Andrea Gambarelli

Toxicity of antimony trioxide nanoparticles on human hematopoietic progenitor cells and comparison to cell lines.

Nanoparticles (NPs) are materials with one dimension in the range of 1-100 nm. The toxicity of NPs remains widely unknown and still poses concerns, due to the peculiar characteristics of materials in the nano-size range. We analyze the toxicity of seven NPs ((Fe2O3, Fe3O4, Sb2O3, Au, TiO2, Co, and Ag) on primary cultures of human hematopoietic progenitor cells from the bone marrow of healthy donors with CFU assays, and show that antimony oxide (Sb2O3) NPs and cobalt (Co) NPs have a toxic effect, while the other NPs have no effect at the tested concentrations (5, 25 and 100 microg/ml). While Co NPs suspension is toxic to both erythroid and granulocytic-monocytic precursors, Sb2O3 NPs at 5 microg/ml are specifically toxic to erythroid colony development, suggesting a highly selective type of toxicity. With liquid culture assays we show that Sb2O3 NPs impair the proliferation of erythroid progenitors, while no toxic effect is observed when Sb2O3 NPs are added during erythroid differentiation. CFU assays and liquid culture assays on seven human cell lines of hematopoietic origin (K562, HL-60, CEM, CEM-R, Thp-1, Jurkat, and Molt-4) show that, contrary to what observed on primary cultures of bone marrow progenitors, Sb2O3 NPs have no toxic effect on proliferation of any of the cell lines, raising concerns about the use of immortalized cell lines for nanotoxicology tests.

Investigation of the Presence of Inorganic Micro- and Nanosized Contaminants in Bread and Biscuits by Environmental Scanning Electron Microscopy

A European project called “Nanopathology” allowed to develop a new diagnostic tool through which the presence of inorganic particulate matter in pathological human tissues of the digestive tract could be shown. This unexpected evidence induced the authors to put forward the hypothesis that that sort of contamination was present in ingested food. In order to demonstrate this hypothesis, 86 samples of wheat bread and 49 of wheat biscuits from 14 different countries were analyzed by means of an Environmental Scanning Electron Microscopy to detect inorganic, micro-, and nano-scaled contaminants. The X-ray microprobe of an Energy Dispersion Spectroscope was employed to identify their chemical composition. The results indicate that 40% of the samples analyzed contained foreign bodies as ceramic and metallic debris of probable environmental or industrial origin. Because of the great variety of chemical composition of the particulate matter, those contaminants were listed according to the most expressed element. The majority of these debris are not biodegradable, some are chemically toxic, and none of them have any nutritive value. The work discusses the possible origin of such a pollution and the role that it can play on human life.

Immunolocalisation of vasoactive intestinal peptide and substance P in the developing gut of Dicentrarchus labrax (L.)

This study was carried out on the sea bass (Dicentrarchus labrax) to follow, during development, the appearance and distribution of substance P (SP) and vasoactive intestinal peptide (VIP), which act on gut motility. The results suggest that SP and VIP play an important role as neuromodulators, influencing the motility of the digestive tract starting from the early stages of gut development, even prior to exotrophic feeding. In the peptidergic nervous system, the appearance of immunoreactivity to SP began at the rectum and followed a distal to proximal gradient, whereas for VIP, it began proximally and progressed along a proximal to distal gradient. The two peptides also appeared in gut epithelial cells. In some regions, all the cells were positive. From this distribution of positive cells, we conclude that these peptides may also have other roles, besides being neurotransmitters in the enteric nervous system and hormones of the gastro-entero-pancreatic system. VIP and SP might have paracrine and/or autocrine activity in the physiological maturation of the gut epithelium, as it has already been hypothesised for other peptides.

Immunolocalization of corticotropin-releasing factor (CRF) and corticotropin-releasing factor receptor 2 (CRF-R2) in the developing gut of the sea bass (Dicentrarchus labrax L.).

Our previous data indicated an important role for adrenocorticotropic (ACTH)-like molecules co-operating with macrophages to control the modifications in body homeostasis during the first period of the life of sea bass (up to 30 days post-hatching) before the lymphoid cells have reached complete maturation. The aim of the study was to determine the immunolocalization of corticotropin-releasing factor (CRF), which is a very important mediator of stress-related responses. Our data showed that immunostaining for CRF is localized already at 8 days after hatching in nerve fibers of the gastrointestinal tract wall from the pharynx to the anterior gut, when the larvae are still feeding on yolk. This pattern of immunolocalization appeared similar to that in 24-day-old larvae, but at this stage there were also large cells immunopositive to CRF located in the wall of the midgut and hindgut. Lipopolysaccharide (LPS) treatment, which is a known stimulator of stress hormone responses, did not modify the CRF immunostaining pattern, though it did affect the immunolocalization of the peripheral CRF receptor, i.e. CRF-R2. Immunolocalization of CRF-R2 appeared in nerve fibers of the gut wall in larvae fixed 1h after the end of lipopolysaccharide (LPS) treatment. The present results suggest that CRF plays important autocrine and/or paracrine roles in the early immune responses at the gut level in the larval stages of sea bass (Dicentrarchus labrax L.) as already proposed for ACTH. Moreover, our studies taken together with other research on fish, in comparison with mammals, suggest a phylogenetically old role of CRF in immune-endocrine interactions.

Structure-function relationships in the integument of Salamandra salamandra during ontogenetic development

Morphological, cytological and transport properties of the integument of Salamandra salamandra were investigated during natural ontogenetic development, from birth to adult. Three stages were operationally defined: I, larvae, from birth to metamorphosis; II, metamorphosis (judged externally by the colour change and loss of the gills); and III, post‐metamorphosis to adult. Pieces of skin were fixed at various stages for immunocytochemical examinations, and the electrical properties were investigated on parallel pieces. Distinct cellular changes take place in the skin during metamorphosis, and lectin (PNA, WGA and ConA) binding indicates profound changes in glycoprotein composition of cell membranes, following metamorphosis. Band 3 and carbonic anhydrase I (CA I) were confined to mitochondria‐rich (MR)‐like cells, and were detected only in the larval stage. CA II on the other hand, was detected both in MR‐like and in MR cells following metamorphosis. The electrical studies show that the skin becomes more tight (transepithelial resistance increases) upon metamorphosis, followed by manifestation of amiloride‐sensitive short‐circuit current (I sc) indicating that functional Na+ uptake has been acquired. The skin of metamorphosed adults had no finite transepithelial Cl− conductance, and band 3 was not detected in its MR cells. The functional properties of MR‐like and MR cells remain to be established.