Immunomodulatory effects of metronomic vinorelbine (mVRL), with or without metronomic capecitabine (mCAPE), in hormone receptor positive (HR+)/HER2-negative metastatic breast cancer (MBC) patients: final results of the exploratory phase 2 Victor-5 study.

Tregs are able of suppressing tumor-specific effector cells, such as lymphocytes CD8+, CD4+ and Natural Killer cells. Different drugs, especially different schedules of administration, like metronomic chemotherapy (mCHT), seem to be able to increase anticancer immunity, by acting on downregulation of Tregs. Most of the data available regarding the immunomodulating effect of mCHT have been obtained with Cyclophosphamide (CTX). Aim of the present study was to explore the effects of mVRL and mCAPE administration, alone or in combination, on T cells. Observation of 13 metastatic breast cancer patients lasted controlling for 56 days, where Treg frequencies and function, spontaneous anti-tumor T-cell responses were monitored, as well as the clinical outcome. No depletion in Treg absolute numbers, or percentage of T lymphocytes, was observed. Only in 5 patients, a modest and transient depletion of Tregs was observed during the first 14 days of treatment. To better describe the effect on Tregs, we subsequently looked at the variations in Memory, Naïve and Activated Treg subpopulations: we observed a trend in reduction for memory Treg (Treg MEM) and an increase for Treg Naïve (Treg NAIVE) and Treg Activated (Treg ACT) components. We finally analyzed the average trend of Treg in the Treg depleted patients and non-depleted ones, without fiding any significant differences. The trend of the Treg MEM appeared different, showing a reduction during the first 14 days, followed by an increase at the levels before treatment at Day 56 in the group of depleted patients and a progressive substantial reduction in the group of non-depleted patients along the entire course of treatment. Opposed to the data known, treatment with mVRL w/o mCAPE did not show any effect on Tregs.

A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation.

Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.

Functional analysis of expression of human ecto-nucleoside triphosphate diphosphohydrolase-1 and/or ecto-5'-nucleotidase in pig endothelial cells.

Adenine nucleosides and nucleotides are important signaling molecules involved in control of key mechanisms of xenotransplant rejection. Extracellular pathway that converts ATP and ADP to AMP, and AMP to adenosine mainly mediated by ecto-nucleoside triphosphate diphosphohydrolase 1, (ENTPD1 or CD39) and ecto-5'-nucleotidase (E5NT or CD73) respectively, is considered as important target for xenograft protection. To clarify feasibility of combined expression of human ENTPD1 and E5NT and to study its functional effect we transfected pig endothelial cell line (PIEC) with both genes together. To do this we have produced a dicistronic construct bearing F2A sequence in frame between human E5NT and human ENTPD1 coding sequences. PIEC cells were mock-transfected as transfection control or transfected with plasmids encoding human ENTPD1 or human E5NT. PIEC cells were exposed to 50 µM ATP or 50 µM ADP or 50 µM AMP. Conversion of extracellular substrates into products (ATP/ADP/AMP/adenosine) was measured by HPLC in the media collected at specific time intervals. Following addition of AMP, production of adenosine in the medium of E5NT/ENTPD1- and E5NT- transfected cells increased to 14.2±1.1 and 24.5±3.4 µM respectively while it remained below 1 µM in controls and in ENTPD1-transfected cells. A marked increase of adenosine formation from ADP or ATP was observed only in E5NT/ENTPD1-transfected cells (11.7±0.1 and 5.7±2.2 µM respectively) but not in any other condition studied. This study indicates feasibility and functionality of combined expression of human E5NT and ENTPD1 in pig endothelial cells using F2A sequence bearing construct.

Quadricuspid morphology of the aortic valve.

Quadricuspid aortic valve is a rare congenital heart defect. It may be isolated or associated to other cardiac anomalies. It may cause aortic valve dysfunction, commonly aortic regurgitation. Management of patients with quadricuspid aortic valve is represented by strict follow-up, because they may require aortic valve replacement in later life. We report the case of a 37-year old male patient, occasionally diagnosed to have quadricuspid aortic valve. Diagnosis and management are discussed.

In vitro production of multigene transgenic blastocysts via sperm-mediated gene transfer allows rapid screening of constructs to be used in xenotransplantation experiments.

Multigene transgenic pigs would be of benefit for large animal models and in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We have previously produced multitransgenic pigs via sperm-mediated gene transfer (SMGT) using integrative constructs expressing 3 different reporter genes. The aim of the present work was to evaluate the efficacy and safety of using 3 integrative constructs carrying 3 different human genes involved in the modulation of inflammatory responses. We developed an in vitro fertilization system to demonstrate that SMGT can be used to efficiently produce multigene transgenic embryos through a 1-step genetic modification using multiple integrative constructs each carrying a different human gene involved in the modulation of inflammatory processes (hHO1, hCD39, and hCD73). The results suggest that this system allowed an effective preliminary test of transgenesis optimization, greatly reducing the number of animals used in the experiments and fulfilling important ethical issues. We performed 5 in vitro fertilization experiments using sperm cells preincubated with all 3 integrative constructs. A total of 1,498 oocytes were fertilized to obtain 775 embryos, among which 340 further developed into blastocysts. We did not observe any toxicity related to the transgenesis procedure that affected normal embryo development. We observed 68.5% transgenesis efficiency. Blastocysts were 48% single, 31% double, and 21% triple transgenic.

Inflammatory activation during coronary artery surgery and its dose-dependent modulation by statin/ACE-inhibitor combination.

BACKGROUND: On-pump coronary artery bypass graft (CABG) surgery triggers an inflammatory response (IR) which may impair revascularization. The study aimed at (1) characterizing the temporal profile of the IR by assaying appropriate markers in both systemic and coronary blood, and (2) determining whether (and which doses of) cardiovascular drugs known to have antiinflammatory properties, namely statins and ACE-inhibitors (ACEI), inhibit the response. METHODS AND RESULTS: Patients scheduled for CABG (n=22) were randomized to statin/ACEI combination treatment at standard doses (STD, ramipril 2.5/simvastatin 20 mg, or atorvastatin 10 mg), or at high doses (HiDo, ramipril 10 mg, or enalapril 20 mg/simvastatin 80 mg, or atorvastatin 40 mg). Plasma levels of interleukin 6, tumor necrosis factor alpha, E-selectin, von Willebrand factor (vWF), and sVCAM-1 were serially assayed (ELISA) before, during, and after CABG. Blood was drawn from an artery, a systemic vein, and the coronary sinus. Myocardial perfusion scans were obtained before and 2 months after surgery in 19 out of 22 subjects. In the STD group both IL-6 and TNF displayed striking increases which were similar at all sites and peaked 10 to 60 minutes after aortic declamping. Such increases were drastically attenuated in the HiDo group. Instead, only modest increases in venous E-selectin, vWF, and sVCAM-1 were observed. Scintigraphic ischemia scores were entirely normalized after versus before CABG in the HiDo but not in the STD treatment group. CONCLUSIONS: On-pump CABG surgery is associated with an intense systemic inflammatory response, which can be almost completely prevented by early treatment with high (but not standard) doses of ACE-inhibitors and statins.

Acute myocardial infarction and rescue percutaneous transluminal coronary angioplasty in a young HIV-infected patient.

We report a case of acute myocardial infarction in an HIV-infected patient without significant coronary artery disease (CAD) risk factors. The patient underwent rescue percutaneous transluminal coronary angioplasty (PTCA), with a successful outcome. We presume a possible pathogenetic role of anti-retroviral therapy and/or direct viral action on ischaemic heart disease progression. We propose that the current approach to management of AIDS-affected patients needs close monitoring for CAD risk factors and symptoms, to improve prognosis and life expectancy.

Angiotensin-converting enzyme inhibition modulates high-glucose-induced extracellular matrix changes in mouse glomerular epithelial cells.

BACKGROUND/AIM: Extracellular matrix alterations are involved in the pathogenesis of diabetic nephropathy. We evaluated the effects of high glucose concentrations and inhibition of angiotensin-converting enzyme on the laminin and fibronectin production by glomerular epithelial cells. METHODS: Glomerular epithelial cells were cultured in 5 and 30 mmol/l glucose, with and without enalaprilat (0.3 mmol/l). Laminin and fibronectin were measured (35S-methionine, immunoprecipitation), and their mRNA expression was evaluated (RT-PCR). RESULTS: The laminin concentration was higher in the cells than in the medium, where an increase of its content was observed under high-glucose conditions (p < 0.01). Fibronectin, found only in the medium, was not modified by the high glucose concentration. Following enalaprilat administration, the laminin concentration was decreased under high-glucose conditions, both in the cell and in the medium (p < 0.001), whereas the fibronectin concentration was increased under high-glucose conditions (p < 0.001). The mRNA expression of laminin and fibronectin under high-glucose conditions only slightly increased. Enalaprilat decreased the fibronectin mRNA synthesis dramatically (>50%, p < 0.0001) under high-glucose conditions. CONCLUSIONS: Enalaprilat normalizes the abnormal, high-glucose-induced concentration of laminin, while it decreases the fibronectin synthesis. The improvement of the renal function in diabetic patients treated with angiotensin-converting enzyme inhibitors may, in part, be due to a modulator effect on extracellular matrix content and composition.

[Thrombo-embolic risk evaluation in patients with atrial fibrillation. Role of echocardiography]. / Stratificazione del rischio tromboembolico nel paziente con fibrillazione atriale. Ruolo dell'ecocardiografia.

Atrial fibrillation (AF) is the most common observed cardiac arrhythmia and is the most frequent condition associated with thromboembolic events in patients with or without mitral valve disease. The source of cardiac emboli is the left atrium, the left atrial appendage or, less frequently, the left ventricle. Emboli may also originate from aortic atherosclerotic plaques. It is important to identify patients at risk in order to perform the appropriate therapy. Risk stratification is multiparametric, being based on clinical, laboratory, and echocardiographic data. Several trials have pointed out the role of echocardiography in the evaluation of anatomic and functional parameters associated with thromboembolic risk. Transthoracic echocardiography (TTE) does not provide sufficient information regarding posterior cardiac structures, being its sensitivity in detecting thrombi relatively low (33-72%). Transesophageal echocardiography (TEE) in contrast, has an almost 100% sensitivity; this technique is, therefore, mandatory in patients with AF for an adequate prevention of thromboembolism. The echocardiographic information joined with clinical features allow to stratify, in a proper way, the risk of every single patient.

Sulfated polysaccharides inhibit leukocyte rolling in rabbit mesentery venules.

Before firm adhesion, leukocytes roll slowly along the walls of small venules at velocities ranging from 0.7 to 36% of mean blood flow velocity. To investigate the nature of the adhesive process underlying leukocyte rolling, synthetic (dextran sulfate) and naturally occurring sulfated polysaccharides (heparin, chondroitin sulfates, keratan sulfate, and heparan sulfate) were infused via glass micropipettes into the lumen of small venules (20-60 microns diam) of the rabbit mesentery. Leukocyte rolling was observed and quantified using both transmitted light and incident fluorescence intravital microscopy. Rolling leukocytes accounted for 27-80% of total leukocyte flux, exhibiting a wide range of individual velocities (0.01-0.84 mm/s) with a mean value of 4% of centerline velocity. Dextran sulfate (Mr 500,000) inhibited leukocyte rolling very effectively [half-effective concentration (ED50) approximately 10 micrograms/ml] and was able to almost completely abolish rolling at 500 micrograms/ml. Heparin (ED50 approximately 50 micrograms/ml), chondroitin 6-sulfate C (ED50 approximately 500 micrograms/ml), and heparan sulfate (ED50 approximately 5 mg/ml) also reduced leukocyte rolling. At 5 mg/ml, chondroitin 4-sulfate B (dermatan sulfate) was marginally effective, but chondroitin 4-sulfate A and keratan sulfate were ineffective. The present data suggest that an adhesion receptor-ligand system distinct from the leukocyte integrins may be underlying transient leukocyte adhesion (rolling). Endothelial glycoproteins or proteoglycans containing sulfated side chains may be involved in mediating this adhesive process.