A DNA methylation atlas of normal human cell types.

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2-5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

Persistent hyperbilirubinemia following preoperative biliary stenting in patients undergoing anatomic hepatectomy predicts serious complications.

BACKGROUND: Biliary obstruction before liver resection is a known risk factor for post-operative complications. The aim of this study was to determine the impact of persistent hyperbilirubinemia following preoperative biliary drainage before liver resection. METHODS: The ACS-NSQIP (2016-2021) database was used to extract patients with cholangiocarcinoma who underwent anatomic liver resection with preoperative biliary drainage comparing those with persistent hyperbilirubinemia (> 1.2 mg/dL) to those with resolution. Patient characteristics and outcomes were compared with bivariate analysis. Multivariable modeling evaluated factors including persistent hyperbilirubinemia to evaluate their independent effect on serious complications, liver failure, and mortality. RESULTS: We evaluated 463 patients with 217 (46.9%) having hyperbilirubinemia (HB) despite biliary stenting. Bivariate analysis demonstrated that patients with HB had a higher rate of serious complications than those with non-HB (80.7% vs 70.3%; P = 0.010) including bile leak (40.9% vs 31.8%; P = 0.045), liver failure (26.7% vs 17.9%; P = 0.022), and bleeding (48.4% vs 36.6%; P = 0.010). Multivariable analysis demonstrated that persistent HB was independently associated with serious complications (OR 1.88, P = 0.020) and mortality (OR 2.39, P = 0.049) but not post-operative liver failure (OR 1.65, P = 0.082). CONCLUSIONS: Failed preoperative biliary decompression is a predictive factor for post-operative complications and mortality in patients undergoing hepatectomy and may be useful for preoperative risk stratification.

Long-term outcomes after normothermic machine perfusion in liver transplantation-Experience at a single North American center.

Normothermic machine perfusion (NMP) has emerged as a valuable tool in the preservation of liver allografts before transplantation. Randomized trials have shown that replacing static cold storage (SCS) with NMP reduces allograft injury and improves graft utilization. The University of Alberta's liver transplant program was one of the early adopters of NMP in North America. Herein, we describe our 7-year experience applying NMP to extend preservation time in liver transplantation using a "back-to-base" approach. From 2015 to 2021, 79 livers were transplanted following NMP, compared with 386 after SCS only. NMP livers were preserved for a median time of minutes compared with minutes in the SCS cohort (P < .0001). Despite this, we observed significantly improved 30-day graft survival (P = .030), although there were no differences in long-term patient survival, major complications, or biliary or vascular complications. We also found that although SCS time was strongly associated with increased graft failure at 1 year in the SCS cohort (P = .006), there was no such association among NMP livers (P = .171). Our experience suggests that NMP can safely extend the total preservation time of liver allografts without increasing complications.

Pancreas and Islet Transplantation: Comparative Outcome Analysis of a Single-centre Cohort Over 20-years.

OBJECTIVE: To provide the largest single-center analysis of islet (ITx) and pancreas (PTx) transplantation. SUMMARY BACKGROUND DATA: Studies describing long-term outcomes with ITx and PTx are scarce. METHODS: We included adults undergoing ITx (n=266) and PTx (n=146) at the University of Alberta from January 1999 to October 2019. Outcomes include patient and graft survival, insulin independence, glycemic control, procedure-related complications, and hospital readmissions. Data are presented as medians (interquartile ranges, IQR) and absolute numbers (percentages, %) and compared using Mann-Whitney and χ2 tests. Kaplan-Meier estimates, Cox proportional hazard models and mixed main effects models were implemented. RESULTS: Crude mortality was 9.4% and 14.4% after ITx and PTx, respectively ( P= 0.141). Sex-adjusted and age-adjusted hazard-ratio for mortality was 2.08 (95% CI, 1.04-4.17, P= 0.038) for PTx versus ITx. Insulin independence occurred in 78.6% and 92.5% in ITx and PTx recipients, respectively ( P= 0.0003), while the total duration of insulin independence was 2.1 (IQR 0.8-4.6) and 6.7 (IQR 2.9-12.4) year for ITx and PTx, respectively ( P= 2.2×10 -22 ). Graft failure ensued in 34.2% and 19.9% after ITx and PTx, respectively ( P =0.002). Glycemic control improved for up to 20-years post-transplant, particularly for PTx recipients (group, P= 7.4×10 -7 , time, P =4.8×10 -6 , group*time, P= 1.2×10 -7 ). Procedure-related complications and hospital readmissions were higher after PTx ( P =2.5×10 -32 and P= 6.4×10 -112 , respectively). CONCLUSIONS: PTx shows higher sex-adjusted and age-adjusted mortality, procedure-related complications and readmissions compared with ITx. Conversely, insulin independence, graft survival and glycemic control are better with PTx. This study provides data to balance risks and benefits with ITx and PTx, which could improve shared decision-making.

Trends in types of graduate degrees and research output for academic general surgeons in Canada.

SummaryThe proportion of general surgeons with graduate degrees in Canada is increasing. We sought to evaluate the types of graduate degree held by surgeons in Canada, and whether differences in publication capacity exist. We evaluated all general surgeons working at English-speaking Canadian academic hospitals to determine the types of degrees achieved, changes over time and research output associated with each degree. We identified 357 surgeons, of whom 163 (45.7 %) had master's degrees and 49 (13.7 %) had PhDs. Achievement of graduate degrees increased over time, with more surgeons earning master's degrees in public health (MPH), clinical epidemiology and education (MEd), and fewer master's degrees in science (MSc) or PhDs. Most publication metrics were similar by degree type, but surgeons with PhDs published more basic science research than those with clinical epidemiology, MEd or MPH degrees (2.0 v. 0.0, p < 0.05); surgeons with clinical epidemiology degrees published more first-author articles than surgeons with MSc degrees (2.0 v. 0.0, p = 0.007). An increasing number of general surgeons hold graduate degrees, with fewer pursuing MSc and PhD degrees, and more holding MPH or clinical epidemiology degrees. Research productivity is similar for all groups. Support to pursue diverse graduate degrees could enable a greater breadth of research.

Estimation of Early Graft Function Using the BETA-2 Score Following Clinical Islet Transplantation.

Little is known about how early islet graft function evolves in the clinical setting. The BETA-2 score is a validated index of islet function that can be calculated from a single blood sample and lends itself to frequent monitoring of graft function. In this study, we characterized early graft function by calculating weekly BETA-2 score in recipients who achieved insulin independence after single transplant (group 1, n = 8) compared to recipients who required a second transplant before achieving insulin independence (group 2, n = 7). We also determined whether graft function 1-week post-transplant was associated with insulin independence in individuals who received initial transplant between 2000-2017 (n = 125). Our results show that graft function increased rapidly reaching a plateau 4-6 weeks post-transplant. The BETA-2 score was higher in group 1 compared to group 2 as early as 1-week post-transplant (15 + 3 vs. 9 + 2, p = 0.001). In an unselected cohort, BETA-2 at 1-week post-transplant was associated with graft survival as defined by insulin independence during median follow up of 12 months (range 2-119 months) with greater survival among those with BETA-2 score >10 (p < 0.001, log-rank test). These findings suggest that primary graft function is established within 4-6 weeks post-transplant and graft function at 1-week post-transplant predicts long-term transplant outcomes.

Current Status, Barriers, and Future Directions for Humanized Mouse Models to Evaluate Stem Cell-Based Islet Cell Transplant.

Islet cell transplant (ITx) continues to improve, with recently published long-term outcomes suggesting nearly 80% graft survival, leading to improvements in glycemic control, reductions in insulin doses, and near-complete abrogation of severe hypoglycemia. Unfortunately, access to ITx remains limited by immunosuppression requirements and donor supply. Discovery of stem cell-derived functional islet-like clusters with the capacity to reverse diabetes offers a renewable, potentially immunosuppression-free solution for future widespread ITx. Evaluation and optimization of these therapies is ongoing, but may one day provide a realistic cure for type 1 diabetes. However, stem cell-based ITx has unique immunologic questions that remain unanswered. Here, we briefly synthesize current approaches for stem cell-derived ITx, review humanized mice models, and elaborate on the potential of humanized mice models for bridging the gap between current small rodent models and human clinical trials for allogeneic and autologous inducible pluripotent stem cell (iPSC)-based ITx while highlighting limitations and future directions.

Association between academic degrees and research productivity: an assessment of Canadian academic general surgeons.

BACKGROUND: For academic hiring committees and surgical trainees, the benefits of a graduate degree are unclear. We sought to identify if graduate degrees or professorship status were associated with increased research productivity among Canadian academic surgeons. METHODS: We included general surgeons from the largest hospitals associated with accredited residency programs. We classified staff surgeons active between 2013 and 2018 by degree (MD only, master's degree, PhD) and professorship (assistant, associate, professor) status. We identified their publications from January 2013 to December 2018. Variables of interest included publications per year, citations per article, journal of publication, CiteScore, author's Hirsch (h) index and the revised h-index (r-index). We used Kruskal-Wallis tests and the Dunn multiple comparison test to assess statistical significance. RESULTS: We identified 3262 publications from 187 surgeons, including 78 (41.7%) with no graduate degree, 84 (44.9%) with master's degrees and 25 (13.4%) with PhDs. Surgeons with graduate degrees had more publications per year, higher CiteScores, more citations per article, and higher h- and r-indices than those without graduate degrees. Surgeons with doctorates had the highest median values in all domains, but differences were not significant compared with surgeons with master's degrees. Seventy-seven (41.8%) surgeons were assistant professors, 63 (34.2%) were associate professors and 44 (23.9%) were full professors. Statistically, full professors had a greater number of publications per year and higher h- and r-indices than their counterparts. CONCLUSION: Surgeons with graduate degrees or more advanced professorships had the greatest research productivity. Surgeons with doctorates trended toward greater research productivity than those holding master's degrees.

Targeting CXCR1/2 in the first multicenter, double-blinded, randomized trial in autologous islet transplant recipients.

Several cytokines and chemokines are elevated after islet infusion in patients undergoing total pancreatectomy with islet autotransplantation (TPIAT), including CXCL8 (also known as interleukin-8), leading to islet loss. We investigated whether use of reparixin for blockade of the CXCL8 pathway would improve islet engraftment and insulin independence after TPIAT. Adults without diabetes scheduled for TPIAT at nine academic centers were randomized to a continuous infusion of reparixin or placebo (double-blinded) for 7 days in the peri-transplant period. Efficacy measures included insulin independence (primary), insulin dose, hemoglobin A1c (HbA1c ), and mixed meal tolerance testing. The intent-to-treat population included 102 participants (age 39.5 ± 12.2 years, 69% female), n = 50 reparixin-treated, n = 52 placebo-treated. The proportion insulin-independent at Day 365 was similar in reparixin and placebo: 20% vs. 21% (p = .542). Twenty-seven of 42 (64.3%) in the reparixin group and 28/45 (62.2%) in the placebo group maintained HbA1c ≤6.5% (p = .842, Day 365). Area under the curve C-peptide from mixed meal testing was similar between groups, as were adverse events. In conclusion, reparixin infusion did not improve diabetes outcomes. CXCL8 inhibition alone may be insufficient to prevent islet damage from innate inflammation in islet autotransplantation. This first multicenter clinical trial in TPIAT highlights the potential for future multicenter collaborations.

Phase 3 trial of human islet-after-kidney transplantation in type 1 diabetes.

Allogeneic islet transplant offers a minimally invasive option for ß cell replacement in the treatment of type 1 diabetes (T1D). The CIT consortium trial of purified human pancreatic islets (PHPI) in patients with T1D after kidney transplant (CIT06), a National Institutes of Health-sponsored phase 3, prospective, open-label, single-arm pivotal trial of PHPI, was conducted in 24 patients with impaired awareness of hypoglycemia while receiving intensive insulin therapy. PHPI were manufactured using standardized processes. PHPI transplantation was effective with 62.5% of patients achieving the primary endpoint of freedom from severe hypoglycemic events and HbA1c  ≤ 6.5% or reduced by ≥ 1 percentage point at 1 year posttransplant. Median HbA1c declined from 8.1% before to 6.0% at 1 year and 6.3% at 2 and 3 years following transplant (P < .001 for all vs baseline), with related improvements in hypoglycemia awareness and glucose variability. The improved metabolic control was associated with better health-related and diabetes-related quality of life. The procedure was safe and kidney allograft function remained stable after 3 years. These results add to evidence establishing allogeneic islet transplant as a safe and effective treatment for patients with T1D and unstable glucose control despite intensive insulin treatment, supporting the indication for PHPI in the post-renal transplant setting.

Frequency of Obliteration of the Dorsal and Ventral Ducts of the Pancreas in Islet Transplantation.

BACKGROUND: Islet isolation is an essential process in every human islet transplantation protocol. Intraductal enzyme delivery followed by adequate distention of the pancreas is the most critical step in islet isolation. Anomalies of the pancreatic duct system can significantly affect this process. Thus, identification and characterization of ductal patency is of paramount importance to achieve optimal islet isolation. AIMS: To investigate the frequency of duct obliteration in the human pancreas and explore donor/patient characteristics associated with specific ductal variations. METHODS: We examined ductal patency of pancreata allocated for islet allotransplantation (n = 597) and autotransplantation (n = 21) after removal of the duodenum during islet isolation procedure. Donor/patient factors were reviewed from the batch files. RESULTS: Among 559 deceased donor pancreata without pancreas divisum (n = 38, 6.4%), both ducts were patent in 50.1%, only ventral duct was patent in 46.7%, and only dorsal duct was patent in 3.2%. Donor age was not associated with the frequency of obliterated dorsal duct. Black race tended to have the higher frequency of patent dorsal duct. As expected, pancreas divisum was more frequent in chronic pancreatitis cases (n = 6, 28.6%). Within 7 cases of chronic pancreatitis with unknown etiology, we encountered one case of ventral duct obliteration. CONCLUSIONS: The minor duodenal papilla and aging do not likely play an important role in the occurrence of dorsal duct obliteration. Although frequency of obliterated ventral duct was low in our population, physicians, including gastroenterologists and endoscopists, as well as islet transplantation researchers should be aware of this possibility.

Update on islet cell transplantation.

PURPOSE OF REVIEW: Chronic diabetes-related complications continue to exert a rapidly growing and unsustainable pressure on healthcare systems worldwide. In type 1 diabetes, glycemic control is particularly challenging, as intensive management substantially increase the risk of severe hypoglycemic episodes. Alternative approaches to address this issue are required. Islet cell transplantation offers the best approach to reduce hypoglycemic risks and glycemic lability, while providing optimal glycemic control. Although ongoing efforts have improved clinical outcomes, the constraints in tissue sources and the need for chronic immunosuppression limit the application of islet cell transplantation as a curative therapy for diabetes. This review provides an update on islet cell transplantation, focusing on recent clinical experience, ongoing research, and future challenges. RECENT FINDINGS: Current evidence demonstrates advances in terms of long-term glycemic control, improved insulin independence rates, and novel approaches to eliminate chronic immunosuppression requirements after islet cell transplantation. Advances in stem cell-based therapies provide a promising path towards truly personalized regenerative therapies, solving both tissue supply shortage and the need for lifelong immunosuppression, enabling widespread use of this potentially curative treatment. However, as these therapies enter the clinical realm, regional access variability and ethical questions regarding commercialization are becoming increasingly important and require a collaborative solution. SUMMARY: In this state-of-the-art review, we discuss current clinical evidence and discuss key aspects on the present and future of islet cell transplantation.

Luseogliflozin increases beta cell proliferation through humoral factors that activate an insulin receptor- and IGF-1 receptor-independent pathway.

AIMS/HYPOTHESIS: Sodium-glucose cotransporter 2 (SGLT2) inhibitors, which prevent the renal reabsorption of glucose, decrease blood glucose levels in an insulin-independent manner. We previously reported creating a mouse model of systemic inhibition of target receptors for both insulin and IGF-1 by treating animals with OSI-906, a dual insulin/IGF-1 receptor inhibitor, for 7 days. The OSI-906-treated mice exhibited an increased beta cell mass, hepatic steatosis and adipose tissue atrophy, accompanied by hyperglycaemia and hyperinsulinaemia. In the present study, we investigated the effects of an SGLT2 inhibitor, luseogliflozin, on these changes in OSI-906-treated mice. METHODS: We treated C57BL/6J male mice either with vehicle, luseogliflozin, OSI-906 or OSI-906 plus luseogliflozin for 7 days, and phenotyping was performed to determine beta cell mass and proliferation. Subsequently, we tested whether serum-derived factors have an effect on beta cell proliferation in genetically engineered beta cells, mouse islets or human islets. RESULTS: SGLT2 inhibition with luseogliflozin significantly ameliorated hyperglycaemia, but not hyperinsulinaemia, in the OSI-906-treated mice. Liver steatosis and adipose tissue atrophy induced by OSI-906 were not altered by treatment with luseogliflozin. Beta cell mass and proliferation were further increased by SGLT2 inhibition with luseogliflozin in the OSI-906-treated mice. Luseogliflozin upregulated gene expression related to the forkhead box M1 (FoxM1)/polo-like kinase 1 (PLK1)/centromere protein A (CENP-A) pathway in the islets of OSI-906-treated mice. The increase in beta cell proliferation was recapitulated in a co-culture of Irs2 knockout and Insr/IR knockout (ßIRKO) beta cells with serum from both luseogliflozin- and OSI-906-treated mice, but not after SGLT2 inhibition in beta cells. Circulating factors in both luseogliflozin- and OSI-906-treated mice promoted beta cell proliferation in both mouse islets and cadaveric human islets. CONCLUSIONS/INTERPRETATION: These results suggest that luseogliflozin can increase beta cell proliferation through the activation of the FoxM1/PLK1/CENP-A pathway via humoral factors that act in an insulin/IGF-1 receptor-independent manner.

Normothermic Preservation of Liver - What Does the Future Hold?

Recent years have demonstrated a surging interest in normothermic ex situ liver perfusion, with iterative experimental and clinical studies establishing this technology as providing obvious advantages over static cold storage. In particular, the safe prolongation of liver graft preservation even up to 1 week opens up possibilities of 'on circuit' interventions, which may radically change the logistics and scope of liver transplant practice. Such approaches are rife with potential, and have yet to be fully explored. Possibilities may include, but are not limited to mitochondrial enhancing strategies, steatotic graft defatting, on circuit addition of anti-aging compounds, altering graft immunogenic potential and gene silencing with siRNA, stem cell and nanoparticle therapies as well as ischemia free liver preservation. Ex situ machine perfusion technology as a platform for advanced graft modification strategies opens up the possibility of very specific, personalized transplant medicine, as well as the possibility of a future where organ grafts are re-used and repaired, providing utility to numerous successive surgical recipients, indefinitely.

Islet cells share promoter hypomethylation independently of expression, but exhibit cell-type-specific methylation in enhancers.

DNA methylation at promoters is an important determinant of gene expression. Earlier studies suggested that the insulin gene promoter is uniquely unmethylated in insulin-expressing pancreatic ß-cells, providing a classic example of this paradigm. Here we show that islet cells expressing insulin, glucagon, or somatostatin share a lack of methylation at the promoters of the insulin and glucagon genes. This is achieved by rapid demethylation of the insulin and glucagon gene promoters during differentiation of Neurogenin3+ embryonic endocrine progenitors, regardless of the specific endocrine cell-type chosen. Similar methylation dynamics were observed in transgenic mice containing a human insulin promoter fragment, pointing to the responsible cis element. Whole-methylome comparison of human α- and ß-cells revealed generality of the findings: genes active in one cell type and silent in the other tend to share demethylated promoters, while methylation differences between α- and ß-cells are concentrated in enhancers. These findings suggest an epigenetic basis for the observed plastic identity of islet cell types, and have implications for ß-cell reprogramming in diabetes and diagnosis of ß-cell death using methylation patterns of circulating DNA.

A Back-to-Base Experience of Human Normothermic Ex Situ Liver Perfusion: Does the Chill Kill?

Normothermic machine perfusion (NMP) has been shown to protect livers from injury between procurement and transplantation in a randomized controlled trial, where the machine was transported to and from the donor center. The aim of this study was to determine whether an alternative, more practical back-to-base approach after initial static cold storage would compromise beneficial outcomes. Between February 2015 and June 2018, a nonrandomized pilot study was performed at a single site. Outcomes of back-to-base livers (n = 26) were compared with those of grafts procured locally that underwent immediate NMP (n = 17). The primary outcome measure (safety) was defined as 30-day patient and graft survival. A total of 46 liver grafts were perfused with NMP, of which 3 were discarded based on poor ex situ perfusion function. The 30-day patient and graft survival in the back-to-base and local NMP groups were both 100% (primary outcome: safety). Despite significantly prolonged mean cold ischemia time (6 versus 3.2 hours; P = 0.001), the back-to-base livers demonstrated no difference in graft function, incidence of complications, or graft and patient survival. In conclusion, the back-to-base approach was safe, did not compromise the overall benefit of NMP, and offers a practical alternative to portable normothermic ex situ machine transport.

Transient Cold Storage Prior to Normothermic Liver Perfusion May Facilitate Adoption of a Novel Technology.

Clinical adoption of normothermic machine perfusion (NMP) may be facilitated by simplifying logistics and reducing costs. This can be achieved by cold storage of livers for transportation to recipient centers before commencing NMP. The purpose of this study was to assess the safety and feasibility of post-static cold storage normothermic machine perfusion (pSCS-NMP) in liver transplantation. In this multicenter prospective study, 31 livers were transplanted. The primary endpoint was 30-day graft survival. Secondary endpoints included the following: peak posttransplant aspartate aminotransferase (AST), early allograft dysfunction (EAD), postreperfusion syndrome (PRS), adverse events, critical care and hospital stay, biliary complications, and 12-month graft survival. The 30-day graft survival rate was 94%. Livers were preserved for a total of 14 hours 10 minutes ± 4 hours 46 minutes, which included 6 hours 1 minute ± 1 hour 19 minutes of static cold storage before 8 hours 24 minutes ± 4 hours 4 minutes of NMP. Median peak serum AST in the first 7 days postoperatively was 457 U/L (92-8669 U/L), and 4 (13%) patients developed EAD. PRS was observed in 3 (10%) livers. The median duration of initial critical care stay was 3 days (1-20 days), and median hospital stay was 13 days (7-31 days). There were 7 (23%) patients who developed complications of grade 3b severity or above, and 2 (6%) patients developed biliary complications: 1 bile leak and 1 anastomotic stricture with no cases of ischemic cholangiopathy. The 12-month overall graft survival rate (including death with a functioning graft) was 84%. In conclusion, this study demonstrates that pSCS-NMP was feasible and safe, which may facilitate clinical adoption.

MiR-184 expression is regulated by AMPK in pancreatic islets.

AMPK is a critical energy sensor and target for widely used antidiabetic drugs. In ß cells, elevated glucose concentrations lower AMPK activity, and the ablation of both catalytic subunits [ß-cell-specific AMPK double-knockout (ßAMPKdKO) mice] impairs insulin secretion in vivo and ß-cell identity. MicroRNAs (miRNAs) are small RNAs that silence gene expression that are essential for pancreatic ß-cell function and identity and altered in diabetes. Here, we have explored the miRNAs acting downstream of AMPK in mouse and human ß cells. We identified 14 down-regulated and 9 up-regulated miRNAs in ßAMPKdKO vs. control islets. Gene ontology analysis of targeted transcripts revealed enrichment in pathways important for ß-cell function and identity. The most down-regulated miRNA was miR-184 (miR-184-3p), an important regulator of ß-cell function and compensatory expansion that is controlled by glucose and reduced in diabetes. We demonstrate that AMPK is a potent regulator and an important mediator of the negative effects of glucose on miR-184 expression. Additionally, we reveal sexual dimorphism in miR-184 expression in mouse and human islets. Collectively, these data demonstrate that glucose-mediated changes in AMPK activity are central for the regulation of miR-184 and other miRNAs in islets and provide a link between energy status and gene expression in ß cells.-Martinez-Sanchez, A., Nguyen-Tu, M.-S., Cebola, I., Yavari, A., Marchetti, P., Piemonti, L., de Koning, E., Shapiro, A. M. J., Johnson, P., Sakamoto, K., Smith, D. M., Leclerc, I., Ashrafian, H., Ferrer, J., Rutter, G. A. MiR-184 expression is regulated by AMPK in pancreatic islets.

Intraoperative continuous renal replacement therapy during liver transplantation: a pilot randomized-controlled trial (INCEPTION).

PURPOSE: To evaluate the feasibility of intraoperative continuous renal replacement therapy (IoCRRT) during liver transplantation (LT), in terms of recruitment, protocol adherence, and ascertainment of follow-up. METHODS: In this pilot randomized open-label controlled trial in adults receiving LT with a Model for End-Stage Liver Disease (MELD) score ≥ 25 and preoperative acute kidney injury (RIFLE - RISK or higher) and/or estimated glomerular filtration rate < 60 mL·min-1·1.73 m-2, patients were randomized to receive IoCRRT or standard of care (SOC). Primary endpoints were feasibility and adverse events. Primary analysis was intention-to-treat (n = 32) and secondary analysis was per-protocol (n = 28). RESULTS: The trial was stopped early because of slow patient accrual and inadequate funding. Sixty patients were enrolled and 32 (53%) were randomized (n = 15 IoCRRT; n = 17 SOC). Mean (standard deviation) MELD was 36 (8), 81% (n = 26) had cirrhosis; 69% (n = 22) received preoperative RRT; 66% (n = 21) received LT from the intensive care unit. Four patients (n = 2 IoCRRT, n = 2 SOC) did not receive LT post-randomization. Seven patients (41%) allocated to SOC crossed over intraoperatively to IoCRRT. Three patients were lost to follow-up at one year. No adverse events occurred related to IoCRRT. There were no differences in survival at one year (IoCRRT, 71% [n = 10/14] vs SOC, 93% [n = 14/15]; risk ratio, 0.77; 95% confidence interval, 0.54 to 1.1). In the per-protocol analysis (n = 28 received IoCRRT after randomization - n = 20 IoCRRT, n = 8 SOC), one-year survival was 92% and perioperative complications were similar between groups. Only one patient was receiving dialysis one year after LT. CONCLUSION: In this pilot randomized trial, IoCRRT was feasible and safe with no difference in complications. Crossover rates were high. Despite high preoperative severity of illness, one-year survival was excellent. These data can inform the design of a larger multicentre trial. TRIAL REGISTRATION: www.clinicalTrials.gov (NCT01575015); registered 12 April, 2012.

RéSUMé: OBJECTIF: Notre but était d'évaluer la faisabilité d'un traitement substitutif peropératoire continu de l'insuffisance rénale pendant une greffe hépatique, notamment en matière de recrutement, d'adhésion au protocole, et de suivi. MéTHODE: Dans cette étude randomisée contrôlée non aveugle pilote réalisée auprès d'adultes recevant une greffe hépatique avec un score MELD (Model for End-Stage Liver Disease) ≥ 25 et une insuffisance rénale aiguë préopératoire (RIFLE - RISQUÉ ou plus élevé) et/ou un taux de filtration glomérulaire estimé < 60 mL·min−1·1,73 m−2, les patients ont été randomisés à recevoir un traitement substitutif peropératoire continu de l'insuffisance rénale (le traitement) ou les soins habituels (la norme). Les critères d'évaluation principaux étaient la faisabilité et les événements indésirables. L'analyse principale était l'analyse du projet thérapeutique (intention-to-treat; n = 32) et l'analyse secondaire était l'analyse selon le protocole (n = 28). RéSULTATS: L'étude a été précocement interrompue en raison du recrutement lent de patients et du manque de fonds. Soixante patients ont été recrutés et 32 (53 %) ont été randomisés (n = 15 traitement; n = 17 norme). Le score MELD moyen (écart type) était de 36 (8), 81 % (n = 26) des patients souffraient de cirrhose; 69 % (n = 22) ont reçu un traitement substitutif de l'insuffisance rénale préopératoire; 66 % (n = 21) ont reçu une greffe hépatique à partir de l'unité de soins intensifs. Quatre patients (n = 2 traitement, n = 2 norme) n'ont pas reçu de greffe hépatique après la randomisation. Sept patients (41 %) alloués au groupe norme sont passés dans le groupe traitement en période peropératoire. Trois patients ont été perdus au suivi au cours de la première année. Aucun événement indésirable n'est survenu en association au traitement substitutif peropératoire continu de l'insuffisance rénale. Aucune différence en matière de survie à un an n'a été observée (traitement, 71 % [n = 10/14] vs norme, 93 % [n = 14/15]; risque relatif, 0,77; intervalle de confiance 95 %, 0,54 à 1,1). Dans l'analyse selon le protocole (n = 28 ont reçu un traitement après la randomisation - n = 20 traitement, n = 8 norme), la survie à un an était de 92 % et les complications périopératoires étaient semblables dans les deux groupes. Un seul patient recevait de la dialyse un an après la greffe hépatique. CONCLUSION: Dans cette étude randomisée pilote, le traitement substitutif peropératoire continu de l'insuffisance rénale s'est avéré faisable et sécuritaire, et aucune différence en matière de complications n'a été observée. Les taux de transfert d'un groupe à l'autre étaient élevés. Malgré une sévérité préopératoire élevée de la maladie, la survie à un an était excellente. Ces données peuvent être utiles pour concevoir une étude multicentrique plus importante. ENREGISTREMENT DE L'éTUDE: www.clinicalTrials.gov (NCT01575015); enregistrée le 12 avril 2012.

Identification of tissue-specific cell death using methylation patterns of circulating DNA.

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic ß-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.