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1.  Age dependent normal horizontal VOR gain of head impulse test as measured with video-oculography 
The head impulse test (HIT) is a recognised clinical sign of the high frequency vestibulo-ocular reflex (VOR), which can be quantified with video-oculography. This measures the VOR gain as the ratio of angular eye velocity to angular head velocity. Although normative data is available for VOR gain with video-oculography, most normal studies in general include small numbers of subjects and do not include analysis of variation of VOR gain with age. The purpose of our study was to establish normative data across 60 control subjects aged 20 to 80 years to represent a population distribution.
Sixty control subjects without any current or previous form of brain disorder or vertigo participated in this study and form the basis for future comparison to patients with vestibular lesions. The relationship between the horizontal vestibulo-ocular reflex (HVOR) velocity gain and age was analysed using a mixed regression model with a random effect for subjects. Differences in testing technique were assessed to ensure reliability in results.
The mean HVOR velocity gain of 60 normal subjects was 0.97 (SD = 0.09) at 80 ms and 0.94 (SD = 0.10) at 60 ms. The 2 SD lower limit of normal HVOR velocity gain was 0.79 at 80 ms and 0.75 at 60 ms. No HVOR velocity gain fell below 0.76 and 0.65 at 80 ms and 60 ms respectively. The HVOR velocity gain declined by 0.012 and 0.017 per decade as age increased at 80 ms and 60 ms respectively. A non-physiologically high horizontal HVOR velocity gain was found to occur in tests where passive HITs were predictable in direction and time and where target distance was below 0.70 m.
Normative data with respect to HVOR velocity gain decreases slightly with age, but with careful attention to methodology the 2 SD lower limit of normal is relatively robust across a wide age range and into the eighth decade, without requirement for adjustment with age.
PMCID: PMC4506627  PMID: 26141721
Head impulse test; Horizontal vestibulo-ocular reflex; Semicircular canal; Eye movements
2.  The Importance of Physiologically Relevant Cell Lines for Studying Virus–Host Interactions 
Viruses  2016;8(11):297.
Viruses interact intimately with the host cell at nearly every stage of replication, and the cell model that is chosen to study virus infection is critically important. Although primary cells reflect the phenotype of healthy cells in vivo better than cell lines, their limited lifespan makes experimental manipulation challenging. However, many tumor-derived and artificially immortalized cell lines have defects in induction of interferon-stimulated genes and other antiviral defenses. These defects can affect virus replication, especially when cells are infected at lower, more physiologically relevant, multiplicities of infection. Understanding the selective pressures and mechanisms underlying the loss of innate signaling pathways is helpful to choose immortalized cell lines without impaired antiviral defense. We describe the trials and tribulations we encountered while searching for an immortalized cell line with intact innate signaling, and how directed immortalization of primary cells avoids many of the pitfalls of spontaneous immortalization.
PMCID: PMC5127011  PMID: 27809273
primary cell; immortalized cell; interferon; interferon regulatory factor 3; interferon-stimulated genes; signal transduction; antiviral defense
3.  Class A scavenger receptor-mediated dsRNA internalization is independent of innate antiviral signaling and does not require PI3K activity1 
Double-stranded RNA is a potent trigger of innate immune signaling, eliciting effects within virally infected cells and following release from dying cells. Given its inherent stability, extracellular dsRNA induces both local and systemic effects. Although the class A scavenger receptors (SR-As)3 mediate dsRNA entry, it is unknown if they contribute to signaling beyond ligand internalization. Here, we investigated if SR-As contribute to innate immune signaling independent of the classic TLR and RLR pathways. We generated a stable A549 human epithelial cell line with inducible expression of the Hepatitis C virus protease NS3/4A, which efficiently cleaves TRIF and IPS-1, adaptors for TLR3 and the RLRs respectively. Cells expressing NS3/4A as well as TLR3/MDA5/IPS-1−/− mouse embryonic fibroblasts completely lacked antiviral activity to extracellular dsRNA relative to control cells, suggesting that SR-As do not possess signaling capacity independent of TLR3 or the RLRs. Previous studies implicated PI3K signaling in SR-A-mediated activities and in downstream production of type I interferon. We found that SR-A-mediated dsRNA internalization occurs independent of PI3K activation, while downstream signaling leading to interferon production was partially dependent on PI3K activity. Overall, these findings suggest that SR-A-mediated dsRNA internalization is independent of innate antiviral signaling.
PMCID: PMC4904834  PMID: 26363049
4.  The NLRP3 inflammasome in pathogenic particle and fibre-associated lung inflammation and diseases 
The concept of the inflammasome, a macromolecular complex sensing cell stress or danger signals and initiating inflammation, was first introduced approximately a decade ago. Priming and activation of these intracellular protein platforms trigger the maturation of pro-inflammatory chemokines and cytokines, most notably, interleukin-1β (IL-1β) and IL-18, to promulgate innate immune defenses. Although classically studied in models of gout, Type II diabetes, Alzheimer’s disease, and multiple sclerosis, the importance and mechanisms of action of inflammasome priming and activation have recently been elucidated in cells of the respiratory tract where they modulate the responses to a number of inhaled pathogenic particles and fibres. Most notably, inflammasome activation appears to regulate the balance between tissue repair and inflammation after inhalation of pathogenic pollutants such as asbestos, crystalline silica (CS), and airborne particulate matter (PM). Different types of fibres and particles may have distinct mechanisms of inflammasome interaction and outcome. This review summarizes the structure and function of inflammasomes, the interplay between various chemokines and cytokines and cell types of the lung and pleura after inflammasome activation, and the events leading to the development of non-malignant (allergic airway disease and chronic obstructive pulmonary disease (COPD), asbestosis, silicosis) and malignant (mesothelioma, lung cancer) diseases by pathogenic particulates. In addition, it emphasizes the importance of communication between cells of the immune system, target cells of these diseases, and components of the extracellular matrix (ECM) in regulation of inflammasome-mediated events.
PMCID: PMC5029018  PMID: 27650313
Inflammation; Fibrosis; Lung cancer; Mesothelioma; Asbestos; Silica; Airborne particulate matter; Nanotubes; Allergic airway disease
5.  Membrane Perturbation-Associated Ca2+ Signaling and Incoming Genome Sensing Are Required for the Host Response to Low-Level Enveloped Virus Particle Entry 
Journal of Virology  2016;90(6):3018-3027.
The type I interferon (IFN) response is an important aspect of innate antiviral defense, and the transcription factor IRF3 plays an important role in its induction. Membrane perturbation during fusion, a necessary step for enveloped virus particle entry, appears sufficient to induce transcription of a subset of IFN-stimulated genes (ISGs) in an IRF3-dependent, IFN-independent fashion. IRF3 is emerging as a central node in host cell stress responses, although it remains unclear how different forms of stress activate IRF3. Here, we investigated the minimum number of Sendai virus (SeV) and human cytomegalovirus (HCMV) particles required to activate IRF3 and trigger an antiviral response. We found that Ca2+ signaling associated with membrane perturbation and recognition of incoming viral genomes by cytosolic nucleic acid receptors are required to activate IRF3 in response to fewer than 13 particles of SeV and 84 particles of HCMV per cell. Moreover, it appears that Ca2+ signaling is important for activation of STING and IRF3 following HCMV particle entry, suggesting that Ca2+ signaling sensitizes cells to recognize genomes within incoming virus particles. To our knowledge, this is the first evidence that cytosolic nucleic acid sensors recognize genomes within incoming virus particles prior to virus replication. These studies highlight the exquisite sensitivity of the cellular response to low-level stimuli and suggest that virus particle entry is sensed as a stress signal.
IMPORTANCE The mechanism by which replicating viruses trigger IRF3 activation and type I IFN induction through the generation and accumulation of viral pathogen-associated molecular patterns has been well characterized. However, the mechanism by which enveloped virus particle entry mediates a stress response, leading to IRF3 activation and the IFN-independent response, remained elusive. Here, we find that Ca2+ signaling associated with membrane perturbation appears to sensitize cells to recognize genomes within incoming virus particles. To our knowledge, this is the first study to show that cytosolic receptors recognize genomes within incoming virus particles prior to virus replication. These findings not only highlight the sensitivity of cellular responses to low-level virus particle stimulation, but provide important insights into how nonreplicating virus vectors or synthetic lipid-based carriers used as clinical delivery vehicles activate innate immune responses.
PMCID: PMC4810640  PMID: 26719279
6.  Mitochondrial-Nuclear Interactions Mediate Sex-Specific Transcriptional Profiles in Drosophila 
Genetics  2016;204(2):613-630.
The assembly and function of mitochondria require coordinated expression from two distinct genomes, the mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Mutations in either genome can be a source of phenotypic variation, yet their coexpression has been largely overlooked as a source of variation, particularly in the emerging paradigm of mitochondrial replacement therapy. Here we tested how the transcriptome responds to mtDNA and nDNA variation, along with mitonuclear interactions (mtDNA × nDNA) in Drosophila melanogaster. We used two mtDNA haplotypes that differ in a substantial number of single nucleotide polymorphisms, with >100 amino acid differences. We placed each haplotype on each of two D. melanogaster nuclear backgrounds and tested for transcription differences in both sexes. We found that large numbers of transcripts were differentially expressed between nuclear backgrounds, and that mtDNA type altered the expression of nDNA genes, suggesting a retrograde, trans effect of mitochondrial genotype. Females were generally more sensitive to genetic perturbation than males, and males demonstrated an asymmetrical effect of mtDNA in each nuclear background; mtDNA effects were nuclear-background specific. mtDNA-sensitive genes were not enriched in male- or female-limited expression space in either sex. Using a variety of differential expression analyses, we show the responses to mitonuclear covariation to be substantially different between the sexes, yet the mtDNA genes were consistently differentially expressed across nuclear backgrounds and sexes. Our results provide evidence that the main mtDNA effects can be consistent across nuclear backgrounds, but the interactions between mtDNA and nDNA can lead to sex-specific global transcript responses.
PMCID: PMC5068850  PMID: 27558138
mtDNA; epistasis; Drosophila; transcriptome; mitonuclear; retrograde signaling
7.  Using in situ management to conserve biodiversity under climate change 
The Journal of Applied Ecology  2016;53(3):885-894.
Successful conservation will increasingly depend on our ability to help species cope with climate change. While there has been much attention on accommodating or assisting range shifts, less has been given to the alternative strategy of helping species survive climate change through in situ management.Here we provide a synthesis of published evidence examining whether habitat management can be used to offset the adverse impacts on biodiversity of changes in temperature, water availability and sea‐level rise. Our focus is on practical methods whereby the local environmental conditions experienced by organisms can be made more suitable.Many studies suggest that manipulating vegetation structure can alter the temperature and moisture conditions experienced by organisms, and several demonstrate that these altered conditions benefit species as regional climatic conditions become unsuitable. The effects of topography on local climatic conditions are even better understood, but the alteration of topography as a climate adaptation tool is not ingrained in conservation practice. Trials of topographic alteration in the field should therefore be a priority for future research.Coastal systems have the natural capacity to keep pace with climate change, but require sufficient sediment supplies and space for landward migration to do so. There is an extensive literature on managed realignment. While the underlying rationale is simple, successful implementation requires careful consideration of elevation and past land use. Even with careful management, restored habitats may not attain the physical and biological attributes of natural habitats. Synthesis and applications. The recent literature provides a compelling case that some of the adverse effects of climate change can be offset by appropriate management. However, much of the evidence for this is indirect and too few studies provide empirical tests of the long‐term effectiveness of these management interventions. It is clear from the existing evidence that some techniques have a higher risk of failure or unexpected outcomes than others and managers will need to make careful choices about which to implement. We have assessed the strength of evidence of these approaches in order to demonstrate to conservation professionals the risks involved.
The recent literature provides a compelling case that some of the adverse effects of climate change can be offset by appropriate management. However, much of the evidence for this is indirect and too few studies provide empirical tests of the long‐term effectiveness of these management interventions. It is clear from the existing evidence that some techniques have a higher risk of failure or unexpected outcomes than others and managers will need to make careful choices about which to implement. We have assessed the strength of evidence of these approaches in order to demonstrate to conservation professionals the risks involved.
PMCID: PMC4991270  PMID: 27609987
adaptive management; biodiversity conservation; climate‐change adaptation; environmental change; global warming; habitat restoration; managed realignment
8.  Inhibition of mesothelioma cancer stem-like cells with adenovirus-mediated NK4 gene therapy 
Malignant mesothelioma (MM) is a highly invasive and chemoresistant malignancy induced by asbestos fibers. NK4, a HGF antagonist and angiogenesis inhibitor, consists of the N-terminal hairpin domain and four kringle domains of the α-chain of HGF. The therapeutic potential of NK4 has been demonstrated in a variety of tumor types. However, the mechanisms by which NK4 inhibits tumor growth have not been well delineated. In this study, we show that the NK4 adenovirus (Ad-NK4) potently inhibits cell viability, invasiveness, and tumorigenicity of human MM cells. Significantly, we demonstrate for the first time that Ad-NK4 inhibits cancer stem-like cell (CSC) properties as assessed by spheroid formation assay, side population analysis, and flow cytometric sorting of CD24 cells. In addition to inhibiting phosphorylation of Met and AKT, Ad-NK4 markedly suppressed the active form of β-catenin, a key mediator of both Wnt and AKT pathways. We further demonstrate that expression of NK4 suppresses β-catenin nuclear localization and transcriptional activity. Intriguingly, the expression levels of Oct4 and Myc, two critical stem cell factors and downstream targets of β-catenin, were also diminished by Ad-NK4. Furthermore, the strong antitumor effect of NK4 was found to be linked to its ability to inhibit CSCs as revealed by immunohistochemical examination of tumor specimens from a mouse xenograft model of human MM. These findings suggest that NK4 acts as a CSC inhibitor by impeding Met/AKT/β-catenin signaling and holds promise for achieving durable therapeutic responses in MM by constraining the CSC component of these aggressive tumors.
PMCID: PMC4428975  PMID: 25501304
malignant mesothelioma; cancer stem-like cells; HGF/Met; NK4; therapy
9.  Practitioner’s Guide to Assessment of Autism Spectrum Disorders in Infants and Toddlers 
Recent advances in clinical research have made it possible to diagnosis autism spectrum disorders (ASD) as early as the second year of life. The diagnostic process early in development is often complex, and thus, familiarity with the most recent findings in clinical assessment instruments, early symptoms, and developmental trajectories of young children with autism is essential. This paper provides an empirically based practitioner’s guide to issues and concerns related to early diagnosis of ASD in very young children, documentation of the course and patterns of ASD symptomatology in infants and toddlers, and current understanding in the field of diagnostic outcomes of children identified in the first and second year of life.
PMCID: PMC4878115  PMID: 22057879
Early diagnosis; Clinical evaluation; Infants and toddlers
10.  Time-Domain Simulation of Three Dimensional Quantum Wires 
PLoS ONE  2016;11(4):e0153802.
A method is presented to calculate the eigenenergies and eigenfunctions of quantum wires. This is a true three-dimensional method based on a direct implementation of the time-dependent Schrödinger equation. It makes no approximations to the Schrödinger equation other than the finite-difference approximation of the space and time derivatives. The accuracy of our method is tested by comparing it to analytical results in a cylindrical wire.
PMCID: PMC4849664  PMID: 27124603
11.  Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication 
Journal of Virology  2015;89(19):9841-9852.
It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment.
IMPORTANCE While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both our understanding of basic cellular biology as well as how this protein is co-opted by HSV.
PMCID: PMC4577907  PMID: 26178983
12.  Mitonuclear Epistasis for Development Time and Its Modification by Diet in Drosophila 
Genetics  2016;203(1):463-484.
Mitochondrial (mtDNA) and nuclear genes have to operate in a coordinated manner to maintain organismal function, and the regulation of this homeostasis presents a substantial source of potential epistatic (G × G) interactions. How these interactions shape the fitness landscape is poorly understood. Here we developed a novel mitonuclear epistasis model, using selected strains of the Drosophila Genetic Reference Panel (DGRP) and mitochondrial genomes from within Drosophila melanogaster and D. simulans to test the hypothesis that mtDNA × nDNA interactions influence fitness. In total we built 72 genotypes (12 nuclear backgrounds × 6 mtDNA haplotypes, with 3 from each species) to dissect the relationship between genotype and phenotype. Each genotype was assayed on four food environments. We found considerable variation in several phenotypes, including development time and egg-to-adult viability, and this variation was partitioned into genetic (G), environmental (E), and higher-order (G × G, G × E, and G × G × E) components. Food type had a significant impact on development time and also modified mitonuclear epistases, evidencing a broad spectrum of G × G × E across these genotypes. Nuclear background effects were substantial, followed by mtDNA effects and their G × G interaction. The species of mtDNA haplotype had negligible effects on phenotypic variation and there was no evidence that mtDNA variation has different effects on male and female fitness traits. Our results demonstrate that mitonuclear epistases are context dependent, suggesting the selective pressure acting on mitonuclear genotypes may vary with food environment in a genotype-specific manner.
PMCID: PMC4858792  PMID: 26966258
MtDNA; epistasis; DGRP; genotype-by-environment interaction; fitness; development time; mitonuclear
13.  Indications for distinct pathogenic mechanisms of asbestos and silica through gene expression profiling of the response of lung epithelial cells 
Human Molecular Genetics  2014;24(5):1374-1389.
Occupational and environmental exposures to airborne asbestos and silica are associated with the development of lung fibrosis in the forms of asbestosis and silicosis, respectively. However, both diseases display distinct pathologic presentations, likely associated with differences in gene expression induced by different mineral structures, composition and bio-persistent properties. We hypothesized that effects of mineral exposure in the airway epithelium may dictate deviating molecular events that may explain the different pathologies of asbestosis versus silicosis. Using robust gene expression-profiling in conjunction with in-depth pathway analysis, we assessed early (24 h) alterations in gene expression associated with crocidolite asbestos or cristobalite silica exposures in primary human bronchial epithelial cells (NHBEs). Observations were confirmed in an immortalized line (BEAS-2B) by QRT-PCR and protein assays. Utilization of overall gene expression, unsupervised hierarchical cluster analysis and integrated pathway analysis revealed gene alterations that were common to both minerals or unique to either mineral. Our findings reveal that both minerals had potent effects on genes governing cell adhesion/migration, inflammation, and cellular stress, key features of fibrosis. Asbestos exposure was most specifically associated with aberrant cell proliferation and carcinogenesis, whereas silica exposure was highly associated with additional inflammatory responses, as well as pattern recognition, and fibrogenesis. These findings illustrate the use of gene-profiling as a means to determine early molecular events that may dictate pathological processes induced by exogenous cellular insults. In addition, it is a useful approach for predicting the pathogenicity of potentially harmful materials.
PMCID: PMC4402341  PMID: 25351596
14.  The Senior Living Lab: an example of nursing leadership 
The Senior Living Lab (SLL) is dedicated to the care of older adults and exemplifies how nursing leadership can influence clinical practice by designing research models capable of configuring interdisciplinary partnerships with the potential of generating innovative practices and better older patient outcomes. Demographic change resulting in growing numbers of older adults requires a societal approach, uniting stakeholders in social innovation processes. The LL approach is an innovative research method that values user perceptions and participation in the cocreation of new products and services. The SLL is crafting a platform responsive to change. It is a learning organization facilitating community-based participatory research methods in the field. Advanced nurse practitioners are well positioned to lead the way forward, fostering interdisciplinary academic collaborations dedicated to healthy aging at home. The SLL demonstrates how nursing science is taking the lead in the field of social innovation.
PMCID: PMC4778789  PMID: 27013869
community-based participatory approach; Living Lab; nursing leadership; nursing practice; Senior Living Lab; social innovation
15.  Spin-momentum coupled Bose-Einstein condensates with lattice band pseudospins 
Nature Communications  2016;7:10867.
The quantum emulation of spin-momentum coupling, a crucial ingredient for the emergence of topological phases, is currently drawing considerable interest. In previous quantum gas experiments, typically two atomic hyperfine states were chosen as pseudospins. Here, we report the observation of a spin-momentum coupling achieved by loading a Bose-Einstein condensate into periodically driven optical lattices. The s and p bands of a static lattice, which act as pseudospins, are coupled through an additional moving lattice that induces a momentum-dependent coupling between the two pseudospins, resulting in s–p hybrid Floquet-Bloch bands. We investigate the band structures by measuring the quasimomentum of the Bose-Einstein condensate for different velocities and strengths of the moving lattice, and compare our measurements to theoretical predictions. The realization of spin-momentum coupling with lattice bands as pseudospins paves the way for engineering novel quantum matter using hybrid orbital bands.
The optical trapping of ultracold atoms allows for the simulation and controlled exploration of phenomena normally found in condensed matter systems. Here, the authors demonstrate spin–orbit coupling between lattice band pseudospins in a Bose-Einstein condensate of ultracold atoms.
PMCID: PMC4773453  PMID: 26924575
16.  Viral Evasion Strategies in Type I IFN Signaling – A Summary of Recent Developments 
The immune system protects the organism against infections and the damage associated with them. The first line of defense against pathogens is the innate immune response. In the case of a viral infection, it induces the interferon (IFN) signaling cascade and eventually the expression of type I IFN, which then causes an antiviral state in the cells. However, many viruses have developed strategies to counteract this mechanism and prevent the production of IFN. In order to modulate or inhibit the IFN signaling cascade in their favor, viruses have found ways to interfere at every single step of the cascade, for example, by inducing protein degradation or cleavage, or by mediate protein polyubiquitination. In this article, we will review examples of viruses that modulate the IFN response and describe the mechanisms they use.
PMCID: PMC5104748  PMID: 27891131
virus; type I interferon; evasion; innate immune signaling; NFκB
17.  Assessing health program performance in low- and middle-income countries: building a feasible, credible, and comprehensive framework 
Many health service delivery models are adapting health services to meet rising demand and evolving health burdens in low- and middle-income countries. While innovative private sector models provide potential benefits to health care delivery, the evidence base on the characteristics and impact of such approaches is limited. We have developed a performance measurement framework that provides credible (relevant aspects of performance), feasible (available data), and comparable (across different organizations) metrics that can be obtained for private health services organizations that operate in resource-constrained settings.
We synthesized existing frameworks to define credible measures. We then examined a purposive sample of 80 health organizations from the Center for Health Market Innovations (CHMI) database ( to identify what the organizations reported about their programs (to determine feasibility of measurement) and what elements could be compared across the sample.
The resulting measurement framework includes fourteen subgroups within three categories of health status, health access, and operations/delivery.
The emphasis on credible, feasible, and comparable measures in the framework can assist funders, program managers, and researchers to support, manage, and evaluate the most promising strategies to improve access to effective health services. Although some of the criteria that the literature views as important – particularly population coverage, pro-poor targeting, and health outcomes – are less frequently reported, the overall comparison provides useful insights.
PMCID: PMC4687324  PMID: 26690660
Performance measurement; Innovation; Framework; Health service delivery; Low- and middle-income countries; Private sector
18.  Consensus guidelines for the detection of immunogenic cell death 
Kepp, Oliver | Senovilla, Laura | Vitale, Ilio | Vacchelli, Erika | Adjemian, Sandy | Agostinis, Patrizia | Apetoh, Lionel | Aranda, Fernando | Barnaba, Vincenzo | Bloy, Norma | Bracci, Laura | Breckpot, Karine | Brough, David | Buqué, Aitziber | Castro, Maria G. | Cirone, Mara | Colombo, Maria I. | Cremer, Isabelle | Demaria, Sandra | Dini, Luciana | Eliopoulos, Aristides G. | Faggioni, Alberto | Formenti, Silvia C. | Fučíková, Jitka | Gabriele, Lucia | Gaipl, Udo S. | Galon, Jérôme | Garg, Abhishek | Ghiringhelli, François | Giese, Nathalia A. | Guo, Zong Sheng | Hemminki, Akseli | Herrmann, Martin | Hodge, James W. | Holdenrieder, Stefan | Honeychurch, Jamie | Hu, Hong-Min | Huang, Xing | Illidge, Tim M. | Kono, Koji | Korbelik, Mladen | Krysko, Dmitri V. | Loi, Sherene | Lowenstein, Pedro R. | Lugli, Enrico | Ma, Yuting | Madeo, Frank | Manfredi, Angelo A. | Martins, Isabelle | Mavilio, Domenico | Menger, Laurie | Merendino, Nicolò | Michaud, Michael | Mignot, Gregoire | Mossman, Karen L. | Multhoff, Gabriele | Oehler, Rudolf | Palombo, Fabio | Panaretakis, Theocharis | Pol, Jonathan | Proietti, Enrico | Ricci, Jean-Ehrland | Riganti, Chiara | Rovere-Querini, Patrizia | Rubartelli, Anna | Sistigu, Antonella | Smyth, Mark J. | Sonnemann, Juergen | Spisek, Radek | Stagg, John | Sukkurwala, Abdul Qader | Tartour, Eric | Thorburn, Andrew | Thorne, Stephen H. | Vandenabeele, Peter | Velotti, Francesca | Workenhe, Samuel T. | Yang, Haining | Zong, Wei-Xing | Zitvogel, Laurence | Kroemer, Guido | Galluzzi, Lorenzo
Oncoimmunology  2014;3(9):e955691.
Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.
PMCID: PMC4292729  PMID: 25941621
ATP release; autophagy; calreticulin; endoplasmic reticulum stress; HMGB1; immunotherapy; APC, antigen-presenting cell; ATF6, activating transcription factor 6; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BCL2, B-cell CLL/lymphoma 2 protein; CALR, calreticulin; CTL, cytotoxic T lymphocyte; Δψm, mitochondrial transmembrane potential; DAMP, damage-associated molecular pattern; DAPI, 4′,6-diamidino-2-phenylindole; DiOC6(3), 3,3′-dihexyloxacarbocyanine iodide; EIF2A, eukaryotic translation initiation factor 2A; ER, endoplasmic reticulum; FLT3LG, fms-related tyrosine kinase 3 ligand; G3BP1, GTPase activating protein (SH3 domain) binding protein 1; GFP, green fluorescent protein; H2B, histone 2B; HMGB1, high mobility group box 1; HSP, heat shock protein; HSV-1, herpes simplex virus type I; ICD, immunogenic cell death; IFN, interferon; IL, interleukin; MOMP, mitochondrial outer membrane permeabilization; PDIA3, protein disulfide isomerase family A; member 3; PI, propidium iodide; RFP, red fluorescent protein; TLR, Toll-like receptor; XBP1, X-box binding protein 1
19.  Correlations of inflammatory gene pathways, corticolimbic functional activities, and aggression in pediatric bipolar disorder: a preliminary study 
Psychiatry research  2014;224(2):107-111.
The mechanisms underlying aggression in adolescents with bipolar disorder have been poorly understood. The present study has investigated the associations among TNF gene expressions, functional brain activations under the frustrative non-reward task, and aggression in adolescents with bipolar disorder. Baseline gene expressions and aggressive tendencies were measured with the RNA-sequencing and Brief Rating of Aggression by Children and Adolescents (BRACHA), respectively. Our results show that activity levels of left subgenual anterior cingulate gyrus (ACG) right amygdala, left Brodmann area 10 (orbitofrontal cortex), and right thalamus were inversely correlated with BRACHA scores and were activated with frustrative non-reward during the affective Posner Task. In addition, eleven TNF related gene expressions were significantly correlated with activation of amygdala or ACG during the affective Posner task. Three TNF gene expressions were inversely correlated with BRACHA score while one TNF gene (TNFAIP3) expression was positively correlated with BRACHA score. Therefore, TNF-related inflammatory cytokine genes may play a role in neural activity associated with frustrative non-reward and aggressive behaviors in pediatric bipolar disorder.
PMCID: PMC4197049  PMID: 25172408
tumor necrosis factor; frustrative non-reward; anterior cingulate gyrus; amygdale; prefrontal cortex; RNA-sequencing
20.  Extracellular Signal-Regulated Kinase 5 and Cyclic AMP Response Element Binding Protein Are Novel Pathways Inhibited by Vandetanib (ZD6474) and Doxorubicin in Mesotheliomas 
Malignant mesothelioma (MM), lung cancers, and asbestosis are hyperproliferative diseases associated with exposures to asbestos. All have a poor prognosis; thus, the need to develop novel and effective therapies is urgent. Vandetanib (Van) (ZD6474, ZACTIMA) is a tyrosine kinase inhibitor that has shown equivocal results in clinical trials for advanced non-small cell lung cancer. However, tyrosine kinase inhibitors alone have shown no significant clinical activity in phase II trials of patients with unresectable MM. Using epithelioid (HMESO) and sarcomatoid (H2373) human MM lines, the efficacy of tumor cell killing and signaling pathways modulated by Van with and without doxorubicin (Dox) was examined. Van alone reduced total cell numbers in HMESO MM and synergistically increased the toxicity of Dox in HMESO and H2373 cells. Most importantly, we identified two novel cell survival/resistance pathways, ERK5 and cyclic AMP response element binding protein (CREB), that were inhibited by Van and Dox. After silencing of either ERK5 or CREB, significant decreases in cell numbers in the Dox-resistant sarcomatoid H2373 line were observed. Results suggest that a plethora of cell signaling pathways associated with cell survival are induced by Dox but inhibited by the addition of Van in MM. Data from our study support the combined efficacy of Van and Dox as a novel approach in the treatment of MM that is further enhanced by blocking ERK5 or CREB signaling cascades.
PMCID: PMC4224081  PMID: 24940987
tyrosine kinases; extracellular signal-regulated kinases; vandetanib; doxorubicin; malignant mesothelioma
21.  Study protocol for “Study of Practices Enabling Implementation and Adaptation in the Safety Net (SPREAD-NET)”: a pragmatic trial comparing implementation strategies 
Little research has directly compared the effectiveness of implementation strategies in any setting, and we know of no prior trials directly comparing how effectively different combinations of strategies support implementation in community health centers. This paper outlines the protocol of the Study of Practices Enabling Implementation and Adaptation in the Safety Net (SPREAD-NET), a trial designed to compare the effectiveness of several common strategies for supporting implementation of an intervention and explore contextual factors that impact the strategies’ effectiveness in the community health center setting.
This cluster-randomized trial compares how three increasingly hands-on implementation strategies support adoption of an evidence-based diabetes quality improvement intervention in 29 community health centers, managed by 12 healthcare organizations. The strategies are as follows: (arm 1) a toolkit, presented in paper and electronic form, which includes a training webinar; (arm 2) toolkit plus in-person training with a focus on practice change and change management strategies; and (arm 3) toolkit, in-person training, plus practice facilitation with on-site visits. We use a mixed methods approach to data collection and analysis: (i) baseline surveys on study clinic characteristics, to explore how these characteristics impact the clinics’ ability to implement the tools and the effectiveness of each implementation strategy; (ii) quantitative data on change in rates of guideline-concordant prescribing; and (iii) qualitative data on the “how” and “why” underlying the quantitative results. The outcomes of interest are clinic-level results, categorized using the Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework, within an interrupted time-series design with segmented regression models. This pragmatic trial will compare how well each implementation strategy works in “real-world” practices.
Having a better understanding of how different strategies support implementation efforts could positively impact the field of implementation science, by comparing practical, generalizable methods for implementing clinical innovations in community health centers. Bridging this gap in the literature is a critical step towards the national long-term goal of effectively disseminating and implementing effective interventions into community health centers.
Trial registration, NCT02325531
PMCID: PMC4609090  PMID: 26474759
Diabetes mellitus; Cardiovascular disease; Quality improvement; Community health centers; Evidence-based; Translational medical research
22.  An Anthropogenic Habitat Facilitates the Establishment of Non-Native Birds by Providing Underexploited Resources 
PLoS ONE  2015;10(8):e0135833.
Anthropogenic modification of habitats may reduce the resources available for native species, leading to population declines and extinction. These same habitats often have the highest richness of non-native species. This pattern may be explained if recently human-modified habitats provide novel resources that are more accessible to non-native species than native species. Using non-native birds in the Iberian Peninsula as a case study, we conduct a large-scale study to investigate whether non-native species are positively associated with human modified habitats, and to investigate whether this positive association may be driven by the presence of resources that are not fully exploited by native species. We do this by comparing the functional diversity and resource use of native and non-native bird communities in a recently human-modified habitat (rice fields) and in more traditional habitats in the Iberian Peninsula. The functional diversity of native bird communities was lower in rice fields, but non-native birds were positively associated with rice fields and plugged this gap. Differences in resource use between native and non-native species allowed non-native species to exploit resources that were plentiful in rice fields, supporting the role of underexploited resources in driving the positive association of non-native birds with rice fields. Our results provide a potential mechanism explaining the positive association of non-native species with anthropogenic habitats, and further work is needed to test if this applies more generally.
PMCID: PMC4537089  PMID: 26273825
23.  Health resource utilization associated with skeletal-related events: results from a retrospective European study 
Bone complications, also known as skeletal-related events (SREs), are common in patients with bone metastases secondary to advanced cancers.
To provide a detailed estimate of the health resource utilization (HRU) burden associated with SREs across eight European countries.
Eligible patients from centers in Austria, the Czech Republic, Finland, Greece, Poland, Portugal, Sweden, and Switzerland with bone metastases or lesions secondary to breast cancer, prostate, or lung cancer or multiple myeloma who had experienced at least one SRE (defined as radiation to bone, long-bone pathologic fracture, other bone pathologic fracture, surgery to bone or spinal cord compression) were entered into this study. HRU data were extracted retrospectively from the patients’ charts from 3.5 months before the index SRE until 3 months after the index SRE (defined as an SRE preceded by an SRE-free period of at least 6.5 months).
Overall, the mean number of inpatient stays per SRE increased from baseline by approximately 0.5–1.5 stays, with increases in the total duration of inpatient stays of approximately 6–37 days per event. All SREs were associated with substantial increases from baseline in the frequency of procedures and the number of outpatient and day-care visits.
SREs are associated with substantial HRU owing to considerable increases in the number and duration of inpatient stays, and in the number of procedures, outpatient visits, and day-care visits. These data collectively provide a valuable summary of the real-world SRE burden on European healthcare systems.
PMCID: PMC4899504  PMID: 26253584
Health resource utilization (HRU); Skeletal-related event (SRE); Bone metastases; Breast cancer; Prostate cancer; Lung cancer
24.  Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene 
PLoS ONE  2015;10(7):e0131129.
Herpes simplex virus 1 (HSV-1) ICP0 is a multi-functional phosphoprotein expressed with immediate early kinetics. An ICP0 deletion mutant, HSV-1 dl1403, has been widely used to study the roles of ICP0 in the HSV-1 replication cycle including gene expression, latency, entry and assembly. We show that HSV-1 dl1403 virions lack detectable levels of envelope protein gC, and that gC is not synthesized in infected cells. Sequencing of the gC gene from HSV-1 dl1403 revealed a single amino acid deletion that results in a frameshift mutation. The HSV-1 dl1403 gC gene is predicted to encode a polypeptide consisting of the original 62 N-terminal amino acids of the gC protein followed by 112 irrelevant, non-gC residues. The mutation was also present in a rescuant virus and in two dl1403-derived viruses, D8 and FXE, but absent from the parental 17+, suggesting that the mutation was introduced during the construction of the dl1403 virus, and not as a result of passage in culture.
PMCID: PMC4505948  PMID: 26186447
25.  Glycoprotein B (gB) vaccines adjuvanted with AS01 or AS02 protect female guinea pigs against cytomegalovirus (CMV) viremia and offspring mortality in a CMV-challenge model 
Vaccine  2013;32(23):2756-2762.
The transmission of cytomegalovirus (CMV) from mother to fetus can give rise to severe neurodevelopment defects in newborns. One strategy to prevent these congenital defects is prophylactic vaccination in young women. A candidate vaccine antigen is glycoprotein B (gB). This antigen is abundant on the virion surface and is a major target of neutralization responses in human infections. Here, we have evaluated in a challenge model of congenital guinea pig CMV (GPCMV) infection, GPCMV-gB vaccines formulated with the clinically-relevant Adjuvant Systems AS01B and AS02V, or with Freund’s adjuvant (FA). Fifty-two GPCMV-seronegative female guinea pigs were administered three vaccine doses before being mated. GPCMV-challenge was performed at Day 45 of pregnancy (of an estimated 65 day gestation). Pup mortality rates in the gB/AS01B, gB/AS02V, and gB/FA groups were 24% (8/34), 10% (4/39) and 36% (12/33), respectively, and in the unvaccinated control group was 65% (37/57). Hence, efficacies against pup mortality were estimated at 64%, 84% and 44% for gB/AS01B (p<0.001), gB/AS02V (p<0.001) and gB/FA (p=0.014), respectively. Efficacies against GPCMV viremia (i.e DNAemia, detected by PCR) were estimated at 88%, 68% and 25% for the same vaccines, respectively, but were only significant for gB/AS01B (p<0.001), and gB/AS02V (p=0.002). In dams with viremia, viral load was approximately 6-fold lower with vaccination than without. All vaccines were highly immunogenic after two and three doses. In light of these results and of other results of AS01-adjuvanted vaccines in clinical development, vaccine immunogenicity was further explored using human CMV-derived gB antigen adjuvanted with either AS01B or the related formulation AS01E. Both adjuvanted vaccines were highly immunogenic after two doses, in contrast to the lower immunogenicity of the unadjuvanted vaccine. In conclusion, the protective efficacy and immunogenicity of adjuvanted vaccines in this guinea pig model are supportive of investigating gB/AS01 and gB/AS02 in the clinic.
PMCID: PMC3894257  PMID: 23867012
Cytomegalovirus; Guinea pig cytomegalovirus; Cytomegalovirus vaccine; Glycoprotein B; Adjuvant; Vaccine efficacy; Guinea pig challenge model

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