Bice Avallone

Chitosan-mediated stimulation of macrophage function

According to the modern definition of biocompatibility, a biocompatible material need not be inert but be bioactive. A benign reactivity implies that the reactivity has to be appropriate for the intended use. Chitosan, a non-acetylated or partially deacetylated chitin (a linear homopolymer of beta (1-4)-linked N-acetylglucosamine) has been proposed as a biomaterial because of its apparent satisfactory biocompatibility. The present investigation demonstrates that chitosan has an in vitro stimulatory effect on both macrophage nitric oxide (NO) production and chemotaxis. The macrophage NO secretion is attributed to the N-acetylglucosamine unit of the chitosan molecule rather than to the glucosamine residue (28 and 15 microM NO respectively). Moreover, the immune stimulatory effect of chitosan was very specific since other glycosaminoglycans, such as N-acetyl-D-mannosamine and N-acetyl-D-galactosamine, had no effects on NO production (5 and 8 respectively). In vivo experiments strengthen this hypothesis. Transmission electron microscopy analysis identifies the presence of many leucocytes in the specimens after 14 d post-implantation, showing poor healing processes (i.e. fibroblast proliferation and collagen deposition) that characterize the tissue repair at this time in our animal model.

Assembly of the Auditory Circuitry by a Hox Genetic Network in the Mouse Brainstem

Rhombomeres (r) contribute to brainstem auditory nuclei during development. Hox genes are determinants of rhombomere-derived fate and neuronal connectivity. Little is known about the contribution of individual rhombomeres and their associated Hox codes to auditory sensorimotor circuitry. Here, we show that r4 contributes to functionally linked sensory and motor components, including the ventral nucleus of lateral lemniscus, posterior ventral cochlear nuclei (VCN), and motor olivocochlear neurons. Assembly of the r4-derived auditory components is involved in sound perception and depends on regulatory interactions between Hoxb1 and Hoxb2. Indeed, in Hoxb1 and Hoxb2 mutant mice the transmission of low-level auditory stimuli is lost, resulting in hearing impairments. On the other hand, Hoxa2 regulates the Rig1 axon guidance receptor and controls contralateral projections from the anterior VCN to the medial nucleus of the trapezoid body, a circuit involved in sound localization. Thus, individual rhombomeres and their associated Hox codes control the assembly of distinct functionally segregated sub-circuits in the developing auditory brainstem.

Detrimental effects of Bartonella henselae are counteracted by L-arginine and nitric oxide in human endothelial progenitor cells.

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae. Nitric oxide (NO) and its precursor l-arginine (l-arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l-arg (1–30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l-arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella-infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l-arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.

Calcification processes in the chick otoconia and calcium binding proteins : patterns of tetracycline incorporation and calbindin-D28K distribution

In order to clarify the otoconia formation and turnover, tetracycline, an antibiotic that precipitates at calcifying fronts and serves as a fluorescent marker, was injected into eggs at different stages of chick embryonic development, as well as into postnatal chicken and into adult animals. The changes in the intensity, location patterns and time course of fluorescent labelling in each examined stage in the otolithic organs was studied. The presence and distribution of calbindin (CB)-D28K, one of the calcium-binding proteins constantly found in the mammalian and chicken cochlea and also in otolithic membrane of some adult mammals, was studied. Results in embryonal stages, postnatal and adult animals allow us to postulate that otoliths are mainly produced during the embryonal phase, but they may also be produced throughout the whole life span. Results also indicate that otoconia are dynamic structures which undergo turnover. The correspondence between the patterns of CB-D28K immunoreactivity and tetracycline fluorescence may indicate that CB-D28K participates in the formation of otoconia.

Structural and functional changes in the zebrafish (Danio rerio) skeletal muscle after cadmium exposure.

This report describes the alterations induced by an environmentally realistic concentration of cadmium in skeletal muscle fibre organization, composition, and function in the teleost zebrafish. Results demonstrate that the ion induces a significant quantitative and qualitative deterioration, disrupting sarcomeric pattern and altering glycoprotein composition. These events, together with a mitochondrial damage, result in a significant reduction in swimming performance. In conclusion, the evidence here collected indicate that in presence of an environmental cadmium contamination, important economic (yields in fisheries/aquaculture), consumer health (fish is an important source of proteins), and ecological (reduced fitness due to reduced swimming performance) consequences can be expected.

Ofd1 Controls Dorso-Ventral Patterning and Axoneme Elongation during Embryonic Brain Development

Oral-facial-digital type I syndrome (OFDI) is a human X-linked dominant-male-lethal developmental disorder caused by mutations in the OFD1 gene. Similar to other inherited disorders associated to ciliary dysfunction OFD type I patients display neurological abnormalities. We characterized the neuronal phenotype that results from Ofd1 inactivation in early phases of mouse embryonic development and at post-natal stages. We determined that Ofd1 plays a crucial role in forebrain development, and in particular, in the control of dorso-ventral patterning and early corticogenesis. We observed abnormal activation of Sonic hedgehog (Shh), a major pathway modulating brain development. Ultrastructural studies demonstrated that early Ofd1 inactivation results in the absence of ciliary axonemes despite the presence of mature basal bodies that are correctly orientated and docked. Ofd1 inducible-mediated inactivation at birth does not affect ciliogenesis in the cortex, suggesting a developmental stage-dependent role for a basal body protein in ciliogenesis. Moreover, we showed defects in cytoskeletal organization and apical-basal polarity in Ofd1 mutant embryos, most likely due to lack of ciliary axonemes. Thus, the present study identifies Ofd1 as a developmental disease gene that is critical for forebrain development and ciliogenesis in embryonic life, and indicates that Ofd1 functions after docking and before elaboration of the axoneme in vivo.

Gentamicin ototoxicity in the saccule of the lizard Podarcis Sicula induces hair cell recovery and regeneration

There is little information available on the susceptibility of reptilian saccule hair cells to ototoxin-induced sensory damage. In this study, we report morphological evidence of hair cell recovery and regeneration after damage induced by gentamicin in the saccule of a lizard. We perform morphological analysis using scanning electron microscopy and confocal laser scanning microscopy with actin and calbindin as markers for hair cells and tubulin as a marker for supporting cells. The data were consistent: gentamicin induced damage in the hair cells, and the damage increased with increasing duration of treatment. Initially, the saccule appeared unhealthy. Subsequently, the sensory hair cells became compromised, with fused stereovilli, followed by widespread loss of hair cell bundles from the hair cells. Finally, numerous hair cells were lost. Morphologically, the saccule appeared normal 28days after gentamicin treatment. Using a mitogenic marker, we tested whether or not there is hair cell regeneration following administration of gentamicin. We found evidence of bromodeoxyuridine incorporation first in supporting cell nuclei and subsequently in hair cell nuclei. This indicates that a process of sensory epithelium repair and hair cell regeneration occurred, in both extrastriolar and striolar regions, and that the recovery was due to both the proliferation of supporting cells and, as seems likely, self-repair of hair cell bundles.

Localization of calbindin D-28K in the otoconia of lizard Podarcis sicula.

The membranous labyrinth of lizard Podarcis sicula contains calcite and aragonite crystals. Saccule, utricle and lagena contain calcite crystals while aragonite crystals are present only in the saccule where they are very abundant. We have recently demonstrated the presence of calbindin D-28K in the organic matrix of lizard P. sicula otoconia. In order to define its localization, since calbindin modulates cellular Ca2+ level, otoconia from utricle and lagena were collected separately from those from saccule and then otoconial proteins were extracted. Immunoblot assay on proteins extracted from the otoconia and confocal laser scanning microscope analyses of otoconia using monoclonal anti-calbindin D-28K antibodies indicated that calbindin D-28K is a protein typical of aragonite crystals.

Calbindin D28K is a component of the organic matrix of lizard Podarcis sicula otoconia

The factors controlling otoconia growth are not well known but it seems that the type of proteins contained in the otoconia regulates the initiation and/or the subsequent rates of crystal growth determining the morphology and the size of the final crystal. In order to clarify the mechanism of otoconia formation and their turnover, major proteins contained in the otoconia from the maculae of the saccule, utricle and lagena of inner ear of lizard Podarcis sicula were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie staining of SDS-PAGE resulted in a major broad band of 15 kDa and four other bands of 21, 28, 45 and 97 kDa. The proteins of 15, 21, 28 and 45 kDa were separated by high-pressure liquid chromatography on a C-4-reverse-phase column and the incubation of blots with monoclonal anti-Calbindin D28K antibodies indicated that the band of 28 kDa was Calbindin D28K, a calcium-binding protein.

Cadmium effects on the retina of adult Danio rerio.

The aim of this work is to describe the effects of cadmium pollution on the vision of adult zebrafish, Danio rerio. Retinal morpho-cytological alterations were investigated by light and electron microscopy, while the functionality of cadmium-exposed retinae was assessed by re-illumination behavioral tests with white or colored light. Our results demonstrate that cadmium toxicity causes significant degeneration and loss of organization at both macro and microscopic levels. These alterations impair functional responses particularly through an increase in light sensitivity. Metallothioneins were not seen to be up-regulated, while the recovery of visual acuity is due to a regenerative process by Müller cells.