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 liver transplantation (LTx) protocols typically use an orthotopic model where the recipients' native liver is removed and replaced with a donor liver. This technically demanding surgical procedure requires advanced micro-surgical skills and is further complicated by lengthy anhepatic and lower body ischemia times. This prompted the development of a less complicated heterotopic method that can be performed faster with no anhepatic or lower body ischemia time, reducing post-surgery stress for the recipient animal. This heterotopic LTx protocol includes two main steps: excising the liver from the donor rat and transplanting the whole liver into the recipient rat. During the excision of the donor liver, the surgeon ligates the supra-hepatic vena cava (SHVC) and hepatic artery (HA). On the recipient side, the surgeon removes the left kidney and positions the donor liver with the portal vein (PV), infra-hepatic vena cava (IHVC), and bile duct facing the renal vessels. Further, the surgeon anastomoses the recipient's renal vein end to end with the IHVC of the liver and arterializes the PV with the renal artery using a stent. A hepaticoureterostomy is utilized for biliary drainage by anastomosing the bile duct to the recipient's ureter, permitting the discharge of bile via the bladder. The average duration of the transplantation was 130 min, cold ischemia duration was around 35 min, and warm ischemia duration was less than 25 min. Hematoxylin and eosin histology of the auxiliary liver from syngeneic transplants showed normal hepatocyte structure with no significant parenchymal alterations 30 days post-transplant. In contrast, 8-day post-transplant allogeneic graft specimens demonstrated extensive lymphocytic infiltration with a Banff Schema rejection activity index score of 9. Therefore, this LTx method facilitates a low morbidity rejection model alternative to orthotopic LTx.

Early allogeneic immune modulation after establishment of donor hematopoietic cell-induced mixed chimerism in a nonhuman primate kidney transplant model.

Background: Mixed lymphohematopoietic chimerism is a proven strategy for achieving operational transplant tolerance, though the underlying immunologic mechanisms are incompletely understood. Methods: A post-transplant, non-myeloablative, tomotherapy-based total lymphoid (TLI) irradiation protocol combined with anti-thymocyte globulin and T cell co-stimulatory blockade (belatacept) induction was applied to a 3-5 MHC antigen mismatched rhesus macaque kidney and hematopoietic cell transplant model. Mechanistic investigations of early (60 days post-transplant) allogeneic immune modulation induced by mixed chimerism were conducted. Results: Chimeric animals demonstrated expansion of circulating and graft-infiltrating CD4+CD25+Foxp3+ regulatory T cells (Tregs), as well as increased differentiation of allo-protective CD8+ T cell phenotypes compared to naïve and non-chimeric animals. In vitro mixed lymphocyte reaction (MLR) responses and donor-specific antibody production were suppressed in animals with mixed chimerism. PD-1 upregulation was observed among CD8+ T effector memory (CD28-CD95+) subsets in chimeric hosts only. PD-1 blockade in donor-specific functional assays augmented MLR and cytotoxic responses and was associated with increased intracellular granzyme B and extracellular IFN-γ production. Conclusions: These studies demonstrated that donor immune cell engraftment was associated with early immunomodulation via mechanisms of homeostatic expansion of Tregs and early PD-1 upregulation among CD8+ T effector memory cells. These responses may contribute to TLI-based mixed chimerism-induced allogenic tolerance.

The Immunological Effect of Oxygen Carriers on Normothermic Ex Vivo Liver Perfusion.

Introduction: Normothermic ex vivo liver perfusion (NEVLP) is an organ preservation method that allows liver graft functional assessment prior to transplantation. One key component of normothermic perfusion solution is an oxygen carrier to provide oxygen to the liver to sustain metabolic activities. Oxygen carriers such as red blood cells (RBCs) or hemoglobin-based oxygen carriers have an unknown effect on the liver-resident immune cells during NEVLP. In this study, we assessed the effects of different oxygen carriers on the phenotype and function of liver-resident immune cells. Methods: Adult Lewis rat livers underwent NEVLP using three different oxygen carriers: human packed RBCs (pRBCs), rat pRBCs, or Oxyglobin (a synthetic hemoglobin-based oxygen carrier). Hourly perfusate samples were collected for downstream analysis, and livers were digested to isolate immune cells. The concentration of common cytokines was measured in the perfusate, and the immune cells underwent phenotypic characterization with flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The stimulatory function of the liver-resident immune cells was assessed using mixed lymphocyte reactions. Results: There were no differences in liver function, liver damage, or histology between the three oxygen carriers. qRT-PCR revealed that the gene expression of nuclear factor κ light chain enhancer of activated B cells (NF-kB), Interleukin (IL-1ß), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 7 (CCL7), and CD14 was significantly upregulated in the human pRBC group compared with that in the naive, whereas the rat pRBC and Oxyglobin groups were not different from that of naive. Flow cytometry demonstrated that the cell surface expression of the immune co-stimulatory protein, CD86, was significantly higher on liver-resident macrophages and plasmacytoid dendritic cells perfused with human pRBC compared to Oxyglobin. Mixed lymphocyte reactions revealed increased allogeneic T-cell proliferation in the human and rat pRBC groups compared to that in the Oxyglobin group. Conclusions: Liver-resident immune cells are important mediators of rejection after transplantation. In this study, we show that the oxygen carrier used in NEVLP solutions can affect the phenotype of these liver-resident immune cells. The synthetic hemoglobin-based oxygen carrier, Oxyglobin, showed the least amount of liver-resident immune cell activation and the least amount of allogeneic proliferation when compared to human or rat pRBCs. To mitigate liver-resident immune cell activation during NEVLP (and subsequent transplantation), Oxyglobin may be an optimal oxygen carrier.

A Novel Intron-Encoded Neuropilin-1 Isoform in Pancreatic Islets Associated With Very Young Age of Onset of Type 1 Diabetes.

Net synthesis of pancreatic ß-cells peaks before 2 years of life. ß-Cell mass is set within the first 5 years of life. In-frame translational readthrough of the NRP1 gene exon 9 into intron 9 generates a truncated neuropilin-1 protein lacking downstream sequence necessary for binding VEGF that stimulates ß-cell replication. VEGF is critical for developing but not adult islet neogenesis. Herein we show that cells in human pancreatic islets containing the full-length neuropilin-1 possess insulin but cells that contain the truncated neuropilin-1 are devoid of insulin. Decreased insulin cells increases susceptibility to onset of type 1 diabetes at a younger age. We also show that the frequency of a genetic marker in NRP1 intron 9 is higher among patients with onset of type 1 diabetes before age 4 years (31.8%), including those with onset at 0.67-2.00 and 2-4 years, compared with that in patients with onset at 4-8 years, at 8-12 years, and after 16 years (16.1%) with frequency equal to that in subjects without diabetes (16.0%). Decreased insulin cells plus the genetic data are consistent with a low effect mechanism that alters the onset of type 1 diabetes to a very young age in some patients, thus supporting the endotype concept that type 1 diabetes is a heterogeneous disease.

A human pancreatic ECM hydrogel optimized for 3-D modeling of the islet microenvironment.

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.

Single-cell RNA sequencing distinguishes dendritic cell subsets in the rat, allowing advanced characterization of the effects of FMS-like tyrosine kinase 3 ligand.

Tissue-resident dendritic cells (DCs) are essential for immunological homeostasis and hold promise for a variety of therapeutic interventions. The rare nature of tissue-resident DCs and their suboptimal description in the lab rat model has limited their characterization. To address this limitation, FMS-like tyrosine kinase 3 ligand (FLT3L) has been utilized to expand these population in vitro and in vivo for investigative or therapeutic purposes. However, conflicting reports have suggested that FLT3L can either promote immune tolerance or enhance immunogenicity, necessitating clarification of the effects of FLT3L on DC phenotype and functionality. We first paired single-cell RNA sequencing with multicolour spectral flow cytometry to provide an updated strategy for the identification of tissue-resident classical and plasmacytoid DCs in the rat model. We then administered FLT3L to Lewis rats in vivo to investigate its effect on tissue-resident DC enumeration and phenotype in the liver, spleen, and mesenteric lymph nodes. We found that FLT3L expands classical DCs (cDCs) 1 and 2 in a dose-dependent manner and that cDC1 and cDC2 in secondary lymphoid organs had altered MHC I, MHC II, CD40, CD80, CD86, and PD-L1 cell-surface expression levels following FLT3L administration. These changes were accompanied by an increase in gene expression levels of toll-like receptors 2, 4, 7, and 9 as well as inflammatory cytokines IL-6 and TNF-α. In conclusion, FLT3L administration in vivo increases cDC enumeration in the liver, spleen, and mesenteric lymph nodes accompanied by a tissue-restricted alteration in expression of antigen presentation machinery and inflammatory mediators.

Complement Blockade in Recipients Prevents Delayed Graft Function and Delays Antibody-mediated Rejection in a Nonhuman Primate Model of Kidney Transplantation.

BACKGROUND: Complement activation in kidney transplantation is implicated in the pathogenesis of delayed graft function (DGF). This study evaluated the therapeutic efficacy of high-dose recombinant human C1 esterase inhibitor (rhC1INH) to prevent DGF in a nonhuman primate model of kidney transplantation after brain death and prolonged cold ischemia. METHODS: Brain death donors underwent 20 h of conventional management. Procured kidneys were stored on ice for 44-48 h, then transplanted into ABO-compatible major histocompatibility complex-mismatched recipients. Recipients were treated with vehicle (n = 5) or rhC1INH 500 U/kg plus heparin 40 U/kg (n = 8) before reperfusion, 12 h, and 24 h posttransplant. Recipients were followed up for 120 d. RESULTS: Of vehicle-treated recipients, 80% (4 of 5) developed DGF versus 12.5% (1 of 8) rhC1INH-treated recipients (P = 0.015). rhC1INH-treated recipients had faster creatinine recovery, superior urinary output, and reduced urinary neutrophil gelatinase-associated lipocalin and tissue inhibitor of metalloproteinases 2-insulin-like growth factor-binding protein 7 throughout the first week, indicating reduced allograft injury. Treated recipients presented lower postreperfusion plasma interleukin (IL)-6, IL-8, tumor necrosis factor-alpha, and IL-18, lower day 4 monocyte chemoattractant protein 1, and trended toward lower C5. Treated recipients exhibited less C3b/C5b-9 deposition on day 7 biopsies. rhC1INH-treated animals also trended toward prolonged mediated rejection-free survival. CONCLUSIONS: Our results recommend high-dose C1INH complement blockade in transplant recipients as an effective strategy to reduce kidney injury and inflammation, prevent DGF, delay antibody-mediated rejection development, and improve transplant outcomes.

Interleukin-10 and Transforming Growth Factor-ß Cytokines Decrease Immune Activation During Normothermic Ex Vivo Machine Perfusion of the Rat Liver.

Normothermic ex vivo liver perfusion (NEVLP) is a novel system for organ preservation that may improve over static cold storage clinically and offers the chance for graft modification prior to transplantation. Although recent studies have shown the presence of inflammatory molecules during perfusion, none have yet shown the effects of NEVLP on liver-resident immune cell activation. We investigated the effects of NEVLP on liver-resident immune cell activation and assessed the ability of anti-inflammatory cytokines interleukin 10 (IL10) and transforming growth factor ß (TGF-ß) to improve organ function and reduce immune activation during perfusion. Rat livers were perfused for 4 hours at 37°C with or without the addition of 20 ng/mL of each IL10 and TGF-ß (n = 7). Naïve and cold storage (4 hours at 4°C) livers served as controls (n = 4). Following preservation, gene expression profiles were assessed through single-cell RNA sequencing; dendritic cell and macrophage activation was measured by flow cytometry; and cytokine production was assessed by enzyme-linked immunosorbent assay. NEVLP induced a global inflammatory gene expression signature, most notably in liver-resident macrophages and dendritic cells, which was accompanied by an increase in cell-surface levels of major histocompatibility complex (MHC) II, CD40, and CD86. Immune activation was partially ameliorated by IL10 and TGF-ß treatment, but no changes were observed in inflammatory cytokine production. Overall levels of liver damage and cellular apoptosis from perfusion were low, and liver function was improved with IL10 and TGF-ß treatment. This is the first study to demonstrate that liver-resident immune cells gain an activated phenotype during NEVLP on both the gene and protein level and that this activation can be reduced through therapeutic intervention with IL10 and TGF-ß.

Optimal Time to Ship Human Islets Post Tissue Culture to Maximize Islet.

Access to functional high-quality pancreatic human islets is critical to advance diabetes research. The Integrated Islet Distribution Program (IIDP), a major source for human islet distribution for over 15 years, conducted a study to evaluate the most advantageous times to ship islets postisolation to maximize islet recovery. For the evaluation, three experienced IIDP Islet Isolation Centers each provided samples from five human islet isolations, shipping 10,000 islet equivalents (IEQ) at four different time periods postislet isolation (no 37°C culture and shipped within 0 to 18 hours; or held in 37°C culture for 18 to 42, 48 to 96, or 144 to 192 hours). A central evaluation center compared samples for islet quantity, quality, and viability for each experimental condition preshipment and postshipment, as well as post 37°C culture 18 to 24 hours after shipment receipt. Additional evaluations included measures of functional potency by static glucose-stimulated insulin release (GSIR), represented as a stimulation index. Comparing the results of the four preshipment holding periods, the greatest IEQ loss postshipment occurred with the shortest preshipment times. Similar patterns emerged when comparing preshipment to postculture losses. In vitro islet function (GSIR) was not adversely impacted by increased tissue culture time. These data indicate that allowing time for islet recovery postisolation, prior to shipping, yields less islet loss during shipment without decreasing islet function.

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

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.

Kidney After Liver Transplantation Matched-pair Analysis: Are Kidneys Allocated to Appropriate Patients to Maximize Their Survival?

BACKGROUND: Kidney after liver transplantation (KALT) is the best therapeutic option for patients with end-stage renal disease after orthotopic liver transplantation (OLT). New allocation policies prioritize kidneys to patients in renal failure within the first year following OLT. There is little data on how kidney quality, measured by kidney donor profile index (KDPI), impacts KALT survival outcomes. METHODS: The United Network for Organ Sharing database was queried for adult KALT recipients from 1988 to 2015 and compared to their paired kidney transplant alone (KTA) recipients. Seven hundred forty-five pairs were stratified into 3 KDPI subgroups and compared patient survival, graft survival, and death-censored graft survival among matched-paired recipients. RESULTS: Overall, KTA recipients had superior patient and graft survival compared with the KALT group. KTA patient survival was superior for all 3 KDPI subgroups analysis. KTA graft survival was superior compared with KALT recipients of KDPI 21%-85% kidneys. Inferior graft half-life was observed in KALT versus KTA recipients with KDPI 21%-85% and >85%. CONCLUSIONS: From a utilitarian perspective, it is important that kidneys are allocated to recipients that are able to maximize their benefit from the full life of the organ. In KTA recipients, graft quality correlates directly to graft survival. However, in KALT patients receiving the matched-pair kidneys of the KTA recipients, patient mortality, rather than kidney quality, dictates graft survival significantly. As allocation practices continue developing, utilization of expanded criteria kidneys that better match anticipated patient and graft survival should be strongly considered to maximize the benefits of limited resources for the greatest number of patients.

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

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.

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

BACKGROUND: 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. METHODS: Brain-dead (n = 12) and sham (n = 5) rhesus macaques were maintained for 20 hours under intensive care unit-level conditions. Samples were collected for immunophenotyping, analysis of plasma proteins, coagulation studies, and gene analysis for changes in immune and metabolic profile with comparison to naive samples (n = 10). RESULTS: We observed an increase in circulating leukocytes and cytokines, activation of complement and coagulation pathways, and upregulation of genes associated with inflammation in both brain-dead and sham subjects relative to naïve controls. Sham demonstrated an intermediate phenotype of inflammation compared to BD. Analysis of gene expression in kidneys from BD kidneys revealed a similar upregulation of inflammatory profile in both BD and sham subjects, but BD presented a distinct reduction in metabolic and respiratory processes compared to sham and naïve kidneys. CONCLUSION: BD is associated with activation of specific pathways of the innate immune system and changes to metabolic gene expression in renal tissue itself; however, sham donors presented an intermediate inflammatory response attributable to the critical care environment. The early onset and penetrating impact of this inflammatory response underscores the need for early intervention to prevent perioperative tissue injury to transplantable organs.

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

INTRODUCTION: 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). METHODS: An IRB-approved single-center retrospective chart review was performed from January 1999 to July 2013. The patients were divided into six groups: DBD KDPI ≤60, DBD KPDI 61-84, DBD KDPI ≥85, DCD KDPI ≤60, DCD KPDI 61-84, and DCD KDPI ≥85. Rates of DGF, patient survival, and graft survival were examined among groups. RESULTS: A total of 2161 kidney transplants were included. DGF rates increased, and graft and patient survival decreased with increasing KDPI (P < .001). DCD kidneys had higher DGF rates than their DBD counterparts (P < .001). In DCD kidneys, a higher KDPI score did not significantly affect the DGF rates (P > .302). There was no significant difference in graft or patient survival in all-comers when comparing DCD and DBD kidneys with equivalent KDPIs (P > .317). Patients with DGF across all categories demonstrated worse graft half-lives. CONCLUSION: The KDPI system is an accurate predictor of donor contributions to transplant outcomes. Recipients of DBD kidneys experience an increase in the rate of DGF as their KDPI increases. DCD kidneys have higher DGF rates than their DBD counterparts with similar KDPIs. Patients with documented post-transplant DGF had between 3- and 5-year shorter graft half-lives when compared to recipients that did not experience DGF. Initiatives to reduce the rate of DGF could provide a significant impact on graft survival and result in a reduction in the number of patients requiring retransplant.

Guidelines for the management of a brain death donor in the rhesus macaque: A translational transplant model.

INTRODUCTION: The development of a translatable brain death animal model has significant potential to advance not only transplant research, but also the understanding of the pathophysiologic changes that occur in brain death and severe traumatic brain injury. The aim of this paper is to describe a rhesus macaque model of brain death designed to simulate the average time and medical management described in the human literature. METHODS: Following approval by the Institutional Animal Care and Use Committee, a brain death model was developed. Non-human primates were monitored and maintained for 20 hours after brain death induction. Vasoactive agents and fluid boluses were administered to maintain hemodynamic stability. Endocrine derangements, particularly diabetes insipidus, were aggressively managed. RESULTS: A total of 9 rhesus macaque animals were included in the study. The expected hemodynamic instability of brain death in a rostral to caudal fashion was documented in terms of blood pressure and heart rate changes. During the maintenance phase of brain death, the animal's temperature and hemodynamics were maintained with goals of mean arterial pressure greater than 60mmHg and heart rate within 20 beats per minute of baseline. Resuscitation protocols are described so that future investigators may reproduce this model. CONCLUSION: We have developed a reproducible large animal primate model of brain death which simulates clinical scenarios and treatment. Our model offers the opportunity for researchers to have translational model to test the efficacy of therapeutic strategies prior to human clinical trials.

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

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-ß1 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.

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

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 ß, 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.

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

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

BACKGROUND: 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). METHODS: Rhesus macaques were assigned to either brain death or control group. BD was induced by inflation of a subdurally placed catheter and confirmed clinically and by cerebral angiography. Animals were monitored for 6 h after BD and managed to maintain hemodynamic stability. RESULTS: Cortisol, epinephrine, nor-epinephrine, and IL-6 levels were elevated immediately after BD induction. Neutrophils and monocytes significantly increased in circulation following BD induction, while dendritic cells were decreased at 6 h post-induction. Flow cytometry revealed increased expression of chemokine receptors CxCR1, CxCR2, CCR2, and CCR5 in peripheral blood leukocytes from NHP subjected to BD. Microarray analysis demonstrated a significant up-regulation of genes related to innate inflammatory responses, toll-like receptor signaling, stress pathways, and apoptosis/cell death in BD subjects. Conversely, pathways related to glucose, lipid, and protein metabolism were down-regulated. In addition, increased expression of SOCS3, S100A8/A9, ICAM-1, MHC class II, neutrophil accumulation, and oxidative stress markers (carboxy-methyl-lysine and hydroxynonenal) were detected by immunoblot and immunohistochemistry. CONCLUSIONS: Activation of the innate immune response after BD in association with a down-regulation of genes associated with cell metabolism pathways in the liver. These findings may provide a potential explanation for the reduced post-transplant function of organs from brain dead donors. In addition, this work suggests potential novel targets to improve donor management strategies.

GPR30, but not estrogen receptor-alpha, is crucial in the treatment of experimental autoimmune encephalomyelitis by oral ethinyl estradiol.

BACKGROUND: Remission of multiple sclerosis during periods of high ovarian hormone secretion (such as pregnancy) has led to a great deal of interest in the potential for estrogens to treat autoimmune disease. Previous work has established that 17beta-estradiol can inhibit onset of experimental autoimmune encephalomyelitis (EAE), while ethinyl estradiol (EE) can reduce the severity of established disease. In the current study, the influence of estrogen receptor-alpha (ERalpha) and the G-protein coupled estrogen receptor (GPR30 or GPER) on EE's ability to treat EAE was explored. RESULTS: EE reduced disease severity in wild-type and ERalpha knockout (ERKO) mice, but did not alter disease in the GPR30KO group. Production of anti-inflammatory IL-10 increased in EE-ERKO mice (which showed reduced disease) but not in EE-GPR30KO mice (who did not have improved disease). CONCLUSIONS: Differential production of IL-10 following EE treatment in ERKO and GPR30KO animals may be responsible for the distinctly different effects on disease severity. Increased IL-10 in ERKO-EE compared to ERKO-Controls is likely to be an important factor in reducing established disease. The inability of EE to reduce disease in GPR30KO mice indicates an important but still undefined role for GPR30 in regulating immune reactivity.