Epstein-Barr Virus Genome Deletions in Epstein-Barr Virus-Positive T/NK Cell Lymphoproliferative Diseases.

The main target cells for Epstein-Barr virus (EBV) infection and persistence are B lymphocytes, although T and NK cells can also become infected. In this paper, we characterize the EBV present in 21 pediatric and adult patients who were treated in France for a range of diseases that involve infection of T or NK cells. Of these 21 cases, 5 pediatric patients (21%) and 11 adult patients (52%) were of Caucasian origin. In about 30% of the cases, some of the EBV genomes contain a large deletion. The deletions are different in every patient but tend to cluster near the BART region of the viral genome. Detailed investigation of a family in which several members have persistent T or NK cell infection by EBV indicates that the virus genome deletions arise or are selected independently in each individual patient. Genome sequence polymorphisms in the EBV in these T or NK cell diseases reflect the geographic origin of the patient and not a distinct type of EBV (the 21 cases studied included examples of both type 1 and type 2 EBV infection). Using virus produced from type 1 or type 2 EBV genomes cloned in bacterial artificial chromosome (BAC) vectors, we demonstrate infection of T cells in cord blood from healthy donors. Our results are consistent with transient infection of some T cells being part of normal asymptomatic infection by EBV in young children. IMPORTANCE EBV contributes to several types of human cancer. Some cancers and nonmalignant lymphoproliferative diseases involving T or NK cells contain EBV. These diseases are relatively frequent in Japan and China and have been shown sometimes to have deletions in the EBV genome in the disease cells. We identify further examples of deletions within the EBV genome associated with T or NK cell diseases, and we provide evidence that the virus genomes with these deletions are most likely selected in the individual cases, rather than being transmitted between people during infection. We demonstrate EBV infection of cord blood T cells by highly characterized, cloned EBV genomes and suggest that transient infection of T cells may be part of normal asymptomatic infection by EBV in young children.

LEF-1 drives aberrant ß-catenin nuclear localization in myeloid leukemia cells.

Canonical Wnt/ß-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized ß-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no nuclear ß-catenin even where cytosolic ß-catenin is abundant. Control of the subcellular localization of ß-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of ß-catenin, we carried out the first nuclear/cytoplasmic proteomic analysis of the ß-catenin interactome in myeloid leukemia cells and identified putative novel ß-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear ß-catenin) versus Wnt-unresponsive cells (low nuclear ß-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of ß-catenin. The relative levels of nuclear LEF-1 and ß-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed ß-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and ß-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first ß-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear ß- catenin level in human myeloid leukemia.

Hospital-acquired complications in intensive care unit patients with diabetes: A before-and-after study of a conventional versus liberal glucose control protocol.

BACKGROUND: Critically ill patients with diabetes mellitus (DM) are at increased risk of in-hospital complications and the optimal glycemic target for such patients remains unclear. A more liberal approach to glucose control has recently been suggested for patients with DM, but uncertainty remains regarding its impact on complications. METHODS: We aimed to test the hypothesis that complications would be more common with a liberal glycemic target in ICU patients with DM. Thus, we compared hospital-acquired complications in the first 400 critically ill patients with DM included in a sequential before-and-after trial of liberal (glucose target: 10-14 mmol/L) vs conventional (glucose target: 6-10 mmol/L) glucose control. RESULTS: Of the 400 patients studied, 165 (82.5%) patients in the liberal and 177 (88.5%) in the conventional-control group were coded for at least one hospital-acquired complication (P = 0.09). When comparing clinically relevant complications diagnosed between ICU admission and hospital discharge, we found no difference in the odds for infectious (adjusted odds ratio [aOR] for liberal-control: 1.15 [95% CI: 0.68-1.96], P = 0.60), cardiovascular (aOR 1.40 [95% CI: 0.63-3.12], P = 0.41) or neurological complications (aOR: 1.07 [95% CI: 0.61-1.86], P = 0.81), acute kidney injury (aOR 0.83 [95% CI: 0.43-1.58], P = 0.56) or hospital mortality (aOR: 1.09 [95% CI: 0.59-2.02], P = 0.77) between the liberal and the conventional-control group. CONCLUSION: In this prospective before-and-after study, liberal glucose control was not associated with an increased risk of hospital-acquired infectious, cardiovascular, renal or neurological complications in critically ill patients with diabetes.

Liberal Glucose Control in ICU Patients With Diabetes: A Before-and-After Study.

OBJECTIVES: To assess the feasibility, biochemical efficacy, and safety of liberal versus conventional glucose control in ICU patients with diabetes. DESIGN: Prospective, open-label, sequential period study. SETTING: A 22-bed mixed ICU of a tertiary hospital in Australia. PATIENTS: We compared 350 consecutive patients with diabetes admitted over 15 months who received liberal glucose control with a preintervention control population of 350 consecutive patients with diabetes who received conventional glucose control. INTERVENTIONS: Liberal control patients received insulin therapy if glucose was greater than 14 mmol/L (target: 10-14 mmol/L [180-252 mg/dL]). Conventional control patients received insulin therapy if glucose was greater than 10 mmol/L (target: 6-10 mmol/L [108-180 mg/dL]). MEASUREMENTS AND MAIN RESULTS: We assessed separation in blood glucose, insulin requirements, occurrence of hypoglycemia (blood glucose ≤ 3.9 mmol/L [70 mg/dL]), creatinine and white cell count levels, and clinical outcomes. The median (interquartile range) time-weighted average blood glucose concentration was significantly higher in the liberal control group (11.0 mmol/L [8.7-12.0 mmol/L]; 198 mg/dL [157-216 mg/dL]) than in the conventional control group (9.6 mmol/L [8.5-11.0 mmol/L]; 173 mg/dL [153-198 mg/dL]; p < 0.001). Overall, 132 liberal control patients (37.7%) and 188 conventional control patients (53.7%) received insulin in ICU (p < 0.001). Hypoglycemia occurred in 6.6% and 8.6%, respectively (p = 0.32). Among 314 patients with glycated hemoglobin A1c greater than or equal to 7%, hypoglycemia occurred in 4.1% and 9.6%, respectively (p = 0.053). Trajectories of creatinine and white cell count were similar in the groups. In multivariable analyses, we found no independent association between glucose control and mortality, duration of mechanical ventilation, or ICU-free days to day 30. CONCLUSIONS: In ICU patients with diabetes, during a period of liberal glucose control, insulin administration, and among patients with hemoglobin A1c greater than or equal to 7%, the prevalence of hypoglycemia was reduced, without negatively affecting serum creatinine, the white cell count response, or other clinical outcomes. (Trial Registration: Australian New Zealand Clinical Trials Registry; ACTRN12615000216516).

Factors affecting the nuclear localization of ß-catenin in normal and malignant tissue.

The canonical Wnt signaling pathway has been the focus of intensive research because of its frequent dysregulation in human cancers. Much of this has been directed towards the aberrant expression and/or activity of the central mediator of this pathway, ß-catenin. In particular, the nuclear localization of ß-catenin and subsequent inappropriate activation of TCF/LEF-mediated transcription appears to be an important process in both the establishment and maintenance of cancer stem cells. Despite this, the exact mechanisms controlling ß-catenin nuclear localization in both normal and malignant cells are poorly understood. This prospect article brings together the many mechanisms previously reported to regulate the nuclear localization of ß-catenin and how they are relevant to cancer.

The PDK1 master kinase is over-expressed in acute myeloid leukemia and promotes PKC-mediated survival of leukemic blasts.

PDK1 is a master kinase that activates at least six protein kinase groups including AKT, PKC and S6K and is a potential target in the treatment of a range of malignancies. Here we show overexpression of PDK1 in over 40% of myelomonocytic acute leukemia patients. Overexpression of PDK1 occurred uniformly throughout the leukemic population, including putative leukemia-initiating cells. Clinical outcome analysis revealed PDK1 overexpression was associated with poorer treatment outcome. Primary acute myeloid leukemia blasts over-expressing PDK1 showed improved in vitro survival and ectopic expression of PDK1 promoted the survival of myeloid cell lines. Analysis of PDK1 target kinases revealed that PDK1 overexpression was most closely associated with increased phosphorylation of PKC isoenzymes and inhibition of PKC strongly inhibited the survival advantage of PDK1 over-expressing cells. Membrane localization studies implicated PKCα as a major target for PDK1 in this disease. PDK1 over-expressing blasts showed differential sensitivity to PDK1 inhibition (in the low micromolar range) suggesting oncogene addiction, whilst normal bone marrow progenitors were refractory to PDK1 inhibition at effective inhibitor concentrations. PDK1 inhibition also targeted subpopulations of leukemic blasts with a putative leukemia-initiating cell phenotype. Together these data show that overexpression of PDK1 is common in acute myelomonocytic leukemia and is associated with poorer treatment outcome, probably arising from the cytoprotective function of PDK1. We also show that therapeutic targeting of PDK1 has the potential to be both an effective and selective treatment for these patients, and is also compatible with current treatment regimes.

Genome Assembly of the Dyeing Poison Frog Provides Insights into the Dynamics of Transposable Element and Genome-Size Evolution.

Genome size varies greatly across the tree of life and transposable elements are an important contributor to this variation. Among vertebrates, amphibians display the greatest variation in genome size, making them ideal models to explore the causes and consequences of genome size variation. However, high-quality genome assemblies for amphibians have, until recently, been rare. Here, we generate a high-quality genome assembly for the dyeing poison frog, Dendrobates tinctorius. We compare this assembly to publicly available frog genomes and find evidence for both large-scale conserved synteny and widespread rearrangements between frog lineages. Comparing conserved orthologs annotated in these genomes revealed a strong correlation between genome size and gene size. To explore the cause of gene-size variation, we quantified the location of transposable elements relative to gene features and find that the accumulation of transposable elements in introns has played an important role in the evolution of gene size in D. tinctorius, while estimates of insertion times suggest that many insertion events are recent and species-specific. Finally, we carry out population-scale mobile-element sequencing and show that the diversity and abundance of transposable elements in poison frog genomes can complicate genotyping from repetitive element sequence anchors. Our results show that transposable elements have clearly played an important role in the evolution of large genome size in D. tinctorius. Future studies are needed to fully understand the dynamics of transposable element evolution and to optimize primer or bait design for cost-effective population-level genotyping in species with large, repetitive genomes.

Targeting ß-catenin in acute myeloid leukaemia: past, present, and future perspectives.

Acute myeloid leukaemia (AML) is an aggressive disease of the bone marrow with a poor prognosis. Evidence suggests long established chemotherapeutic regimens used to treat AML are reaching the limits of their efficacy, necessitating the urgent development of novel targeted therapies. Canonical Wnt signalling is an evolutionary conserved cascade heavily implicated in normal developmental and disease processes in humans. For over 15 years its been known that the central mediator of this pathway, ß-catenin, is dysregulated in AML promoting the emergence, maintenance, and drug resistance of leukaemia stem cells. Yet, despite this knowledge, and subsequent studies demonstrating the therapeutic potential of targeting Wnt activity in haematological cancers, ß-catenin inhibitors have not yet reached the clinic. The aim of this review is to summarise the current understanding regarding the role and mechanistic dysregulation of ß-catenin in AML, and assess the therapeutic merit of pharmacologically targeting this molecule, drawing on lessons from other disease contexts.

BCL-3 loss sensitises colorectal cancer cells to DNA damage by targeting homologous recombination.

The proto-oncogene BCL-3 is upregulated in a subset of colorectal cancers (CRC), where it has been shown to enhance tumour cell survival. However, although increased expression correlates with poor patient prognosis, the role of BCL-3 in determining therapeutic response remains largely unknown. In this study, we use combined approaches in multiple cell lines and pre-clinical mouse models to investigate the function of BCL-3 in the DNA damage response. We show that suppression of BCL-3 increases γH2AX foci formation and decreases homologous recombination in CRC cells, resulting in reduced RAD51 foci number and increased sensitivity to PARP inhibition. Importantly, a similar phenotype is seen in Bcl3-/- mice, where Bcl3-/- mouse crypts also exhibit sensitivity to DNA damage with increased γH2AX foci compared to wild type mice. Additionally, Apc.Kras-mutant x Bcl3-/- mice are more sensitive to cisplatin chemotherapy compared to wild type mice. Taken together, our results identify BCL-3 as a regulator of the cellular response to DNA damage and suggests that elevated BCL-3 expression, as observed in CRC, could increase resistance of tumour cells to DNA damaging agents including radiotherapy. These findings offer a rationale for targeting BCL-3 in CRC as an adjunct to conventional therapies and suggest that BCL-3 expression in tumours could be a useful biomarker in stratification of rectal cancer patients for neo-adjuvant chemoradiotherapy.

Using Flow Cytometry to Detect and Measure Intracellular Thiol Redox Status in Viable T Cells from Heterogeneous Populations.

Increased production of reactive oxygen species (ROS) and deficiencies in cellular antioxidant defenses are the principal causes of cellular oxidative stress. ROS can react with a variety intracellular molecules, including redox active cysteine thiols (-SH) within proteins. Cysteine thiols can occupy several redox states and conversion between them is highly dynamic during, for example, cell growth, resulting in modification and subsequent loss of the "reduced thiol" form (-SH or -S-). The challenge lies with detecting and measuring thiol redox status inside viable heterogeneous cell populations (e.g., peripheral blood mononuclear cells (PBMCs)). Here we describe a flow cytometric approach for the evaluation of intracellular thiol redox status in human CD3+ T cells within a viable PBMC preparation. Using the thiol reactive probe, fluorescein-5 maleimide (F5M), we demonstrate that loss of reduced intracellular thiol correlates with a decrease in F5M fluorescence. We also detected a loss of F5M fluorescence in Jurkat cell cultures exposed to exogenous H2O2 generated by glucose oxidase. Since F5M binds irreversibly to reduced cysteine thiols, cells may be sorted based on F5M fluorescence intensity and redox active proteins can subsequently be extracted and separated using SDS-PAGE. This final step facilitates identification of redox active proteins from individual cell populations in live heterogeneous cell mixes using proteomic analysis.

Inhibition of GATA2 restrains cell proliferation and enhances apoptosis and chemotherapy mediated apoptosis in human GATA2 overexpressing AML cells.

GATA2, a zinc finger transcription factor predominantly expressed in hematopoietic cells, acts as an essential regulator of hematopoietic stem cell generation, survival and functionality. Loss and gain of GATA2 expression has been implicated in myelodysplastic syndrome and acute myeloid leukemia (AML) yet the precise biological impact of GATA2 expression on human AML cell fate decisions remains ambiguous. Herein, we performed large-scale bioinformatics that demonstrated relatively frequent GATA2 overexpression in AML patients as well as select human AML (or AML-like) cell lines. By using shRNAi to target GATA2 in these AML cell lines, and an AML cell line expressing normal levels of GATA2, we found that inhibition of GATA2 caused attenuated cell proliferation and enhanced apoptosis exclusively in AML cell lines that overexpress GATA2. We proceeded to pharmacologically inhibit GATA2 in concert with AML chemotherapeutics and found this augmented cell killing in AML cell lines that overexpress GATA2, but not in an AML cell line expressing normal levels of GATA2. These data indicate that inhibition of GATA2 enhances chemotherapy-mediated apoptosis in human AML cells overexpressing GATA2. Thus, we define novel insights into the oncogenic role of GATA2 in human AML cells and suggest the potential utilization of transient GATA2 therapeutic targeting in AML.

Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations: An optimised protocol for digital flow cytometers.

Flow cytometric methods for detecting and quantifying reduced intracellular thiol content using fluorescein-5-maleimide (F5M) in viable eukaryotic cells date back to 1983 (Durand and Olive [1]). There has been little development in these methodologies since that time, a period that has witnessed huge technological advances, particularly with the emergence of digital multi-parameter flow cytometric systems. Concurrent advancement in our understanding of redox regulation within eukaryotic cellular systems has also followed, whereby it is now accepted that cysteine thiols partake in redox reactions, which regulate protein activity and function (Groitl and Jakob (2014), Won et al. (2012)). Moreover, we are at the dawn of a new era in redox biology whereby the importance of 'reductive stress' in eukaryotic cellular systems is gathering momentum (Wadley et al. (2018) [4]). It is therefore critical that methods be continually advanced to better understand these concepts in more detail at the cellular level. Flow cytometry is a powerful technique that may be used for this purpose. Henceforth we have rejuvenated these methods to address modern scientific questions. In this paper, essential detail is provided on: •The adaption of a protocol initially described by Durand and Olive [1] for use with modern digital flow cytometer configurations. Here we provide optimal conditions for labelling intracellular thiols with F5M for detection using digital flow cytometers. Our modifications avoid the use of methanol fixation thus preserving cell viability in single cell suspension cultures.•Demonstration that flow cytometry can detect the gain and loss of reduced intracellular thiols in cells exposed to physiological doses of hydrogen peroxide mediated by glucose oxidase (Hole et al. (2013) [5]).•Validation of F5M protein labelling by coupling method to confocal microscopy and downstream proteomics, thus permitting a powerful experimental platform for potential use with next generation flow cytometry e.g. CyTOF (Lin and Maecker (2018) [6]).

Preliminary evidence of reductive stress in human cytotoxic T cells following exercise.

This study investigated immunophenotypic differences in intracellular thiol redox state of peripheral blood mononuclear cells (PBMCs) isolated from trained [ n = 9, means ± SD: age 28 ± 5 yr; (body mass index) BMI 23.2 ± 2.6 kg/m2; V̇o2max (maximal oxygen intake)56.9 ± 6.1 ml·kg-1·min-1] and recreationally active (RA, n = 11, means ± SD: age 27 ± 6 yr; BMI 24.2 ± 3.7 kg/m2; V̇o2max 45.1 ± 6.4 ml·kg-1·min-1) participants before and after a maximal aerobic exercise tolerance test. Blood samples were taken before (Pre), during (sample acquired at 70% maximum heart rate), immediately after (Post + 0), and 15 min postexercise (Post + 15). PBMCs were isolated, and reduced thiol analysis [fluorescein-5 maleimide (F5M)] by immunophenotype [cluster of differentiation (CD)3+, CD4+, and CD8+] was performed using flow cytometry. A significant increase in cellular F5M fluorescence was observed in CD3+ T cells at Post + 0, with changes driven to a greater extent by CD8+ T cells (fold change in both groups CD4: +2.3, CD8: +2.8; P < 0.05). Further analysis revealed a population of highly reduced CD8+ T cells (CD8+T-reduced+) that significantly increased from Pre to Post + 0 in RA participants only (RA: +272 cell/µl, P < 0.05). To understand these results further, CD8+T-reduced+ and CD8+T-reduced- cells were analyzed for immunophenotype in response to the same exercise protocol ( n = 6, means ± SD: age 24 ± 5 yr; BMI 25.7 ± 4.1 kg·m-2; V̇o2max 41.33 ± 7.63 ml·kg-1·min-1). CD8+T-reduced+ had significantly less lymphoid homing potential (chemokine receptor type 7) Post + 0 compared with Pre. This study is the first, to our knowledge, to demonstrate that lymphocyte populations become more reductive in response to acute exercise. NEW & NOTEWORTHY The study presented provides the first evidence to suggest that cytotoxic T cells become transiently reductive in healthy individuals following a single bout of cycling. Detection of these cells was enabled via the use of a flow cytometric assay that incorporates the thiol reactive probe fluorescein-5 maleimide. Using this method, transient reductive stress in viable T cells is permissible and provides the basis for further research in the area of exercise immunology.

The design and production of Co-Cr alloy implants with controlled surface topography by CAD-CAM method and their effects on osseointegration.

Improved fixation and increased longevity are still important performance criteria in the development of orthopaedic prostheses. The osseointegration of a series of implant designs made of conventional cobalt-chromium alloy was investigated, the shape of each implant being the critical variable. The shape was defined by computer-aided design with a view to maximising interdigitation of new bone with the implant. Two different process routes, conventional casting and selective laser sintering were employed, each process yielded implants that had identical surface topology but different microstructures. Hydroxyapatite (HA) was used to coat some samples by plasma spraying. Bone formation associated with each implant design was delineated through the administration of fluorescent vital dyes at three time points following their implantation into New Zealand white rabbits. After one month, specimens were harvested, resin embedded, serial sectioned and examined under fluorescent light microscopy. The amount of bone growth was quantified using image analysis. Plasma spray HA-coated samples promoted better osteogenesis and integration than uncoated samples. The extent of bone growth associated with identically shaped specimens fabricated by the SLS route was markedly greater, attributed to the microstructure of these implants.

LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/ß-catenin signalling in neuroblastoma.

LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of ß-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5.