Potency and Islet Transplantation Outcomes.

Brain death (BD)-associated inflammation has been implicated in decreased kidney allograft function and survival, but the underlying mechanisms have not been well distinguished from the conditions of critical care itself. We have developed a clinically translatable model to separate and investigate strategies to improve donor management and critical care.

Brain Death Enhances Activation of the Innate Immune System and Leads to Reduced Renal Metabolic Gene Expression.

Pancreatic steatosis is thought to be a negative risk factor for pancreas transplant outcomes. Despite considering donor body mass index (BMI) and the visualization of intercalated fat as indicators of donor pancreas lipid content, transplant surgeons do not use a quantitative method to directly measure steatosis when deciding to transplant a pancreas. In this study, we used nondiabetic human pancreata donated for research to measure the pancreatic and islet-specific lipid content to determine which clinical markers correlate best with lipid content. Interestingly, we found that BMI and age correlate with increased pancreatic lipid content (Panc-LC) in men, but not women. Our findings further suggest that total Panc-LC correlates with an increase in islet lipid content for both men and women. We noted that pancreata donated from individuals with a history of hypertension have increased Panc-LC independent of donor BMI or sex. Moreover, we identify hypertension as a risk factor for reduced islet function after islet isolation. Together, our findings emphasize differences in pancreas graft quality related to pancreatic and islet lipid content, which may not be predicted by assessing BMI alone but may be influenced by a donor history of hypertension.

Hypertension, but not body mass index, is predictive of increased pancreatic lipid content and islet dysfunction.

Delayed graft function (DGF) in renal transplant is associated with reduced graft survival and increased immunogenicity. The complement-driven inflammatory response after brain death (BD) and posttransplant reperfusion injury play significant roles in the pathogenesis of DGF. In a nonhuman primate model, we tested complement-blockade in BD donors to prevent DGF and improve graft survival. BD donors were maintained for 20 hours; kidneys were procured and stored at 4°C for 43-48 hours prior to implantation into ABO-compatible, nonsensitized, MHC-mismatched recipients. Animals were divided into 3 donor-treatment groups: G1 - vehicle, G2 - rhC1INH+heparin, and G3 - heparin. G2 donors showed significant reduction in classical complement pathway activation and decreased levels of tumor necrosis factor α and monocyte chemoattractant protein 1. DGF was diagnosed in 4/6 (67%) G1 recipients, 3/3 (100%) G3 recipients, and 0/6 (0%) G2 recipients (P = .008). In addition, G2 recipients showed superior renal function, reduced sC5b-9, and reduced urinary neutrophil gelatinase-associated lipocalin in the first week posttransplant. We observed no differences in incidence or severity of graft rejection between groups. Collectively, the data indicate that donor-management targeting complement activation prevents the development of DGF. Our results suggest a pivotal role for complement activation in BD-induced renal injury and postulate complement blockade as a promising strategy for the prevention of DGF after transplantation.

Targeted donor complement blockade after brain death prevents delayed graft function in a nonhuman primate model of kidney transplantation.

Extracellular matrix (ECM) plays a multitude of roles, including supporting cells through structural and biochemical interactions. ECM is damaged in the process of isolating human islets for clinical transplantation and basic research. A platform in which islets can be cultured in contact with natural pancreatic ECM is desirable to better understand and support islet health, and to recapitulate the native islet environment. Our study demonstrates the derivation of a practical and durable hydrogel from decellularized human pancreas that supports human islet survival and function. Islets embedded in this hydrogel show increased glucose- and KCl-stimulated insulin secretion, and improved mitochondrial function compared to islets cultured without pancreatic matrix. In extended culture, hydrogel co-culture significantly reduced levels of apoptosis compared to suspension culture and preserved controlled glucose-responsive function. Isolated islets displayed altered endocrine and non-endocrine cell arrangement compared to in situ islets; hydrogel preserved an islet architecture more similar to that observed in situ. RNA sequencing confirmed that gene expression differences between islets cultured in suspension and hydrogel largely fell within gene ontology terms related to extracellular signaling and adhesion. Natural pancreatic ECM improves the survival and physiology of isolated human islets.

Article	Year
Early activation of the inflammatory response in the liver of brain-dead non-human primates. The Journal of surgical research\| PubMed ID: 22440934	2012
Donor Pretreatment With IL-1 Receptor Antagonist Attenuates Inflammation and Improves Functional Potency in Islets From Brain-Dead Nonhuman Primates. Cell transplantation\| PubMed ID: 24759633	2015
Complement inhibition attenuates acute kidney injury after ischemia-reperfusion and limits progression to renal fibrosis in mice. PloS one\| PubMed ID: 28832655	2017
The impact of kidney donor profile index on delayed graft function and transplant outcomes: A single-center analysis. Clinical transplantation\| PubMed ID: 29314286	2018
Novel Fusion Protein Targeting Mitochondrial DNA Improves Pancreatic Islet Functional Potency and Islet Transplantation Outcomes. Cell transplantation\| PubMed ID: 29338388	2017
Brain Death Enhances Activation of the Innate Immune System and Leads to Reduced Renal Metabolic Gene Expression. Transplantation\| PubMed ID: 30964836	2019
Hypertension, but not body mass index, is predictive of increased pancreatic lipid content and islet dysfunction. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons\| PubMed ID: 31715064	2020
Targeted donor complement blockade after brain death prevents delayed graft function in a nonhuman primate model of kidney transplantation. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons\| PubMed ID: 31922336	2020
A human pancreatic ECM hydrogel optimized for 3-D modeling of the islet microenvironment. Scientific reports\| PubMed ID: 35504932	2022

Peter Chlebeck

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