SatSel: A Satellite Selection Algorithm to reduce delivery time in DTN-Nanosatellite Networks for Internet Access in Rural Areas.

There are some different ways to connect rural areas to the Internet. One of these provides the use of a nanosatellite constellation. This type of network allows people in rural areas to enjoy all services the Internet can offer keeping low the cost of Internet access. One of the critical aspect is related to the delivery time, because LEO satellite links are not always up. This means that the system must be able to deal with periodic disruptions and high delays in the path from the source to the destination, considering that data could be stored in nanosatellite, Internet gateway (also called hot spot), and rural gateway (also called cold spot) buffers also for several seconds or minutes waiting to be forwarded. In the path from rural areas to the Internet, it is possible to reduce data delivery time acting on rural gateways. We propose SatSel: a selection algorithm which allows the cold spots to choose the nanosatellite to whom upload data in order to reduce the data delivery tim

Bioprocess engineering of insect cells for accelerating vaccines development

The majority of novel protein-based biologics require animal cell technology for discovery and production. The costs and complexity associated to these production platforms are commonly high making mandatory further improvements in product yields. Technological breakthroughs and/or new producer hosts allied to classical systems/molecular biology tools and engineering methodologies are therefore necessary for accelerating biologics manufacturing process, including vaccines. Two case studies will be presented where bioprocess engineering assisted/accelerated vaccines development. In the first case study, the aim was to generate an Influenza VLP in which the surface antigens are presented in their native conformation as membrane-bound proteins and are comprised of several hemagglutinin (HA) variants specifically designed to target B cells capable of mounting broad neutralising antibodies. The platform herein adopted for production of enveloped Influenza VLP enclosed certain drawbacks, namely (1) instability of baculovirus vectors encompassing multiple HA genes, and (2) low expression levels and recovery yields. To tackle these issues, a set of bioprocess engineering schemes were designed and subsequently implemented, which included (1) combination of stable and baculovirus-mediated expression of HA in insect High Five cells for production of difficult to express multi-HA VLPs (pentavalent VLP of H3 subtype), (2) DoE for identifying best infection strategy and evolutionary engineering of insect cells phenotype, and (3) development of a scalable, “universal” and “All-Filtration” purification platform of Influenza VLPs. In the second case study, the aim was to develop a fast and flexible insect cell platform for production of enveloped VLPs pseudo-typed with membrane proteins of interest. Stable insect cell lines have been successfully generated using site-specific gene integration based on flipase-mediated cassette exchange (FMCE) technology. Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept demonstrated using two membrane proteins, the Influenza HA protein (e.g. for vaccines) and the human beta-2 adrenergic receptor (e.g. for drug screening or antibody discovery). Bioprocess engineering schemes have been designed (adaptive laboratory evolution to hypothermic culture conditions and supplementation with productivity enhancers), allowing to improve Gag-VLP production in the developed stable insect cells. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist/accelerate vaccines development. Acknowledgments: This work was supported by European Commission (Project EDUFLUVAC, Grant nr. 602640) and by Portuguese “Fundação para a Ciência e a Tecnologia” through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project EXPL/BBBBIO/1541/2013, and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/201

Insect cell platforms for production of pseudo-typed VLPs for drug and vaccine development

Conformational-complex membrane proteins (MPs) are vaccine/drug targets in many diseases, but drug and vaccine development has been slowed down by the lack of efficient production tools. Co-expression of MPs with matrix proteins from enveloped viruses is a promising approach to obtain correctly folded proteins at the surface of ordered nanoscale architectures such as virus-like particles (VLPs), preserving their native lipidic environment. In this work, we implemented an innovative site-specific recombination strategy based on flipase-mediated cassette exchange technology to establish reusable insect cell platforms for fast production of enveloped VLPs pseudo-typed with target MPs. Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept (PoC) demonstrated using two membrane proteins, the influenza HA protein (e.g. for vaccines) and the human beta-2 adrenergic receptor (e.g. for drug screening or antibody discovery). Bioprocess engineering schemes were designed (adaptive laboratory evolution to hypothermic culture conditions and supplementation with productivity enhancers), allowing to improve HIV Gag-VLPs production in the developed stable insect cells. Under hypothermic culture conditions, adapted cells expressed up to 30-fold more HIV Gag-VLPs than non-adapted cells. Noteworthy, the element driving such increase in productivity is the adaptation process and not the temperature shift as the later alone leads to lower production yields. A more modest increase in productivity (up to 7-fold) was observed when supplementing non-adapted cell cultures with productivity enhancers NaBu and DMSO. PoC was successfully demonstrated in 0.5 L stirred-tank bioreactors. Profiting from the platforms developed above, a modular system comprising stable and baculovirus-mediated expression in insect cells was established for the production of a multi-HA influenza VLP as vaccine candidate that otherwise could not be obtained due to baculovirus vector instability. By combining stable with transient expression systems, we could rationally distribute the number of genes to be expressed per platform and thus generate the target VLP for subsequent animal studies. In addition, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth resulting in a 4-fold increase in HA titers per mL. PoC was successfully demonstrated in 2 L stirred-tank bioreactors. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist/accelerate drug and vaccine development. Acknowledgments: This work was supported by European Commission (Project EDUFLUVAC, Grant nr. 602640) and by Portuguese “Fundação para a Ciência e a Tecnologia” through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project EXPL/BBB-BIO/1541/2013, and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012

CAR-T therapy: the role of the hematopoietic stem cell processing laboratory

Purpose of this paper is to review all the phases that in-volved, for the first time, the SC of Hematology and the SC of Transfu-sion Medicine, specifically, the CSE Processing laboratory, in imple-menting an innovative operative protocol of gene immunotherapydue to the inclusion of the first patient, affected by diffuse large B-celllymphoma (DLBCL), stage IV, into the CAR-T therapeutic program.The program took place within the SS Antonio e Biagio e Cesare Ar-rigo Hospital of Alessandria

Selective Proinflammatory Activation of Astrocytes by High-Mobility Group Box 1 Protein Signaling

Abstract Extracellular high-mobility group box 1 protein (HMGB1) triggers inflammatory events in the brain. We demonstrate that astrocytes, the main glial cells in the brain, acquire a specific reactive phenotype when exposed to HMGB1. This cell activation, which involves the receptor for advanced glycation end-products and the MAPK/ERK1/2 cascade, results in the transcriptional/translational induction of a restricted number of inflammatory mediators, including cyclooxygenase-2, matrix metalloproteinase-9, and several chemokines of the CC and CXC families. The mixture of factors released by HMGB1-reactive astrocytes displays a potent chemotactic activity on human monocytic cells. This study is the first to suggest that HMGB1/astrocyte interaction plays a specific functional role in the progression of inflammatory processes in the CNS by facilitating local leukocyte infiltration

A Retrospective Epidemiological Study on the Association of Bullous Pemphigoid and Neurological Diseases

Bullous pemphigoid is a rare chronic recurrent dermatosis that is often reported in association with various neurological diseases. No investigation involving a large number of patients has ever been carried out to demonstrate such an association. This study was accomplished by analysing the discharge diagnosis of all hospitalized patients, both day-patients and inpatients, during a 5-year period (1995–2000) covering a total population group of 934,023 living in a region of Italy that has approximately 1,200,000 inhabitants. The results support the hypothesis of an association between bullous pemphigoid, multiple sclerosis and Parkinson's disease on a highly significant statistical basis. The aetiopathogenic mechanisms and the causes that induce the loss of immunological tolerance are not yet understood

Insect cells platforms for fast production of Pseudo-Typed VLPs for drug and vaccine development

Expression systems capable of delivering high concentrations of membrane proteins in their native structure are essential in the vaccine field as well as in drug discovery. In this work, we took advantage of insect cell expression and site-specific gene integration based on flipase-mediated cassette exchange (FMCE) technology to generate cell platforms for efficient production of membrane proteins on the surface of a protein scaffold, namely enveloped virus-like particles (VLPs). The expression of membrane proteins concomitantly with capsid proteins of enveloped viruses (e.g. HIV Gag or influenza M1) will enable their capturing in lipid rafts of the cellular plasma membrane and their display on the surface of budding VLPs, thus providing a native conformation for downstream assays. Parental insect Sf-9 and High Five cells were randomly tagged with GFP-fused Gag or M1 proteins and FACS enriched with cells tagged in genomic “hot-spots” supporting high expression. A linker including a Flp recognition target (FRT) site was used to allow posterior removal of the marker gene from the particle through cassette exchange. By confocal microscopy we could observe that Gag localizes preferentially at the plasma membrane whereas M1 disperses within the cell. Upon promoting Flp-mediated recombination in the tagging populations, cassette exchange was well succeeded, allowing to recover cells tagged in loci supporting FMCE. We are currently evaluating the capability of both core proteins as scaffolds to display GPCRs (e.g. beta-2 adrenergic receptor) and Influenza HA proteins. For the latter, we will present recent results on the feasibility of combining stable and baculovirus-mediated expression of HA in insect High Five cells for production of multi-HA influenza enveloped VLPs towards the development of an “universal” vaccine. This strategy surpasses standard methods for production of multivalent Influenza VLPs such as coinfections or the use of larger, unstable vectors. Overall, modular insect cells platforms are being generated to be readily adaptable for production of a broad range of VLP-based vaccines as well as receptor display particles for drug screening or antibody discovery. Acknowledgments: Funding from European Commission (Project EDUFLUVAC; Grant nr. 602640) and Fundação para a Ciência e a Tecnologia through the project EXPL/BBB-BIO/1541/2013 and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012

IL-27, but not IL-35, inhibits neuroinflammation through modulating GM-CSF expression

IL-27 and IL-35 are heterodimeric cytokines, members of the IL-12 family and considered to have immunomodulatory properties. Their role during neuroinflammation had been investigated using mutant mice devoid of either one of their subunits or lacking components of their receptors, yielding conflicting results. We sought to understand the therapeutic potential of IL-27 and IL-35 delivered by gene therapy in neuroinflammation. We constructed lentiviral vectors expressing IL-27 and IL-35 from a single polypeptide chain, and we validated in vitro their biological activity. We injected IL-27 and IL-35-expressing lentiviral vectors into the cerebrospinal fluid (CSF) of mice affected by experimental neuroinflammation (EAE), and performed clinical, neuropathological and immunological analyses. Both cytokines interfere with neuroinflammation, but only IL-27 significantly modulates disease development, both clinically and neuropathologically. IL-27 protects from autoimmune inflammation by inhibiting granulocyte macrophages colony-stimulating factor (GM-CSF) expression in CD4+ T cells and by inducing program death-ligand 1 (PD-L1) expression in both CNS-resident and CNS-infiltrating myeloid cells. We demonstrate here that IL-27 holds therapeutic potential during neuroinflammation and that IL-27 inhibits GM-CSF and induces pd-l1 mRNA in vivo

Cross-cultural adaptation and psychometric evaluation of the Juvenile Arthritis Multidimensional Assessment Report (JAMAR) in 54 languages across 52 countries: review of the general methodology

The aim of this project was to cross-culturally adapt and validate the Juvenile Arthritis Multidimensional Assessment Report (JAMAR) questionnaire in 54 languages across 52 different countries that are members of the Paediatric Rheumatology International Trials Organisation (PRINTO). This effort was part of a wider project named Epidemiology and Outcome of Children with Arthritis (EPOCA) to obtain information on the frequency of juvenile idiopathic arthritis (JIA) categories in different geographic areas, the therapeutic approaches adopted, and the disease status of children with JIA currently followed worldwide. A total of 13,843 subjects were enrolled from the 49 countries that took part both in the cross-cultural adaptation phase and in the related validation and data collection: Algeria, Argentina, Belgium, Brazil, Bulgaria, Canada, Chile, Colombia, Croatia, Czech Republic, Denmark, Ecuador, Egypt, Estonia, Finland, France, Georgia, Germany, Greece, Hungary, India, Islamic Republic of Iran, Israel, Italy, Latvia, Libya, Lithuania, Mexico, Netherlands, Norway, Oman, Paraguay, Poland, Portugal, Romania, Russian Federation, Saudi Arabia, Serbia, Slovakia, Slovenia, South Africa, Spain, Sweden, Switzerland, Thailand, Turkey, Ukraine, United Kingdom and United States of America. 9021 patients had JIA (10.7% systemic arthritis, 41.9% oligoarthritis, 23.5% RF negative polyarthritis, 4.2% RF positive polyarthritis, 3.4% psoriatic arthritis, 10.6% enthesitis-related arthritis and 5.7% undifferentiated arthritis) while 4822 were healthy children. This introductory paper describes the overall methodology; results pertaining to each country are fully described in the accompanying manuscripts. In conclusion, the JAMAR translations were found to have satisfactory psychometric properties and it is thus a reliable and valid tool for the multidimensional assessment of children with JIA