MOLECULAR AND CELLULAR MECHANISMS IN ASTROCYTE-T CELL CROSS-TALK

Migration of encephalitogenic T cells into the brain parenchima through the blood\u2013brain barrier (BBB) is a crucial feature for initiating tissue injury in different neuroinflammatory diseases. The BBB is comprised of astrocyte processes and endothelial cells, which form the lumen of the brain microvasculature and help in maintaining immune quiescence through contact-dependent mechanisms as well as release of soluble factors. Activated CD4+ T cells may establish physical contacts with astrocytes, thereby reciprocally influencing cellular activity and functions. In addition, astrocytes and CD4 T cells may communicate through the secretion of soluble signaling molecules during contact. Among molecules secreted by astrocyte, ATP is a key messenger which can also signal to CD4 T cell through purinergic P2 receptors. Our results show that activated CD4+ T cells inhibit calcium oscillations in astrocytes through direct modulation of extracellular ATP levels. This effect correlated with the expression of plasma membrane ectonucleoside triphosphate diphosphohydrolase CD39, which is induced by contact of the activated T cell with astrocyte. In addition, T cell contact with astrocyte results in the upregulation of the ecto-5\u2019-nucleotidase CD73, which converts AMP to adenosine. This effect was peculiar of T cell contact with astrocyte since it did not occur with microglia or peritoneal macrophages. Pharmacological inhibition of Ca2+ oscillations in astrocyte completely prevented CD73 induction on T cell, thus suggesting that a gliostrasmitter released by astrocyte in a Ca2+-dependent fashion might be responsible of this effect. Since degradation of ATP to adenosine by CD73 regulates BBB permeability and leukocytes infiltration into the brain this regulatory circuit might have important pathogenetic implications in multiple sclerosis and other neuroinflammatory conditions. Finally, functional characterization of T cell upon contact with astrocyte allowed us to assess a proinflammatory phenotype and Th17 skewing albeit with important differences, such as CD39 and CD73 expression, with respect to conventionally activated cells. Thus, we characterized an astrocyte specific signature of T cell activation, which might be important in the pathogenesis of neuroinflammatory disorders

An interactive tool for the identification of airborne and food fungi

The growth of fungi may result in several kinds of food-spoilage: off-flavours, discolouration, rotting and formation of pathogenic or allergenic propagules. Moreover many foodborne fungi produce mycotoxins and thus fungal growth in foods and feeds should be avoided. Much interest has also grown for the fungi present in indoor environments, since exposure to airborne biological agents in both the occupational and residential environments could be associated with a wide range of adverse health effects with major public health impact, including infectious diseases, acute toxic effects, allergies and cancer. An interactive identification tool was created for food- and airborne microfungi at the genus and/or species level, based on morphological and physiological data, using the software FRIDA. The interactive key can also be stored on CD- or DVD-roms, or used on media such as PocketPCs of Smartphones. Our key allows the identification of 59 genera/groups and 217 species belonging mainly to Zygomycota and anamorphic and teleomorphic Ascomycota. The database comes with a set of detailed descriptions of each genus and species, a rich archive of images, a glossary of the most frequent mycological terms, and references to descriptions; in addition, culture condition requirements for identification are provided

Strategies and tools for studying microglial-mediated synapse elimination and refinement

The role of microglia in controlling synapse homeostasis is becoming increasingly recognized by the scientific community. In particular, the microglia-mediated elimination of supernumerary synapses during development lays the basis for the correct formation of neuronal circuits in adulthood, while the possible reactivation of this process in pathological conditions, such as schizophrenia or Alzheimer\u27s Disease, provides a promising target for future therapeutic strategies. The methodological approaches to investigate microglial synaptic engulfment include differen

Fine tuning of a sequencing protocol for species identification of Mytilus spp. specimens

The genus Mytilus comprises eight species of mussels of commercial relevance, which have different geographic origin and distribution and can hybridize in case of coexistence (1). To date, events of habitat contamination with allochthonous species occur with increasing frequency, due mainly to anthropogenic transfer, posing threats to local ecosystems (2). Moreover, such events favour species mislabeling, which is reported as the most frequent issue affecting seafood products (3). To prevent these problems, EU Regulation 1379/2013 promotes the use of DNA-based methods. PCR-RFLP is to date one of the most efficient techniques used in Mytilus spp. species identification (4-5). However, it has some drawbacks linked to limited resolution and subjective interpretation of the results. To improve such aspects, an optimized protocol for the sequencing of short fragments was developed. A total of 50 samples were analyzed. Of them, 12 were DNA samples belonging to various Mytilus spp. that were already identified by PCR-RFLP in a previous study (5). The other 38 were tissue samples, of which 10 belonging to specimens directly collected from production sites in Chile, and 28 belonging to fresh and pre-cooked specimens collected from national market. For these samples, the total DNA was extracted using the DNeasy Mericon Food kit (Qiagen). The target PAP region was amplified from all the 50 samples (DNA and tissue) using the primer pair designed by Satto et al. (4). All PCR products were analyzed through capillary electrophoresis to verify the successful amplification, and then sequenced using Sanger technique. Twenty samples (41%), of which 10 from production site and 10 from market samples, were randomly selected to perform the RFLP analysis to be compared to the sequencing results. Ten out of the 12 species (83.3%) identified by PCR-RFLP in the previous study were confirmed by sequencing. The remaining 2 samples, identified as M. chilensis x M. edulis by PCR-RFLP were instead identified as M. chilensis x M. trossulus by capillary electrophoresis and confirmed by sequencing. For the remaining 38 samples, sequencing identified 20 tissue samples (52.6%) as M. galloprovincialis, 7 as M. chilensis (18.4%), 6 as hybrids M. chilensis x M. galloprovincialis (15.8%), 2 as M. edulis (5.3%), 2 as hybrids M. chilensis x M. trossulus (5.3%) and 1 as hybrid M. galloprovincialis x M. edulis (2.6%). The PCR-RFLP confirmed the sequencing results for 19 of the tested samples and failed the unambiguous identification of one sample which was identified as hybrid by sequencing. Overall, the species identification by PCRRFLP failed in 3 out 32 (9.4%) tested samples (12 DNA and 20 tissue). These findings suggest that the optimized protocol relying on Sanger sequencing has some practical advantages. First, the use of capillary electrophoresis to visualize the PCR products allowed the clear distinction of M. edulis and M. trossulus which, having a difference in amplicon length of only 12 bp (177 bp vs 165 bp), are very hard to discriminate with a standard agarose gel electrophoresis. Moreover, the use of sequencing allowed the unambiguous identification of pure species and hybrids, especially in the case of M. galloprovincialis x M. edulis, in which a double peak at the SNP site is clearly visible. Considering the ever-dropping costs linked to sequence-based technology, we propose this sequencing protocol as a valid, consistent, and reliable alternative to the currently used methods

Soluble TREM2: Innocent bystander or active player in neurological diseases?

Triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune receptor expressed by macrophages and microglia in the central nervous system (CNS). TREM2 has attracted a lot of interest in the past decade for its critical role in modulating microglia functions under homeostatic conditions and in neurodegenerative diseases. Genetic variation in TREM2 is sufficient to cause Nasu-Hakola disease, a rare pre-senile dementia with bone cysts, and to increase risk for Alzheimer\u27s disease, frontotemporal dementia, and other neurodegenerative disorders. Beyond the role played by TREM2 genetic variants in these diseases, TREM2 engagement is a key step in microglia activation in response to different types of tissue injury (e.g. β-Amyloid deposition, demyelination, apoptotic cell death) leading to enhanced microglia metabolism, phagocytosis, proliferation and survival. TREM2 also exists as a soluble form (sTREM2), generated from receptor shedding or alternative splicing, which is detectable in plasma and cerebrospinal fluid (CSF). Genetic variation, physiological conditions and disease status impact CSF sTREM2 levels. Clinical and preclinical studies suggest that targeting and/or monitoring sTREM2 could have clinical and therapeutic implications. Despite the critical role of sTREM2 in neurologic disease, its function remains poorly understood. Here, we review the current literature on sTREM2 regarding its origin, genetic variation, and possible functions as a biomarker in neurological disorders and as a potential active player in CNS diseases and target for therapies