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Approximately 20% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients are currently incurable due to primary or secondary resistance to glucocorticoid-based therapies. Here we employed an integrated approach to selectively kill T-ALL cells by increasing mitochondrial reactive oxygen species (ROS) using NS1619, a benzimidazolone that activates the K + (BK) channel, and dehydroepiandrosterone (DHEA), which blunts ROS scavenging through inhibition of the pentose phosphate pathway. These compounds selectively killed T-ALL cell lines, patient-derived xenografts and primary cells from patients with refractory T-ALL, but did not kill normal human thymocytes. T-ALL cells treated with NS1619 and DHEA showed activation of the ROS-responsive transcription factor NRF2, indicating engagement of antioxidant pathways, as well as increased cleavage of OPA1, a mitochondrial protein that promotes mitochondrial fusion and regulates apoptosis. Consistent with these observations, transmission electron microscopy analysis indicated that NS1619 and DHEA increased mitochondrial fission. OPA1 cleavage and cell death were inhibited by ROS scavengers and by siRNA-mediated knockdown of the mitochondrial protease OMA1, indicating the engagement of a ROS-OMA1-OPA1 axis in T-ALL cells. Furthermore, NS1619 and DHEA sensitized T-ALL cells to TRAIL-induced apoptosis. In vivo, the combination of dexamethasone and NS1619 significantly reduced the growth of a glucocorticoid-resistant patient-derived T-ALL xenograft. Taken together, our findings provide proof-of-principle for an integrated ROS-based pharmacological approach to target refractory T-ALL.

Adult T cell Leukemia/Lymphoma (ATLL) is a mature T cell malignancy associated with Human T cell Leukemia Virus type 1 (HTLV-1) infection. Among its four main clinical subtypes, the prognosis of acute and lymphoma variants remains poor. The long latency (3-6 decades) and low incidence (3-5%) of ATLL imply the involvement of viral and host factors in full-blown malignancy. Despite multiple preclinical and clinical studies, the contribution of the stromal microenvironment in ATLL development is not yet completely unraveled. The aims of this study were to investigate the role of the host microenvironment, and specifically fibroblasts, in ATLL pathogenesis and to propose a murine model for the lymphoma subtype. Here we present evidence that the oncogenic capacity of HTLV-1-immortalized C91/PL cells is enhanced when they are xenotransplanted together with human foreskin fibroblasts (HFF) in immunocompromised BALB/c Rag2 -/- γ c -/- mice. Moreover, cell lines derived from a developed lymphoma and their subsequent in vivo passages acquired the stable property to induce aggressive T cell lymphomas. In particular, one of these cell lines, C91/III cells, consistently induced aggressive lymphomas also in NOD/SCID/IL2Rγ c KO (NSG) mice. To dissect the mechanisms linked to this enhanced tumorigenic ability, we quantified 45 soluble factors released by these cell lines and found that 21 of them, mainly pro-inflammatory cytokines and chemokines, were significantly increased in C91/III cells compared to the parental C91/PL cells. Moreover, many of the increased factors were also released by human fibroblasts and belonged to the known secretory pattern of ATLL cells. C91/PL cells co-cultured with HFF showed features reminiscent of those observed in C91/III cells, including a similar secretory pattern and a more aggressive behavior in vivo . On the whole, our data provide evidence that fibroblasts, one of the major stromal components, might enhance tumorigenesis of HTLV-1-infected and immortalized T cells, thus throwing light on the role of microenvironment contribution in ATLL pathogenesis. We also propose that the lymphoma induced in NSG mice by injection with C91/III cells represents a new murine preclinical ATLL model that could be adopted to test novel therapeutic interventions for the aggressive lymphoma subtype.

Human T-lymphotropic virus 1 (HTLV-1) immortalizes T-cells and is the causative agent of adult T-cell leukemia/lymphoma (ATLL). HTLV-1 replication and transformation are governed by multiple interactions between viral regulatory proteins and host cell factors that remain to be fully elucidated. The present study investigated the impact of HTLV-1 infection on the expression of miR-34a, a microRNA whose expression is downregulated in many types of cancer. Results of RT-PCR assays showed that five out of six HTLV-1-positive cell lines expressed higher levels of miR-34a compared to normal PBMC or purified CD4+ T-cells. ATLL cell line ED, which did not express miR-34a, showed methylation of the miR-34a promoter. Newly infected PBMC and samples from 10 ATLL patients also showed a prominent increase in miR-34a expression compared to PBMC controls. The primary miR-34a transcript expressed in infected cell line C91PL contained binding motifs for NF-κB and p53. Pharmacological inhibition of NF-κB with Bay 11-7082 indicated that this pathway contributes to sustain miR-34a levels in infected cells. Treatment of infected cell lines with the p53 activator nutlin-3a resulted in a further increase in miR-34a levels, thus confirming it as a transcriptional target of p53. Nutlin-3a-treated cells showed downregulation of known miR-34a targets including the deacetylase SIRT1, which was accompanied by increased acetylation of p53, a substrate of SIRT1. Transfection of C91PL cells with a miR-34a mimic also led to downregulation of mRNA targets including SIRT1 as well as the pro-apoptotic factor BAX. Unlike nutlin-3a, the miR-34a mimic did not cause cell cycle arrest or reduce cell viability. On the other hand, sequestration of miR-34a with a sponge construct resulted in an increase in death of C91PL cells. These findings provide evidence for a functional role for miR-34a in fine-tuning the expression of target genes that influence the turnover of HTLV-1-infected cells.

No comprehensive data are available on the role of platelet indexes (PI) in the periprocedural risk stratification of patients who underwent percutaneous coronary intervention (PCI). The aim of this study was to investigate the relation of PI to platelet reactivity (PR) and periprocedural myocardial infarction (PMI) in patients receiving PCI. A total of 502 PCI patients had preprocedural measurement of PI and PR, the latter assessed by VerifyNow P2Y12 assay. Study end points were incidence of PMI and high platelet reactivity (HPR) according to tertiles of PI and evaluation of PI in HPR patients. Incidence of PMI in the overall population was 6.6%. Rates of PMI were not different in PI tertiles: platelet count (I: 6.0%, II: 7.1%, III: 6.5%; p = 0.74), mean platelet volume (MPV, I: 6.6%, II: 7.3%, III: 5.8%;p = 0.86), platelet distribution width (I: 7.2%, II: 7.2%, III: 5.8%;p = 0.74), and MPV/P ratio (I: 6.6%, II: 6.0%, III: 7.1%; p = 0.91). The occurrence of PMI was significantly different in PR tertiles (I: 3%, II: 5.4%, III: 11.4%; p = 0.006). Platelet count and MPV/P ratio were significantly different in patients with and without HPR (221.8 ± 58.6 × 103/µL vs207 ± 59.4 × 103/µL, p = 0.008; 51.73 ± 15.17 vs 56.7 ± 18.3, p = 0.002).In conclusion, this study showed no relation between PI and PMI in PCI patients but confirms the association of HPR with increased incidence of PMI; thus, PI seem to be not able to identify patients at higher periprocedural risk, but monitoring PR by a bedside assay remains a useful tool for risk stratification., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We investigated the impact of suboptimal platelet reactivity on clinical outcomes after percutaneous coronary intervention (PCI). We enrolled 500 patients with stable coronary artery disease undergoing elective PCI. Platelet reactivity was measured before PCI using the VerifyNow P2Y12 assay. Primary endpoint was the incidence of ischemic or bleeding events at 1 month and 5 years. Patients with high platelet reactivity (HPR) showed significantly higher rates of ischemic events both during the 1st month after PCI (HR 2.06, 95% CI 1.02-4.06), and beyond 1 month compared with patients without HPR (HR 1.73, 95% CI 1.02-2.95). Conversely, compared with patients without low platelet reactivity (LPR), patients with LPR presented significantly higher rates of bleeding only during the 1st month (HR 3.67, 95% CI 1.68-8.02). In conclusion, pre-procedural HPR is associated with ischemic events even beyond the 1st month after PCI. The association of LPR with bleeding events seems to be confined to the periprocedural period.

Background: Patients with atrial fibrillation (AF) who interrupt anticoagulation are at high risk of thromboembolism and death., Methods and Results: Patients enrolled in the ENGAGE AF-TIMI 48 trial (randomized comparison of edoxaban vs. warfarin) who interrupted study anticoagulant for >3 days were identified. Clinical events (ischemic stroke/systemic embolism, major cardiac and cerebrovascular events [MACCE]) were analyzed from day 4 after interruption until day 34 or study drug resumption. During 2.8 years median follow-up, 13,311 (63%) patients interrupted study drug for >3 days. After excluding those who received open-label anticoagulation during the at-risk window, the population for analysis included 9148 patients. The rates of ischemic stroke/systemic embolism and MACCE post interruption were substantially greater than in patients who never interrupted (15.42 vs. 0.26 and 60.82 vs. 0.36 per 100 patient-years, respectively, p adj  < .001). Patients who interrupted study drug for an adverse event (44.1% of the cohort), compared to those who interrupted for other reasons, had an increased risk of MACCE (HR adj 2.75; 95% CI 2.02-3.74, p < .0001), but similar rates of ischemic stroke/systemic embolism. Rates of clinical events after interruption of warfarin and edoxaban were similar., Conclusion: Interruption of study drug was frequent in patients with AF and was associated with a substantial risk of major cardiac and cerebrovascular events over the ensuing 30 days. This risk was particularly high in patients who interrupted as a result of an adverse event; these patients deserve close monitoring and resumption of anticoagulation as soon as it is safe to do so., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In patients with atrial fibrillation undergoing percutaneous coronary intervention (PCI), the effectiveness and safety of dual compared with triple antithrombotic therapy are a matter of debate, especially when considering the prevention of end points at low incidence, such as myocardial infarction (MI), stent thrombosis, or mortality. This study-level meta-analysis included 4 controlled randomized trials and 6,036 patients with a clinical indication to chronic oral anticoagulation (OAC) after PCI, mainly for atrial fibrillation. Patients receiving dual therapy with a single antiplatelet agent, essentially a P2Y 12 inhibitor, plus OAC were compared with those treated with triple therapy (aspirin, a P2Y 12 inhibitor, and OAC). The incidence of the following outcomes was evaluated: Thrombolysis In Myocardial Infarction major and minor bleeding, MI, stent thrombosis, stroke, cardiovascular, and all-cause death. Occurrence of Thrombolysis In Myocardial Infarction major bleeding was significantly lower in patients treated with dual therapy: 1.97% versus 3.53% in those on triple therapy (odds ratios 0.55, 95% confidence interval 0.39 to 0.78, p = 0.0007); rates of minor bleeding were also decreased in the former (57% relative reduction). With dual therapy, there was not a statistically significant difference in all-cause and cardiovascular mortality (3.81% vs 4.01%, p = 0.37 and 1.62% vs 2.02%, p = 0.42, respectively). Incidence of MI (3.25% vs 2.78%, p = 0.61), definite stent thrombosis (0.92% vs 0.66%, p = 0.46), and stroke (1.28% vs 1.32%, p = 0.85) was similar in the 2 treatment strategies. In patients with long-term indication to OAC after PCI, compared with triple therapy, dual antithrombotic therapy reduces bleeding, without an excess in thromboembolic and ischemic cardiac events., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Observational studies suggest that symptomatic atherosclerosis may be associated with risk of venous thromboembolism (VTE). Prior randomized studies have demonstrated a significant reduction in recurrent VTE with aspirin monotherapy. Whether VTE risk is associated with more severe symptomatic atherosclerosis and more intensive antiplatelet therapy reduces VTE risk beyond aspirin monotherapy is unknown., Methods: TRA2P-TIMI 50 (Thrombin Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events-Thrombolysis in Myocardial Infarction) (vorapaxar) and PEGASUS-TIMI 54 (Prevention of Cardiovascular Events in Patients With Prior Heart Attack Using Ticagrelor Compared to Placebo on a Background of Aspirin-Thrombolysis in Myocardial Infarction 54) (ticagrelor) were blinded, randomized placebo-controlled trials of antiplatelet therapy for the prevention of ischemic events in stable patients with symptomatic atherosclerosis. Two blinded vascular specialists systematically identified symptomatic venous thromboembolic events in both trials., Results: Of 47 611 patients with stable vascular disease followed for 3 years in both studies there were 343 VTE events in 301 patients (Kaplan-Meier rate at 3 years, 0.9% for placebo). The risk of VTE was independently associated with age, body mass index, polyvascular disease, chronic obstructive pulmonary disease, and malignancy. The burden of atherosclerosis manifested as an increasing number of symptomatic vascular territories was associated with a graded increase in the 3-year rates of VTE (0.76% for 1, 1.53% for 2, and 2.45% for 3 territories). More intensive antiplatelet therapy (vorapaxar and ticagrelor pooled) significantly reduced the risk of VTE by 29% compared with background antiplatelet therapy, from 0.93% to 0.64% at 3 years (hazard ratio, 0.71; 95% confidence interval, 0.56-0.89; P =0.003)., Conclusions: The rate of VTE in patients with atherosclerosis is ≈0.3% per year while on treatment with ≥1 antiplatelet agent, with increased risk independently associated with the number of symptomatic vascular territories. More intensive antiplatelet therapy reduces the risk of VTE. These data suggest a relationship between atherosclerosis burden and VTE risk, and they support inclusion of VTE as a prospective end point in long-term secondary prevention trials evaluating the risks and benefits of antiplatelet therapies in patients with atherosclerosis., Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01225562., (© 2017 American Heart Association, Inc.)

Viruses must exploit the cellular biosynthetic machinery and evade cellular defense systems to complete their life cycles. Due to their crucial roles in cellular bioenergetics, apoptosis, innate immunity and redox balance, mitochondria are important functional targets of many viruses, including tumor viruses. The present review describes the interactions between mitochondria and proteins coded by the human tumor viruses human T-cell leukemia virus type 1, Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, human hepatitis viruses B and C, and human papillomavirus, and highlights how these interactions contribute to viral replication, persistence and transformation.

Elderly patients with atrial fibrillation are at a higher risk of both ischemic and bleeding events compared with younger patients; therefore, balancing risks and benefits of antithrombotic strategies in this population is crucial. Recent studies have shown that because the risk of stroke increases with age more than the risk of bleeding, the absolute benefit of oral anticoagulation is the highest in elderly patients in whom it outweighs the risk of bleeding. Direct oral anticoagulants (DOACs) have been developed as a treatment for the prevention of cardioembolic stroke to overcome some limitations of warfarin, such as the need for frequent monitoring, labile INR values requiring frequent dose adjustment, dietary and drugs interactions, and increased risk of intracranial bleeding. Despite the better safety profiles of DOACs compared with warfarin, elderly patients often remain undertreated because of the fear of bleeding complications. This review summarizes current evidence regarding the risks of thromboembolisms and bleeding in different antithrombotic strategies in elderly patients (aged ≥75 years) with atrial fibrillation, including data from the warfarin-controlled phase 3 DOACs trials.

Vitamin D has been suggested as a protective compound for diabetes mellitus. Several mechanisms linking vitamin D to the regulation of the immune response support a role for vitamin D in the pathogenesis of autoimmune diabetes. Epidemiological evidence and observational studies suggesting that adequate vitamin D status is related to decreased risk of developing type 1 diabetes further corroborates this concept. However, only few and mostly underpowered randomized clinical trials have been conducted to test the effectiveness of vitamin D supplementation in autoimmune diabetes, with disappointing results. Similarly, recent evidence linking vitamin D action to insulin secretion and sensitivity led to the hypothesis that this compound may play a key role in the regulation of glucose homeostasis in both pre-diabetes and overt type 2 diabetes (T2D). However, the main clinical trials evaluating the efficacy of vitamin D supplementation for the control of glucose homeostasis in people at risk for or affected by T2D have yielded inconsistent results. The aim of this review is to summarize the rationale and results of randomized clinical trials testing vitamin D and its analogs in both autoimmune and T2D., (© 2018 S. Karger AG, Basel.)

There are limited real-world data on prevalence and predictors of dual antiplatelet therapy (DAPT) prolongation beyond one year after acute coronary syndrome (ACS). We have explored such issue in the START ANTIPLATELET Registry, which is a prospective, observational, multicenter, Italian registry performed in seven Italian cardiology institutions including patients admitted for ACS and followed up to one year. Out of a total population of 840 ACS patients, 596 patients had completed 12-month follow-up being on DAPT. Decision to prolong DAPT beyond one year was taken in 79 patients (13%), whereas in 517 patients DAPT was stopped. The strongest predictors of DAPT continuation were a new cardiovascular events after the index admission event (OR 3.3, 95% CI 1.4-7.7), no bleeding complications (OR 3.2, 95% CI 1.2-8.3) and no anemia during one-year follow-up (OR 2.6, 95% CI 1.1-5.9); other independent predictors were renal failure (OR 2.5, 95% CI 1.3-5.0) and peripheral artery disease (OR 1.8, 95% CI 1.1-3.0). The choice of DAPT prolongation was not correlated with younger ager, presence of diabetes mellitus, coronary angioplasty as initial treatment strategy or type of implanted stent (drug-eluting vs bare metal). In conclusion, this study provides a real-world snapshot on the factors influencing the option to continue DAPT beyond one year after ACS; a low bleeding risk seems to influence the choice to prolong DAPT more than a high ischemic risk.

Background: Increasing age predisposes to both thromboembolic and bleeding events in patients with atrial fibrillation; therefore, balancing risks and benefits of antithrombotic strategies in older populations is crucial. We investigated 1-year outcome with different antithrombotic approaches in very elderly atrial fibrillation patients (age ≥85 years) compared with younger patients., Methods and Results: We accessed individual patients' data from the prospective PREFER in AF (PREvention oF thromboembolic events-European Registry in Atrial Fibrillation), compared outcomes with and without oral anticoagulation (OAC), and estimated weighed net clinical benefit in different age groups. A total of 6412 patients, 505 of whom were aged ≥85 years, were analyzed. In patients aged <85 years, the incidence of thromboembolic events was 2.8%/year without OAC versus 2.3%/year with OAC (0.5% absolute reduction); in patients aged ≥85 years, it was 6.3%/year versus 4.3%/year (2% absolute reduction). In very elderly patients, the risk of major bleeding was higher than in younger patients, but similar in patients on OAC and in those on antiplatelet therapy or without antithrombotic treatment (4.0%/year versus 4.2%/year; P =0.77). OAC was overall associated with weighted net clinical benefit, assigning weights to nonfatal events according to their prognostic implication for subsequent death (-2.19%; CI, -4.23%, -0.15%; P =0.036). We found a significant gradient of this benefit as a function of age, with the oldest patients deriving the highest benefit., Conclusions: Because the risk of stroke increases with age more than the risk of bleeding, the absolute benefit of OAC is highest in very elderly patients, where it, by far, outweighs the risk of bleeding, with the greatest net clinical benefit in such patients., (© 2017 The Authors and Daiichi Sankyo Europe GmbH. Published on behalf of the American Heart Association, Inc., by Wiley.)

Malignant Pleural Mesothelioma (MPM) is a chemoresistant tumor characterized by low rate of p53 mutation and upregulation of Murine Double Minute 2 (MDM2), suggesting that it may be effectively targeted using MDM2 inhibitors. In the present study, we investigated the anticancer activity of the MDM2 inhibitors Nutlin 3a (in vitro) and RG7112 (in vivo), as single agents or in combination with rhTRAIL.In vitro studies were performed using MPM cell lines derived from epithelioid (ZL55, M14K), biphasic (MSTO211H) and sarcomatoid (ZL34) MPMs. In vivo studies were conducted on a sarcomatoid MPM mouse model.In all the cell lines tested (with the exception of ZL55, which carries a biallelic loss-of-function mutation of p53), Nutlin 3a enhanced p21, MDM2 and DR5 expression, and decreased survivin expression. These changes were associated to cell cycle arrest but not to a significant induction of apoptosis. A synergistic pro-apoptotic effect was obtained through the association of rhTRAIL in all the cell lines harboring functional p53. This synergistic interaction of MDM2 inhibitor and TRAIL agonist was confirmed using a mouse preclinical model. Our results suggest that the combined targeting of MDM2 and TRAIL might provide a novel therapeutic option for treatment of MPM patients, particularly in the case of sarcomatoid MPM with MDM2 overexpression and functional inactivation of wild-type p53.

The left atrial appendage (LAA) is the main source of thromboembolism in patients with non-valvular atrial fibrillation (AF). As such, the LAA can be the target of specific occluding device therapies. Optimal management of patients with AF includes a comprehensive knowledge of the many aspects related to LAA structure and thrombosis. Here we provide baseline notions on the anatomy and function of the LAA, and then focus on current imaging tools for the identification of anatomical varieties. We also describe pathogenetic mechanisms of LAA thrombosis in AF patients, and examine the available evidence on treatment strategies for LAA thrombosis, including the use of non-vitamin K antagonist oral anticoagulants and interventional approaches., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

In patients with atrial fibrillation (AF), the safety and efficacy of nonvitamin K antagonist oral anticoagulants (NOACs) vs warfarin according to diabetes mellitus (DM) status are not completely characterized. We performed a meta-analysis to clarify whether in these patients the strategy of oral anticoagulation should be tailored to diabetes status. In this study-level meta-analysis, we included 4 randomized phase III trials comparing NOACs and warfarin in patients with nonvalvular AF; a total of 18 134 patients with DM and 40 454 without DM were overall considered. Incidence of the following outcome measures was evaluated during the follow-up: stroke or systemic embolism, ischemic stroke, major bleeding, intracranial bleeding, and vascular death. Use of NOACs compared with warfarin reduced stroke/systemic embolism in diabetic (Risk Ratios [RR] 0.80, 95% CI 0.68-0.93; P = .004) and nondiabetic patients (RR 0.83, 0.73-0.93; P = .001) (P for interaction .72). No interaction between diabetes status and benefits of NOACs was found for the occurrence of ischemic stroke, major bleeding, or intracranial bleeding (P for interaction >.05 for each comparison). Reduction of vascular death rates with NOACs was significant in diabetic patients (4.97% vs 5.99% with warfarin; RR 0.83, 0.72-0.96; P = .01), in whom absolute the reduction of this outcome measure was higher than in nondiabetics (1.02% vs 0.27%), although no interaction was present (P = .23). Results of this meta-analysis support the safety and efficacy of NOACs compared with warfarin in diabetic patients with nonvalvular AF., (Copyright © 2016 John Wiley & Sons, Ltd.)

Background: Diabetes is a known risk predictor for thromboembolic events in patients with atrial fibrillation (AF), but no study has explored the prognostic weight of insulin in this setting., Objectives: This study evaluated the differential role of insulin versus no insulin therapy on thromboembolic risk in patients with diabetes and AF., Methods: We accessed individual patient data from the prospective, real-world, multicenter, PREFER in AF (European Prevention of thromboembolic events-European Registry in Atrial Fibrillation). We compared the rates of stroke/systemic embolism at 1 year according to diabetes status (no diabetes, diabetes without insulin therapy, diabetes on insulin therapy)., Results: In an overall population of 5,717 patients, 1,288 had diabetes, 22.4% of whom were on insulin. For patients with diabetes who were on insulin, there was a significantly increased risk of stroke/systemic embolism at 1 year versus either no diabetes (5.2% vs. 1.9%; hazard ratio: 2.89; 95% confidence interval: 1.67 to 5.02; p = 0.0002) or diabetes without insulin treatment (5.2% vs. 1.8%; hazard ratio: 2.96; 95% confidence interval: 1.49 to 5.87; p = 0.0019). Notably, rates of stroke/embolism were similar in patients with diabetes not receiving insulin versus patients without diabetes (hazard ratio: 0.97; 95% confidence interval: 0.58 to 1.61; p = 0.90). The selective predictive role of insulin-requiring diabetes was independent of potential confounders, including diabetes duration, and was maintained in various subpopulations, including the subgroup receiving anticoagulant therapy., Conclusions: In this cohort of anticoagulated patients with AF, the sole presence of diabetes not requiring insulin did not imply an increased thromboembolic risk. Conversely, insulin-requiring diabetes contributed most, if not exclusively, to the overall increase of thromboembolic risk in AF., (Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Human T-cell leukemia virus type 1 (HTLV-1) infects about 20 million people world-wide. Around 5% of the infected individuals develop adult T-cell leukemia (ATL) or a neurological disease termed tropical spastic paraparesis (TSP) after a clinical latency of years to decades. Through the use of two promoters and alternative splicing HTLV-1 expresses at least 12 different proteins. HTLV-1 establishes a life-long persistent infection by inducing the clonal expansion of infected cells, a property largely ascribed to the viral genes Tax and HBZ. However, the fact that ATL arises in a minority of infected individuals after a long clinical latency suggests the existence of factors counterbalancing the oncogenic potential of HTLV-1 in the context of natural infection.To study the role of the different HTLV-1 gene products in the HTLV-1 life cycle, we optimized a transfection protocol for primary T-cells using an approach based on the electroporation of in vitro-transcribed RNA. Results showed that the RNA transfection technique combines a high transfection efficiency with low toxicity, not only in Jurkat T-cells but also in primary T-cells. These findings suggest that RNA electroporation is preferable for experiments aimed at investigating the role of HTLV-1 gene products in the context of primary T-cells, which represent the main target of HTLV-1 in vivo.

Patients with prior myocardial infarction (MI) are at long-term heightened risk for recurrent ischemic events. Several large randomized controlled trials have demonstrated the benefit of more intensive antiplatelet strategies for long-term secondary prevention of cardiovascular death, recurrent MI, and stroke in patients with a history of MI at a cost of increased bleeding. The bleeding risk associated with long-term intensive antiplatelet strategies requires careful patient selection and involvement of patients in shared decision making regarding risks and benefits of therapy. Clinical characteristics, adherence to therapy, and integrated risk scores may aid clinicians in translating clinical trials into individualized therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Neutrophil to lymphocyte ratio (NLR) has been proposed as a marker of cardiovascular risk. The potential relation between NLR and periprocedural myocardial damage after percutaneous coronary intervention (PCI) is unclear. We enrolled 502 consecutive patients with stable coronary artery disease undergoing elective PCI. Blood samples were drawn in all patients at baseline, 6 hours, and 24 hours after PCI for complete blood cell count and cardiac biomarkers (creatine kinase-MB and troponin T [Tn-T]) assessment. NLR was calculated as the ratio between the absolute number of neutrophil over the absolute number of lymphocyte. Periprocedural myocardial infarction (PMI) was defined according to the 2012 universal definition of myocardial infarction. In the overall population, a significant postprocedural increase in NLR was observed (3.255 [2.763 to 3.995] at baseline, 4.430 [3.390 to 6.020] at 6 hours, 4.720 [3.940 to 5.750] at 24 hours, p <0.0001). PMI occurred in 33 patients (6.6%). Baseline NLR was similar in patients with and without PMI (3.250 [2.820 to 3.885] vs 3.260 [2.750 to 4.000], p = 0.898); however, patients who developed PMI showed significantly higher NLR both at 6 hours (5.750 [4.360 to 9.095] vs 4.370 [3.370 to 5.950], p <0.001) and 24 hours (5.180 [4.440 to 8.065] vs 4.670 [3.920 to 5.710], p = 0.003). Among patients who developed PMI, periprocedural NLR increase showed a moderate positive correlation with both creatine kinase-MB (rho = 0.377, p = 0.031) and troponin T increase (rho = 0.506, p = 0.003). In conclusion, preprocedural NLR values do not impact on the occurrence of PMI during elective PCI; however, PCI procedures induce a significant increase in NLR that seems to be proportional to the magnitude of periprocedural myocardial damage., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Body circumferences have been proposed as potential anthropometric measures for the assessment of cardiometabolic risk as they are independently associated with insulin resistance and diabetes. The aim of this study was to validate neck and wrist circumference and waist-to-hip ratio as practical markers of metabolic dysfunction and atherosclerosis; 120 subjects who underwent coronary angiography and carotid Doppler ultrasound were enrolled in this cross-sectional study. Exclusion criteria were history of diabetes, acute myocardial infarction, body mass index (BMI) <18.5 or ≥45.0 kg/m(2). Metabolic dysfunction was ascertained by the calculation of visceral adiposity index (VAI) and by diagnosis of metabolic syndrome (MS). Advanced atherosclerotic disease was defined as ≥70% coronary lumen and/or ≥50% carotid lumen stenosis. No association between body circumferences and VAI or MS was found in subjects with BMI <25 kg/m(2). VAI was significantly related to waist-to-hip ratio (R(2) = 0.09, p = 0.008), neck (R(2) = 0.09, p = 0.007), and wrist circumferences (R(2) = 0.05, p = 0.041) in subjects with BMI ≥25 kg/m(2). In overweight subjects, higher gender-specific tertiles of wrist circumference were independently associated with an increased risk of MS (odds ratio 2.57, 95% confidence interval 1.11 to 5.96, p = 0.028). VAI was independently associated with carotid intima-media thickness: β = 0.104, R(2) = 0.118, p = 0.003. Carotid intima-media thickness and MS, but not body circumferences, were associated with advanced atherosclerosis. In conclusion, these data indicate that anthropometric measurements, in particular wrist circumference, can be used as practical tools for assessment of metabolic risk in overweight-obese subjects but not as markers of advanced atherosclerosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: In 1990 the American Heart Association (AHA) established a standard 0.05 to 150Hz bandwidth for the routine recording of 12-lead electrocardiograms (ECGs). However, subsequent studies have indicated a very high prevalence of deviations from the recommended cutoffs., Objective: This prospective observational study investigates the impact of 40Hz compared to 150Hz high-frequency cutoffs on ECG quality and clinical interpretation in a single-center surgical outpatient population., Methods: 1582 consecutive adult patients underwent two standard 12-lead ECG tracings using different high-frequency cutoffs (40Hz and 150Hz). Two blinded cardiologists randomly reviewed and interpreted the recordings according to pre-defined parameters (PR and ST segment, Q and T wave abnormalities). An arbitrary score, ranging from 1 to 3, was established to evaluate the perceived quality of the recordings and the non-interpretable ECGs were noted. The tracings were then matched to compare interpretations between 40 and 150Hz filters., Results: A 40Hz high-frequency cutoff resulted in an increased rate of optimal quality ECGs compared to the 150Hz cutoff (93.4% vs 54.6%; p<0.001) and a lower rate of non-interpretable traces (0.25% vs 4.80%; p<0.001). Analyzing the morphologic parameters, no significant differences between the filter settings were found, except for a higher incidence of the J-point elevation in the 40Hz high-frequency cutoff (p=0.007) and a higher incidence of left ventricular hypertrophy in the 150Hz high-frequency cutoff (7.4% vs 5.4%, p<0.001). The latter was noted only in ECGs with borderline QRS amplitudes (between 3.3 and 3.7mV; p<0.001)., Conclusion: Despite current recommendations, the large deviation from standard high-frequency cutoff in clinical practice does not seem to significantly affect ECG clinical interpretation and a 40Hz high-frequency cutoff of the band-pass filtering may be acceptable in a low risk population, allowing for a better quality of tracings., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Whether dual antiplatelet therapy (DAPT) is beneficial beyond 1 year after myocardial infarction (MI) is not demonstrated; in particular, available studies may be individually underpowered for end points at low incidence, that is, major and fatal bleeding or mortality. We thus assessed the effectiveness and safety of prolonged DAPT after MI over the long term., Methods: We conducted a systematic search to identify randomized trials on the topic; 3 studies and 21,534 post-MI patients receiving placebo or aspirin plus P2Y12 inhibition for ≥2 years were included. Incidence of the following outcome measures was evaluated: major adverse cardiac events (MACE), major bleeding, fatal bleeding, and cardiovascular and noncardiovascular death., Results: Occurrence of MACE was lower in patients treated with prolonged DAPT: 6.3% vs 7.9% in those without prolonged DAPT (odds ratios 0.74, 95% CI 0.60-0.91, P = .005); in the former, there was also a significant 16% reduction in cardiovascular mortality. Increase in major bleeding with extended duration DAPT was not significant in the overall analysis (1.5% vs 1.0%; P = .10), but became significant in the analysis restricted to patients receiving ticagrelor or prasugrel as second antiplatelet agent (odds ratios 2.16, 95% CI 1.63-2.86); prolonged use of DAPT did not raise rates of fatal bleeding or noncardiovascular mortality., Conclusion: Prolonged DAPT after MI reduces MACE and cardiovascular mortality over the long term; this was paralleled by higher risk of nonfatal major bleeding mainly with the newer, more potent P2Y12 antagonists. Tailoring duration of DAPT after MI on the comparative evaluation of both ischemic and bleeding risk is mandatory in this setting., (Copyright © 2016. Published by Elsevier Inc.)

Background: There is poor evidence on clinical outcome of switching from clopidogrel to prasugrel in patients undergoing percutaneous coronary intervention., Objectives: Data on the topic are limited and we performed a study-level meta-analysis to assess safety and efficacy of such strategy., Methods: A total of 15 studies and 3974 patients were included. The following comparisons were performed: prasugrel switching versus prasugrel only therapy; and prasugrel switching versus clopidogrel only therapy. Outcome measures were overall bleeding, major bleeding, and major adverse cardiac events (MACE)., Results: There was no statistically significant increased bleeding risk in the prasugrel switching versus prasugrel only group [overall bleeding: OR 1.07, 95% confidence interval (CI), 0.69-1.66; P = 0.77; major bleeding: OR 0.69, 95% CI, 0.32-1.49; P = 0.34]; MACE rates were also comparable. Incidence of safety end points was similar in the prasugrel switching and clopidogrel only groups (overall bleeding: OR 1.27, 95% CI, 0.75-2.15; P = 0.37; major bleeding: OR 0.70, 95% CI, 0.29-1.68; P = 0.42); occurrence of MACE was 3.8% in the prasugrel switching versus 8.3% in the clopidogrel only group (P = 0.23). No statistically significant difference in the safety outcomes was present stratifying by clinical presentation., Conclusions: Switching from clopidogrel to prasugrel does not increase bleeding complications during follow-up of patients undergoing percutaneous coronary intervention; however, the strength of the data is not sufficient to make definitive clinical recommendations.

There is uncertainty regarding the optimal therapy for preventing thromboembolic stroke in patients with atrial fibrillation and CHA(2)DS(2)-VASc score 1. In fact, no extensive data on this topic are available, and the latest guidelines provide different recommendations. In this article, we examine current results on the use of various antithrombotic agents, including the newer oral anticoagulant agents, in those patients. Several factors must be considered and weighted in this setting and may influence the choice of the antithrombotic approach: the expected incidence of both thromboembolic stroke and bleeding complications as well as their impact in terms of morbidity and mortality, the patient's bleeding risk profile, an accurate, further stratification of the thromboembolic risk beyond the CHA(2)DS(2)-VASc score, and socioeconomic issues., (Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Human T-cell leukemia virus type 1 (HTLV-1) expression depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of viral expression. In the present study, we investigated the Rex dependence of the complete set of alternatively spliced HTLV-1 mRNAs. Analyses of cells transfected with Rex-wild-type and Rex-knockout HTLV-1 molecular clones using splice site-specific quantitative reverse transcription (qRT)-PCR revealed that mRNAs encoding the p30Tof, p13, and p12/8 proteins were Rex dependent, while the p21rex mRNA was Rex independent. These findings provide a rational explanation for the intermediate-late temporal pattern of expression of the p30tof, p13, and p12/8 mRNAs described in previous studies. All the Rex-dependent mRNAs contained a 75-nucleotide intronic region that increased the nuclear retention and degradation of a reporter mRNA in the absence of other viral sequences. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis revealed that this sequence formed a stable hairpin structure. Cell cycle synchronization experiments indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. These findings indicate a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system., Importance: HTLV-1 is a complex retrovirus that causes two distinct pathologies termed adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy in about 5% of infected individuals. Expression of the virus depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of virus expression. The findings reported in this study revealed a novel cis-acting regulatory element and indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. Our results add a layer of complexity to the mechanisms controlling the expression of alternatively spliced HTLV-1 mRNAs and suggest a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Human T cell leukemia virus type 1 (HTLV-1) gene expression is controlled by the key regulatory proteins Tax and Rex. The concerted action of these proteins results in a two-phase kinetics of viral expression that depends on a time delay between their action. However, it is difficult to explain this delay, as Tax and Rex are produced from the same mRNA. In the present study we investigated whether HTLV-1 may produce novel mRNA species capable of expressing Rex and Tax independently., Findings: Results revealed the expression of three alternatively spliced transcripts coding for novel Rex isoforms in infected cell lines and in primary samples from infected patients. One mRNA coded for a Tax isoform and a Rex isoform, and two mRNAs coded for Rex isoforms but not Tax. Functional assays showed that these Rex isoforms exhibit activity comparable to canonic Rex. An analysis of the temporal expression of these transcripts upon ex vivo culture of cells from infected patients and cell lines transfected with a molecular clone of HTLV-1 revealed early expression of the dicistronic tax/rex mRNAs followed by the monocistronic mRNAs coding for Rex isoforms., Conclusion: The production of monocistronic HTLV-1 mRNAs encoding Rex isoforms with comparable activity to canonical Rex, but with distinct timing, would support a prolonged duration of Rex function with gradual loss of Tax, and is consistent with the two-phase expression kinetics. A thorough understanding of these regulatory circuits will shed light on the basis of viral latency and provide groundwork to develop strategies for eradicating persistent infections.

Aims: Statin pretreatment in patients undergoing cardiac surgery is understood to prevent postoperative atrial fibrillation (AF). However, this is based on observational and limited randomized trial evidence, resulting in uncertainty about any genuine anti-arrhythmic benefits of these agents in this setting. We therefore aimed to quantify precisely the association between statin pretreatment and postoperative AF among patients undergoing cardiac surgery., Methods and Results: A detailed search of MEDLINE and PubMed databases (1st January 1996 to 31st July 2012) was conducted, followed by a review of the reference lists of published studies and correspondence with trial investigators to obtain individual-participant data for meta-analysis. Evidence was combined across prospective, randomized clinical trials that compared the risk of postoperative AF among individuals randomized to statin pretreatment or placebo/control medication before elective cardiac surgery. Postoperative AF was defined as episodes of AF lasting ≥5 min. Overall, 1105 participants from 11 trials were included; of them, 552 received statin therapy preoperatively. Postoperative AF occurred in 19% of these participants when compared with 36% of those not treated with statins (odds ratio 0.41, 95% confidence interval 0.31-0.54, P < 0.00001, using a random-effects model). Atrial fibrillation prevention by statin pretreatment was consistent across different subgroups., Conclusion: Short-term statin pretreatment may reduce the risk of postoperative AF among patients undergoing cardiac surgery., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Transthoracic echocardiography is not a routine test in the pre-operative cardiac evaluation of patients undergoing non-cardiac surgery but may be considered in those with known heart failure and valvular heart disease or complaining cardiac symptoms. In this setting, hand-held echocardiography (HHE) could find a potential application as an alternative to standard echocardiography in selected patients; however, its utility in this context has not been investigated. The aim of this pilot study was to evaluate the conclusiveness of HHE compared to standard echocardiography in this subset of patients. 100 patients scheduled for non-cardiac surgery were randomized to receive a standard exam with a Philips Ie33 or a bedside evaluation with a pocket-size imaging device (Opti-Go, Philips Medical System). The primary endpoint was the percentage of satisfactory diagnosis at the end of the examination referred as conclusiveness. Secondary endpoints were the mean duration time and the mean waiting time to perform the exams. No significant difference in terms of conclusiveness between HHE and standard echo was found (86 vs 96%; P = 0.08). Mean duration time of the examinations was 6.1 ± 1.2 min with HHE and 13.1 ± 2.6 min with standard echocardiography (P < 0.001). HHE resulted in a consistent save of waiting time because it was performed the same day of clinical evaluation whereas patients waited 10.1 ± 6.1 days for a standard echocardiography (P < 0.001). This study suggests the potential role of HHE for pre-operative evaluation of selected patients undergoing non-cardiac surgery, since it provided similar information but it was faster and earlier performed compared to standard echocardiography.

Despite dual antiplatelet treatment with aspirin and clopidogrel, patients with acute coronary syndromes (ACSs) remain at risk for recurrent cardiovascular events. This may be due, at least in part, to an incomplete response to clopidogrel, which is more frequent in ACS patients compared to stable patients because of massive platelet activation and increased platelet turnover. Currently, numerous laboratory-based methods and point-of-care tests are available to assess platelet reactivity. Several studies have tried to establish a standardized definition of high on-treatment platelet reactivity and to evaluate a correlation between this aggregometric phenomenon and clinical outcomes. Indeed a strong relationship between high on-treatment platelet reactivity and ischemic events was found, especially in high-risk ACS patients undergoing percutaneous coronary revascularization. Therefore, evaluation of platelet reactivity in this subset of patients may guide physicians to choose the best antiplatelet regimen for the individual patient avoiding both ischemic and bleeding complications. This was the rationale for tailored antiplatelet therapy pursued by utilization of different clopidogrel regimens, more potent P2Y12 receptor antagonists, or more extensive administration of glycoprotein IIb/IIIa inhibitors. To date, data from randomized studies addressing the concept of tailored antiplatelet therapy did not show any clinical benefit from a strategy based on platelet reactivity monitoring. However, as predominantly elective and stable patients were included into the latter studies, these results cannot be completely transferred into an ACS setting. This review summarizes current evidence about the potential role of platelet reactivity in the therapeutic management of patients with ACS.

Introduction: Malignant pleural mesothelioma (MPM) is an aggressive, currently incurable tumor with increasing incidence in industrialized countries. Tumor necrosis factor-related, apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which induces cancer cell death through extrinsic apoptotic pathway, while sparing normal cells. The aim of this study was to investigate the antitumor activity of recombinant human Apo2L/TRAIL (dulanermin) in combination with chemotherapy in MPM in vitro and in vivo., Methods: In the present studies, we employed a panel of MPM cell lines to test the antitumor activity of recombinant human Apo2L/TRAIL (T) in combination with carboplatin and pemetrexed (CP) in vitro and SCID mice., Results: Results demonstrated a significant increase of apoptosis in cell lines treated with CPT compared with those receiving CP or T as single agents. This synergistic effect was dependent on the ability of CP to increase the expression of the TRAIL receptors DR4 and DR5 in a p53 manner. The CPT combination was also effective in blocking the growth of MPM cell lines in a SCID mice preclinical model., Conclusions: CPT increases MPM cell death in vitro and in vivo compared with CP. In vitro results suggest that chemotherapy sensitizes MPM to TRAIL-dependent apoptosis through p53 activation and subsequent upregulation of DRs.

The impact of chronic kidney disease (CKD) on residual platelet reactivity (PR) in patients undergoing percutaneous coronary intervention (PCI) is still debatable. We sought to investigate the interaction between PR and renal function and the related clinical outcomes in patients with coronary artery disease treated with PCI. Immediately before PCI, we measured PR (as P2Y12 reaction units [PRUs]) in 800 patients on clopidogrel with the VerifyNow P2Y12 assay. High PR was defined as a PRU value of ≥240 and low PR as a PRU value of ≤178. Based on a glomerular filtration rate of < or ≥60 ml/min/1.73 m2, patients were respectively grouped into those with or without moderate-to-severe CKD. Primary end point was the incidence of 30-day net adverse clinical events (NACEs). Patients with moderate-to-severe CKD (n=173, 21.6%) and those without showed similar PRU values (208±67 vs 207±75, p=0.819). Yet, NACEs were significantly higher in patients with moderate-to-severe CKD (19.7% vs 9.1%, p<0.001), in terms of both ischemic (12.1% vs 7.2%, p=0.036) and bleeding events (8.7% vs 2.1%, p<0.001). NACEs were significantly higher when moderate-to-severe CKD was associated with either high PR or low PR (25.4%, p for trend<0.001); this association was the strongest predictor of NACE at multivariate analysis (odds ratio 3.4, 95% confidence interval 2.0 to 5.6, p<0.001). In conclusion, we did not find an association between moderate-to-severe CKD and residual PR on clopidogrel. However, the association of moderate-to-severe CKD with either high or low PR was a strong determinant of adverse events after PCI., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Like other complex retroviruses such as HIV-1, HTLV-1 encodes several regulatory and auxiliary non-structural proteins from overlapping open reading frames through the generation of alternatively spliced mRNAs. HTLV-1 expression is orchestrated by the Tax and Rex regulatory proteins; Tax drives the transcription of the viral genome, while Rex acts at the posttranscriptional level by enhancing the nuclear export and expression of unspliced and incompletely spliced mRNAs. The present chapter is focused on the techniques employed to quantitate HTLV-1 mRNAs in the nuclear and cytoplasmic compartments. To ensure a quantitative transcript-specific detection of the levels of individual HTLV-1 mRNAs in a complex mixture of closely related species, splice junction-specific primers and TaqMan probes were used. As HTLV-1 gene regulation is based on the controlled nucleo-cytoplasmic export of the different viral mRNAs, we quantitated the individual viral transcripts in the nuclear and cytoplasmic fractions.

Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are delta retroviruses that share a common overall genetic organization, splicing pattern, and ability to infect and immortalize T-cells in vitro. However, HTLV-1 and HTLV-2 exhibit a clearly distinct pathogenic potential in infected patients. To find clues to the possible viral determinants of the biology of these viruses, recent studies investigated the timing of expression and the intracellular compartmentalization of viral transcripts in ex-vivo samples from infected patients. Results of these studies revealed a common overall pattern of expression of HTLV-1 and -2 with a two-phase kinetics of expression and a nuclear accumulation of minus-strand transcripts. Studies in cells transfected with HTLV-1 molecular clones demonstrated the strict Rex-dependency of this "two-phase" kinetics. These studies also highlighted interesting differences in the relative abundance of transcripts encoding the Tax and Rex regulatory proteins, and that of the accessory proteins controlling Rex expression and function, thus suggesting a potential basis for the different pathobiology of the two viruses.

The incremental predictive value of high inflammatory status and high on-treatment platelet reactivity (HPR) on the occurrence of periprocedural myocardial infarction (PMI) after percutaneous coronary intervention (PCI) has not been characterized. The aim of this study was to evaluate the correlation of elevated C-reactive protein (CRP) level and/or HPR with the incidence of PMI in patients who undergo PCI. Five hundred consecutive patients treated with clopidogrel who underwent PCI had preprocedural measurement of CRP levels and platelet reactivity using the point-of-care VerifyNow P2Y12 assay. Elevated inflammatory status was defined as CRP >3 mg/L and HPR as P2Y12 reactivity units ≥240. The primary end point was the incidence of PMI in relation to platelet reactivity and/or inflammatory status. Rates of PMI were increased in patients with CRP levels >3 mg/L (10.9% vs 4.6% in those with normal levels, odds ratio 2.4, 95% confidence interval 1.2 to 4.5, p = 0.015) and in patients with HPR (11% vs 5.5% in those without HPR, odds ratio 2.2, 95% confidence interval 1.2-4.4, p = 0.018). The occurrence of PMI was highest in the subgroup with HPR and high inflammatory status (16.6% vs 3.6% in patients with CRP ≤3 mg/L and P2Y12 reactivity units <240, odds ratio 4.3, 95% confidence interval 1.5 to 12.6, p = 0.008). HPR in association with elevated CRP levels resulted in a significant increase in the discriminatory power of a model including clinical and procedural variables in predicting PMI (area under the curve 0.811, p = 0.041). In conclusion, in patients who undergo PCI, baseline stratification according to platelet reactivity and inflammatory status may identify those at higher risk for PMI., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Human T cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are genetically related complex retroviruses that are capable of immortalizing human T-cells in vitro and establish life-long persistent infections in vivo. In spite of these apparent similarities, HTLV-1 and HTLV-2 exhibit a significantly different pathogenic potential. HTLV-1 is recognized as the causative agent of adult T-cell leukemia/lymphoma (ATLL) and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). In contrast, HTLV-2 has not been causally linked to human malignancy, although it may increase the risk of developing inflammatory neuropathies and infectious diseases. The present paper is focused on the studies aimed at defining the viral genetic determinants of the pathobiology of HTLV-1 and HTLV-2 through a comparison of the expression strategies and functional properties of the different gene products of the two viruses.

The discovery of human retroviruses in the early 1980s revealed the existence of viral-encoded non-structural genes that were not evident in previously described animal retroviruses. Based on the absence or presence of these additional genes retroviruses were classified as 'simple' and 'complex', respectively. Expression of most of these extra genes is achieved through the generation of alternatively spliced mRNAs. The present review summarizes the genetic organization and expression strategies of human complex retroviruses and highlights the converging mechanisms controlling their life cycles.

Human T-cell leukemia virus type 1 (HTLV-1) codes for 9 alternatively spliced transcripts and 2 major regulatory proteins named Tax and Rex that function at the transcriptional and posttranscriptional levels, respectively. We investigated the temporal sequence of HTLV-1 gene expression in primary cells from infected patients using splice site-specific quantitative RT-PCR. The results indicated a two-phase kinetics with the tax/rex mRNA preceding expression of other viral transcripts. Analysis of mRNA compartmentalization in cells transfected with HTLV-1 molecular clones demonstrated the strict Rex-dependency of the two-phase kinetics and revealed strong nuclear retention of HBZ mRNAs, supporting their function as noncoding transcripts. Mathematical modeling underscored the importance of a delay between the functions of Tax and Rex, which was supported by experimental evidence of the longer half-life of Rex. These data provide evidence for a temporal pattern of HTLV-1 expression and reveal major differences in the intracellular compartmentalization of HTLV-1 transcripts.

The present study investigated the function of p13, a mitochondrial protein of human T-cell leukemia virus type 1 (HTLV-1). Although necessary for viral propagation in vivo, the mechanism of function of p13 is incompletely understood. Drawing from studies in isolated mitochondria, we analyzed the effects of p13 on mitochondrial reactive oxygen species (ROS) in transformed and primary T cells. In transformed cells (Jurkat, HeLa), p13 did not affect ROS unless the cells were subjected to glucose deprivation, which led to a p13-dependent increase in ROS and cell death. Using RNA interference we confirmed that expression of p13 also influences glucose starvation-induced cell death in the context of HTLV-1-infected cells. ROS measurements showed an increasing gradient from resting to mitogen-activated primary T cells to transformed T cells (Jurkat). Expression of p13 in primary T cells resulted in their activation, an effect that was abrogated by ROS scavengers. These findings suggest that p13 may have a distinct impact on cell turnover depending on the inherent ROS levels; in the context of the HTLV-1 propagation strategy, p13 could increase the pool of "normal" infected cells while culling cells acquiring a transformed phenotype, thus favoring lifelong persistence of the virus in the host.

Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K(+) current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K(+) in the assay medium. The effects of p13 on ROS are mimicked by the K(+) ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K(+) vs. H(+) currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K(+) permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.

Loss of menin, a tumor suppressor coded by the MEN1 gene, is a key factor in the pathogenesis of multiple endocrine neoplasia type I and in a percentage of sporadic endocrine tumors of the pancreas and parathyroid glands. This study investigated expression of the menin protein in the normal exocrine pancreas and in pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic tumor. Immunofluorescence (IF) analyses showed that menin is expressed at high levels in normal acinar and duct cells. Examination of 24 clinical samples of PDAC revealed a pronounced decrease in menin expression in all tumors examined. To identify alterations underlying this defect, we searched for disruption and epigenetic silencing of the MEN1 gene. Analysis of nine laser-microdissected tumors revealed loss of heterozygosity of intragenic (one tumor) or adjacent (three tumors) MEN1 microsatellite markers. Methylation of CpG sites in the MEN1 promoter was documented in five of 24 tumors. IF analyses also revealed low to undetectable menin expression in the PDAC cell lines MiaPaCa-2 and Panc-1. Ectopic expression of menin in these cells resulted in a marked alteration of the cell cycle, with an increase in the G1/S+G2 ratio. These findings represent the first evidence that the MEN1 gene is a target of mutation and methylation in PDAC and that menin influences the cell cycle profile of duct cells., (Copyright 2009 Wiley-Liss,Inc.)

Human T cell leukemia virus type 1 encodes an "accessory" protein named p13(II) that is targeted to mitochondria and triggers a rapid flux of K(+) and Ca(2+) across the inner membrane. In this study, we investigated the effects of p13(II) on tumorigenicity in vivo and on cell growth in vitro. Results showed that p13(II) significantly reduced the incidence and growth rate of tumors arising from c-myc and Ha-ras-cotransfected rat embryo fibroblasts. Consistent with these findings, HeLa-derived cell lines stably expressing p13(II) exhibited markedly reduced tumorigenicity, as well as reduced proliferation at high density in vitro. Mixed culture assays revealed that the phenotype of the p13(II) cell lines was dominant over that of control lines and was mediated by a heat-labile soluble factor. The p13(II) cell lines exhibited an enhanced response to Ca(2+)-mediated stimuli, as measured by increased sensitivity to C2-ceramide-induced apoptosis and by cAMP-responsive element-binding protein (CREB) phosphorylation in response to histamine. p13(II)-expressing Jurkat T cells also exhibited reduced proliferation, suggesting that the protein might exert similar effects in T cells, the primary target of HTLV-1 infection. These findings provide clues into the function of p13(II) as a negative regulator of cell growth and underscore a link between mitochondria, Ca(2+) signaling, and tumorigenicity.

Multiple endocrine neoplasia type 1 (MEN1) is a hereditary syndrome linked to mutations in the MEN1 gene, which encodes a 610-amino-acid nuclear protein termed menin. Because of the lack of a suitable detection protocol, the in situ expression pattern of menin in human tissues remains to be determined. In this study, we have developed an antimenin monoclonal antibody and an indirect immunofluorescence/laser-scanning microscopy protocol for analyzing menin expression in frozen tissue sections. Because neuroendocrine pancreatic tumors represent a key feature of MEN1, we focused this study on nontumoral pancreas and a small panel of neuroendocrine pancreatic tumors. Results showed that menin was readily detected in nontumoral exocrine cells. In contrast, most islet cells expressing insulin, glucagon, or somatostatin showed considerably weaker levels of menin expression; however, a subpopulation of pancreatic polypeptide-positive cells exhibited a signal comparable with that detected in adjacent exocrine cells. Sporadic endocrine tumors showed variable levels of menin expression, whereas a MEN1-/- gastrinoma scored negative. This report thus provides the first description of the expression pattern of menin in human pancreas in situ and lays the groundwork for further studies of other tissues and tumors.

Human T-cell leukemia virus type 1 encodes a number of "accessory" proteins of unclear function; one of these proteins, p13(II), is targeted to mitochondria and disrupts mitochondrial morphology. The present study was undertaken to unravel the function of p13(II) through (i) determination of its submitochondrial localization and sequences required to alter mitochondrial morphology and (ii) an assessment of the biophysical and biological properties of synthetic peptides spanning residues 9-41 (p13(9-41)), which include the amphipathic mitochondrial-targeting sequence of the protein. p13(9-41) folded into an alpha helix in micellar environments. Fractionation and immunogold labeling indicated that full-length p13(II) accumulates in the inner mitochondrial membrane. p13(9-41) induced energy-dependent swelling of isolated mitochondria by increasing inner membrane permeability to small cations (Na(+), K(+)) and released Ca(2+) from Ca(2+)-preloaded mitochondria. These effects as well as the ability of full-length p13(II) to alter mitochondrial morphology in cells required the presence of four arginines, forming the charged face of the targeting signal. The mitochondrial effects of p13(9-41) were insensitive to cyclosporin A, suggesting that full-length p13(II) might alter mitochondrial permeability through a permeability transition pore-independent mechanism, thus distinguishing it from the mitochondrial proteins Vpr and X of human immunodeficiency virus type 1 and hepatitis B virus, respectively.