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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.