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In the aging world population, the occurrence of neuromotor deficits arising from stroke and other medical conditions is expected to grow, demanding the design of new and more effective approaches to rehabilitation. In this paper, we show how the combination of robotic technologies with progress in exergaming methodologies may lead to the creation of new rehabilitation protocols favoring motor re-learning. To this end, we introduce the Track-Hold system for neuromotor rehabilitation based on a passive robotic arm and integrated software. A special configuration of weights on the robotic arm fully balances the weight of the patients’ arm, allowing them to perform a purely neurological task, overcoming the muscular effort of similar free-hand exercises. A set of adaptive and configurable exercises are proposed to patients through a large display and a graphical user interface. Common everyday tasks are also proposed for patients to learn again the associated actions in a persistent way, thus improving life independence. A data analysis module was also designed to monitor progress and compute indices of post-stroke neurological damage and Parkinsonian-type disorders. The system was tested in the lab and in a pilot project involving five patients in the post-stroke chronic stage with partial paralysis of the right upper limb, showing encouraging preliminary results.

This paper analyzes the potentialities to classify vessels detected through optical and synthetic-aperture radar (SAR) satellite-borne platforms and estimate their motion. For classification, the discriminative power of a set of geometric features extracted from segmented remote-sensed images is evaluated by clustering data derived from a set of accurate footprints belonging to either tanker or cargo ships. The same procedure is repeated on a few dozens of real, remote-sensed optical images. Concerning velocity estimation, which in this context is based on the detection and analysis of the wake pattern generated by the ship motion, a discussion concerning the accuracy of the wake detection task is presented. In particular, since wake patterns are usually hard to detect, a method is proposed to enhance the wake signal-to-noise ratio, based on a dedicated pre-filtering stage. Results returned by the proposed method are compared with those obtained adopting a standard literature approach, eventually observing that the introduction of the pre-filtering stage improves the wake detection accuracy. A maritime surveillance system based on a pipeline of the modules described here represents a useful tool to support the authorities in charge of monitoring maritime traffic with safety, security and law enforcement purposes.

The main purpose of this paper is to describe a software platform dedicated to sea surveillance, capable of detecting and identifying illegal maritime traffic. This platform results from the cascade pipeline of several image processing algorithms that input Radar or Optical imagery captured by satellite-borne sensors and try to identify vessel targets in the scene and provide quantitative descriptors about their shape and motion. This platform is innovative since it integrates in its architecture heterogeneous data and data processing solutions with the goal of identifying navigating vessels in a unique and completely automatic processing streamline. More in detail, the processing chain consists of: (i) the detection of target vessels in an input map, (ii) the estimation of each vessel&rsquo, s most descriptive geometrical and scatterometric (for radar images) features, (iii) the estimation of the kinematics of each vessel, (iv) the prediction of each vessel&rsquo, s forthcoming route, and (v) the visualization of the results in a dedicated webGIS interface. The resulting platform represents a novel tool to counteract unauthorized fishing and tackle irregular migration and the related smuggling activities.

Time-correlated single photon counting (TCSPC) is popular in the resolved techniques due to its prominent performance such as ultra-high time resolution and ultra-high sensitivity. This paper presents advance signal processing techniques on the optical TCSPC signals obtained from the series of experiments on fabricated tissue like phantom. A pulsed laser sources at a wavelength of 830 rim transmits the light through the surface of phantom and finally at receiver side, photon counting device generates the histogram of the receiving signal. The noisy data obtained from the photon counter is processed with the splitting based denoising method. The method divide the signal into different subsets based on the transitions. Each subset is then processed individually and final merging of all subsets gives noise free signal. The main objective of this work is to analyze the signal obtained from photon counter in context of skin blood absorption. We had examined the signal obtained by varying the distance between transmitter and receiver to extract the features. Experimental results with our prototype shows more scattering with the increase in the distance at 3dB level and hence less absorption with increase in the distance.

The main purpose of the work described in this paper concerns the development of a platform dedicated to sea surveillance, capable of detecting and identifying illegal maritime traffic. This platform results from the cascade implementation of several image processing algorithms that take as input Radar or Optical maps captured by satellite-borne sensors. More in detail, the processing chain is dedicated to (i) the detection of vessel targets in the input map, (ii) the refined estimation of the vessel most descriptive geometrical features and, finally, (iii) the estimation of the kinematic status of the vessel. This platform will represent a new tool for combating unauthorized fishing, irregular migration and related smuggling activities.

We present a strategy to estimate the heading, the length overall and the beam overall of targets already detected as ships in a wide-swath SAR image acquired by a satellite platform. Such images are often affected by distortions due to marine clutter, spectral leakage, or antenna sidelobes. These can mask the target image, thus hampering the possibility of evaluating the size and the behaviour of the ship. Even in the presence of strong artefacts, we found that the principal inertia axes can help the estimation of the target heading and be included in an iterative procedure to erode the false target features, so to enable a more accurate evaluation of the overall measurements of the ship. Here we introduce our idea and present some results obtained from real SAR images.

Defects of the angiogenic process occur in the brain of twitcher mouse, an authentic model of human Krabbe disease caused by genetic deficiency of lysosomal β-galactosylceramidase (GALC), leading to lethal neurological dysfunctions and accumulation of neurotoxic psychosine in the central nervous system. Here, quantitative computational analysis was used to explore the alterations of brain angioarchitecture in twitcher mice. To this aim, customized ImageJ routines were used to assess calibers, amounts, lengths and spatial dispersion of CD31(+) vessels in 3D volumes from the postnatal frontal cortex of twitcher animals. The results showed a decrease in CD31 immunoreactivity in twitcher brain with a marked reduction in total vessel lengths coupled with increased vessel fragmentation. No significant changes were instead observed for the spatial dispersion of brain vessels throughout volumes or in vascular calibers. Notably, no CD31(+) vessel changes were detected in twitcher kidneys in which psychosine accumulates at very low levels, thus confirming the specificity of the effect. Microvascular corrosion casting followed by scanning electron microscopy morphometry confirmed the presence of significant alterations of the functional angioarchitecture of the brain cortex of twitcher mice with reduction in microvascular density, vascular branch remodeling and intussusceptive angiogenesis. Intussusceptive microvascular growth, confirmed by histological analysis, was paralleled by alterations of the expression of intussusception-related genes in twitcher brain. Our data support the hypothesis that a marked decrease in vascular development concurs to the onset of neuropathological lesions in twitcher brain and suggest that neuroinflammation-driven intussusceptive responses may represent an attempt to compensate impaired sprouting angiogenesis.

A person's well-being status is reflected by their face through a combination of facial expressions and physical signs. The SEMEOTICONS project translates the semeiotic code of the human face into measurements and computational descriptors that are automatically extracted from images, videos, and three-dimensional scans of the face. SEMEOTICONS developed a multisensory platform in the form of a smart mirror to identify signs related to cardio-metabolic risk. The aim was to enable users to self-monitor their well-being status over time and guide them to improve their lifestyle. Significant scientific and technological challenges have been addressed to build the multisensory mirror, from touchless data acquisition, to real-time processing and integration of multimodal data.

Here we describe the implementation of the first prototype of the Wize Sniffer 1.x (WS 1.x), a low cost, portable electronic device for breath analysis. The device is being developed in the framework of the Collaborative European Project SEMEOTICONS (SEMEiotic Oriented Technology for Individuals CardiOmetabolic risk self-assessmeNt and Self-monitoring). In the frame of SEMEOTICONS project, the Wize Sniffer will help the user monitor his/her state of health, in particular giving feedbacks about those noxious habits for cardio-metabolic risk, such as alcohol intake and smoking. The low cost and compactness of the device allows for a daily screening that, even if without a real diagnostic meaning, could represent a pre-monitoring, useful for an optimal selection of more sophisticated and standard medical analysis.

Calcitonin Gene-Related Peptide (CGRP) inhibits microglia inflammatory activation in vitro. We here analyzed the involvement of CGRP and Receptor Component Protein (RCP) in experimental autoimmune encephalomyelitis (EAE). Alpha-CGRP deficiency increased EAE scores which followed the scale alpha-CGRP null>heterozygote>wild type. In wild type mice, CGRP delivery into the cerebrospinal fluid (CSF) 1) reduced chronic EAE (C-EAE) signs, 2) inhibited microglia activation (revealed by quantitative shape analysis), and 3) did not alter GFAP expression, cell density, lymphocyte infiltration, and peripheral lymphocyte production of IFN-gamma, TNF-alpha, IL-17, IL-2, and IL-4. RCP (probe for receptor involvement) was expressed in white matter microglia, astrocytes, oligodendrocytes, and vascular-endothelial cells: in EAE, also in infiltrating lymphocytes. In relapsing-remitting EAE (R-EAE) RCP increased during relapse, without correlation with lymphocyte density. RCP nuclear localization (stimulated by CGRP in vitro) was I) increased in microglia and decreased in astrocytes (R-EAE), and II) increased in microglia by CGRP CSF delivery (C-EAE). Calcitonin like receptor was rarely localized in nuclei of control and relapse mice. CGRP increased in motoneurons. In conclusion, CGRP can inhibit microglia activation in vivo in EAE. CGRP and its receptor may represent novel protective factors in EAE, apparently acting through the differential cell-specific intracellular translocation of RCP.

The proapoptotic death receptor 5 (DR5) expressed by tumor associated endothelial cells (TECs) mediates vascular disrupting effects of human CD34(+) cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL (+) cells) in mice. Indeed, lack of DR5 on TECs causes resistance to CD34-TRAIL (+) cells. By xenografting in nonobese diabetic/severe combined immunodeficient mice the TRAIL-resistant lymphoma cell line SU-DHL-4V, which generates tumors lacking endothelial DR5 expression, here we demonstrate for the first time that the Akt inhibitor perifosine induces in vivo DR5 expression on TECs, thereby overcoming tumor resistance to the vascular disruption activity of CD34-TRAIL (+) cells. In fact, CD34-TRAIL (+) cells combined with perifosine, but not CD34-TRAIL (+) cells alone, exerted marked antivascular effects and caused a threefold increase of hemorrhagic necrosis in SU-DHL-4V tumors. Consistent with lack of DR5 expression, CD34-TRAIL (+) cells failed to affect the growth of SU-DHL-4V tumors, but CD34-TRAIL (+) cells plus perifosine reduced tumor volumes by 60 % compared with controls. In view of future clinical studies using membrane-bound TRAIL, our results highlight a strategy to rescue patients with primary or acquired resistance due to the lack of DR5 expression in tumor vasculature.

PRIAR (Pattern Recognition Image Augmented Resolution) is an innovative approach to single-frame super-resolution that combines common single-frame super-resolution with pattern-recognition algorithms. PRIAR uses the information gained through pattern-recognition to enhance resolution for low quality images, and to allow the end user to explore, recognize and super-resolve low-resolution images. In this paper, we present the basic functionality of the PRIAR algorithm that we have implemented. The program is modular and each module is easily combined. In addition, such modularity permits us to work on images where single modules can be changed in order to resolve different classes of problems. In this paper, we firstly present the features of the PRIAR program processing images reproducing animal cells recorded with a scanning probe microscope.

We present a simple and general-purpose method able to combine high-resolution procedure with the classification and identification of objects of interest from microscopy imaging. The method is composed of two stages. First (pattern recognition), promising components (possible objects of interest) in the image are detected and small regions containing the objects of interest are extracted using a feature finder. Second, high-resolution algorithms are applied to such identified components in order to approach a multiple scales of resolution. Although the method is indeed to be applied to any microscopy technique, in this paper, we have focused the attention on biological systems, like animal cells, recorded with an atomic force microscopy.

In the last years, the complexity paradigm is gaining momentum in many research fields where large multidimensional datasets are made available by the advancements in instrumental technology. A complex system is a multi-component system with a large number of units characterized by cooperative behavior and, consequently, emergence of well-defined self-organized structures, such as communities in a complex network. The self-organizing behavior of the brain neural network is probably the most important prototype of complexity and is studied by means of physiological signals such as the ElectroEncephaloGram (EEG). Physiological signals are typically intermittent, i.e., display non-smooth rapid variations or crucial events (e.g., cusps or abrupt jumps) that occur randomly in time, or whose frequency changes randomly. In this work, we introduce a complexity-based approach to the analysis and modeling of physiological data that is focused on the characterization of intermittent events. Recent findings about self-similar or fractal intermittency in human EEG are reviewed. The definition of brain event is a crucial aspect of this approach that is discussed in the last part of the paper, where we also propose and discuss a first version of a general-purpose event detection algorithm for EEG signals.

The use of associations with dream reports, although being diffused and greatly useful in psychotherapy, has so far played a limited role in dream research not directly finalized to therapy. On the other hand, it is difficult to investigate a basic property of dreaming, that of establishing connections, without searching for information beyond the dream report: indeed, the simplest and likely most effective way of obtaining this information consists in asking the dreamer for associations. Well-defined and rigorous methods are however necessary for research approaches which aim to achieve a better understanding of how the dreaming mind operates by processing and combining the dreamer's memory sources: we propose an experimental protocol aiming to give a contribution in this direction. This protocol consists in asking the dreamer to complete a form which has been implemented so as to make it easier to recognize the sources of single dreams and to describe the link patterns among the sources; this form has been oriented to the identification of episodic sources, definite in space and time. To be more effective, the method takes advantage of the presence of a researcher while the dreamer is completing the form: the terms of the interaction between dreamer and researcher are subjected to marked restrictions and rigorously defined. The protocol was applied to a homogeneous group of participants; the observed link patterns presented interesting phenomena, whose occurrence was characterized by high statistical significance.

What if you could discover your health status by looking at yourself in the mirror? Since November 2013, the EU FP7 Project SEMEOTICONS is working to make this possible. The Project is building a multi-sensory device, having the form of a conventional mirror, able to read the semeiotic code of the face and detect possible evidence of the onset of cardio-metabolic diseases. The device, called Wize Mirror, integrates unobtrusive imaging sensors used to capture videos, images and 3D scans of the face. These are processed to assess the risk of a cardio-metabolic disease and thereby suggest possible strategies to prevent its onset.

Recently, we have suggested a simple and general-purpose method able to combine high-resolution analysis with the classification and identification of components of microscopy imaging. The method named PRIAR (Pattern Recognition Image Augumented Resolution) is a tool developed by the authors that gives the possibility to enhance spatial and photometric resolution of low-res images. The implemented algorithm follows the scheme: 1) image classification; 2) blind super-resolution on single frame; 3) pattern-analysis; 4) reconstruction of the discovered pattern. In this paper, we suggest some improvements of the PRIAR algorithm, in particular, the definition of a segmentation method which is based on homomorphism between a processed image and a graph describing the image itself, able to identify object of interest in complex patterns. The case study is the identification of organs inside biological cells acquired with Atomic Force Microscopy Technique.

The paper describes a system for light spectrum detection of an one-way mirror transmittance by line spectrometry. The system is composed of a focusing lens, a dispersion device and a monochromatic camera. The focusing lens system determines the Region Of Interest (ROI), consisting of a slight rectangular area that can be considered as a linear array of adjacent cells, and sent the light emitted by the region to the entrance slit of the dispersion device. The light rays emerging from the dispersion device hit the area sensor of the camera, that produces an image composed by the spectra of the elementary cells of the ROI. The paper reports a processing procedure for transforming raw data produced by the camera into transmittance or reflectance data. The system allows to measure spectra in the range 380-780 nm, with spectral resolution of about 5 nm. The spatial resolution, that is the dimension of the cells of the ROI, can be set by the imaging lens. The paper describes also an application realized to determine the spectral transmittance of transparent or semi-reflective mirrors and plates.

ICT solutions to foster behavioural change have been shown to be effective in implementing primary prevention in terms of a healthy lifestyle. Primary prevention is the most viable approach to reduce the socio-economic burden of chronic and widespread diseases, such as cardiovascular and metabolic diseases. In this paper, we present a novel multisensory device, the Wize Mirror, which is under development in the EU FP7 Project SEMEOTICONS. The Wize Mirror detects and monitors over time semeiotic face signs related to cardio-metabolic risk, and encourages users to reduce their risk by improving their lifestyle.

3-Iodothyronamine (T1AM) is an endogenous biogenic amine, structurally related to thyroid hormone, which is regarded as a novel chemical messenger. The molecular mechanisms underlying T1AM effects are not known, but it is possible to envisage changes in gene expression, since delayed and long-lasting phenotypic effects have been reported, particularly with regard to the modulation of lipid metabolism and body weight. To test this hypothesis we analysed gene expression profiles in adipose tissue and liver of eight rats chronically treated with T1AM (10 mg/Kg twice a day for five days) as compared with eight untreated rats. In vivo T1AM administration produced significant transcriptional effects, since 378 genes were differentially expressed in adipose tissue, and 114 in liver. The reported changes in gene expression are expected to stimulate lipolysis and beta-oxidation, while inhibiting adipogenesis. T1AM also influenced the expression of several genes linked to lipoprotein metabolism suggesting that it may play an important role in the regulation of cholesterol homeostasis. No effect on the expression of genes linked to toxicity was observed. The assay of tissue T1AM showed that in treated animals its endogenous concentration increased by about one order of magnitude, without significant changes in tissue thyroid hormone concentration. Therefore, the effects that we observed might have physiological or pathophysiological importance. Our results provide the basis for the reported effectiveness of T1AM as a lipolytic agent and gain importance in view of a possible clinical use of T1AM in obesity and/or dyslipidaemia.

Image acquisition systems integrated with laboratory automation produce multi-dimensional datasets. An effective computational approach for automatic analysis of image datasets is given by pattern recognition methods; in some cases, it can be advantageous to accomplish pattern recognition with image super-resolution procedures. In this paper, we define a method derived from pattern recognition techniques for the recognition of artefacts and noise on set of images combined with super-resolution algorithms. The advantage of our approach is automatic artefacts recognition, opening the possibility to build a general framework for artefact recognition independently by the specific application where it is used.

The anti-lymphoma activity and mechanism(s) of action of the multikinase inhibitor sorafenib were investigated using a panel of lymphoma cell lines, including SU-DHL-4V, Granta-519, HD-MyZ, and KMS-11 cell lines. In vitro, sorafenib significantly decreased cell proliferation and phosphorylation levels of MAPK and PI3K/Akt pathways while increased apoptotic cell death. In vivo, sorafenib treatment resulted in a cytostatic rather than cytotoxic effect on tumor cell growth associated with a limited inhibition of tumor volumes. However, sorafenib induced an average 50% reduction of tumor vessel density and a 2-fold increase of necrotic areas. Upon sorafenib treatment, endothelial and tumor cells from SU-DHL-4V, Granta-519, and KMS-11 nodules showed a potent inhibition of either phospho-ERK or phospho-AKT, whereas a concomitant inhibition of phospho-ERK and phospho-AKT was only observed in HD-MyZ nodules. In conclusion, sorafenib affects the growth of lymphoid cell lines by triggering antiangiogenic mechanism(s) and directly targeting tumor cells.

The Thesaurus Project, funded by the Regione Toscana, combines humanistic and technological research aiming at developing a new generation of cooperating Autonomous Underwater Vehicles and at documenting ancient and modern Tuscany shipwrecks. Technological research will allow performing an archaeological exploration mission through the use of a swarm of autonomous, smart and self-organizing underwater vehicles. Using acoustic communications, these vehicles will be able to exchange each other data related to the state of the exploration and then to adapt their behavior to improve the survey. The archival research and archaeological survey aim at collecting all reports related to the underwater evidences and the events of sinking occurred in the sea of Tuscany. The collected data will be organized in a specific database suitably modeled.

Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/β-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated β-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)γc(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.

Several lines of evidence indicate that neuromuscular junction (NMJ) destruction and disassembly is an early phenomenon in amyotrophic lateral sclerosis (ALS). Here we analyzed by confocal and electron microscopy the NMJ structure in the diaphragm of SOD1G93A mice at symptom onset. In these mice, which provide a model for familial ALS, diaphragm denervation (~50%) as well as gastrocnemius denervation (~40%) was found. In addition, the size of the synaptic vesicle pool was reduced and alterations of mitochondria were observed in approximately 40% of the remaining presynaptic terminals. Chronic treatment of SOD1G93A mice with the anabolic steroid nandrolone during the presymptomatic stage preserved the diaphragm muscle mass and features indicative of synaptic activity. These features were represented by the number of vesicles docked within 200 nm from the presynaptic membrane and area of acetylcholine receptor clusters. Structural preservation of mitochondria was documented in presynaptic terminals. However, innervation of diaphragm muscle fibers was only slightly increased in nandrolone-treated SOD1-mutant mice. Altogether the results point out and define fine structural alterations of diaphragm NMJs in the murine model of familial ALS at symptom onset, and indicate that nandrolone may prevent or delay structural alterations in NMJ mitochondria and stimulate presynaptic activity but does not prevent muscle denervation during the disease.

POF1B is a candidate gene for premature ovarian failure (POF); it is mainly expressed in polarised epithelial tissues, but its function in these tissues and the relationship with the disorder are unknown. Here we show colocalisation of POF1B with markers of both adherens and tight junctions in human jejunum. The tight junction localisation was maintained by the human POF1B stably expressed in the MDCK polarised epithelial cell line, whereas it was lost by the POF1B R329Q variant associated with POF. Localisation of apico-basal polarity markers and ultrastructure of the tight junctions were maintained in cells expressing the mutant. However, tight junction assembly was altered, cells were dysmorphic and the monolayer organisation was also altered in three-dimensional culture systems. Moreover, cells expressing the POF1B R329Q variant showed defects in ciliogenesis and cystogenesis as a result of misorientation of primary cilia and mitotic division. All of these defects were explained by interference of the mutant with the content and organisation of F-actin at the junctions. A role for POF1B in the regulation of the actin cytoskeleton was further verified by shRNA silencing of the endogenous protein in human intestinal Caco-2 cells. Taken together, these data indicate that localisation of POF1B to tight junctions has a key role in the organisation of epithelial monolayers by regulating the actin cytoskeleton. © 2011. Published by The Company of Biologists Ltd.

C-tail-anchored (TA) proteins constitute a heterogeneous group of membrane proteins that are inserted into membranes by unique post-translational mechanisms and that play key roles within cells. During recent years, bioinformatic screens on eukaryotic genomes have helped to obtain comprehensive pictures of the number, intracellular distribution and functions of TA proteins, but similar screens had not yet been carried out on prokaryotic cells. Here, we report the results of a bioinformatic screen of the genomes of two bacteria and one archeon. We find that all three of these prokaryotes contain TA proteins in proportions approaching those found in eukaryotic cells, indicating that this protein group is present in all three domains of life. Although some of our hits correspond to proteins of unknown function, others are enzymes with hydrophobic substrates or have functions carried out at the inner face of the cytoplasmic membrane. To generate hypotheses on the insertion mechanisms of prokaryotic TA proteins, we compared the sequences of the prokaryotic and eukaryotic versions of Asna1/Trc40/GET3, a cytosolic ATPase that plays a key role in TA protein post-translational delivery to membranes in eukaryotic cells. We found that hydrophobic residues involved in TA binding by the eukaryotic chaperone (Mateja et al., Nature 2009;461:361–366) are generally replaced with equally hydrophobic amino acids in the archeal homologue (ArsA), whereas this is not the case for the bacterial protein. Thus, eukaryotes may have inherited the GET3 targeting pathway from our archeal ancestor, while the bacterial homologue may be exclusively dedicated to heavy metal resistance.

Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor–related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 105 tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell–derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling–stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

Experimental approaches currently used to quantify the activity of antiangiogenic treatments in cancer therapy do not generally address the importance of spatial distribution of microvessels in target tissues. We report a new computerized method to assess tumor vascularization by quantifying the distribution of functional microvessels as revealed by in vivo staining with sulfosuccinimidyl-6-(biotinamido) hexanoate. Our approach was based on pixel dilation of digital images of blood vessels and addressed the space-filling property of the vessel layouts. This was practically achieved computing the number of dilation cycles (Halo index) needed to permeate a pre-defined amount of each image. Our approach was validated on human tumor xenografts in nonobese diabetic/severe combined immunodeficient mice treated with the antiangiogenic drug sorafenib. For each experimental model, area normalization allowed the unbiased comparison of several hundreds of images showing different amounts of vascular tissue. In two different tumor types, comparison of Halo values showed statistically significant differences between control and sorafenib-treated samples. Conversely, this effect was not observed in samples from an additional xenograft known to resist the antiangiogenic treatment. By separating the analysis of vessel area from the quantification of vessel distributions, our approach can potentially contribute to a better evaluation of the antiangiogenic or vascular-disrupting activity of new drugs or treatments.

The transcription factor C/EBPβ is believed to play a fundamental role in regulating activated macrophage functions. However, the molecular mechanisms and the target genes involved have been, so far, poorly characterized, partly due to the difficulty of reproducibly obtaining homogeneous and abundant primary macrophage populations. In this study, we describe the generation and characterization of immortalized macrophage-like cell lines from C/EBPβ-deficient and wild-type mice. Using these cells, we were able to identify a number of genes involved in activated macrophage functions whose induction was affected in the C/EBPβ−/− cells. IFN-γ/LPS-dependent induction of IL-6, IL-1β, TNF-α, inducible NO synthase, and plasminogen activator inhibitor-1 mRNAs was variably impaired, while IL-12 p40, RANTES and macrophage inflammatory protein-1β mRNAs were up-regulated in the absence of C/EBPβ. The differential mRNA expression correlated with differential transcription levels of the corresponding genes, and was in most cases confirmed in primary macrophage populations. Moreover, in sharp contrast to the enhanced induction of IL-12 p40 mRNA, C/EBPβ−/− primary macrophages derived from both the bone marrow and the peritoneal cavity displayed totally defective expression of IL-12 p35 mRNA. Therefore, the IL-12 p35 gene represents a novel obligatory target for C/EBPβ in macrophages and this may explain the defective production of bioactive IL-12 and the impaired Th1 responses of C/EBPβ-deficient mice to Candida albicans infection observed in previous work.

Quantitative characterization of the in vivo effects of vascular-targeted therapies on tumor vessels is hampered by the absence of useful 3D vascular network descriptors aside from microvessel density. In this study, we extended the quantification of planar vessel distribution to the analysis of vascular volumes by studying the effects of antiangiogenic (sorafenib and sunitinib) or antivascular (combretastatin A4 phosphate) treatments on the quantity and spatial distributions of thin microvessels. These observations were restricted to perinecrotic areas of treated human multiple myeloma tumors xenografted in immunodeficient mice and to microvessels with an approximate cross-sectional area lower than 75 µm(2). Finally, vessel skeletonization minimized artifacts due to possible differential wall staining and allowed a comparison of the various treatment effects. Antiangiogenic drug treatment reduced the number of vessels of every caliber (at least 2-fold fewer vessels vs. controls; p

Introduction. We have previously demonstrated that adenovirus-transduced CD34+ cells expressing membrane-bound (m)TRAIL (CD34-TRAIL+ cells) exert potent antitumor activity against a variety of hematopoietic tumors by targeting both tumor cells and tumor vasculature (Blood, 115:2231-40, 2010). Recently, we have identified non-Hodgkin lymphoma cell lines which are resistant to the in vivo antitumor activity of mTRAIL. Perifosine has been shown to increase the toxicity of soluble TRAIL against cancer cell lines by inhibiting the PI3K/Akt pathway as well as enhancing pro-apoptotic TRAIL receptors. We therefore investigated the efficacy of Perifosine in modulating the antitumor activity of CD34-TRAIL+ cells using as model systems the TRAIL-resistant SU-DHL-4V and the TRAIL-sensitive KMS-11 cell lines. Methods and Results. In vitro, Perifosine significantly enhanced the cytotoxic activity of CD34-TRAIL+ cells against both SU-DHL-4V and KMS-11 cell lines by increasing the expression of TRAIL receptors and down-modulating phospho-Akt, cFLIP and Mcl-1 expression. In vivo, in NOD/SCID mice bearing subcutaneous nodules, Perifosine significantly increased the antitumor activity of CD34-TRAIL+ cells against both tumor types. In fact, CD34-TRAIL+ cells used as single agent exerted a limited if any activity against TRAIL-resistant SU-DHL-4V nodules, whereas, when combined with Perifosine, transduced cells reduced SU-DHL-4V growth by 43% (p< .001) over controls. TRAIL-sensitive KMS-11 nodules were reduced by 39% (p< .001) following treatment with CD34-TRAIL+ cells, and addition of Perifosine further reduced tumor growth by 65% (p Conclusions. Our results demonstrate that: (i) TRAIL-R2 expression by TECs correlates with the in vivo antivascular activity of CD34-TRAIL+ cells; (ii) Perifosine potentiates the antitumor activity of TRAIL-armed CD34+ cells and is able to overcome in vivo resistance to mTRAIL by inducing TRAIL-R2 expression on tumor endothelial cells. These results may open new perspectives in view of clinical applications of CD34-TRAIL+ cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 640. doi:10.1158/1538-7445.AM2011-640

The nucleotide sequence of a PstI fragment prepared from a cloned MH2 virus genome, pMH2-Hd, has been deduced using chemical and enzymatic methods. This fragment, 1862 nucleotides in length, starts with the gag gene, encodes the v-mil sequence and stops within the v-myc gene. This sequence shows that the v-mil gene is fused to the gag gene giving rise to a fused polyprotein of 98 000 daltons: 515 amino acids at the amino terminus would correspond to p10, p19, p27 and part of p12 determinants, 347 amino acids at the carboxy terminus correspond to the v-mil specific sequence. The mil protein shares homology with a number of onc proteins such as src, fes, fms, mos, yes, fps and erbB, as well as with the catalytic chain of the cAMP-dependent protein kinase. This PstI fragment also encodes the beginning of the myc gene which was integrated in MH2 along with the 3' end of the preceding intron placing an acceptor splice site in front of the used open reading frame. As deduced from the sequence, the MH2 myc protein is not identical to the MC29 myc protein. It differs at its amino terminus, which contains little or no gag determinants, depending on the ATG used to initiate translation.

The rearrangement and transcription of the antigen receptor alpha, beta and gamma genes were investigated in murine antigen-specific suppressor T cell lines, to establish whether the suppressor T cell subset expresses the same antigen receptor transcripts previously found in helper and cytotoxic T lymphocytes. The genomic organization of the alpha, beta and gamma chain loci was investigated using probes representative of the entire gene or fragments from variable, joining and constant regions. The present results show that in functional suppressor T cells the three antigen receptor genes are all rearranged. The beta gene is expressed in all the tested cell lines, while the expression of the alpha and gamma genes is variable. In one cell line (LH8) alpha and gamma genes are not efficiently transcribed; in the other cell line (LA41) the gamma mRNA is found in amounts similar to beta mRNA, whereas the alpha gene is expressed at low levels. These data suggest that in suppressor T cells no direct correlation exists between the expression of alpha, beta and gamma antigen receptor genes and the effector function.