Expansion Of Pool Research Articles

Transplanting organs from hepatitis B positive donors: Is it safe? Is it ethical?

Liver transplant centres throughout the USA face a huge shortage of liver organs for their wait-listed patients. Various types of innovations are being considered for expansion of this donor pool. Organs that were previously deemed to be high risk are now being considered for transplantation. For the last 25years, hepatitis B core antibody (anti-HBc+) organs have been used for liver transplantation. While the initial transplantations did reveal a high incidence of de novo hepatitis (DNH) in the recipients, the medical knowledge and experience have evolved and this risk has been markedly decreased. In this paper, medical literature evaluating the safety of such organ transplants has been reviewed. There is strong evidence to suggest that using anti-HBc+ organs with appropriate prophylaxis after transplant is a safe practice with good patient and graft survivals. In the second half of the paper, we discuss whether it is ethical to use anti-HBc+ organs. We argue that the use of such organs is in compliance with the principles of medical ethics and that society at large benefits from the use of these organs. Hence, we recommend that the use of such organs is both safe and ethical and this practice should be continued in the future.

Strain differences in thymic atrophy in rats immunized for EAE correlate with the clinical outcome of immunization

An accumulating body of evidence suggests that development of autoimmune pathologies leads to thymic dysfunction and changes in peripheral T-cell compartment, which, in turn, perpetuate their pathogenesis. To test this hypothesis, thymocyte differentiation/maturation in rats susceptible (Dark Agouti, DA) and relatively resistant (Albino Oxford, AO) to experimental autoimmune encephalomyelitis (EAE) induction was examined. Irrespective of strain, immunization for EAE (i) increased the circulating levels of IL-6, a cytokine causally linked with thymic atrophy, and (ii) led to thymic atrophy reflecting partly enhanced thymocyte apoptosis associated with downregulated thymic IL-7 expression. Additionally, immunization diminished the expression of Thy-1, a negative regulator of TCRαβ-mediated signaling and activation thresholds, on CD4+CD8+ TCRαβlo/hi thymocytes undergoing selection and thereby impaired thymocyte selection/survival. This diminished the generation of mature CD4+ and CD8+ single positive TCRαβhi thymocytes and, consequently, CD4+ and CD8+ recent thymic emigrants. In immunized rats, thymic differentiation of natural regulatory CD4+Foxp3+CD25+ T cells (nTregs) was particularly affected reflecting a diminished expression of IL-7, IL-2 and IL-15. The decline in the overall thymic T-cell output and nTreg generation was more pronounced in DA than AO rats. Additionally, differently from immunized AO rats, in DA ones the frequency of CD28- cells secreting cytolytic enzymes within peripheral blood CD4+ T lymphocytes increased, as a consequence of thymic atrophy-related replicative stress (mirrored in CD4+ cell memory pool expansion and p16INK4a accumulation). The higher circulating level of TNF-α in DA compared with AO rats could also contribute to this difference. Consistently, higher frequency of cytolytic CD4+ granzyme B+ cells (associated with greater tissue damage) was found in spinal cord of immunized DA rats compared with their AO counterparts. In conclusion, the study indicated that strain differences in immunization-induced changes in thymopoiesis and peripheral CD4+CD28- T-cell generation could contribute to rat strain-specific clinical outcomes of immunization for EAE.

Diverging impact of cell fate determinants Scrib and Llgl1 on adhesion and migration of hematopoietic stem cells.

Cell fate determinants Scrib and Llgl1 influence self-renewal capacity of hematopoietic stem cells (HSCs). Scrib-deficient HSCs are functionally impaired and lack sufficient repopulation capacity during serial transplantation and stress. In contrast, loss of Llgl1 leads to increased HSC fitness, gain of self-renewal capacity and expansion of the stem cell pool. Here, we sought to assess for shared and unique molecular functions of Llgl1 and Scrib by analyzing their interactome in hematopoietic cells. Interactome analysis was performed by affinity purification followed by mass spectrometry. Motility, migration and adhesion were assessed on primary murine HSCs, which were isolated by FACS sorting following conditional deletion of Scrib or Llgl1, respectively. Imaging of Scrib-deficient HSCs was performed by intravital 2-photon microscopy. Comparison of Scrib and Llgl1 interactome analyses revealed involvement in common and unique cellular functions. Migration and adhesion were among the cellular functions connected to Scrib but not to Llgl1. Functional validation of these findings confirmed alterations in cell adhesion and migration of Scrib-deficient HSCs in vitro and in vivo. In contrast, genetic inactivation of Llgl1 did not affect adhesion or migratory capacity of hematopoietic stem cells. Our data provide first evidence for an evolutionarily conserved role of the cell fate determinant Scrib in HSC adhesion and migration in vitro and in vivo, a unique function that is not shared with its putative complex partner Llgl1.

Abstract 543: Uncovering the Mechanisms by Which Fatty Acid Oxidation Suppresses Cardiomyocyte Hypertrophy

In cardiac hypertrophy, the adult heart switches from mainly using fatty acids to an increased reliance on glucose to maintain its energetic demands. We have previously shown that preserving fatty acid oxidation (FAO) by cardiac-specific deletion of Acetyl-CoA Carboxylase 2 (ACC2) prevents the shift of substrate preference towards glucose, preserves cardiac function and reduces cardiomyocyte hypertrophy during chronic pressure overload. To determine whether maintaining FAO specifically prevented cardiomyocyte hypertrophy, we treated adult rat cardiomyocytes (CMs) with and without adenoviral-mediated ACC2 knock-down (KD) with phenylephrine (PE, 10 μM). ACC2 KD effectively prevented CM hypertrophy after PE stimulation compared to control CMs (+9±6% vs. 42±6%) in medium supplemented with fatty acids (FA) (5.5 mM glucose, 0.4 mM mixed long-chain FAs and 0.1 mU/ml insulin). Whereas PE stimulation in control CMs increased glucose uptake (+28±8%), accumulation of glycolytic intermediates and lactate (+52±15%), this was normalized after ACC2 KD. Inhibiting FAO by etomoxir or increasing glucose utilization by dichloroacetate abolished the antihypertrophic effects of ACC2 KD after PE stimulation. However, replacing glucose with pyruvate or propionate restored the anti-hypertrophic effect of ACC2 KD. The expansion of TCA cycle pool caused by PE stimulation was also suppressed by ACC KD. Furthermore, ACC2 KD resulted in decreased intracellular amino acid levels, consistent to what has been seen in ACC2 knock-out hearts in vivo. This was accompanied by reduced glutamine consumption from the media (-60±25%) in ACC2 KD CMs, indicating reduced glutamine reliance. Taken together, our data suggest that ACC2 KD reduces the utilization of glucose and glutamine; both substrates known to be required for cell growth in vitro and in vivo. This further supports the hypothesis that maintaining FAO prevents the metabolic switch and CM hypertrophy.

Reactivation of RNA metabolism underlies somatic restoration after adult reproductive diapause in C. elegans.

The mechanisms underlying biological aging are becoming recognized as therapeutic targets to delay the onset of multiple age-related morbidities. Even greater health benefits can potentially be achieved by halting or reversing age-associated changes. C. elegans restore their tissues and normal longevity upon exit from prolonged adult reproductive diapause, but the mechanisms underlying this phenomenon remain unknown. Here, we focused on the mechanisms controlling recovery from adult diapause. Here, we show that functional improvement of post-mitotic somatic tissues does not require germline signaling, germline stem cells, or replication of nuclear or mitochondrial DNA. Instead a large expansion of the somatic RNA pool is necessary for restoration of youthful function and longevity. Treating animals with the drug 5-fluoro-2'-deoxyuridine prevents this restoration by blocking reactivation of RNA metabolism. These observations define a critical early step during exit from adult reproductive diapause that is required for somatic rejuvenation of an adult metazoan animal.

3042 - Visualizing the Clonal Complexity of the Hematopoietic System during the Natural Lifespan of the Mouse and after Serial Transplantation

Multiple genome sequencing studies have identified the prevalence of clonal hematopoiesis of indeterminate potential (CHIP) in aged human populations. Still, little is known about the contribution and dynamics of hematopoietic clones during the lifespan of an individual. Here, we used a non-invasive in vivo color labeling system to observe changes in hematopoietic clonal complexity during the natural murine lifespan. During steady-state hematopoiesis, the clonal complexity of the peripheral blood (PB) was essentially stable until 20–24 months, at which point complexity decreased by 30% (p = 0.04). Meanwhile, the clonal complexity of different hematopoietic stem and progenitor cell bone marrow compartments (HSPC) in the bone marrow of aged mice decreased much more dramatically by 70–77% (p < 0.001). This discrepancy in complexity between the PB and the HSPC may be reflective of a substantial clonal expansion of the HSPC pool during aging, creating a mixture of HSPC in which the majority are compromised in their stem-like ability to contribute to the PB. This compromised population would likely mask the smallerbut more diverseHSPCs that actively contribute to the PB. Indeed, we detected an 18-fold increase in the number of immunophenotypic HSC in aged bone marrow (p = 0.001), yet aged HSC displayed reduced repopulating activity compared to young HSC. Further, our in vivo color labelling system allowed us to track the clonal dynamics of PB and bone marrow repopulation as young and aged bone marrow were serially transplanted. By tertiary and quaternary transplantation, most recipients displayed mono- or di-color PB and HSPC pools, reflective of clonal hematopoiesis. Whole exome sequencing is underway to identify molecular drivers of these predominant clones. Our study represents the first analysis of the evolving clonal complexity of hematopoiesis throughout the natural lifespan of the mouse.

Longitudinal profile of circulating T follicular helper lymphocytes parallels anti-HLA sensitization in renal transplant recipients.

Antibody-mediated rejection is responsible for 30%-50% of renal graft failures. Differentiation of B cells into antibody-producing plasmablasts depends on the collaboration of follicular helper T cells (Tfh). We analyzed circulating Tfh (cTfh) in kidney recipients and studied cTfh relationship with anti-HLA antibody production and graft outcome. cTfh were longitudinally analyzed in a prospective cohort of patients (n=206), pre- and posttransplantation. Clinical data, HLA sensitization, and cTfh function were recorded. Both pretransplant and 6-month posttransplant cTfh were able to derive IgG-producing plasmablasts. Pretransplant cTfh was decreased in patients, especially in those who received dialysis. However, these cells were increased in patients with previous allograft or transfusions and in HLA-sensitized recipients. After transplantation cTfh expanded, significantly more in patients who developed de novo anti-HLA antibodies than in patients who remained unsensitized. Augmented pretransplant cTfh positively correlated with higher intensity of pretransplant anti-HLA class I and with de novo anti-HLA class I and anti-HLA class II antibodies. Consistently, pretransplantation cTfh were higher in patients who experienced acute rejection (HR=1.14 [1.04-1.25]). Thus, we show a role for Tfh in anti-HLA sensitization and rejection. Multicenter studies with additional patient cohorts are needed to validate these results. Immunosuppressive drugs targeting Tfh could be useful to improve outcomes.

Oxygen consumption during hypothermic and subnormothermic machine perfusions of porcine liver grafts after cardiac death.

The use of grafts donated after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. Machine perfusion (MP) is a promising technology to improve DCD liver grafts. Several perfusion technologies under various temperature and oxygenation conditions have been suggested and are still debated. It is important to confirm the relationship between oxygen consumption and organ conditions during MP. In this study, we analyzed oxygen consumption during oxygenated MP of porcine DCD liver grafts under different temperature conditions: hypothermic and subnormothermic. Grafts exposed to 60min of warm ischemia were perfused for 4h with a modified UW-gluconate perfusate under hypothermic (HMP) and subnormothermic conditions. Oxygen consumption, pressures of the portal vein and hepatic artery, and effluent enzymes were analyzed. Oxygen consumption was strongly related to the graft temperature during MP. Effluent enzyme level of the LDH were lower in the high oxygen consumption group than in the low oxygen consumption group during MP. In summary, we found that high oxygen consumption under subnormothermic temperature conditions has several advantages over HMP for DCD liver graft preservation.

PO-286 Protein kinase D3: a crucial regulator of breast cancer stem cell survival

IntroductionTriple negative breast cancer (TNBC) accounts for 15%–20% of all breast carcinomas in women. TNBC is particularly aggressive, highly metastatic and associated with poor prognosis. We previously identified Protein Kinase D3 (PKD3) to be upregulated in TNBC. PKD3 is a serine-threonine kinase that is best known for its functions in membrane trafficking and actin remodelling. Here we identify a novel role for PKD3 in breast cancer stem cell (CSC) maintenance. CSCs are endowed with tumor-initiating capacity, they possess increased chemoresistance and are known to initiate metastasis, and therefore are responsible for tumour relapse.Material and methodsIn the TNBC cell line MDA-MB-231 with a stable PKD3 knockdown, we performed a flow cytometry based screen of cell surface proteins, revealing the downregulation of several membrane proteins associated with CSCs. The importance of PKD3 for driving a CSC gene expression signature, maintaining CSC numbers and function was tested by qRT-PCR analysis of CSC markers, by measuring aldehyde dehydrogenase (ALDH) activity and performing mammosphere assays, respectively. Paclitaxel sensitivity of TNBC cell lines was assessed in combination with the selective PKD inhibitor CRT0066101. Whether the ectopic expression of PKD3 was sufficient to induce the expansion of the CSC pool was analysed in the non-transformed breast epithelial cell line MCF10A.Results and discussionsIn MDA-MB-231 cells with PKD3 knockdown, the CSC markers (CD184, Notch4, Sox2, Oct3/4) were downregulated. This was associated with reduced CSC numbers based on ALDH positivity and sphere-forming efficiency in mammosphere assays. Pharmacological PKD inhibition in the TNBC cell lines MDA-MB-231, BT549 and MDA-MB-436 verified these results. By contrast, inducible PKD3 expression in MCF10A cells elevated mammosphere formation and stem cell marker expression. Importantly, the combination of paclitaxel with CRT0066101 was superior over the single treatments in suppressing the survival of MDA-MB-231, BT549 and MDA-MB-436 cells in both colony formation and mammosphere assays.ConclusionOur results establish PKD3 as a crucial regulator of CSC maintenance and demonstrate that paclitaxel treatment in combination with PKD inhibition provides a potential novel strategy for future therapeutic applications in TNBC treatment.

Abstract 1143: Prox1 and Notch mark distinct colorectal cancer stem cell populations

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality. Signaling pathways, such as the Wnt and Notch pathways, are essential for the maintenance and differentiation of wild-type intestinal stem cells. We have shown that the Prox1 transcription promotes expansion of the colorectal cancer stem cell pool and that a subpopulation of the Prox1+ CRC cells display stem cell activity. Because of these results we have further analyzed the role of Prox1 in the regulation of CRC stem cell. Here, we report that Prox1 regulates CRC stem-like cells via bidirectional interaction with Notch1 during CRC initiation and progression. Using genetic in vivo models and ex vivo 3D organoid cultures, we show that Notch inhibition decreases the number of Lgr5+ stem cells whereas it increases expression of the Prox1. Although Notch inhibition led to increased proliferation of the Prox1 positive cells, it did not affect their ability to give rise to differentiated Prox1 negative progeny. Ectopic overexpression of the active fragment of Notch1 suppressed Prox1 expression and inhibited stem cell activity in the CRC cells. On the other hand, the PROX1-NuRD complex suppressed the Notch signaling pathway and Prox1 deletion increased Notch target gene expression and promoter activity, indicating reciprocal regulation between Prox1 and Notch1. Thus, although Prox1 and Notch suppress each other in colorectal cancer cells, Prox1+ cells can function as a stem cell population without the need for Notch pathway activity. Citation Format: Jenny Högström, Sarika Heino, Pauliina Kallio, Marianne Lähde, Veli-Matti Leppänen, Seppo Kaijalainen, Diego Balboa, Timo Otonkoski, Sylvie Robine, Zoltan Wiener, Kari Alitalo. Prox1 and Notch mark distinct colorectal cancer stem cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1143.

Trends in Transplantation with Older Liver Donors in the United States

As the United States population ages, older liver donors (OLDs) represent a potential expansion of the donor pool. Historically, grafts from OLDs have been associated with poor outcomes and higher rates of discard. We sought to evaluate trends in demographics, discard, and outcomes of OLDs. Methods We identified 4127 OLDs (aged≥70) and 3350 liver-only OLD graft recipients using data from the Scientific Registry of Transplant Recipients in the United States (1/1/2003-12/31/2016). We studied temporal changes in OLD graft characteristics, utilization, and recipient characteristics. Modified Poisson regression was used to estimate the annual discard rate. Cuzick test of trend was used to compare changes in OLD transplants performed over the study period. Kaplan-Meier methods were also used to create unadjusted cumulative incidence curves of mortality and all-cause graft loss. Cox proportional hazards models were used to estimate mortality and graft loss for OLD graft recipients. Results From 2003 to 2016, the discard of OLDs increased from 11.6% to 15.4% with the highest discard rate in 2008 at 24.5%. Discarded OLDs were more likely to become younger (74.3 years in 2013-2016 vs. 75.6 years in 2003-2006, p=0.004), have a higher BMI (28.7 vs. 26.7, p=0.008), and less likely to be Caucasian (73.7 vs. 80.8, p=0.03). Since 2003 the percentage of OLD transplants performed out of all adult liver transplants has decreased from 6.0% to 3.2% (p=0.001). The average age of OLD recipients increased from 55.9 years in 2003 to 59.8 years in 2016 (p<0.001). Since 2003, the indication for liver transplant in recipients of OLD grafts has changed. OLD recipients became more likely to have non-alcoholic steatohepatitis (16.9% in 2013-2016 vs 4.1% in 2003-2006) or HCC (22.6% vs 10.6%) as their indication for LT. Also, the average cold ischemia time decreased from 7.7 hours in 2003-2006 to 5.7 hours in 2013-2016 (p<0.001). Graft and patient survival for OLD graft recipients improved since 2003: OLD graft recipients from 2013-2016, mortality was 60% lower (aHR:0.40,95%CI:0.31-0.52,p<0.001) and all-cause graft loss was 55% lower (aHR:0.45,95%CI:0.36-0.57,p<0.001) than between 2003-2006. Conclusion Up to 25% of OLDs are discarded annually across the US, and the number of OLD transplants performed has decreased. However, there is a significant improvement in graft and patient survival for OLD recipients since 2003. Particularly in the setting of an aging population, these trends in improved outcomes can guide OLD use and decrease OLD discard to possibly expand the donor pool. National Institutes of Health.

PO-173 Sialylation of integrin alpha 2 promotes ovarian cancer cells metastasis

IntroductionThe emergence of migratory cell populations within solid tumours is a critical early stage during cancer metastasis. We have previously reported the enhanced migratory properties of one such population, marked by CD66 expression, in cervical cancers. It is not clear what mechanisms drive such migratory populations, and how these cells would retain a memory of initial migration-inducing cues during metastasis. Here, we sought to assess whether an altered heterochromatin state, controlled by chromatin regulator Suv39H1, drives migratory populations in cervical cancer.Material and methodsTo avoid an assumption regarding the role of an Epithelial to Mesenchymal Transition (EMT) in migration, cervical cancer cell line SiHa was sorted based on migratory ability in vitro. Following this, Suv39H1 function was assessed in vitro using RNAi. These approaches were complemented by a clinicopathological approach, using histopathology of clinical biopsies to examine cancer progression, along with a meta-analysis of RNA-Seq and clinical data obtained from The Cancer Genome Atlas (TCGA). Lastly, to infer putative mechanisms involved, RNA-Seq, confocal imaging and H3K9me3 ChIP-Seq were performed on sorted migrated cell populations.Results and discussionsWe observe that migrated populations show low Suv39H1, and that Suv39H1 knockdown enhances cell migration. Using histopathology, we observed the proliferative expansion of a Suv39H1high pool of cells in early stages of clinical progression, followed by the emergence of a sarcomatoid, migratory Suv39H1low cell population in advanced stages. Additionally, a meta-analysis of TCGA data revealed that low tumoural Suv39H1 also correlated with lower patient survival, and with gene expression signatures of migration, TGF-β signalling, and links with a CD66 cell signature. Lastly, using genome wide profiling and imaging on migrated populations in vitro, we observed that these populations show Suv39H1-linked migratory expression signatures, decondensed nuclei and a broad loss of H3K9me3, particularly across gene promoter elements. Our findings suggest a role for broad Suv39H1-mediated heterochromatin changes in regulating fate plasticity in migrated populations, contrasting with locus-specific heterochromatin changes previously described in EMT-based models.ConclusionThus, we report a Suv39H1-low heterochromatin state as a driver of migratory cell populations in cervical cancers. The understanding of such drivers may be valuable in efforts to develop anti-metastatic strategies.

Living Kidney Donation Is Recipient Age Sensitive and Has a High Rate of Donor Organ Disqualifications

BackgroundLiving donor kidney transplantation (LDKT) is the best therapy for patients with chronic renal failure. Its advantages, compared with cadaveric transplantation, include the possibility of avoiding dialysis, the likelihood of best outcome, and donor pool expansion. Careful assessment of potential donors is important to minimize the risks and ensure success. However, the proportion of donors disqualified has been poorly investigated. The aim of this work is to describe our experience and present the main reasons for missed donation. MethodsThis was a single-center, retrospective study of all potential donors and recipients evaluated for LDKT between January 2008 and December 2017. ResultsDuring the period of study, 81 donor-recipient pairs were evaluated. Of these, 45.7% were disqualified and 37 LDKTs were carried out. LDKT was the first choice in 68% of cases and preemptive in 20%; 60% of transplants were among family members. Sex distribution revealed a prevalence of females in the donor group (69%) and males in the recipient group (70%). The mean living donor age was 53 ± 9.5 years; the mean recipient age was lower in recipients listed in the living transplant program than those listed for cadaver transplantation (45.8 ± 13.4 vs 54.2 ± 11.08; P < .0001). Reasons for denial included hypertension (18.9%), deceased donor transplant performed during the study period (16.2%), urologic pathology (13.5%), incompatibility (13.5%), withdrawal of consent by donor or recipient (13.5%), psychological unsuitability (8.1%), donor cancer (5.4%), and reduced renal clearance (2.7%). ConclusionLDKT is considered an option especially for younger recipients. Of the potential kidney living donors, 45.7% were disqualified during the evaluation, with medical reasons being the primary cause.

Mig6 Is Required for Pancreas Development in Zebrafish

Strategies to promote β cell neogenesis could be used to restore functional β cell mass in both type 1 and type 2 diabetes. Here we examine the role of mitogen-inducible gene 6 (mig6, a.k.a. erffi1) in the regulation of functional β cell mass. mig6 is evolutionarily conserved and encodes a protein that inhibits the epidermal growth factor receptor (EGFR) in a classic feedback mechanism, suggesting a potential role in cellular growth and development. Based on functional studies in mouse and human, we hypothesized that mig6 is required in pancreatic ducts for pancreatogenesis and β cell function. We used morpholino knockdown in zebrafish to define the role of mig6 in both exocrine pancreas and islet development. We established that mig6 knockdown (mig6MO) embryos have a disrupted pancreas morphogenesis, particularly, a truncated exocrine pancreas and fewer intra-pancreatic duct cells. Additionally, mig6MO-injected animals had fewer β cells than controls. Together, our data lead us to hypothesize that mig6 may facilitate the expansion of the pancreas progenitor pool by blocking EGFR activation. We further investigated the role of mig6 in islet regeneration. Neogenic β cells may arise from two sources in the developing zebrafish, which can be distinguished by the retention of a fluorescent mark made at the one-cell stage. β cells derived via transdifferentiation from other postmitotic endocrine cells remain labeled whereas those derived from duct-associated progenitors lose their label via proliferation. We found that although the number of regenerated β cells was reduced overall in mig6MO-injected embryos, most new β cells were not labeled, suggesting that a significant progenitor pool remains in the knockdown embryos. Together, our data demonstrate that mig6 is essential for pancreas development and that it might be exploited as a switch between progenitor differentiation and endocrine transdifferentiation to increase β cell mass in diabetic patients. Disclosure K. El: None. P.T. Fueger: None. R.M. Anderson: None.

Accepting hepatitis C virus-infected donor hearts for transplantation: Multistep consent, unrealized opportunity, and the Stanford experience.

The current mismatch between supply and demand of organs has prompted transplant clinicians to consider innovative solutions to broaden the donor pool. Advancements of direct-acting antiviral agent (DAA) therapy for hepatitis C virus (HCV) have allowed entertaining the use of viremic donor organs in nonviremic recipients. In this report, we describe the evolution of HCV treatment, ethics and informed consent, cost-effectiveness of HCV medications in treating acute HCV post-transplantation, and the Stanford experience with two HCV-viremic donor heart transplantations. We describe excellent short-term outcomes post-heart transplantation with HCV NAT-positive organs. The availability of this therapy may expand the donor pool. While we await larger-scale clinical data on the effectiveness and safety of DAA therapy in patients after heart transplantation, many transplant centers have already started accepting organs from HCV-infected donors, balancing the unknown long-term risks versus the benefits of shorter wait times and expansion of the donor pool. Protocols and multidisciplinary teams are needed to effectively communicate risk to potential recipients, to ensure timely DAA access, and to implement appropriate clinical follow-up in order to achieve excellent clinical outcomes and to maximize the donor pool by utilizing HCV-infected organs for heart transplantation.

Proliferation of hippocampal progenitors relies on p27-dependent regulation of Cdk6 kinase activity.

Neural stem cells give rise to granule dentate neurons throughout life in the hippocampus. Upon activation, these stem cells generate fast proliferating progenitors that complete several rounds of divisions before differentiating into neurons. Although the mechanisms regulating the activation of stem cells have been intensively studied, little attention has been given so far to the intrinsic machinery allowing the expansion of the progenitor pool. The cell cycle protein Cdk6 positively regulates the proliferation of hippocampal progenitors, but the mechanism involved remains elusive. Whereas Cdk6 functions primarily as a cell cycle kinase, it can also act as transcriptional regulator in cancer cells and hematopoietic stem cells. Using mouse genetics, we show here that the function of Cdk6 in hippocampal neurogenesis relies specifically on its kinase activity. The present study also reveals a specific regulatory mechanism for Cdk6 in hippocampal progenitors. In contrast to the classical model of the cell cycle, we observe that the Cip/Kip family member p27, rather than the Ink4 family, negatively regulates Cdk6 in the adult hippocampus. Altogether, our data uncover a unique, cell type-specific regulatory mechanism controlling the expansion of hippocampal progenitors, where Cdk6 kinase activity is modulated by p27.

DGCR8 Promotes Neural Progenitor Expansion and Represses Neurogenesis in the Mouse Embryonic Neocortex.

DGCR8 and DROSHA are the minimal functional core of the Microprocessor complex essential for biogenesis of canonical microRNAs and for the processing of other RNAs. Conditional deletion of Dgcr8 and Drosha in the murine telencephalon indicated that these proteins exert crucial functions in corticogenesis. The identification of mechanisms of DGCR8- or DROSHA-dependent regulation of gene expression in conditional knockout mice are often complicated by massive apoptosis. Here, to investigate DGCR8 functions on amplification/differentiation of neural progenitors cells (NPCs) in corticogenesis, we overexpress Dgcr8 in the mouse telencephalon, by in utero electroporation (IUEp). We find that DGCR8 promotes the expansion of NPC pools and represses neurogenesis, in absence of apoptosis, thus overcoming the usual limitations of Dgcr8 knockout-based approach. Interestingly, DGCR8 selectively promotes basal progenitor amplification at later developmental stages, entailing intriguing implications for neocortical expansion in evolution. Finally, despite a 3- to 5-fold increase of DGCR8 level in the mouse telencephalon, the composition, target preference and function of the DROSHA-dependent Microprocessor complex remain unaltered. Thus, we propose that DGCR8-dependent modulation of gene expression in corticogenesis is more complex than previously known, and possibly DROSHA-independent.

1312 Nrf2 activation enhances the healing of cutaneous wounds through the activation of hair follicle stem cells

The transcription factor Nrf2 is a key regulator of the cellular stress response through the regulation of antioxidant enzymes, cytoprotective proteins and various transporters. Strong genetic activation of Nrf2 in keratinocytes leads to a pilosebaceous phenotype, characterized by hyperplasia of the sebaceous glands and infundibula, hyperkeratosis, and seborrhea, implicating Nrf2 in several other key processes in the epidermis. Here we show that Nrf2 activation in keratinocytes promotes the proliferation and expansion of the junctional zone (JZ) and upper isthmus (UI) hair follicle stem cells, while bulge stem cells are only mildly affected. This was observed to be functionally important for wound repair, since Nrf2 activation also led to the faster closure of excisional wounds through the formation of a longer and larger wound epithelium. This however, was neither due to changes in proliferation or apoptosis of keratinocytes in the wound epithelium, nor their migration as measured in vitro. Instead, an increased number and proliferation of follicular JZ and UI stem cells were observed peripheral to the wound. An enhancement of wound healing in Nrf2 transgenic mice following tape stripping of the epidermis revealed a functional link between the Nrf2-mediated expansion of hair follicles stem cells and accelerated re-epithelialization. The effect of Nrf2 activation on JZ and UI stem cells resulted from the Nrf2-mediated up-regulation of the EGF family member Epigen and subsequent EGF receptor activation. These results suggest pharmacological Nrf2 activation as a promising approach for the enhancement of wound healing through expansion of hair follicle stem cell pools.

Treatment and management options for the hepatitis C virus infected kidney transplant candidate.

A substantial body of literature has unequivocally established that prevalent hepatitis C virus infection in chronic kidney disease (CKD), end stage renal disease (ESRD) and kidney transplant recipients is associated with a negative impact on patient survival. As a consequence of remarkable work that explained the details of the hepatitis C virus (HCV) genome, a class of drugs referred to as the direct-acting antiviral (DAA) agents were developed that targeted specific key sites in viral replication. Large clinical trials in the HCV-infected general population followed soon after that demonstrated cure rates exceeding 95%. Treatment paradigms have been further refined and expanded to populations of patients that were initially excluded from the large pivotal trials. This includes the CKD and ESRD patients for whom there are now safe and effective DAAs available as well. In this context, the focus of decision making has shifted from initially demonstrating safety and efficacy to now identifying which patient should receive therapy and at what point in their CKD/ESRD journey. The specific issue of timing of treatment is particularly relevant to the HCV-infected ESRD patient who is being considered for kidney transplantation. The option of treating with DAAs prior to the transplant or alternatively delaying therapy and treating in the posttransplant period will be influenced by several factors, including patient preference, the extent of liver injury, the availability of a living or deceased donor, and more recently the option of transplanting a kidney from HCV-positive donor. The latter has been associated with the advantage of shortened waiting times and expansion of the organ donor pool. The optimal timing and choice of therapy will be the result of a decision that has been individualized for each patient as a consequence of a process of clear communication involving the patient, primary care physician, nephrologist, gastroenterologist (GI)/hepatologist, and local transplant center.

Reduction of Reperfusion Cardiac Injury in Donation After Circulatory Death Hearts Through Modulation of Electron Transport

INTODUCTIONHeart transplantation (HT) is a valuable option to prolong life in an end stage heart failure (HF) patient, but this option is limited due to the availability of donor hearts, which currently come from brain death donors only. Expansion of the heart donor pool is urgently needed. One potential source of donor hearts is donation after circulatory death (DCD) donors. Since the DCD process involves substantial ischemia (ISC) and reperfusion (REP) injury to heart, strategies are needed to reduce cardiac injury in DCD hearts for HT.HYPOTHESISAmobarbital (AMO) give during REP decreases cardiac injury. Based on the current human DCD protocol, the duration of ISC prior to heart procurement varies from 25 to 40 min. Thus, we examined if AMO given at the onset of REP can decrease cardiac injury in DCD heart following 45 min of global ISC.METHODSAfter 45 min of ISC, DCD hearts received AMO (2 mM) at the onset of REP for 5 min followed by buffer perfusion for 55 min; or buffer perfusion only for 60 min. Cardiac injury was measured by the left ventricle (LV) infarct size. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria(IFM) were isolated at 10 min of REP to measure calcium retention capacity (CRC, nmol calcium/mg protein) to assess mitochondrial permeability transition pore (MPTP) opening and rate of oxidative phosphorylation (nAO/min/mg).RESULTSAMO given during REP decreased the infarct size by 35% compared to untreated hearts. AMO improved the CRC in both SSM and IFM (Table), indicating a decreased susceptibility to MPTP opening during early REP. AMO did not influence oxidative phosphorylation with either complex I (glutamate) or complex II substrates (succinate+rotenone), suggesting that the protection of AMO is not due to improved oxidative phosphorylation.CONCLUSIONModulation of mitochondrial respiration at REP is a potential strategy to decrease cardiac injury in DCD hearts through inhibition of MPTP opening.Support or Funding InformationScientist development grant (MQ) from American Heart Association, Merit Review Award ( 2IO1BX001355‐01 EJ), R21AG054975‐01 (QC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.