Assessment of pesticides risk for bees: methods for PNEC measurements

Background: An individual honeybee shows a complex behavioral structure. Each bee takes part in the collective behavioral set up that ensures bee colony survival and development. Contaminants are likely to have effects on individual bees’ behavior with consequences at the level of the whole colony. They also are likely to alter bees’ physiology, including lifespan, fertility or fecundity, leading to colony weakness or colony collapse. Results: Peer-reviewed scientific literature provides a wide range of methods used for testing honeybees’ behavioral or physiological parameters. Apart from alterations that may appear during the conduction of acute or chronic toxicity tests, specific tests could be conducted to complement the risk assessment in order to evaluate the impact of sublethal doses of contaminants on bees. Such tests can be developed both in laboratory conditions or as part of the semi-field and field tests that are currently required as higher tier tests of risk assessment schemes. Conclusion: The purpose of this work is to review some of these methods and discuss their relevance in the evaluation of pesticide active substances and/or products in view to propose their future inclusion in pesticides risk assessment to bees. Keywords: honey bee, sublethal effects, risk assessmen

A Non-Cytosolic Protein of Trypanosoma evansi Induces CD45-Dependent Lymphocyte Death

In a recent study dealing with a mouse model of Trypanosoma evansi-associated disease, a remarkable synchrony between the parasitaemia peak and the white-blood-cell count nadir was noticed. The present study was designed to establish whether there is a direct causal link between the parasite load during its exponential phase of growth and the disappearance of peripheral blood leukocytes. In vitro experiments performed with trypanosomes and purified peripheral blood mononucleated cells revealed the existence of a lymphotoxin embedded in the T. evansi membrane: a protein sensitive to serine proteases, with a molecular mass of less than 30 kDa. Lymphocytes death induced by this protein was found to depend on the intervention of a lymphocytic protein tyrosine phosphatase. When lymphocytes were exposed to increasing quantities of a monoclonal antibody raised against the extracellular portion of CD45, a transmembrane protein tyrosine phosphatase covering over 10% of the lymphocyte surface, T. evansi membrane extracts showed a dose-dependent decrease in cytotoxicity. As the regulatory functions of CD45 concern not only the fate of lymphocytes but also the activation threshold of the TCR-dependent signal and the amplitude and nature of cytokinic effects, this demonstration of its involvement in T. evansi-dependent lymphotoxicity suggests that T. evansi might manipulate, via CD45, the host's cytokinic and adaptive responses

Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27×10−9 M versus 2.69×10−7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol

Testing candidate effectors contributing to resistance to pneumoviruses

Pneumoviruses are members of the Paramyxoviridae family, negative sense single stranded RNA viruses. The 3 major members of the pneumovirus genus are human respiratory syncytial virus (huRSV), bovine respiratory syncytial virus (boRSV) and their murine counterpart, pneumonia virus of mice (PVM). In humans, huRSV mainly infects young children and can lead, in the worst cases, to infantile bronchiolitis, which is the first cause of children hospitalization and concerns 2.3% of children under the age of one. Many laboratories are studying this disease in order to better understand the role played by the immune system on its pathogenesis and to create an efficient and safe vaccine that does not exist for now. In bovine, boRSV generates stable epidemics in calves under 6 months of age and causes important economic losses in terms of veterinary costs and loss of productivity. These viruses infect the respiratory tract and generate a disease that importantly depends on the genetic background of the host. The quality of the immune response plays a considerable role in the expression as well as in the outcome of the disease, especially during its first instants. The study of pneumoviruses diseases has been widely developed using murine models of infection by heterologous huRSV. It is now assumed that this model, although providing interesting information, only weakly reproduces human disease on the contrary to infection of mice with their homologous pneumovirus: PVM. This model has been broadly developed in our laboratory, by highlighting the similarities between murine, human and bovine diseases, i.e. an important morbidity, intra-pulmonary granulocytic influx, viral amplification and evolution toward acute respiratory distress syndrome. The present work consisted in studying potential effectors of the resistance of mice against PVM infection: two proteic and two cellular effectors. A first working hypothesis was to evaluate the role of Carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam1) as a potential receptor for PVM. This membrane protein present at the surface of respiratory epithelial cells is known to be mutated in SJL/J mice, the most resistant strain to PVM infection. We have thus infected highly PVM-susceptible 129/Sv mice and compared their susceptibility to Ceacam1a knock-out 129/Sv mice. Our results have permitted to discard Ceacam1a as a receptor to PVM. The second tested protein is bovine Mx1 (boMx1) GTPase. We wished to assess if its expression could lead, as it is the case for numerous negative sense single stranded RNA viruses, to a resistance to PVM. Transgenic mice producing boMx1, engineerd in our laboratory in an FVB/J background, have been infected and their susceptibility has been compared to wild-type FVB/J mice. We could evidently demonstrated that boMx1 induces resistance to PVM. We have thus, for the first time, demonstrated that an Mx protein induces resistance to a pneumovirus. We have then compared 2 inbred mice strains presenting opposite phenotypes concerning their susceptibility to PVM: SJL/J (resistant) and 129/Sv (susceptible). Our goal was to evaluate if the difference in resistance is present during the first moments of infection. Therefore, we have focussed on both cell types that first interact with the virus: respiratory epithelial cells and alveolar macrophages. Virus tropism and respiratory epithelial permissivity to viral amplification were compared between both strains. In the same way, alveolar macrophages’ phenotypes (phagocytosis, susceptibility to viral infection, cytokine production) were compared between SJL/J and 129/Sv. Our results could not demonstrate any difference concerning respiratory epithelium. On the other hand, a pivotal role could be attributed to alveolar macrophages in the first instants of the infection. SJL/J alveolar macrophages presented greater phagocytic capacity, increased resistance to virus replication and earlier and higher cytokine production. Our results concerning the role of alveolar macrophages as a key effector of the immune response during the infection of mice by PVM raises new interrogations. Differences observed between SJL/J and 129/Sv alveolar macrophages regarding their resistance to viral infection, their greater phagocytic capacity and their cytokine secretion will have to be further investigated in order to understand the relative importance of these three characteristics in the resistance process of SJL/J to PVM infection. A particular attention will have to be given to the candidate cytokines in order to determine if one of them plays a particular role in the disease outcome. Moreover, the anti-PVM property of boMx1 raises the question of why boMx1 possesses an anti-PVM property but no anti-boRSV effect. Comparison of the interaction of boMx1 with PVM or boRSV could help understanding this anti-PVM effect.TESTING CANDIDATE EFFECTORS CONTRIBUTING TO RESISTANCE TO PNEUMOVIRUSE

In vivo modulation of the innate response to pneumovirus by type-I and -III interferon-induced Bos taurus Mx1

The respiratory syncytial virus (RSV) is a major pathogen of the human species. This pneumovirus is a prominent cause of airway morbidity in children and maintains an excessive hospitalization rate despite decades of research. As involvement of a genetic vulnerability is a possibility supported by recent data, we addressed the question of whether the Mx gene products, the typical target of which consists in single-stranded negative-polarity RNA viruses, could alter the course of pneumovirus-associated disease in vivo. Wild-type and Bos taurus Mx1-expressing transgenic FVB/J mice were inoculated with the mouse counterpart and closest phylogenetic relative of RSV, pneumonia virus of mice. Survival data and follow-up of body weight, histological scores, lung virus spread and lung viral load unequivocally showed that the viral infection was severely repressed in Mx-transgenic mice, thus suggesting that pneumoviruses belong to the antiviral spectrum of mammalian Mx GTPases. Elucidating the underlying mechanisms at the molecular level could reveal critical information for the development of new anti-RSV molecules

Solving the Enforcement Dilemma of EU Fiscal Rules

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Solving the enforcement dilemma of the EU fiscal rules

To ensure a smooth functioning of the Economic and Monetary Union, the Stability and Growth Pact defines limits on national fiscal policies and encompasses the possibility to impose sanctions. This short piece explores the much under-examined issue of enforcement of the EU fiscal rules. In the ongoing reform debate, a more consistent recourse to financial sanctions under the Stability and Growth Pact is presented as a counterweight to more flexible and tailor-made rules. At the same time, in light of past experience and in the absence of concrete changes to EU governance, many observers take a rather negative stance on the enforceability of EU fiscal rules. We clarify a number of crucial concepts with the aim of debunking the politically appealing but risky view that in the EU imposing sanctions on sovereigns is nearly impossibl