Di Paolo Research Articles

The Combination of Entospletinib and Vincristine Demonstrates Synergistic Activity in a Broad Panel of Hematological Cancer Cell Lines and Anti-Tumor Efficacy in a DLBCL Xenograft Model

BackgroundEntospletinib (GS-9973) selectively inhibits spleen tyrosine kinase (SYK), a critical signaling component of the BCR pathway that is expressed primarily in cells of hematopoietic lineage including normal and malignant B-lymphocytes. Entospletinib is currently in phase II clinical trials, where it has demonstrated both a high degree of safety as well as efficacy against chronic lymphocytic leukemia (Sharman, J., et al. Blood, 2015) and other B cell malignancies. Despite these successes, new therapeutic options, including combinations with standard of care agents, are needed in order to achieve the goal of curing disease through finite treatment. We show here that the combination of entospletinib and vincristine causes synergistic apoptosis in vitro in a broad panel of cell lines derived from hematological cancers including diffuse large B cell lymphoma (DLBCL), acute lymphocytic leukemia, follicular lymphom), multiple myeloma, and acute myelogenous leukemia. We also evaluated and compared the in vivo efficacy of entospletinib and vincristine as singe agents and in combination in a DLBCL tumor xenograft model using the SU-DHL-10 cell line.MethodsIn vitro growth inhibition of a panel of malignant hematological cell lines was assessed using CellTiter-Glo™ Assay (Promega) after 72h incubation with entospletinib or vincristine alone or in combination. Synergy was evaluated using the Bliss model of independence (Meletiadis, J., et al., Med Mycol, 2005). In vivo, SU-DHL-10 cells (5 x 106 cells) were implanted subcutaneously in the axilla in male SCID beige mice. All mice were sorted into study groups on Day 16 such that each group's mean tumor volume fell within 10% of the overall mean (197mm3). Dosing was initiated on Day 16 and animals were dosed for 17 days. Plasma concentrations of entospletinib and vincristine were assessed on Day 19, and the entospletinib 75 mg/kg dose was lowered on Day 22 to 50 mg/kg to approximate the human achievable SYK target coverage of EC80. Efficacy and tolerability were evaluated by tumor measurements and body weight monitored three times weekly. Tumor burden data were analyzed by the application of a two-way analysis of variance (ANOVA), with post-hoc analysis.ResultsIn vitro combinations of entospletinib with low concentrations of vincristine resulted in marked inhibition of cell proliferation and induction of apoptosis in a broad panel of 19 tumor cell lines representing major B cell malignancies including DLBCL. The combination of entospletinib with vincristine had a profound inhibitory effect on proliferation in all subtypes of DLBCL. Entospletinib was evaluated at a concentration equivalent to the Cminof the clinical dose and vincristine was used at concentrations (≤ 10 nM) that had little to no significant single agent effect in these cell lines.In vivo in a SU-DHL-10 xenograft model, entospletinib dosed alone at 25 or 75/50 mg/kg significantly inhibited tumor growth, causing 39% and 20% tumor growth inhibition (TGI), respectively, compared to the vehicle-treated control group. Vincristine administered at either 0.15 and 0.5 mg/kg Q7D x 3 also resulted in significant TGI (42% and 85% TGI, respectively). The addition of entospletinib (75/50 mg/kg) to 0.5 mg/kg or 0.15 mg/kg vincristine resulted in a significant increase in TGI from 85% to 96% (p= 0.001) and 42% to 71% (p< 0.0001), respectively. The addition of entospletinib (25 mg/kg) to vincristine did not significantly increase the tumor growth inhibition. While the groups receiving either entospletinib or vincristine as single agents had no complete or partial tumor regression, 50% of the mice receiving the combination of 75/50 mg/kg entospletinib with 0.5 mg/kg vincristine had partial responses, 8% had complete regression and 8% were tumor free at the end of study (Figure 1).ConclusionEntospletinib and vincristine demonstrated efficacy and tolerability both alone and in combination in the SU-DHL-10 DLBCL cell line xenograft model in SCID beige mice. Vincristine combinations with entospletinib showed significantly greater efficacy than vincristine alone. These data support the further clinical development of entospletinib in combination with vincristine for the treatment of DLBCL.a ENTO: PO: Q12H x 2 (Day 16-32)b VCR: IV: Q7D x 3 (Days 18, 25, 32) [Display omitted] DisclosuresAxelrod:Gilead Sciences: Employment, Equity Ownership. Fowles:Gilead Sciences: Employment, Equity Ownership. Silverman:Gilead Sciences: Employment, Equity Ownership. Clarke:Gilead Sciences: Employment, Equity Ownership. Tang:Gilead Sciences: Employment, Equity Ownership. Rousseau:Gilead Sciences: Employment, Equity Ownership. Webb:Gilead Sciences: Employment, Equity Ownership. Di Paolo:Gilead Sciences: Employment, Equity Ownership.

Combination Effects of B Cell Receptor Pathway Inhibitors (Entospletinib, ONO/GS-4059, and Idelalisib) and a BCL-2 Inhibitor in Primary CLL Cells

BackgroundCompared to single-agent therapy, combination therapy in chronic lymphocytic leukemia (CLL) has the goal to achieve complete responses with greater durability and ultimately to achieve minimal residual disease negativity and cure with finite therapy. The investigational agents entospletinib, ONO/GS-4059, and ABT-199 are selective, oral, small molecule inhibitors of SYK, BTK, and BCL-2, respectively, and currently are each being developed for the treatment of CLL. Idelalisib (Zydelig™), a first-in-class, selective, oral inhibitor of PI3Kδ, is approved for the treatment of relapsed chronic lymphocytic leukemia (CLL) in combination with rituximab. Though significant clinical efficacy has been achieved in CLL with currently approved agents, improvement in outcome could be enhanced with combinations of novel agents. We investigated the activity of combinations of the B cell receptor pathway inhibitors, entospletinib, ONO/GS-4059 and idelalisib, and the BCL-2 inhibitor, ABT-199, in vitro in primary CLL cells to define the potential utility of these combinations for clinical investigation in patients.MethodsPeripheral blood mononuclear cells (PBMCs) prepared from CLL patients were stimulated with αIgM/αIgG and αCD40 with or without co-culture with the human bone marrow stromal cell line, HS-5. Entospletinib, ONO/GS-4059, idelalisib, or ABT-199 were combined and incubated with cells for 66-72 hours at clinically achievable concentrations of each. Apoptosis was assessed in CD5+ CD19+ CLL cells by Annexin V/Live-Deadassay (Promega). Combinations of the compounds were assessed using a range of concentrations in an 8 × 3 or 3 × 3 matrix format and assayed for apoptosis using a minimum of 8 individual CLL donors after 66-72 hours. Synergy was evaluated using the Bliss model of independence (Meletiadis, J., et al., Med Mycol, 2005).ResultsStimulation of CLL cells by αIgM/αIgG/αCD40 led to a variable, but statistically significant, protection of primary CLL cells in vitro (p=0001), and co-culture with the stromal cell line, HS-5, in conjunction with αIgM/αIgG/αCD40 stimulation further reduced the observed level of apoptosis (p=0.0051). Entospletinib, ONO/GS-4059, idelalisib, and ABT-199, were able to induce apoptosis in primary αIgM/αIgG/αCD40 stimulated CLL cells with an average maximal proportion of apoptotic cells of 50.2%, 26.1%, 23%, and 91.4%, respectively. Addition of ONO/GS-4059 + entospletinib or idelalisib, had an additive effect on induction of apoptosis in primary CLL cells from 5/9 and 6/8 patients, respectively (Figure 1). The addition of ABT-199 to entospletinib, ONO/GS-4059, or idelalisib showed additive to synergistic effects on induction of apoptosis in primary CLL cells from all patients tested, and increased the maximal level of apoptosis (Figure 1).Primary CLL cells were exposed to pairwise combinations of ONO/GS-4059 with entospletinib or idelalisib (A) or ONO/GS-4059 entospletinib, or idelalisib with ABT-199 (B) using a range of concentrations in an 8 x 3 or 3 x 3 matrix format for 66-72 h. Each point represents the mean result for each pairwise combination for each compound at each concentration tested. The percent apoptosis was assessed by AnnexinV/Live-Dead cell staining. The plots show the measured apoptosis versus the calculated, Bliss predicted score for each combination at each concentration tested. Observed apoptosis greater than the bliss predicted score indicates a synergistic relationship. Area within the dashed lines, indicate additive responses.SummaryCombination treatment with the B cell receptor pathway inhibitors resulted in additive increases in apoptosis in primary CLL cells compared to treatment with the individual agents. The combination of the BCL2 inhibitor ABT-199 with entospletinib, ONO/GS-4059 or idelalisib resulted in an additive to synergistic increase in apoptosis in these primary CLL cells and achieved the greatest maximal levels of apoptosis. These in vitro data support clinical investigation of these combinations in patients with CLL. [Display omitted] [Display omitted] DisclosuresJones:Gilead Sciences: Employment, Equity Ownership. Axelrod:Gilead Sciences: Employment, Equity Ownership. Tumas:Gilead Sciences: Employment, Equity Ownership. Quéva:Gilead Sciences: Employment, Equity Ownership. Di Paolo:Gilead Sciences: Employment, Equity Ownership.

Book Review: Contemporary Sensorimotor Theory

Consciousness, with its irreducible subjective character, was almost exclusively a philosophical topic until relatively recently. Today, however, the problem of explaining the felt quality of experience has also become relevant to science and engineering, including robotics andAI: “What would we have to build into a robot so that it really felt the touch of a finger, the redness of red, or the hurt of a pain?” (O’Regan, 2014, p. 23). Yet a practical response still requires an adequate theory of consciousness, which brings us back to the hard problem: how canwe account, from a scientific point of view, for the phenomenological character of experience? Over a decade ago, O’Regan and Noe (2001) proposed a new approach to these questions, the so-called sensorimotor approach to perceptual experience. How far has this approach come and what are its outstanding challenges? The volumeContemporary Sensorimotor Theory, edited by Bishop and Martin, takes stock of the current state of the field. The book starts with Bishop andMartin (2014) presenting different facets of sensorimotor theory, highlighting, for example, that O’Regan (2011) and Noe (2004) ended up developing different ideas concerning the applicability of the theory to robots: a positive account appealing to higherorder cognitive capacities versus a skeptical stance citing the necessity of life for mind, respectively. Ambiguous labeling does not help the current situation. According to Hutto and Myin (2013), the sensorimotor approach of O’Regan and Noe (2001) is also “enactive,” a label which Noe (2004) himself began to adopt, but from which Pascal and O’Regan (2008) distanced themselves. In fact, several overlapping approaches may be distinguished in addition to the classic sensorimotor approach, including sensorimotor enactivism (Varela et al., 1991; Noe, 2004), which turned into autopoietic enactivism (Thompson, 2005, 2007; Noe, 2009; Froese and Di Paolo, 2011), and which is distinguished from radical enactivism by Hutto and Myin (2013). The book’s contributions range over all of them. Noe did not contribute to this volume, but his absence is compensated by other submissions. Pepper (2014) points out some conceptual difficulties with Noe’s theory of perception, which could be resolved withMerleau-Ponty’s phenomenology of the body schema and sedimentation.Wadham (2014) claims that Noe’s theory implies the invisibility of perspectival properties, which requires a revision of his theory of perspectival content. O’Regan (2014) reports on his sensorimotor approach. He proposes that “experiencing a sensation involves being engaged in sensorimotor interaction” but that “being conscious of something [. . .] requires appeal to a form of ‘higher-order’ cognitive access” (p. 34). In contrast, Rainey (2014) argues that consciousness is non-conceptual while experience is conceptual, and that consciousness is, therefore, the enabling ground for the possibility of experience.

The Normative Turn in Enactive Theory: An Examination of Its Roots and Implications

This paper traces the development of enactive concepts of value and normativity from their roots in the canonical work of Varela et al. (Embodied mind: cognitive science and human experience. MIT Press, Cambridge, 1991) through more recent works of Ezequiel Di Paolo and others. It aims to show the central importance of these concepts for enactive theory while exposing a potentially troublesome ambiguity in their definition. Most definitions of enactive normativity are purely proscriptive, but it seems that enactive theories of cognitive agency and experience demand something more. On the other hand, it is not clear that anything other than proscriptive normativity can be made compatible with the enactive tenet of autonomy and the rejection of representations.

Editorial: Pattern Recognition Receptors and Cancer

Editorial: Pattern Recognition Receptors and Cancer

Editorial: Habits: plasticity, learning and freedom.

Editorial: Habits: plasticity, learning and freedom.

Abstract 1575: Serum circulating miR–Test application: Standardized protocol for miR–Test clinical application

Abstract Lung cancer is leading cause of cancer death worldwide. Being lung cancer asymptomatic in its early stages, the majority of patients are diagnosed with advanced disease. Therefore, it is vital that screening programs and novel diagnostic tools are developed to increase lung cancer early detection. The development of a minimally invasive blood-based diagnostic tool would be ideal as a first-line screening procedure. An increasing number of studies are demonstrating that fluctuations of circulating miRNAs are associated to lung cancer. Recently, we described a serum circulating miRNA signature (miR-Test) diagnostic for asymptomatic, early stage, lung cancer, that was validated in a large cohort of individuals (N = 1115) enrolled in the lung cancer screening program COSMOS (Continuous Observation of SMOking Subjects). However, the transfer to the clinic of a blood test based on circ-miRNAs requires the establishment of standardized operating procedures (SOPs), working instructions and guidelines for all pre-analytical and analytical procedures. We identified possible sources of variability affecting circulating miRNAs, analyzed their impact on the miR-Test performance, and defined a standardized protocol to optimize miR-Test application. Analysis of all possible technical and biological variation affecting circ-miRNAs level, revealed two main sources of variability: one related to analytical procedures for miRNAs extraction and quantification, and the other due to pre-analytical conditions, on how samples are prepared. The extraction causes the main source of analytical imprecision. In conclusion, we identified an optimal protocol for the application of miR-Test for lung cancer early diagnosis. Citation Format: Francesca Montani, Matteo Marzi, Fabio Dezi, Elisa Dama, Rose Mary Carletti, Giulia Veronesi, Francesco Nicassio, Pier Paolo Di Fiore, Fabrizio Bianchi. Serum circulating miR–Test application: Standardized protocol for miR–Test clinical application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1575. doi:10.1158/1538-7445.AM2015-1575

Abstract 1414: Molecular and functional characterization of ovarian cancer stem cells

Abstract Epithelial Ovarian Cancer (OC) is one of the leading causes of death for women, and no significant therapeutic progress has been made in the last decades. Recent data suggest that one of the mechanisms accounting for resistant and/or relapsing disease is a subpopulation of cells in human tumors with stem-like characteristics (cancer stem cells, CSCs). CSCs are defined as a small subpopulation of cells within the tumor bulk that possess the capacity, on one hand, to self-renew and, on the other hand, to give rise to all heterogeneous cancer cell lineages that compose the tumor of origin. The CSC hypothesis provides an attractive cellular mechanism to explain the therapeutic refractoriness, dormant behavior, and relapse of the disease. Our study aims at assessing ovarian cancer stem cells (OCSC) as causal players in OC etiology and progression and at defining their molecular and functional profile. Specifically, we are pursuing the following objectives through the accomplishment of these milestones: 1) collection of normal and pathological samples; 2) identification of OCSC based on functional properties; 3) comparison of gene expression profiles between cancer stem cells and their normal counterpart; 4) characterization of novel genes/pathways involved in OCSC function (clonogenicity, tumorigenicity, quiescence, chemoresistance, etc.). This workflow is being applied to a series of fresh surgical samples of OC as well as to normal ovarian surface epithelium (OSE) and fallopian tube epithelium (FTE), namely the tissues of origin of OC. Thus, we obtained a collection of primary cells that recapitulate many traits of the original tissue both in vitro and in vivo. The gene expression profiles of sphere-forming SC has been compared to that of adherent, parental cells, aimed at identifying stemness-associated genes. This screening has been extended to OC, OSE, and FTE, resulting in a set of genes that are differentially expressed in OCSC as compared to their normal counterparts. A subset of such genes has been selected for the subsequent validation, both as OCSC biomarkers that could have clinical applications and as drivers in the biological and pathogenic function of OCSC. Our study might set the stage for innovative therapeutic approaches aimed at the selective elimination of OCSC, thus preventing tumor recurrence and chemoresistance, namely the two main causes of ovarian cancer lethality. Citation Format: Michela Lupia, Giovanni Bertalot, Nicoletta Colombo, Stefano Confalonieri, Pier Paolo Di Fiore, Ugo Cavallaro. Molecular and functional characterization of ovarian cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1414. doi:10.1158/1538-7445.AM2015-1414

Abstract 233: Mining cancer gene expression databases for latent information on intronic microRNAs

Abstract In recent years, enormous effort has been dedicated to transcriptomic profiling of various physiological and pathological conditions, in particular in cancer biology field. Microarray gene expression (mRNA) datasets of thousands of human tumors are now publicly available and can be used to get insights of cancer processes by systems-based analysis and to identify biomarkers for improvement of cancer therapy. However, the high complexity of human transcriptome may be difficult to handle. In this regard, shifting the attention to the miRNome could be an advantage, since its complexity is at least 20-fold lower than that of a reference transcriptome (∼1000 miRNAs vs. ∼20,000 genes). Importantly, patterns of distinct miRNA expression profiles were observed in tumors and there is a growing interest in miRNAs, behaving as potential cancer determinants and biomarkers. Knowing that almost 50% of human miRNA genes are located within introns of coding genes and that they usually share transcriptional regulation, those publicly available datasets are likely to contain “latent” information on intronic-miRNA expression. In other words, it could be possible to predict the regulation of intronic-miRNA expression by simply analyzing their host genes profile (miR-HG). The aim of our work is to take advantage of cancer datasets, focusing mainly on breast cancer datasets, to provide proof of principle evidences that meta-analysis of miR-HG expression profiles can pinpoint intronic-miRNAs with a role in breast cancer cell biology, and a potential utility as cancer biomarkers. Using this approach, we successfully discovered a diagnostic microRNA signature enabling the identification of breast cancer molecular subtypes. Importantly, qRT-PCR analysis of just three intronic-miRNAs, using FFPE samples, was sufficient to identify more aggressive breast tumor subtypes (i.e. basal, HER2 and luminal B subtypes), with a ∼80% of accuracy, in patients with moderately differentiated breast cancer. Unexpectedly, in a number of cases, the regulation of expression of intronic-miRNAs was more relevant to cancer phenotypes than the expression of their host genes. In line with these encouraging results, we propose our data mining strategy as a valid tool for cancer research and other biomedical fields. Citation Format: Simona Monterisi, Giovanni D'Ario, Elisa Dama, Nicole Rotmensz, Stefano Confalonieri, Chiara Tordonato, Flavia Troglio, Giovanni Bertalot, Patrick Maisonneuve, Giuseppe Viale, Francesco Nicassio, Pier Paolo Di Fiore, Fabrizio Bianchi. Mining cancer gene expression databases for latent information on intronic microRNAs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 233. doi:10.1158/1538-7445.AM2015-233

Abstract 1573: miR-Test: a blood test for lung cancer early detection

Abstract Background: Lung cancer is the leading cause of cancer death worldwide. As lung cancer is asymptomatic in its early stages, the majority of patients are diagnosed with advanced disease, when the tumor is unresectable. Consequently, the survival rate is very low: 15% at 5 years. It is vital, therefore, that screening programs and novel diagnostic tools are developed, which will increase the detection of lung cancer in its early stages (stage I-II), when the tumor is still curable, to reduce lung cancer mortality. Recently, we described a serum microRNA signature diagnostic for asymptomatic, early stage, lung cancer. The availability of reliable biomarkers to identify high-risk individuals might help to reduce the size of the target population for LDCT-based programs, thereby reducing costs and probably increasing compliance Methods: We performed a large-scale validation study of a miRNA blood test based on our signature (the miR-Test) in a population of high-risk individuals (N = 1115) enrolled in the lung cancer screening program COSMOS (Continuous Observation of SMOking Subjects), and other 74 lung cancer patients diagnosed outside of screening. Results: The miR-Test showed overall accuracy, specificity and sensitivity of 75%, 78%, and 75%, respectively, with an AUC of 0.85. The test appears to have a dual origin: the first from epithelial cells (the epithelial-like component); the second from cells of hematopoietic origin (the inflammatory-like component). Of note, we found that both components are needed to maintain a good performance of the miR-Test. Conclusions: The relatively high sensitivity of the miR-Test in detecting asymptomatic lung cancer and its high negative predictive value (NPV &amp;gt; 99%), confirm the effectiveness of the test, both interms of its ability to identify asymptomatic lung cancer patients and to reduce significantly unnecessary CTs on healthy individuals. Citation Format: Francesca Montani, Matteo Jacopo Marzi, Fabio Dezi, Elisa Dama, Rose Mary Carletti, Giuseppina Bonizzi, Raffaella Bertolotti, Massimo Bellomi, Cristiano Rampinelli, Patrick Maisonneuve, Lorenzo Spaggiari, Giulia Veronesi, Francesco Nicassio, Pier Paolo Di Fiore, Fabrizio Bianchi. miR-Test: a blood test for lung cancer early detection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1573. doi:10.1158/1538-7445.AM2015-1573

Enactive Affectivity, Extended

In this paper I advance an enactive view of affectivity that does not imply that affectivity must stop at the boundaries of the organism. I first review the enactive notion of “sense-making”, and argue that it entails that cognition is inherently affective. Then I review the proposal, advanced by Di Paolo (Topoi 28:9–21, 2009), that the enactive approach allows living systems to “extend”. Drawing out the implications of this proposal, I argue that, if enactivism allows living systems to extend, then it must also allow sense-making, and thus cognition as well as affectivity, to extend—in the specific sense of allowing the physical processes (vehicles) underpinning these phenomena to include, as constitutive parts, non-organic environmental processes. Finally I suggest that enactivism might also allow specific human affective states, such as moods, to extend.

Editorial: Phosphoinositides and their phosphatases: Linking electrical and chemical signals in biological processes.

Editorial: Phosphoinositides and their phosphatases: Linking electrical and chemical signals in biological processes.

Worlds of Western Anarchism and Syndicalism: Class Struggle, Transnationalism, Violence and Anti-imperialism, 1870s–1940s

The French Anarchists in London, 1880-1914: Exile arid Transnationalism in the First Globalisation, by Constance Bantman. Liverpool, Liverpool University Press, 2013. 256 pp. $99.95 US (cloth). The Knights Errant of Anarchy: London and the Italian Anarchist Diaspora (1880-1917), by Pietro Di Paola. Liverpool, Liverpool University Press, 2013. 256 pp. $99.95 US (cloth). The Haymarket Conspiracy: Transatlantic Anarchist Networks, by Timothy Messer-Kruse. Urbana, Chicago, Springfield, University of Illinois Press, 2012. ix, 236 pp. $30.00 US (paper), $85.00 US (cloth). Black Flag Boricuas: Anarchism, Authoritarianism and the Left in Puerto Rico, 1897-1921, by K.R. Schaffer. Urbana, Chicago and Springfield, University of Illinois Press, 2013. 240 pp. $65.00 US (cloth). Many political movements--anarchism included--have draped themselves in the clothes of antiquity, by imagining a historical lineage fading back into the mists of history. Anarchism is, in fact, a younger movement than Marxism: an integral part of modern socialism, it as an active political movement within the First (or International Workingmen's Association), a coalition of unions, political groups and clubs, and cooperatives that ran from 1864-1877 (Bantman pp. 1, 7-8). From the 1890s into the 1920s, it was in many contexts the dominant force on the revolutionary left, with a substantial impact on unions, popular and anti-imperialist movements worldwide. Even outside of this glorious period, anarchism and its union offshoots, anarcho- and revolutionary syndicalism, was (and is) an important current. (1) Its initial rise coincided, and not accidentally, with the first modern globalization of the 1880s to the 1910s, a period characterized by massive international flows of labour and capital, a transportation and telecommunications revolution, and the rapid spread of industrialization. Anarchism was a global movement not only in aspiration; in practice too, it was global and transnational. Migration--both voluntary and forced (due to persecutions)--and a widely circulating press facilitated the exchange of ideas, struggle repertoires and key militants, which in turn linked and created international activist communities. Stereotypes of an action-first outlook notwithstanding, the movement an intensely bookish culture stressing publication, theory, and debate (Bantman p. 20). Repression, aimed at suppressing radical ideas and formations, including the First International, often had the opposite effect, as exiles spread their ideas abroad and developed, debated and applied these ideas in different contexts on their travels. One effect was the massive dissemination of radical ideas in Europe and abroad: Italian anarchists, for example, were active in the Balkans, the Middle East and North Africa, West Europe, and both Americas (Di Paolo p. 3). Exile enriched radicalism, and many ideas were developed, even forged, abroad. Thus exile exerted a remarkable influence on the development of socialist ideas in Italy and in other countries (Di Paola p. 2), and proved very fruitful despite the pressures and frustrations that exile inevitably brought (Bantman p. 73). A growing literature continues to shed new light on this historical and contemporary current that--despite its importance--remains strikingly under-researched. The four books under review are part of the welcome recent upsurge in research. A core part of three of the volumes under consideration centres on the operations of international anarchist and syndicalist networks as they emerged in the context of this increased radicalism. Di Paola and Bantman focus on anarchist exile communities--Italian and French respectively--in late nineteenth- and early twentieth-century London, where relatively tolerant British immigration and political asylum policies enabled the flourishing of a range of overlapping, polyglot radical milieus. …

Commentary on: "The body social: an enactive approach to the self". A tool for merging bodily and social self in immobile individuals.

Commentary on: "The body social: an enactive approach to the self". A tool for merging bodily and social self in immobile individuals.

Your body, my body, our coupling moves our bodies.

Sensitivity to temporal contingencies appears early in life and plays a key role in the ontogeny of socio-cognitive abilities in humans (Nadel et al., 1999; Gratier and Apter-Danon, 2009). The tendency for rhythmic coordination, sometimes referred to as “entrainment,” requires sensory-motor coupling (Phillips-Silver et al., 2010). In most of the fields of cognitive science, action-perception and agent-world coupling views are replacing the classical stimulus-response dichotomy (Marsh et al., 2009; Silberstein and Chemero, 2012; Schilbach et al., 2013; Novembre and Keller, 2014). Such conceptual frameworks are well suited to study coordination phenomena as they emphasize the dynamical nature of cognition (Varela et al., 1993; Kelso, 1995; Buzsaki and Draguhn, 2004; Lehmann and Schonwiesner, 2014). Moreover, they leave room for the balance of autonomy, a central feature of complex biological systems, and interactive coupling, through which such systems relate to—and make sense of—their environment (Di Paolo, 2005; Barandiaran et al., 2009; Buhrmann et al., 2013). A naturalistic study of autonomy and coupling requires both embracing ecological situations and considering first-person perspective. Furthermore, many social coordination phenomena cannot be observed in the laboratory without the interaction of at least two subjects. We propose to consider linking first- and third-person measures, and even relate them across multiple interacting individuals. We will discuss how these concepts are intertwined in coordination phenomena, and outline existing methods to address those issues.

Pietro Di Paola. The Knights Errant of Anarchy: London and the Italian Anarchist Diaspora (1880–1917).

Pietro Di Paolo has written an important study on the Italian anarchist diaspora in London in the decades prior to World War I. The author's primary goal is “to contribute to the historiography of diasporic anarchism by exploring the practical and ideological aspects of the Italian anarchists—their everyday lives as well as their ideological thought and its development—in London, one of the most significant nodes of the transnational anarchist network” (p. 5). By this measure, Di Paolo has clearly achieved his objective and much more. Di Paolo points out the limitations of nation-state studies that do not account for anarchist activity abroad, particularly when such movements are experiencing political repression at home. Likewise, the nation-state focus impedes understanding of the dissemination of anarchist militants and ideas across the globe as a transnational movement. The book, for example, gives solid evidence to the continuity of an Italian anarchist movement from the 1880s to World War I, even when anarchist activity in Italy waxed and waned in the wake of fierce state repression.

Why call bodily sense making "languaging"?

Why call bodily sense making "languaging"?

Abstract 4205: Mass-spectroscopy phosphorylation profiling identified differential activation of signaling molecules via spleen tyrosine kinase in B-cell receptor signaling and stromal cell stimulation in DLBCL and CLL cells that are inhibited by GS-9973

Abstract Dysregulation of protein kinase activity leading to aberrant or excessive downstream signaling is widely believed to underlay the pathogenesis of multiple cancers. The purpose of this study was to identify the spleen tyrosine kinase (SYK)-dependent phosphoproteins that were activated in a diffuse large B-cell lymphoma (DLBCL) cell line, DHL4, and primary chronic lymphocytic leukemia (CLL) cells, upon either B-cell receptor (BCR) activation, stromal cell contact, or in a resting state and demonstrate inhibition with a selective SYK inhibitor, GS-9973. A commercial PTMScan Direct procedure from Cell Signaling Technology was utilized in this assessment. Cell lysates generated from resting, αIgG treatment or HS-5 human stromal cell line contact were digested with trypsin and the resultant peptides were enriched by immunoprecipitation with either an anti-phosphotyrosine or anti-phosphoserine/threonine antibody cocktail, and subjected to mass spectrometric analysis to identify and quantify phosphorylated peptides. The phosphorylated peptide profile was then compared to a similarly treated lysate after incubation with 1 μM GS-9973. Protein phosphorylations that were significantly changed by &amp;lt; or &amp;gt; 2.5-fold with GS-9973 treatment were identified and confirmed, where possible, by western blot of DHL-4 lysates or in primary CLL samples with antibodies specific for the identified sites. PTMScan Direct analysis showed overlapping, but not equivalent, patterns of phosphorylated peptides upon αIgG or stromal cell activation. A shared set of GS-9973 inhibited phosphoproteins were identified including pSyk, pAkt, pMek, pErk, pJnk2, pPKCδ, and pS6 kinase. Western blot analysis validated 13 out of 14 proteins as being modulated by 1 μM GS-9973 treatment. Furthermore, dose-response inhibition was demonstrated for pAkt-T473, pBCR-Y177, pBlnk-Y84, pMek-S217/221, and pErk-T202/Y204 with EC50s ranging from 23 to 50 nM. These data support the concept that Syk inhibition can block multiple signaling pathways known to be important in malignant B-cells function. Furthermore, identification of these sites elucidates possible pharmacodynamic signatures of GS-9973 inhibition that may be followed in clinical investigations of GS-9973 in oncology. Citation Format: Julie A. Di Paolo, Astrid Clarke, Hong Rong, Emma Rousseau. Mass-spectroscopy phosphorylation profiling identified differential activation of signaling molecules via spleen tyrosine kinase in B-cell receptor signaling and stromal cell stimulation in DLBCL and CLL cells that are inhibited by GS-9973. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4205. doi:10.1158/1538-7445.AM2014-4205

Abstract 1778: Characterization and anti-tumor functionality of a neuroblastoma-specific peptide, either free or conjugated to nanocarriers

Abstract Introduction. The identification of peptide ligands specific for solid tumors is expected to provide targeting moieties to improve delivery and to decrease toxicity of chemotherapy. We have recently identified the peptide HSYWLRS as a specific ligand for neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. Experimental procedures. The capability of peptide HSYWLRS to recognize NB cells was evaluated by coupling Qdot fluorescent nanoparticles with HSYWLRS or its scrambled version (SCR). NB cell association and internalization of HSYWLRS-targeted liposomes were tested by FACS and confocal microscopy studies. We further evaluated a potential role of this peptide in perturbing tumor-stroma interactions and tumor growth. NB cell lines stably transfected with eGFP were mixed with endothelial cells in the presence of either SCR or HSYWLRS peptides. Cell morphology and reciprocal cellular interactions were evaluated by optical and fluorescence microscopy. We finally performed therapeutic experiments with mice orthotopically injected with luc-trasfected NB cells and treated with HSYWLRS-targeted, doxorubicin-loaded liposomes (HSYWLRS-SL[DXR]). Anti-tumor efficacy was evaluated by BLI imaging. In vivo imaging was also performed by injecting mice with a bolus of fluorodeoxyglucose during a list mode acquisition lasting one hour using a dedicated micro-PET system. After framing rate optimization, tumor glucose consumption was measured using Patlak graphical approach and normalizing the slope of regression line for serum glucose level. Results. FACS analysis showed that HSYWLRS-Qdot and SCR-Qdot bound NB cells in a dose-dependent manner, however with different intensity, being HSYWLRS-Qdot the more potent. The binding of HSYWLRS-Qdot was efficiently inhibited by an excess of HSYWLRS, but not by control SCR peptide. In contrast, the binding of SCR-Qdot was not inhibited neither by an excess of SCR nor by HSYWLRS peptide, suggesting that the binding of SCR-Qdot is not specific. Again, the specific peptide-driven binding of HSYWLRS-SL to NB cells was inhibited by an excess of HSYWLRS peptide. In all cases, HSYWLRS specifically altered in vitro the interactions of NB cells with endothelium. Similarly, this peptide statistically decreased tumor take and growth when co-injected with tumor cells in the adrenal gland of nude mice. Preliminary in vivo results obtained by BLI and micro-PET devises indicated that HSYWLRS-SL[DXR] decrease tumor growth through a reduction of tumor glucose consumption, leading to an enhanced life span in treated mice. Conclusion. Our findings demonstrate that HSYWLRS peptide recognizes NB cells and is functional in the design of nanocarriers with therapeutic efficacy paving the way to its clinical development. Citation Format: Alice Bartolini, Monica Loi, Daniela Di Paolo, Laura Emionite, Angelina Sacchi, Flavio Curnis, Gianluca Bottoni, Michela Massollo, Cristina Gagliani, Silvia Bruno, Alessandro Gori, Renato Longhi, Michele Cilli, Carlo Tacchetti, Angelo Corti, Gianmario Sambuceti, Mirco Ponzoni, Serena Marchiò, Fabio Pastorino. Characterization and anti-tumor functionality of a neuroblastoma-specific peptide, either free or conjugated to nanocarriers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1778. doi:10.1158/1538-7445.AM2014-1778

Abstract 1453: MicroRNA replacement and RNAi-mediated silencing of ALK as combined targeted therapies for neuroblastoma

Abstract The effective treatment of advanced Neuroblastoma (NB) is still a challenge in pediatric oncology, because the clinical use of most therapeutics is limited by insufficient drug delivery to the tumor and high systemic toxicity. The discovery of the RNAi has great promise for anti-cancer therapeutics but, as in high-grade solid tumors a single ‘oncogene addiction’ is rare, multi-'gene' target combinations are required and a targeted delivery system is mandatory to successfully translate RNAi-based therapeutics into the clinics. It is now ascertained the master role of ALK and related genes such as PHOX2B, able to promote its transcription, in NB growth and survival, opening new perspectives for RNAi-mediated therapies for NB. Nevertheless, even if the counterpart of tumor suppressor (TS) regulators in NB is not completely known, a number of miRNAs with TS functions have been found aberrantly under-expressed in NB and associated with aggressive phenotypes. To develop a new generation of targeted and multi-RNAi-directed therapeutics for NB, we are exploiting our well-established NB-specific delivery system, the GD2-Targeted Liposomes entrapping specific siRNAs, TL[siRNA], or miRNAs mimics, TL[miRNA]. The final purpose is to get simultaneously the silencing of the oncogenic functions of ALK and related genes and the replacement of miRNAs with TS functions in clinically relevant mouse models of NB. We have planned to test different combinations of individually entrapped TL[siRNA] or TL[miRNA] for in vivo therapeutic efficacy. In a pilot study, NB-bearing mice were treated with different TL[siRNA]. After 65 days from i.v. injection of HTLA-230 (WT ALK) cells, we observed: about 50% of TL[siALK]-treated mice still disease-free, marginal effects with TL[siPHOX2A] and moderate effects with TL[siPHOX2B] formulations alone and a significant increased mice survival with an equimolar combination of TL[siRNA] of each gene. The enhanced efficacy of ALK and PHOX2 genes knockdown combination highlights a powerful therapeutic potential. After pilot transfection studies of SH-SY5Y (F1174L mut ALK) and GI-LI-N (WT ALK) cell lines, TS miRNAs mimics were screened and selected. Cell proliferation was evaluated at different days post-transfection. miR-96, miR-34a and let7b replacement resulted the most effective in inhibiting NB cell growth. Liposomal preparation of miRNA-NC and the above mimics for in vivo studies was tuned up according to the number of negative charges of the stem-loop sequence with an entrapment efficiency of 80-90%. Currently, an ongoing in vivo trial indicates promising results after replacement of miR-34a and let7b. We believe that the use of tumor-targeted biocompatible carriers of RNAi molecules able to replace loss-of-functions and to knockdown gain-of-functions in cancer cells at the same time is likely to represent an innovative modality for a more effective gene therapy. Citation Format: Patrizia Perri, Daniela Di Paolo, Leslie Priddy, Annarita Di Fiore, Chiara Brignole, Fabio Pastorino, David Brown, Mirco Ponzoni. MicroRNA replacement and RNAi-mediated silencing of ALK as combined targeted therapies for neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1453. doi:10.1158/1538-7445.AM2014-1453