The Use of Tail as a Minimal-Invasive Method to Detect a Large Set of Biochemical Responses in the Italian Wall Lizard Podarcis siculus (Rafinesque, 1810)

Conventional methods to analyze biochemical processes related to contaminant toxicity usually require the sacrifice of animals to collect tissues and organs. However, for ethical reasons and especially for endangered species, non- or minimal-invasive methods should be preferred. Among vertebrates, reptiles show a general decline worldwide and therefore the use of non- or minimal-invasive methods to measure some biochemical processes in these animals are encouraged. It is well known that most lizards use a common safety behavior implying the natural loss of tail in the case of predation events. Therefore, if common analyses testing contaminant toxicity could be performed in tail tissue, this method, not implying the sacrifice of the animals, could be considered as a good minimal-invasive method. The aim of this study is to test on wild Italian wall lizard Podarcis siculus the use of tail to detect a large set of biomarkers including oxidative stress (TOSCAROO, TOSCAOH, CAT, tGSH, MDA), biotransformation processes (EROD, GSTs) and neurotoxicity (AChE, BChE). All the biochemical responses, excluding EROD and MDA, resulted to be analytically detectable in tail tissues of P. siculus, although the mean values obtained with this minimal-invasive method were significantly lower than those obtained with invasive one

Zinc transport and metallothionein secretion in the intestinal human cell line Caco-2.

Caco-2, a human cell line, displays several biochemical and morphological characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concentrations of the metal. High pressure liquid chromatography fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins), were present in the apical and basal media of controls as well as in cells grown in the presence of high concentrations of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling experiments with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concentrations of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells

USE OF CAGED MUSSEL MYTILUS GALLOPROVINCIALIS IN AN ECOTOXICOLOGICAL APPROACH TO ASSESS ENVIRONMENTAL IMPACT IN OFF-SHORE ACTIVITIES

Abstract Mediterranean mussels, Mytilus galloprovincialis, are well recognized bioindicator organisms which can be easily caged in investigated areas to assess the impact of anthropogenic activities. In this work a monitoring protocol was developed for off-shore installations in the Adriatic sea. Integration of chemical analyses with a wide range of biomarkers analysed in mussels caged at 2 platforms, allowed to evaluate the biological disturbance and confirmed the utility of the ecotoxicological approach for monitoring off-shore activities. Keywords : Bio-indicators, Adriatic Sea. Several environmental issues are associated with the off-shore oil and gas industry, from the impact caused during installation to various form of disturbance related to daily ship traffic, extraction activities, maintenance of structures and, finally, decommissioning of old platforms. During the last year a monitoring protocol with caged mussels, Mytilus galloprovincialis, has been developed, to evaluate the potential ecotoxicological effects caused from the off-shore platform "Giovanna" in the Adriatic sea. Obtained results allowed to exclude marked biological disturbance and demonstrated the suitability of this approach. In this respect considering "Giovanna" as model platform, the monitoring protocol with caged mussel has been extended including also another off-shore installation, the "Emilio" platform. In this work native mussels were collected on a seasonal basis from a reference site on the Adriatic coast (Portonovo, Ancona) and transplanted for 4-6 weeks in both the sampling area and to the investigated platform "Giovanna" (42 • 46' 060N, 14 • 27' 750E) and Emilio (42 • 56' 305 N; 14 • 13' 915 E). After the translocation period, mussels were recovered dissected tissues frozen in liquid nitrogen and maintained at -80 • C until analyses. Chemical analyses on trace metals (arsenic, cadmium, chromium, copper, iron, mercury, manganese, nickel, lead, zinc) in mussels tissues An overall evaluation of results confirmed the absence of marked biological effects caused by the activities of "Giovanna" platform, as already demonstrated during the previous monitoring project. More variations were observed in mussels translocated to "Emilio", i.e. higher activities of glutathione S-transferases, catalase and peroxisomal proliferation decrease of oxyradical scavenging capacity toward hydroxyl and peroxyl radicals and lysosomal destabilization (inhibition of neutral red retention time), indicating an onset of impairment condition in the organisms. Compared to mussels transplanted at the reference site, those from "Emilio" platform did not exhibit more elevated concentrations for the various metals and only for zinc and cadmium an higher bioavailability was detected close to the platform, suggesting the influence of galvanic anodes for cathodic protection. The overall results of this work confirmed the utility of using caged mussels as an additional contribution for monitoring off-shore activities and provided an ecotoxicological protocol based on cellular biomarkers for the early detection of biological disturbance

Endogenous antigen processing drives the primary CD4+ T cell response to influenza.

By convention, CD4+ T lymphocytes recognize foreign and self peptides derived from internalized antigens in combination with major histocompatibility complex class II molecules. Alternative pathways of epitope production have been identified, but their contributions to host defense have not been established. We show here in a mouse infection model that the CD4+ T cell response to influenza, critical for durable protection from the virus, is driven principally by unconventional processing of antigen synthesized within the infected antigen-presenting cell, not by classical processing of endocytosed virions or material from infected cells. Investigation of the cellular components involved, including the H2-M molecular chaperone, the proteasome and γ-interferon-inducible lysosomal thiol reductase revealed considerable heterogeneity in the generation of individual epitopes, an arrangement that ensures peptide diversity and broad CD4+ T cell engagement. These results could fundamentally revise strategies for rational vaccine design and may lead to key insights into the induction of autoimmune and anti-tumor responses

Alteration of endosomal trafficking is associated with early-onset parkinsonism caused by SYNJ1 mutations

Recently, a new form of autosomal recessive early-onset parkinsonism (PARK20), due to mutations in the gene encoding the phosphoinositide phosphatase, Synaptojanin 1 (Synj1), has been reported. Several genes responsible for hereditary forms of Parkinson's disease are implicated in distinct steps of the endolysosomal pathway. However, the nature and the degree of endocytic membrane trafficking impairment in early-onset parkinsonism remains elusive. Here, we show that depletion of Synj1 causes drastic alterations of early endosomes, which become enlarged and more numerous, while it does not affect the morphology of late endosomes both in non-neuronal and neuronal cells. Moreover, Synj1 loss impairs the recycling of transferrin, while it does not alter the trafficking of the epidermal growth factor receptor. The ectopic expression of Synj1 restores the functions of early endosomes, and rescues these trafficking defects in depleted cells. Importantly, the same alterations of early endosomal compartments and trafficking defects occur in fibroblasts of PARK20 patients. Our data indicate that Synj1 plays a crucial role in regulating the homeostasis and functions of early endosomal compartments in different cell types, and highlight defective cellular pathways in PARK20. In addition, they strengthen the link between endosomal trafficking and Parkinson's disease

Unique Type I Interferon Responses Determine the Functional Fate of Migratory Lung Dendritic Cells during Influenza Virus Infection

Migratory lung dendritic cells (DCs) transport viral antigen from the lungs to the draining mediastinal lymph nodes (MLNs) during influenza virus infection to initiate the adaptive immune response. Two major migratory DC subsets, CD103+ DCs and CD11bhigh DCs participate in this function and it is not clear if these antigen presenting cell (APC) populations become directly infected and if so whether their activity is influenced by the infection. In these experiments we show that both subpopulations can become infected and migrate to the draining MLN but a difference in their response to type I interferon (I-IFN) signaling dictates the capacity of the virus to replicate. CD103+ DCs allow the virus to replicate to significantly higher levels than do the CD11bhigh DCs, and they release infectious virus in the MLNs and when cultured ex-vivo. Virus replication in CD11bhigh DCs is inhibited by I-IFNs, since ablation of the I-IFN receptor (IFNAR) signaling permits virus to replicate vigorously and productively in this subset. Interestingly, CD103+ DCs are less sensitive to I-IFNs upregulating interferon-induced genes to a lesser extent than CD11bhigh DCs. The attenuated IFNAR signaling by CD103+ DCs correlates with their described superior antigen presentation capacity for naïve CD8+ T cells when compared to CD11bhigh DCs. Indeed ablation of IFNAR signaling equalizes the competency of the antigen presenting function for the two subpopulations. Thus, antigen presentation by lung DCs is proportional to virus replication and this is tightly constrained by I-IFN. The “interferon-resistant” CD103+ DCs may have evolved to ensure the presentation of viral antigens to T cells in I-IFN rich environments. Conversely, this trait may be exploitable by viral pathogens as a mechanism for systemic dissemination

Modeling Within-Host Dynamics of Influenza Virus Infection Including Immune Responses

Influenza virus infection remains a public health problem worldwide. The mechanisms underlying viral control during an uncomplicated influenza virus infection are not fully understood. Here, we developed a mathematical model including both innate and adaptive immune responses to study the within-host dynamics of equine influenza virus infection in horses. By comparing modeling predictions with both interferon and viral kinetic data, we examined the relative roles of target cell availability, and innate and adaptive immune responses in controlling the virus. Our results show that the rapid and substantial viral decline (about 2 to 4 logs within 1 day) after the peak can be explained by the killing of infected cells mediated by interferon activated cells, such as natural killer cells, during the innate immune response. After the viral load declines to a lower level, the loss of interferon-induced antiviral effect and an increased availability of target cells due to loss of the antiviral state can explain the observed short phase of viral plateau in which the viral level remains unchanged or even experiences a minor second peak in some animals. An adaptive immune response is needed in our model to explain the eventual viral clearance. This study provides a quantitative understanding of the biological factors that can explain the viral and interferon kinetics during a typical influenza virus infection

Meeting of the Ecosystem Approach Correspondence Group on on Pollution Monitoring (CorMon Pollution)

In accordance with the UNEP/MAP Programme of Work adopted by COP 21 for the biennium 2020-2021, the United Nations Environment Programme/Mediterranean Action Plan-Barcelona Convention Secretariat (UNEP/MAP) and its Programme for the Assessment and Control of Marine Pollution in the Mediterranean (MED POL) organized the Meeting of the Ecosystem Approach Correspondence Group on Pollution Monitoring (CorMon on Pollution Monitoring). The Meeting was held via videoconference on 26-27 April 2021. 2. The main objectives of the Meeting were to: a) Review the Monitoring Guidelines/Protocols for IMAP Common Indicator 18, as well as the Monitoring Guidelines/Protocols for Analytical Quality Assurance and Reporting of Monitoring Data for IMAP Common Indicators 13, 14, 17, 18 and 20; b) Take stock of the state of play of inter-laboratory testing and good laboratory practice related to IMAP Ecological Objectives 5 and 9; c) Analyze the proposal for the integration and aggregation rules for IMAP Ecological Objectives 5, 9 and 10 and assessment criteria for contaminants and nutrients; d) Recommend the ways and means to strengthen implementation of IMAP Pollution Cluster towards preparation of the 2023 MED Quality Status Report

The effector T cell response to influenza infection

Influenza virus infection induces a potent initial innate immune response, which serves to limit the extent of viral replication and virus spread. However, efficient (and eventual) viral clearance within the respiratory tract requires the subsequent activation, rapid proliferation, recruitment, and expression of effector activities by the adaptive immune system, consisting of antibody producing B cells and influenza-specific T lymphocytes with diverse functions. The ensuing effector activities of these T lymphocytes ultimately determine (along with antibodies) the capacity of the host to eliminate the viruses and the extent of tissue damage. In this review, we describe this effector T cell response to influenza virus infection. Based on information largely obtained in experimental settings (i.e., murine models), we will illustrate the factors regulating the induction of adaptive immune T cell responses to influenza, the effector activities displayed by these activated T cells, the mechanisms underlying the expression of these effector mechanisms, and the control of the activation/differentiation of these T cells, in situ, in the infected lungs