Les phlébotomes (Diptera-Psychodidae) de l'île de Chypre.II - Presence de Leishmania (Leishmania) infantum Nicolle, 1908 (zymodeme MON 1) chez Phlebotomus (Larroussius) tobbi Adler et Theodor, 1930

Dans le foyer leishmanien cypriote, les auteurs ont disseque 2910 femelles de phlebotomes appartenant a 11 especes : Phlebotomus papatasi, P. sergenti, P. jacusieli, P. alexandri, P. tobbi, P. galilaeus, P. mascittii, P. economidesi, Sergentomyia fallax, S. minuta et S. azizi . Les deux larroussius (P. galilaeus et P. tobbi ) sont les especes les plus abondantes. Elles representent plus de 60 % des captures realisees avec des pieges CDC. Des promastigotes ont ete observees chez un seul specimen appartenant a l'espece P. tobbi . Elles ont ete mises en culture puis identifiees selon la methode isoenzymatique. La souche de Leishmania isolee appartient a l'espece Leishmania infantum , zymodeme MON 1. Le meme zymodeme a ete aussi isole et identifie chez quatre chiens de l'ile. En l'absence des vecteurs habituels de L. infantum dans l'est mediterraneen (P. neglectus ef P. syriacus) et en raison de sa repartition a Chypre, P. tobbi constitue vraisemblablement un bon vecteur local. Sa faible anthropophilie expliquerait peut-etre le tres faible nombre de cas humains. Le role de P. galilaeus dans la transmission de la leishmaniose a Chypre reste a preciser

Promotion of Hendra virus replication by microRNA 146a

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease

Naturally existing isoforms of miR-222 have distinct functions

Deep-sequencing reveals extensive variation in the sequence of endogenously expressed microRNAs (termed 'isomiRs') in human cell lines and tissues, especially in relation to the 3' end. From the immunoprecipitation of the microRNA-binding protein Argonaute and the sequencing of associated small RNAs, we observe extensive 3'-isomiR variation, including for miR-222 where the majority of endogenously expressed miR-222 is extended by 1-5 nt compared to the canonical sequence. We demonstrate this 3' heterogeneity has dramatic implications for the phenotype of miR-222 transfected cells, with longer isoforms promoting apoptosis in a size (but not 3' sequence)-dependent manner. The transfection of longer miR-222 isomiRs did not induce an interferon response, but did downregulate the expression of many components of the pro-survival PI3K-AKT pathway including PIK3R3, a regulatory subunit whose knockdown phenocopied the expression of longer 222 isoforms in terms of apoptosis and the inhibition of other PI3K-AKT genes. As this work demonstrates the capacity for 3' isomiRs to mediate differential functions, we contend more attention needs to be given to 3' variance given the prevalence of this class of isomiR.Feng Yu, Katherine A. Pillman, Corine T. Neilsen, John Toubia, David M. Lawrence, Anna Tsykin, Michael P. Gantier, David F. Callen, Gregory J. Goodall and Cameron P. Bracke

Novel downstream process and analytical tools developed for Influenza VLP vaccine

Vaccination remains the most effective way to prevent the infection with Influenza viruses. However, their constant antigenic drift implies that current human Influenza vaccines need to be annually updated with high inherent costs. Virus-like particles (VLPs) have become widely used as vaccine candidates because of their versatility, immunogenicity, and safety profile. In this iBET project we are attempting to produce a candidate for a universal vaccine for which 35 different VLPs (mono, trivalent and pentavalent) were purified. Here we describe three recent advances on Influenza VLPs bioprocessing: two new analytical tools and the development of an integrated all filtration purification process, inserted in the “anything but chromatography” concept. The first method is a label-free tool that uses Biolayer interferometry technology applied on an Octet platform to quantify Influenza VLPs at all stages of DSP. Human and avian sialic acid receptors were used, in order to quantify hemagglutinin (HA) content in several mono- and multivalent Influenza VLP strains. The applied method was able to detect and quantify HA from crude sample up to final VLP product with high throughput, real-time results and improved detection limits, when compared to traditional approaches, crucial for in-line monitoring of DSP. Using a click-chemistry approach that involves Azidohomoalanine incorporation and functionalization, Influenza VLPs were selectively and fluorescently tagged. Taking into account that this chemical tag does not affect particle size, charge and biological activity we report here a valuable tool to online/at-line product monitoring during DSP optimization of virus related biopharmaceuticals. Moreover, using this tool coupled with FACS we were able to discriminate between VLPs and baculovirus, the major impurity of the system. The proposed all-filtration process will be described, with special focus on the clarification stage, followed by multiple ultrafiltration and diafiltration steps to achieve the needed concentration and purity specifications. Using this all-filtration platform, we are able to speed up the time process, to improve the scale-up and to reduce costs due to the removal of chromatographic steps

miR451 and AMPK Mutual Antagonism in Glioma Cell Migration and Proliferation: A Mathematical Model

Glioblastoma multiforme (GBM) is the most common and the most aggressive type of brain cancer; the median survival time from the time of diagnosis is approximately one year. GBM is characterized by the hallmarks of rapid proliferation and aggressive invasion. miR-451 is known to play a key role in glioblastoma by modulating the balance of active proliferation and invasion in response to metabolic stress in the microenvironment. The present paper develops a mathematical model of GBM evolution which focuses on the relative balance of growth and invasion. In the present work we represent the miR-451/AMPK pathway by a simple model and show how the effects of glucose on cells need to be “refined” by taking into account the recent history of glucose variations. The simulations show how variations in glucose significantly affect the level of miR-451 and, in turn, cell migration. The model predicts that oscillations in the levels of glucose increase the growth of the primary tumor. The model also suggests that drugs which upregulate miR-451, or block other components of the CAB39/AMPK pathway, will slow down glioma cell migration. The model provides an explanation for the growth-invasion cycling patterns of glioma cells in response to high/low glucose uptake in microenvironment in vitro, and suggests new targets for drugs, associated with miR-451 upregulation

Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA

U1-snRNA is an integral part of the U1 ribonucleoprotein pivotal for pre-mRNA splicing. Toll-like receptor (TLR) signaling has recently been associated with immunoregulatory capacities of U1-snRNA. Using lung A549 epithelial/carcinoma cells, we report for the first time on interferon regulatory factor (IRF)-3 activation initiated by endosomally delivered U1-snRNA. This was associated with expression of the IRF3-inducible genes interferon-β (IFN-β), CXCL10/IP-10 and indoleamine 2,3-dioxygenase. Mutational analysis of the U1-snRNA-activated IFN-β promoter confirmed the crucial role of the PRDIII element, previously proven pivotal for promoter activation by IRF3. Notably, expression of these parameters was suppressed by bafilomycin A1, an inhibitor of endosomal acidification, implicating endosomal TLR activation. Since resiquimod, an agonist of TLR7/8, failed to stimulate A549 cells, data suggest TLR3 to be of prime relevance for cellular activation. To assess the overall regulatory potential of U1-snRNA-activated epithelial cells on cytokine production, co-cultivation with peripheral blood mononuclear cells (PBMC) was performed. Interestingly, A549 cells activated by U1-snRNA reinforced phytohemagglutinin-induced interleukin-10 release by PBMC but suppressed that of tumor necrosis factor-α, indicating an anti-inflammatory potential of U1-snRNA. Since U1-snRNA is enriched in apoptotic bodies and epithelial cells are capable of performing efferocytosis, the present data in particular connect to immunobiological aspects of apoptosis at host/environment interfaces

An Interspecific Nicotiana Hybrid as a Useful and Cost-Effective Platform for Production of Animal Vaccines

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs

TLR7 gain-of-function genetic variation causes human lupus

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA and binds to guanosine. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP1 and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition

Small Interfering RNA Targeted to IGF-IR Delays Tumor Growth and Induces Proinflammatory Cytokines in a Mouse Breast Cancer Model

Insulin-like growth factor I (IGF-I) and its type I receptor (IGF-IR) play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs) for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2′-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD). Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2′-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines