Ariel - Volume 4 Number 6

Editors David A. Jacoby Eugenia Miller Tom Williams Associate Editors Paul Bialas Terry Burt Michael Leo Gail Tenikat Editor Emeritus and Business Manager Richard J. Bonnano Movie Editor Robert Breckenridge Staff Richard Blutstein Mary F. Buechler J.D. Kanofsky Rocket Weber David Maye

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Mousses et lichens, bioindicateurs (s.l.) de l’état des zones humides : exemples de quatre sites protégés du département de L’Isère (France)

In four protected wetland areas of the department of Isère (marshes and alluvial forests), epiphytic lichens and bryophytes were studied in order to evaluate the floristic diversity (inventories and relations with plant communities) and set a monitoring of the diffuse atmospheric pollution (by analysis of Cl, N, P, Hg, Pb contents). The diversity of these cryptogams is lowered whereas, especially for lichens, the percentage of nitrophilous species augments with increasing anthropization and eutrophization. The important sensitiveness of these groups of plants to environmental changes would permit to use them to adapt conservation management and also to foresee some ecosystem alterations not easily detected by the mere observation of vascular plants. In spite of a low number of analyses, results of the present study show a low total pollution, except for a trend towards contamination by phosphorus in agricultural areas and by nitrogenous compounds nearly everywhere. It will be necessary to increase measurements in these four areas and extend them to other areas in the department, in order to develop a biomonitoring network.Dans quatre zones humides protégées de l’Isère (marais et forêts alluviales), une étude des lichens corticoles et des bryophytes a été entreprise avec deux objectifs : (1) évaluer la diversité floristique (inventaires, corrélations avec les communautés de végétaux supérieurs), (2) mettre en place un suivi de la pollution atmosphérique diffuse (analyses des teneurs en Cl, N, P, Hg, Pb). La diversité cryptogamique diminue tandis que le taux d’espèces nitrophiles, surtout pour les lichens, augmente avec l’accroissement de l’anthropisation et de l’eutrophisation. La forte sensibilité aux modifications externes de ces cryptogames devrait permettre de les utiliser pour adapter les modes de gestion conservatoire et pour prévoir certaines altérations des écosystèmes non décelables facilement par l’observation des végétaux vasculaires. Malgré le faible nombre d’analyses effectuées, les résultats montrent un taux global de pollution faible dans ces sites, à l’exception d’une tendance à la contamination par le phosphore dans les sites en contexte agricole et par les composés azotés presque partout, à des degrés divers. Il s’avère nécessaire d’augmenter les mesures sur ces sites et de les étendre à d’autres sites du département pour développer un réseau de biosurveillance.Agnello Gregory, Manneville Olivier, Asta Juliette. Mousses et lichens, bioindicateurs (s.l.) de l’état des zones humides : exemples de quatre sites protégés du département de L’Isère (France) . In: Revue d'Écologie (La Terre et La Vie), tome 59, n°1-2, 2004. pp. 147-162

A comparative study of atmospheric deposits and lichen populations in a protected alpine area in the Grenoble region (France)

International audienc

Isolation of technogenic magnetic particles

International audienceTechnogenic magnetic particles (TMPs) emitted by various industrial sources, such as smelting plants, end up after atmospheric transfer on the soil surface. In the present study, we characterised the origin and composition of such particles emitted by a large iron smelting plant and deposited on particular substrates, namely tombstones, which act as a very interesting and appropriate matrix when compared to soil, tree bark, lichens or attic dust. The isolation and subsequent description of TMPs require a critical step of separation between different components of the sample and the magnetic particles; here, we described an efficient protocol that fulfils such a requirement: it resorts to water suspension, sonication, repeated magnetic extraction, sedimentation, sieving and organic matter destruction at 550 °C in some instances. The isolated TMPs displayed a noticeable crystalline shape with variable compositions: a) pure iron oxides, b) iron + Cr, Ni or Zn, and c) a complex structure containing Ca, Si, Mg, and Mn. Using Scanning Electron Microscope Energy Dispersive X-ray (SEM–EDX), we obtained profiles of various and distinct magnetic particles, which allowed us to identify the source of the TMPs

Are coarse particles unexpected common reservoirs for some atmospheric anthropogenic trace elements? A case study

International audienceWithout specific experimental equipment, it is very difficult to sample long-term atmospheric deposits on a pure state. That is why the composition of air-transferred solid material accumulated for 40 years in the 2 m-high walls, pierced with numerous holes of an outdoor public shelter, Grenoble city, France, was studied. An appropriate fractionation procedure allowed to obtain several fractions which were i) a sand fraction (8.3%) (fraction A), ii) a large mass of organic matter corresponding mostly to large fragments (>250 μm) of plant origin (66.7%) (fraction B) or to pollen fraction C (0.4%), iii) a slowly depositing organo-clay fraction (20%) (fractions D1 and D2) and iv) a solution mixed with non-settable particles (4.3%) (fraction E). The composition of each fraction was determined for 20 elements. The sand fraction showed very high concentrations specifically in Cu, Pb and Fe corresponding respectively to 81.5, 48.2 and 35.2% of the samples content in these elements. In contrast, Cd and Zn were mainly accumulated in the fraction B (67.5 and 62.2%, respectively). The scanning electron microscopy coupled to energy dispersive X-ray analysis (SEM-EDX) study of the fraction A showed the presence of large particles bearing Pb and Fe, particles rich in Cu and typical fly ashes originating mostly from iron industry. Most of these particles had a crystalline shape suggesting that they were formed after emission at a high temperature. The Pb–Fe–Cu deposit seen in fraction A likely originates from the neighbouring road surface contaminated by car traffic for several decades. The 206Pb/207Pb ratio (1.146 ± 0.004) showed that in the coarse sandy fraction A, Pb was represented at 65% by non-gasoline lead and 35% by “gasoline” lead emitted before 1999. The fraction A particles can only be transported on a limited distance by high magnitude events. They constitute a large reservoir for Cu and Pb and may play a major role in the long-term contamination of urban soils

Metal exposure in cows grazing pasture contamined by iron industry : Insights from magnetic particles used as tracers

International audienceMagnetic particles (MP) emitted by an iron smelter were used to investigate the exposure of cows grazing on a grassland polluted by these MP and by large amounts of potentially toxic elements (PTE). The morphology as well as the chemical composition of the MP separated from cow dung were studied. Large amounts of typical MP were found (1.1 g kg−1 dry weight) in the cow dung sampled from the exposed site, whereas these particles were absent from the reference unpolluted site. The ingested MP were mainly technogenic magnetic particles (TMP) emitted by the smelter. Considering the MP concentration in the grazed grass on the exposed site, it was concluded that cows absorb the MP not only from the grass but also from the soil surface. The results of a mild acidic leaching of the MP suggested that the particles were possibly submitted to a superficial dissolution in the abomasum, pointing at a potential route of transfer of the PTE originating from the TMP and leading into food chains. TMP were only a small part of the anthropogenic contamination having affected the soil and the dung. However, due to their unequivocal signature, TMP are a powerful tracer of the distribution of PTE in the different compartments constituting the food chains and the ecosystems. Furthermore, the measurement of the particle sizes gave evidence that a noticeable proportion of the MP could enter the respiratory tract