PO-032 The knock-down of ferritin heavy subunit induces xenobiotic-resistance in k562 cells through the activation of nf-kb pathway

Introduction The transcriptional factor NF-κB, composed by five subunits (RelA/p65, c-Rel, RelB, p50, p52), is largely involved in many facets of cellular physiology such as innate and adaptive immunity as well as inflammation. In addition, NF-kB play a central role in cancer cell survival and chemoresistance partly by its implication in cross-talks with redox-regulating proteins. Ferritin is the major iron storage protein; it is composed by a variable assembly of Heavy (FHC) and Light (FLC) subunits. FHC, in particular, has been widely demonstrated to be devoted in iron uptake and release thus controlling the redox homeostasis. Material and methods K562 erythroleukemia cells were stably silenced for FHC by using the shRNA method. Then FHC reconstitution was achieved by transient transfection of a FHC specific expression vector. ROS were determined by incubating cells with the redox-sensitive probe 2'−7'-DCF. NAC was used to inhibit ROS production. MTT assay was performed to analyse cell viability. Increasing concentrations of Doxorubicin, ranging from 0 to 5 µM, were used to treat K562 cells. Results and discussions The results of this study highlighted that FHC amounts negatively affect NF-kB activation in K562 cells. FHC silencing was accompanied by an increased expression of the nuclear NF-kB subunit p65, FHC rescue determined nuclear p65 decrease. FHC silencing is responsible for intracellular ROS production and ROS are implicated in NF-kB pathway. To elucidate the relationship between ROS amount and nuclear p65 content, we determined ROS amounts in our in vitro model and evaluated p65 nuclear expression after treatment with the ROS scavenger NAC. First, we observed that, as expected, ROS levels increased upon FHC silencing and return to basal levels upon NAC treatment. Interestingly, NAC was also able to decrease nuclear p65 amount in FHC-silenced K562 cells. Considering the effect of NF-kB activated pathway on cell survival, we analysed the effect of FHC silencing-mediated p65 increase in K562 cells upon treatment with increasing doses of Doxorubicin. Cell viability assay highlighted that FHC-silencing was accompanied by an increased resistance to the drug with an IC 50 about doubled compared to that of the K562 control cells at each the time points. This resistance of FHC-silenced cells was reverted upon NF-kB inhibitor transfection. Conclusion FHC silencing induced NF-kB activation in K562 cells through the modulation of intracellular ROS content. This regulatory axis can be used to modulate K562 chemoresistance

po 119 ferritin heavy subunit enhances apoptosis of non small cell lung cancer cells through modulation of a mir 125b p53 axis

Introduction The ferritin molecule is a nanocage composed by the variable assembly of 24 heavy and light subunits. As major intracellular iron storage protein, ferritin has been studied for many years in the context of iron metabolism. However, recent evidences highlight its role, and particularly that of the heavy subunit (FHC), in pathways related to cancer development and progression, such as cell proliferation, epithelial-mesenchymal transition, cell death and angiogenesis. This new role of FHC is largely due to its ability to regulate a repertoire of oncogenes and oncomiRNAs. Moreover, the existence of a feedback loop between FHC and the tumour suppressor p53 has been observed in different cell types. Material and methods FHC was overexpressed in A549 and H460 non-small cell lung cancer (NSCLC) cells through transient transfection of a specific expression vector. FHC, p53 and miR-125b levels were measured by q-PCR and TaqMan analyses in cancer cell lines as well as in tumour tissue specimens. The analysis of the methylation status of miR-125b promoter region was achieved by Methylation Specific PCR (MSP). FHC-overexpressing and control A549 and H460 cells were monitored for changes in proliferation and apoptosis through PI and Annexin/7-AAD flow cytometry assays. Intrinsic and extrinsic apoptosis biomarkers were measured by Western Blot. Statistical analysis was performed by Student t-test or non-parametric Wilcoxon signed-rank test. Results and discussions The major finding of this study was that FHC is able to enhance p53 expression through the down-regulation of miR-125b in A549 and H460 NSCLC cell lines. Indeed, we found that FHC overexpression induces hypermethylation and thus the down-regulation of miR-125b which, in turn, is a direct repressor of the tumour suppressor p53. Absolute q-PCR highlighted a significant correlation among these three key molecules also in human tumour tissue specimens thus strongly suggesting the existence of a new regulatory axis In vitro, FHC overexpression also triggered p53-mediated cell apoptosis that is partially reverted by miR-125b reconstitution. Up-regulation of BAX, a pro-apoptotic member of Bcl-2 family, and the enhanced cleavage of caspase-9 demonstrated the activation of the intrinsic apoptotic pathway. Conclusion Overall, the identification of a FHC/miR-125b/p53 regulatory axis may provide a novel molecular strategy for the regulation of the apoptotic cell death in non small cell lung cancer

Coronary flow reserve in stress-echo lab. From pathophysiologic toy to diagnostic tool

The assessment of coronary flow reserve by transthoracic echocardiography has recently been introduced into clinical practice with gratifying results for the diagnosis of left anterior descending artery disease simultaneously reported by several independent laboratories. This technological novelty is changing the practice of stress echo for 3 main reasons. First, adding coronary flow reserve to regional wall motion allows us to have – in the same sitting – high specificity (regional wall motion) and a high sensitivity (coronary flow reserve) diagnostic marker, with an obvious improvement in overall diagnostic accuracy. Second, the technicalities of coronary flow reserve shift the balance of stress choice in favour of vasodilators, which are a more robust hyperemic stress and are substantially easier to perform with dual imaging than dobutamine or exercise. Third, the coronary flow reserve adds a quantitative support to the exquisitely qualitative assessment of wall motion analysis, thereby facilitating the communication of stress echo results to the cardiological world outside the echo lab. The next challenges involve the need to expand the exploration of coronary flow reserve to the right and circumflex coronary artery and to prove the additional prognostic value – if any – of coronary flow reserve over regional wall motion analysis, which remains the cornerstone of clinically-driven diagnosis in the stress echo lab

Recovery of distal coronary flow reserve in LAD and LCx after Y-Graft intervention assessed by transthoracic echocardiography

<p>Abstract</p> <p>Background</p> <p>Y- graft (Y-G) is a graft formed by the Left Internal Mammary Artery (LIMA) connected to the Left Anterior Descending Artery (LAD) and by a free Right Internal Mammary Artery (RIMA) connected to LIMA and to a Marginal artery of Left Circumflex Artery (LCx). Aim of the work was to study the flow of this graft during a six months follow-up to assess whether the graft was able to meet the request of all the left coronary circulation, and to assess whether it could be done by evaluation of coronary flow reserve (CFR).</p> <p>Methods</p> <p>In 13 consecutive patients submitted to Y-G (13 men), CFR was measured in distal LAD and in distal LCx from 1 week after , every two months, up to six months after operation (a total of 8 tests for each patient) by means of transthoracic echocardiography (TTE) and Adenosine infusion (140 mcg/kg/min for 3-6 min). A Sequoia 256, Acuson-Siemens, was used. Contrast was used when necessary (Levovist 300 mg/ml solution at a rate of 0,5-1 ml/min). Max coronary flow diastolic velocity post-/pre-test ≥2 was considered normal CFR.</p> <p>Results</p> <p>Coronary arteriography revealed patency of both branches of Y-G after six months. Accuracy of TTE was 100% for LAD and 85% for LCx. Feasibility was 100% for LAD and 85% for LCx. CFR improved from baseline in LAD (2.21 ± 0.5 to 2.6 ± 0.5, p = 0.03) and in LCx (1.7 ± 1 to 2.12 ± 1, p = 0.05). CFR was under normal at baseline in 30% of patients <it>vs </it>8% after six months in LAD (p = 0.027), and in 69% of patients <it>vs </it>30% after six months in LCx (p = 0.066).</p> <p>Conclusion</p> <p>CFR in Y-G is sometimes reduced in both left territories postoperatively but it improves at six months follow-up. A follow-up can be done non-invasively by TTE and CFR evaluation.</p

Diagnostic value of harmonic transthoracic echocardiography in native valve infective endocarditis: comparison with transesophageal echocardiography

<p>Abstract</p> <p>Background</p> <p>Although echocardiography has been incorporated into the diagnostic algorithm of patients with suspected infective endocarditis (IE), systematic usage in clinical practice remains ill defined. To determine the diagnostic accuracy of detecting vegetations using harmonic transthoracic echocardiography (hTTE) as compared to transesophageal echocardiography (TEE) in patients with an intermediate likelihood of native valve IE.</p> <p>Methods</p> <p>Between 2004 and 2005, 36 consecutive inpatients with an intermediate likelihood of disease were prospectively evaluated by hTTE and TEE.</p> <p>Results</p> <p>Of 36 patients (21 males with a mean age of 57 ± 15 years, range 32 to 86 years), 19 patients had definite IE by TEE. The sensitivity for the detection of vegetations by hTTE was 84%, specificity of 88%, positive predictive value (PPV) of 89% and negative predictive value (NPV) of 82%. The association between hTTE and TTE interpretation for the presence and absence of vegetations were high (kappa = 0.90 and 0.85 respectively).</p> <p>Conclusion</p> <p>In patients with an intermediate likelihood of native valve IE, TTE with harmonic imaging provides diagnostic quality images in the majority of cases, has excellent concordance with TEE and should be recommended as the first line test.</p

Standard of Practice for the Endovascular Treatment of Thoracic Aortic Aneurysms and Type B Dissections

Thoracic endovascular aortic repair (TEVAR) represents a minimally invasive technique alternative to conventional open surgical reconstruction for the treatment of thoracic aortic pathologies. Rapid advances in endovascular technology and procedural breakthroughs have contributed to a dramatic transformation of the entire field of thoracic aortic surgery. TEVAR procedures can be challenging and, at times, extraordinarily difficult. They require seasoned endovascular experience and refined skills. Of all endovascular procedures, meticulous assessment of anatomy and preoperative procedure planning are absolutely paramount to produce optimal outcomes. These guidelines are intended for use in quality-improvement programs that assess the standard of care expected from all physicians who perform TEVAR procedures

Echocardiography practice, training and accreditation in the intensive care: document for the World Interactive Network Focused on Critical Ultrasound (WINFOCUS)

Echocardiography is increasingly used in the management of the critically ill patient as a non-invasive diagnostic and monitoring tool. Whilst in few countries specialized national training schemes for intensive care unit (ICU) echocardiography have been developed, specific guidelines for ICU physicians wishing to incorporate echocardiography into their clinical practice are lacking. Further, existing echocardiography accreditation does not reflect the requirements of the ICU practitioner. The WINFOCUS (World Interactive Network Focused On Critical UltraSound) ECHO-ICU Group drew up a document aimed at providing guidance to individual physicians, trainers and the relevant societies of the requirements for the development of skills in echocardiography in the ICU setting. The document is based on recommendations published by the Royal College of Radiologists, British Society of Echocardiography, European Association of Echocardiography and American Society of Echocardiography, together with international input from established practitioners of ICU echocardiography. The recommendations contained in this document are concerned with theoretical basis of ultrasonography, the practical aspects of building an ICU-based echocardiography service as well as the key components of standard adult TTE and TEE studies to be performed on the ICU. Specific issues regarding echocardiography in different ICU clinical scenarios are then described