Search

Fil: Quintero, Cristian. Laboratorio de Biología Celular y Molecular. Área de Ciencia y Técnica. Universidad Juan Agustín Maza. Mendoza. República Argentina. Fil: Miranda, Daniel. Rectorado. Universidad Juan Agustín Maza. Mendoza. República Argentina. Fil: Vejling, Lizzet. Área de Ciencia y Técnica. Universidad Juan Agustín Maza. Mendoza. República Argentina. Fil: Zamorano, Mario. Área de Comunicación. Universidad Juan Agustín Maza. Mendoza. República Argentina. Fil: Muñoz, Gabriela. Área de Comunicación. Universidad Juan Agustín Maza. Mendoza. República Argentina. Fil: Piazza, Marcela. Área de Comunicación. Universidad Juan Agustín Maza. Mendoza. República Argentina. Fil: Franceschini, Valeria. Área de Comunicación. Universidad Juan Agustín Maza. Mendoza. República Argentina. Fil: López, Florencia. Área de Comunicación. Universidad Juan Agustín Maza. Mendoza. República Argentina. Fil: Chaud, Gabriel. Área de Comunicación. Universidad Juan Agustín Maza. Mendoza. República Argentina. Fil: Piffaretti, Julieta. Área de Comunicación. Universidad Juan Agustín Maza. Mendoza. República Argentina. Este video fue editado por el Área de Comunicación en base al Informe Técnico emanado por el Área de Ciencia y Técnica, ambas áreas de la Universidad Juan Agustín Maza. Busca la concientización de la población en acciones preventivas contra la propagación del virus COVID-19.

Germ cells play a unique role in heredity and evolution as carriers of genetic information across generations, but the study of the early stage of germ cell specification is still an overlooked issue.In this study, we documented the earliest stages of germ cell specification in Ruditapes philippinarum (the Manila clam), describing both the mechanism and timing. Initially, we focused our investigation on OSKAR, the necessary and sufficient factor involved in the earliest stage of Drosophila germline specification. On the basis of bioinformatic analyses we identified a Tudor domain-containing protein 7 (TDRD7) as the possible best candidate acting in the corresponding stage of germline specification in R. philippinarum. This hypothesis is supported by domain similarities between OSKAR and TDRD7, as well as in situ localization of the latter in putative germ cells in their earliest stage of differentiation from germline stem cells.In this light, germline specification appears to show evolutionarily conserved features, while maintaining some kind of plasticity, thus allowing differences among different taxa. Although OSKAR orthologues are not known outside insects, a corresponding role in other animals has to be carried out, possibly by different proteins with similar functions. We propose TDRD7 as a germline factor acting at early stages of germline specification in R. philippinarum, thus a good candidate as OSKAR functional-homolog.

Mitochondrial heteroplasmy is the presence of more than one type of mitochondrial genome (mtDNA) within an individual, and in most of the reported cases it seems to be an unfavourable condition. For example, heteroplasmy in humans increases the risk of common age-related disorders as neurodegenerative diseases. The only evolutionarily stable and natural heteroplasmic system in Metazoa is the Doubly Uniparental inheritance (DUI), typical of some bivalves, in which two mitochondrial lineages are present: one transmitted through eggs (F-type) and the other through sperm (M-type). While females are homoplasmic for the F-type, males have M-type-homoplasmic gametes, but heteroplasmic soma. So far, no study has investigated mitochondrial heteroplasmy at the protein level, and no analysis has been performed to clarify if it is present at tissue, cell, or organelle level. We characterized the expression of three mitochondrially-encoded proteins (ND5, CYTB, COX3) in the DUI species Ruditapes philippinarum. Specific antibodies were produced to discriminate, with immunolocalization, between the F and M form of the same protein (variants highly divergent in DUI species) in germ line and somatic tissues of females and males in different developmental stages. Unexpectedly, M-type antibodies labelled mitochondria in female primordial stem cells (PriSCs), undifferentiated germ cells, and early oocytes, while mature eggs and female somatic cells expressed only the F-type. M- and F-type expression in male somatic tissues showed mitochondrial heteroplasmy at the cell level. Interestingly, F-labelled mitochondria were detected in male PriSCs and early germ cells. We hypothesize that PriSCs and undifferentiated germ cells carry both types of mtDNA, but during gamete maturation one of the two genomes disappears, and only the sexspecific mtDNA remains, maintaining the homoplasmy of the germ line. This would imply a selective degradation of mitochondria in adulthood.

The life cycle of many organism features an early moment in which cells split between two lineages, the somatic line and the germ line. These represent, respectively, the beginning of body architecture in the present generation, and the beginning of body architecture in the following generation. The separation of germ cells is a key point for the development of an organism and occurs by specification. This process has two main modes of action, the ancestral one, called epigenesis, and the derived one, called preformation. In this work we investigated bivalves (Mollusca Bivalvia), a clade in which preformation has evolved. Despite the relevance of these animals in marine ecosystems, their mechanism of germ line speci cation and seasonal gonad reconstitution lacks a detailed study. We thus analyzed bivalve germ line development by tracking the expression of VASA, a protein often used as germ line marker. We used specific antisera produced against the VASA homolog of Ruditapes philippinarum (Subclass Heterodonta, Family Veneridae). We compared the known developmental pattern of R. philippinarum to that of two species of the Subclass Pteriomorphia, Anadara kagoshimensis (Family Arcidae) and Crassostrea gigas (Family Ostreidae), and another species of the Subclass Heterodonta, Mya arenaria (Family Myidae). The immunohistological data obtained support for the two Subclasses a similar mechanism of primordial germ cells proliferation among the columnar epithelium of the gut. Indeed, given the taxonomic separation of the analyzed species, pertaining to two highly divergent clades, we suggest that the observed seasonal migration to the reconstituting gonad, associated with the timing of gonad maturation, could be a shared feature of bivalve molluscs. The comparative study of bivalve reproductive biology using the germ line determinant VASA can add details on the preformation mechanism and help to depict a more comprehensive picture of the role that this protein plays during cell specification.

Acoustic organ of European anchovy from Adriatic Sea was described through histological and immunohistochemical analyses, in order to define a new approach for future studies of quali-quantitative variations in specimens according to sex, age and distribution.

Quali-quantitative ultrastructural changes during ontogenesis of the sagitta otoliths of the inner ear European anchovy from the Adriatic Sea were analyzed by SEM. Crystalline arrangement area presented a complex modification of the crystal growth dimension (TL around 40 mm) and crystalline arrangement complexity. These changes in the ear could be related to ecological changes in mobility and feeding.

Chronic renal disease causes olfactory impairment through an unknown mechanism. Chondrichthyes, a class of vertebrates characterized by high plasma urea concentration, lack olfactory cilia and the G protein alpha subunit olfactory-type (Gαolf - marker of the olfactory receptor neurons in vertebrates) in their olfactory neuroepithelium (ONE). Despite of that, there is no data in the literature about a direct action of urea on the ONE. In this study, Danio rerio embryos were exposed to 75 mM urea concentrations from 24 to 96 hours post fertilization. The endocytosis of Neutral Red dye in the olfactory mucosa were detected in control and urea-exposed larvae as a marker of binding and internalization of the Olfactory Receptors. The presence and distribution of Gαolf were investigated in the olfactory epithelium of control and urea-exposed larvae, using a commercial antibody. As Nitric oxide (NO) has important roles in the ONE and urea is known to affect its production, we evaluated, in control and urea-treated larvae, the presence of NO synthase (NOS) in the ONE using immunohistochemistry and a histoenzymatic reaction. We also exposed D. rerio larvae to a NOS-inhibitory molecule, NG-L-Nitro-Arginine (L-NArg), comparing them to the control and urea-treated fishes. Both Gαolf and NOS were increased in urea- and L-NArg-treated fishes than in the control, while the Neutral Red internalization was unaffected. Different hypotheses can be made about the effect of urea on NO production in the olfactory epithelium and, more speculatively, also about the role of NO on the Gαolf synthesis/degradation. Further investigations are needed in order to understand the real effects of urea on the olfactory system of pathologically and physiologically uremic vertebrates.

Crypt cells are the most recently described morphotype of fish olfactory neurons, not found in tetrapods. These cells are less abundant than ciliated and microvillar ones in the sensory epithelium. They are suspected to be involved in reproductive behaviours, even if electrophysiological investigations demonstrated some sensitivity to amino acids but failed to identify response to pheromones. In adult Poecilia reticulata the number of crypt cells is different in the two sexes. Using immunohistochemical markers, Gαolf, calretinin and S100 to identify ciliated, microvillar and crypt cells, we compared the size of their populations in juvenile guppies at various steps from birth to 90 days, when their gonads reach maturity. We observed that the number of crypt cells is sex specific, with independent developmental dynamics between males and females, while ciliated and microvillar cell populations have similar pathways. In the guppy calretinin appeared to be expressed also in a subfraction of S100-positive crypt cells (calretinin(+)-S100(-) crypt cells were not observed). We examined the changes in the amount of calretinin(+)-crypt neurons during growth: cell density reflects the variations observed in the whole population of S100(+) crypt cells in both sexes in the first 45 days. However it decreases reaching 3 months of age, in contrast with the increment registered for calretinin(-)-S100(+) cells. We estimated the total number of calretinin(+) crypt neurons and we observed that it remains very close to the value calculated for all crypt cells until 21 days, but it only slightly rises in the next 2 months, while calretinin(-) crypt neurons sensibly increment their number. We hypothesize that this new identified phenotype, percentually more represented in the first weeks of life than later, could be involved in early non-reproductive function. The known sensitivity for amino acids might indicate a role in the perception of food-related stimuli. On the contrary, calretinin(-) crypt cells become predominant reaching sexual maturity, in line with their supposed involvement in reproductive activity.

Chronic renal disease is known to alter olfactory function. Of the uraemic toxins, high urea blood levels have been suspected to induce a hyposmic condition. However, with the exception of olfactometric analysis on human subjects, no other examinations have been conducted, particularly histomorphological observations of the olfactory organ. Even if hematic administration could better mimic pathologic conditions, as first approach we opted for direct nasal exposure, to minimize potential systemic interference. In this study, we describe the effects of environmental exposure to elevated concentrations of urea (7 g/L, 13.5 g/L and 20 g/L) on the sensory mucosa of zebrafish (considered a good predictive model of mammalian toxicity) in acute (48h and 96h) and chronic (30 days) toxicity tests. We observed that lamellae maintained structural integrity and epithelial thickness was slightly reduced only after 30 days at highest urea concentration. However the ratio between the volumes of sensory and non-sensory epithelial regions sensibly decreased during exposure. Pan-neuronal labelling with anti-Hu was negatively correlated with high doses of urea (13.5 g/L and 20 g/L), thus, we investigated whether distinct neuron subtypes were equally sensitive to the toxicant. Using densitometric analysis we evaluated and compared the immunolabelling of Golf -, TRPC2- and S100-expressing cells, as representatives of ciliated, microvillous and crypt neurons, respectively. The three subpopulations responded differently to urea. In particular, crypt cells were more severely affected than the other cell types, and Golf -immunoreactivity increased when fish were exposed to low doses of urea. The moderate sensory toxicity of urea seems to be in accordance with the olfactometric measurements reported in the literature. We counted also the dividing PCNA+-cells, whose density remained constant: it is possible that other non-neuronal cells substituted olfactory neurons preserving epithelial integrity.

Introduction Urea treatment in Danio rerio affects the expression or degradation of the G protein alpha subunit olfactory type (Gαolf), involved in olfactory transduction, causing an increased presence of this protein in the olfactory cilia (Ferrando et al, 2014; Bettini et al 2015). The effects of uremia on human olfactory functions have been evaluated in some clinical studies (Landis et al 2011). We here attempt to determine a specific action of urea, among all the uremic toxins, on the olfactory capability of a vertebrate.The OAT on CTRL showed a sudden burst of swimming. The U5m fishes showed a reaction to the stimuli similar to the CTRL. The U20m did not show an appreciable response to the stimuli. The unresponsiveness lasted during the 5 days of treatment. A recovery of 15 min. in clean water did not restore the responsiveness, while 1h was enough to show an appreciable response to the stimuli, still not equal to the CTRL. Although HIC is not quantitative, it was possible to observe that the Gαolf immunoreactivity (ir) was similar in the CTRL and in the U20m fishes, while it was more intense in the U5d and the U5dR1h adults.Urea altered the response to the OAT of D. rerio with a quite rapid and reversible effect. This effect is probably independent from a mere interference of urea on the receptor-ligand binding (Ferrando et al 2014). The Gαolf ir level after 5 days was higher than control and 5-20 min. This suggests that two different mechanisms would be involved in effect of urea on olfaction capability in the long and short term. Since in the rat striatum the activation of Gαolf cause its degradation (Hervé et al 2001) we speculate that in our experimental setting the Gαolf ir increase could depend by the lack of Gαolf activation after the receptor binding with odorant molecules or by an alteration of further post signal transduction cascade.

Le cellule staminali costituiscono la sorgente indifferenziata di tutti i possibili citotipi. Esse sono alla base delle varie fasi dello sviluppo, dai processi embriogenetici e morfogenetici alla crescita dell’organismo. Nell’adulto sono state rinvenute in pressocchè tutti i tessuti, non solo quelli di origine mesenchimale, compreso quello nervoso, e intervengono nel mantenimento di popolazioni cellulari sottoposte a turnover o nella rigenerazione. Dall’embrione allo stadio adulto la loro capacità differenziativa decresce progressivamente, passando dalla totipotenza tipica dei primissimi stadi della segmentazione, alla pluripotenza, vincolata al foglietto embrionale di origine, alla multipotenza, limitata ad alcune linee cellulari (addirittura una sola nel caso dell’unipotenza). Tuttavia è stato recentemente dimostrato che anche le cellule staminali adulte possiedono plasticità: il microambiente di residenza, infatti, guida il loro destino attraverso l’espressione di specifici fattori di crescita. Modificando artificialmente la composizione biochimica della nicchia staminale (per esempio variando il terreno di coltura in vitro o trapiantando le cellule in altri tessuti) è possibile indurre cellule multipotenti a transdifferenziare verso nuove linee. Questa scoperta ha rilanciato l’interesse nella medicina rigenerativa, precedentemente orientata maggiormente verso lo studio delle cellule embrionali, ma frenata da implicazioni etiche. Tecniche sempre più innovative mirano ad ampliarne le potenzialità e gli usi: dalla cura di malattie degenerative al trapianto autologo di tessuti ed organi (ingegneria tissutale), dal recupero funzionale in seguito a paralisi o traumi alle cure anti-invecchiamento, fino alla terapia antitumorale. Nuove prospettive nascono anche dall’individuazione di nuove fonti più facilmente accessibili di cellule staminali pluripotenti, come il sangue di cordone ombelicale, il liquido amniotico ed il tessuto adiposo, e dallo studio delle iPSC, cellule staminali riprogrammate da elementi somatici adulti.

Objectives. Mesenchymal stem cells (MSC) are currently being investigated in numerous pre-clinical and clinical settings of regenerative medicine. We had previously reported (Revoltella et al., Cell Transplant. 2008; 17, 665-678) that human umbilical cord blood CD133+ stem cells transplanted IV into pre-irradiated nod-scid mice made permanently deaf by ototoxic treatment with kanamycin or intense sound, were able to engraft the cochlea and contribute to inner ear restoration, in vivo. We further investigated here whether human adult MSC derived from either bone marrow or fat (lipid suction), if injected IV to deafened nod-scid mice pre-treated with kanamycin , were able to engraft the damaged cochlea regaining hearing. Materials, Methods & Results. We tested HLA-DQa1 DNA and three human microsatellites (CODIS) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the Organ of Corti in the damaged cochlea, at 7, 31 and 60 days following MSC transplant. After 31 days, histology, immunohistochemistry, and lectin staining confirmed the repair process and stimulation ex novo of morphological recovery in the inner ear, contrasting with the lack of morphological repair in control similarly injured but non-transplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating inner ear with a very limited number of chimaeric cells. Dual color FISH analysis provided evidence of a limited positive engraftment in the inner ear and in other mouse tissues, also revealing small numbers of possible heterokaryons, probably resulting from unstable clones derived from fusion of donor with endogenous cells, up to 2 months following transplantation. Stem cells and differentiation pathways focused PCR arrays favoured to select MSC inducing the best response. Conclusions. These findings support the concept that transplanted MSC migrating to the damaged inner ear area may provide conditions for the resumption of a damaged cochlea , emerging as a potential strategy for hearing rehabilitation.

L'uomo, pur essendo un animale microsmatico, ha sempre affidato all'olfatto importantissimi compiti per la propria sopravvivenza quali la difesa dai pericoli, la marcatura del territorio, la ricerca del cibo e la riproduzione. L'olfatto costituisce un modello privilegiato per lo studio e la comprensione dei processi molecolari della trasduzione dell'informazione sensoriale laddove la disosmia sembra avere un valore semeiotico forte come sintomo di allarme per molte malattie non solo rinosinusali ma anche neurodegenerative.

The olfactory epithelium of fish includes three main types of olfactory sensory neurons (OSNs). Whereas ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs) are common to all vertebrates, a third, smaller group, the crypt cells, is exclusive for fish. Dissolved pollutants reach OSNs, thus resulting in impairment of the olfactory function with possible neurobehavioral damages, and nickel represents a diffuse olfactory toxicant. We studied the effects of three sublethal Ni2+ concentrations on the different OSN populations of zebrafish that is a widely used biological model. We applied image analysis with cell count and quantification of histochemically-detected markers of the different types of OSNs. The present study shows clear evidence of a differential responses of OSN populations to treatments. Densitometric values for Gα olf, a marker of cOSNs, decreased compared to control and showed a concentration-dependent effect in the ventral half of the olfactory rosette. The densitometric analysis of TRPC2, a marker of mOSNs, revealed a statistically significant reduction compared to control, smaller than the decrease for Gα olf and without concentration-dependent effects. After exposure, olfactory epithelium stained with anti-calretinin, a marker of c- and mOSNs, revealed a decrease in thickness while the sensory area appeared unchanged. The thickness reduction together with increased densitometric values for HuC/D, a marker of mature and immature neurons, suggests that the decrements in Gα olf and TRPC2 immunostaining may depend on cell death. However, reductions in the number of apical processes and of antigen expression could be a further explanation. We hypothesize that cOSNs are more sensitive than mOSNs to Ni2+ exposure. Difference between subpopulations of OSNs or differences in water flux throughout the olfactory cavity could account for the greater susceptibility of the OSNs located in the ventral half of the olfactory rosette. Cell count of anti-TrkA immunopositive cells reveals that Ni2+ exposure does not affect crypt cells. The results of this immunohistochemical study are not in line with those obtained by electro-olfactogram.

Hearing loss, or deafness, affects 360 million people worldwide of which about 32 million are children. Deafness is irreversible when it involves sensory hair cell death because the regenerative ability of these cells is lost in mammals after embryo development. The therapeutic strategies for deafness include hearing aids and/or implantable devices. However, not all patients are eligible or truly benefit from these medical devices. Regenerative medicine based on stem cell application could play a role in both improvement of extant medical devices and in vivo recovery of auditory function by regeneration of inner ear cells and neurons. A review of recent literature on the subject indicates that two promising approaches to renewal and differentiation of cochlear tissues are transplantation of stem cells and in situ administration of growth factors. Rather than directly regenerating dead cells, these procedures apparently induce, through various pathways, differentiation of resident cochlear cells. More studies on the possible adverse effects of transplanted cells and the recovery of tonotopic sensorineural activity or required. To date, no reliable clinical results have been obtained in the field of cochlear regeneration.

Reconstitution and renewal of tissues are key topics in developmental biology. In this brief work, we analyzed the wintry spent phase of the reproductive cycle in the Manila clam Ruditapes philippinarum (Bivalvia, Veneridae) in order to study the gonad rebuilding that in this species occurs at the beginning of the warmer months. We labeled VASA homolog protein-a germ cell marker-and compared the histological observations of the spent phase with those of the previously analyzed gametogenic phase. In R. philippinarum, during the reproductive season, most of the body mass is represented by sack-like structures (acini) full of developing gametes. In that period, VASA-stained cells are present at the basal pole of the gut epithelium, in the connective tissue, and around the acini. We here show that during the spent phase large portions of the intestine lack such cell type, except for some areas showing a few faintly VASA-stained cells. Cells with similar nuclear morphology are present among loosely organized cells of connective tissue, sometimes as single units, sometimes in small groups, rarely partially organized in primordial gonadic structures. These observations match the findings of RNA-targeting studies that during the spent phase identified the source of bivalve germ cells within the connective tissue in the form of quiescent units and add new information on the possible maintenance of VASA-stained, multipotent cells among the batiprismatic cells of the intestine during the whole life span of these bivalves.

Background Cutaneous squamous cell carcinoma (cSCC) is an uncommon disease affecting koi carp (Cyprinus carpio). Cutaneous papilloma (carp pox) is a benign epidermal proliferation reported in koi and has been shown to be caused by Cyprinid Herpesvirus 1 (CyHV1). Hypothesis/Objectives Histological, ultrastructural and molecular investigations were carried out aiming to investigate the aetiology of cSCC within archived tissue samples. Animals Surgical samples of masses located on the integument, fins and lips of 13 koi carp belonging to different private owners were included in this retrospective study. Methods CyHV1 DNA and RNA presence were investigated in five cSCC formalin-fixed paraffin-embedded tissue samples to recognize CyHV1 presence and its replication activity. Results All cases were histologically diagnosed as cSCC. The ultrastructural observations confirmed the squamous differentiation of neoplastic epithelial cells, which showed abundant tonofilament bundles and desmosomes. Although no virus particles were revealed ultrastructurally, the molecular investigation detected viral DNA in five epidemiologically unrelated cSCC. Viral transcript analysis revealed no evidence for viral replication in the tested cSCC, which could be consistent with latent infection. Conclusions and clinical importance These findings illustrate the frequent association of carp cSCC with CyHV1, although a direct cause–effect relationship cannot be established at this time. Therefore, surveillance programmes should take into account the suspected viral origin of cSCC to better inform prevention and control of CyHV1 in the future.

Local drug delivery based on nanoparticles (NP) represents a novel strategy to improve inner ear treatments. The intratympanic delivery of NP may be suitable to treat or prevent hearing loss originating from damage to hair cells and spiral ganglion neurons in the cochlea. Numerous experimental studies support in vitro and in vivo the biocompatibility of NP, their physical stability, target specificity, cell/tissue uptake and ability to internalize therapeutic agents. The topical use of NP helps to reduce the amount of drug required and avoid systemic side effects. This review focuses on recent findings and applications of different NP systems locally delivered to the inner ear. The perspectives for clinical application of NP in inner ear drug delivery are also discussed.

Germ line segregation can occur during embryogenesis or after embryogenesis completion, with multipotent cells able to give rise to both germ and somatic cells in the developing juvenile or even in adulthood. These undifferentiated cells, in some animals, are self-renewing stem cells. In all these cell lineages, the same set of genes, among which vasa, appears to be expressed. We traced VASA expression during the peculiar gonad rebuilding of bivalves to verify its presence from undifferentiated germ cells to mature gametes in an animal taxon in which the mechanism of germ line establishment is still under investigation. We utilized antibodies produced against VASPH, VASA homolog of Ruditapes philippinarum (Subclass Heterodonta), to compare the known expression pattern of R. philippinarum to two species of the Subclass Pteriomorphia, Anadara kagoshimensis and Crassostrea gigas, and another species of the Subclass Heterodonta, Mya arenaria. The immunohistological data obtained support a conserved mechanism of proliferation of “primordial stem cells” among the simple columnar epithelium of the gut, as well as in the connective tissue, contributing to the seasonal gonad reconstitution. Given the taxonomic separation of the analyzed species, we suggest that the process could be shared in bivalve molluscs. The presence of germ cell precursors in the gut epithelium appears to be a feature in common with model organisms, such as mouse, fruit fly, and human. Thus, the comparative study of germ line establishment can add details on bivalve development, but can also help to clarify the role that VASA plays during germ cell specification. Electronic supplementary material The online version of this article (doi:10.1007/s00418-017-1560-x) contains supplementary material, which is available to authorized users.

The peripheral olfactory system of fish is in direct contact with the external aqueous environment, so dissolved contaminants can easily impair sensory functions and cause neurobehavioral injuries. The olfactory epithelium of fish is arranged in lamellae forming a rosette in the olfactory cavity and contains three main types of olfactory sensory neurons (OSNs): ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs), common to all vertebrates, and a third minor group of olfactory neurons, crypt cells, absent in tetrapods. Since copper is a ubiquitously diffusing olfactory toxicant and a spreading contaminant in urban runoff, we investigated the effect of low copper concentration on the three different OSNs in the olfactory epithelium of zebrafish, a model system widely used in biological research. Image analysis was applied for morphometry and quantification of immunohistochemically detected OSNs. Copper exposure resulted in an evident decrease in olfactory epithelium thickness. Moreover, after exposure, the lamellae of the dorsal and ventral halves of the olfactory rosettes showed a different increase in their sensory areas, suggesting a lateral migration of new cells into non-sensory regions. The results of the present study provide clear evidence of a differential response of the three neural cell populations of zebrafish olfactory mucosa after 96 h of exposure to copper ions at the sublethal concentration of 30 µg/L-1. Densitometric values of cONS, immunostained with anti-G αolf, decreased of about 60% compared to the control. When the fish were transferred to water without copper addition and examined after 3, 10 and 30 days, we observed a partial restoration of anti-Gαolf staining intensity to normal condition. The recovery of cOSNs appeared sustained by neuronal proliferation, quantified with anti-PCNA immunostaining, in particular in the early days after exposure. The densitometric analysis applied to mOSNs, immunostained with anti-TRPC2, revealed a statistically significant decrease of about 30% compared to the control. For cOSNs and mOSNs, the decrement in staining intensity may be indicative of cell death, but reduction in antigen expression may not be excluded. In the post-exposure period of 1 month we did not find recovery of mOSNs. We hypothesize that cOSNs are more sensitive than mOSNs to copper treatment, but also more prompted to tissue repair. Anti-TrkA-immunopositive crypt cells appeared not to be affected by copper exposure since statistical analysis excluded any significant difference between the control and treated fish. Comparative studies on OSNs would greatly enhance our understanding of the mechanisms of olfaction.

The olfactory and vomeronasal systems of vertebrates are characterised by neurogenesis occurring throughout life. The regenerative ability of olfactory receptor neurons relies on specific glial cells, the olfactory and vomeronasal axon-surrounding cells. Numerous studies have examined mammalian olfactory ensheathing cells which are considered potential candidates for spinal cord injury repair using cell-based therapy. With regard to non-mammalian vertebrates, limited information is available on these glial cells in fish, and there is no information on them in terrestrial anamniotes, the amphibians. In the present research, we studied the immunocytochemical characteristics of axon-surrounding cells in Ambystoma mexicanum. Urodeles have relatively simple olfactory and vomeronasal systems, and represent a good model for studying ensheathing cells in extant representatives of basal tetrapods. Sections from the decalcified heads of A. mexicanum were immunocytochemically processed for the detection of proteins used in research on mammalian olfactory-ensheathing cells. S100, GFAP and NCAM were clearly observed. p75NTR, Gal-1 and PSA-NCAM showed weak staining. No vimentin immunopositivity was observed. The corresponding areas of the olfactory and vomeronasal pathways displayed the same staining characteristics, with the exception of Gal-1, p75NTR and PSA-NCAM in the mucosae. The degree of marker expression was not uniform throughout the sensory pathways. In contrast to fish, both olfactory and vomeronasal nerves displayed uniform staining intensity. This study showed that some markers for mammalian and fish-ensheathing glia are also applicable in urodeles. The olfactory systems of vertebrates show similarities, and also clear dissimilarities. Further investigations are required to ascertain the functional significance of these regional and interspecific differences.

Monitoring of BCR-ABL1 levels by quantitative PCR (qPCR) is essential for the management of CML patients treated with TKIs. Because of intrinsic limits, qPCR does not appear to be an optimal assay to select the best candidates to TKIs discontinuation. Up to 40% of CML patients treated with TKIs can achieve a Deep Molecular Response (DMR), but only 50% maintain a stable Treatment Free Remission (TFR) after therapy discontinuation. Digital PCR (dPCR), giving an absolute quantification of BCR-ABL1, is expected to be more sensitive and accurate than qPCR in the assessment of molecular MRD. dPCR performed on a QuantStudio 3D Digital PCR System (Life Technologies) by using a TaqMan-MGB probes targeting the BCR-ABL1 transcript was used to comparatively analyse 102 CML patients with Major (MR3.0 = 31 cases) or Deep (MR4.0= 33 cases; MR4.5 = 24 cases and MR5.0 = 14 cases) molecular response. Preliminary results showed that: a) ≥77% of deep responders (MR4.0, MR4.5 and MR5.0) fell under the value of 0.468 BCR-ABL1 copies/▢l indicated by the ROC analysis as the value below which the patients with lower levels of MRD might be dissected (spec.=71%, sens.=77%; AUC=0,79); b) BCR-ABL1 transcript levels were detectable by dPCR also in cases resulted undetectable by qPCR. In this study, we analysed the BCR-ABL1 transcript levels by dPCR in 207 samples related to 102 CML patients. Fourteen (14%) out of 102 CML patients discontinued the TKIs therapy. Among them, 3 patients (21%) lost DMR and all of them showed dPCR values > 0.468 BCR-ABL1 copies/▢l (previously described as cut-off for a deep MRD), while 11 (79%) maintained a stable DMR and 9 of them (82%) fell under the value of 0.468 BCR-ABL1 copies/▢l. These latter patients stratified in different DMR classes by qPCR and all had undetectable level of BCR-ABL1 by qPCR. In 149 out of 207, qPCR revealed DMR. They were: MR4.0= 59 samples; MR4.5= 61 cases; MR5.0 = 29 cases. One hundred twenty-five (84%) fell under the value of 0.468 BCR-ABL1 copies/▢l. A linear regression analysis in these samples did not show any correlation between BCR-ABL1 copies/▢l detected by qPCR with the ones detected by dPCR (R2 < 0.01). On the basis of our preliminary results, TFR seems to be correlated to the maintenance of dPCR values < 0.468 BCR-ABL1 copies/▢l. The concordance between qPCR and dPCR quantification in patients with DMR and with BCR-ABL1 copies/▢l value < 0.468 was poor. Patients with dPCR values < 0.468 BCR-ABL1 copies/▢l may have 75% of probability to maintain TFR status. These results suggest that dPCR may be more sensitive to detect the MRD and could be more useful for DMR monitoring and for dissecting the best candidate to discontinuation of therapy with TKIs. Disclosures Tiribelli: Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Castagnetti:Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; ARIAD Pharmaceuticals: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Soverini:Novartis: Consultancy; Ariad: Consultancy; Bristol-Myers Squibb: Consultancy. Rosti:Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Martinelli:Roche: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Genentech: Consultancy; Amgen: Consultancy; MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Genentech: Consultancy; Amgen: Consultancy; Novartis: Speakers Bureau; MSD: Consultancy.

Chronic renal disease is known to alter olfactory function, but the specific changes induced in olfactory organs during this process remain unclear. Of the uraemic toxins generated during renal disease, high levels of urea are known to induce hyposmic conditions. In this study, the effects of environmental exposure to elevated concentrations of urea (7, 13.5 and 20 g L(-1)) on the sensory mucosa of zebrafish in acute toxicity and chronic toxicity tests were described. It was observed that lamellae maintained structural integrity and epithelial thickness was slightly reduced, but only following exposure to the highest concentrations of urea. Pan-neuronal labelling with anti-Hu revealed a negative correlation with levels of urea, leading to investigation of whether distinct neuronal subtypes were equally sensitive. Using densitometric analysis of immunolabelled tissues, numbers of Gα olf-, TRPC2- and TrkA-expressing cells were compared, representing ciliated, microvillous and crypt neurons, respectively. The three neuronal subpopulations responded differently to increasing levels of urea. In particular, crypt cells were more severely affected than the other cell types, and Gα olf-immunoreactivity was found to increase when fish were exposed to low doses of urea. It can be concluded that exposure to moderate levels of urea leads to sensory toxicity directly affecting olfactory organs, in accordance with the functional olfactometric measurements previously reported in the literature.