Pasqualina Maria Picciotti

Trafficking and Postsecretory Events Responsible for the Formation of Secreted Human Salivary Peptides A Proteomics Approach

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and “S type” Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.

Pathogenesis of presbycusis in animal models: A review

Presbycusis is the most common cause of hearing loss in aged subjects, reducing individuals communicative skills. Age related hearing loss can be defined as a progressive, bilateral, symmetrical hearing loss due to age related degeneration and it can be considered a multifactorial complex disorder, with both environmental and genetic factors contributing to the aetiology of the disease. The decline in hearing sensitivity caused by ageing is related to the damage at different levels of the auditory system (central and peripheral). Histologically, the aged cochlea shows degeneration of the stria vascularis, the sensorineural epithelium, and neurons of the central auditory pathways. The mechanisms responsible for age-associated hearing loss are still incompletely characterized. This work aims to give a broad overview of the scientific findings related to presbycusis, focusing mainly on experimental studies in animal models.

Vascular endothelial growth factor (VEGF) expression in noise-induced hearing loss.

Noise-induced hearing loss has been associated with alterations in cochlear blood flow. Our study analyzed the expression of Vascular Endothelial Growth Factor (VEGF) and its functional receptors, Flt-1 and Flk-1, in the cochlear structures of noise-exposed and unexposed guinea pigs. VEGF is a prototypical angiogenic agent, with multiple functions on vascular biology, ranging from vascular permeability to endothelial cell migration, proliferation, differentiation, and survival. Acoustic trauma was induced by a continuous pure tone of 6 kHz, at 120 dB SPL for 30 min. Auditory function was evaluated by electrocochleographic recordings at 2-20 kHz for 7 days. Noise-induced cochlear morphological changes were studied by immunohistochemistry and scanning electron microscopy. The expression of VEGF and its receptors was examined by immunohistochemistry and western blotting analysis. The hearing threshold shift reached a level of 60 dB SPL on day 1 after trauma and underwent a partial recovery over time, reaching a value of about 20 dB SPL on day 7. Outer hair cell loss was more prominent in the area located 14-16 mm from the apex. Increased cochlear VEGF expression was observed in noise-exposed animals, in particular at the level of stria vascularis, spiral ligament, and spiral ganglion cells. No changes were observed in the expression of VEGF-receptors. Our data suggest a role for VEGF in the regulation of the vascular network in the inner ear after acoustic trauma and during auditory recovery, with potentially important clinical and therapeutic implications.

Age-dependent modifications of expression level of VEGF and its receptors in the inner ear

The mechanisms responsible for age-associated hearing loss are still incompletely characterized. In this study, we used a murine model of age-dependent hearing loss and evaluated whether this condition is associated with vascular modifications of the structures of the inner ear. We used old C57BL/6J mice that are affected by rapid and severe age-related hearing loss, and analyzed the expression pattern of vascular endothelial growth factor (VEGF), a prototypical angiogenic cytokine, and its receptors Flt-1 and Flk-1 in the inner ear. We report for the first time morphological and quantitative data about the expression of these crucial angiogenic molecules in the murine cochlea. We also show that in this animal model, cochlear VEGF expression is significantly reduced as a function of age. Our findings provide new evidence of possible interdependent relationships between aging, VEGF, and presbycusis, suggesting that vascular abnormalities might play a role in aging-associated hearing loss, with potentially important fundamental and clinical implications.

Postural control in horizontal benign paroxysmal positional vertigo.

Abstract Sixteen patients affected by benign paroxysmal positional vertigo of the horizontal semicircular canal (BPPV-HSC) were investigated by means of dynamic posturography (DP) and during bithermal caloric stimulation. Data were compared to data from 40 patients with benign paroxysmal positional vertigo of the posterior semicircular canal (BPPV-PSC) and 20 healthy controls. No postural deficit was observed before or after a liberative Lempert’s manoeuvre when patients were compared to control subjects. BPPV-PSC postural scores were significantly impaired compared to scores from the BPPV-HSC group. A residual significant postural impairment was also observed after a successful liberative manoeuvre in the BPPV-PSC group. Electronystagmographic recordings before recovery revealed significant hypoexcitability of the affected ear in 8/16 patients of the BPPV-HSC group. After the liberative manoeuvre, a symmetric bilateral response to caloric stimulation was recorded in all patients. Three main conclusions can be drawn from the present data. First, disorders of the horizontal semicircular canal do not change postural control. Second, dynamic posturography can detect the postural imbalance due to posterior semicircular canal dysfunction even after resolution of paroxysmal vertigo attacks. Third, utricular dysfunction can be ruled out as a cause of the residual postural deficit observed in BPPV-PSC patients. Therefore the recovery delay observed even 1 month after the liberative manoeuvre in the BPPV-PSC-group might be due to the persistence of small amounts of residual debris in the canal, to paralysis of ampullar receptors, or to the time needed for central vestibular re-adaptation.

Chrono-Proteomics of Human Saliva: Variations of the Salivary Proteome during Human Development

An important contribution to the variability of any proteome is given by the time dimension that should be carefully considered to define physiological modifications. To this purpose, whole saliva proteome was investigated in a wide age range. Whole saliva was collected from 17 preterm newborns with a postconceptional age at birth of 178-217 days. In these subjects sample collection was performed serially starting immediately after birth and within about 1 year follow-up, gathering a total of 111 specimens. Furthermore, whole saliva was collected from 182 subjects aged between 0 and 17 years and from 23 adults aged between 27 and 57 years. The naturally occurring intact salivary proteome of the 316 samples was analyzed by low- and high-resolution HPLC-ESI-MS platforms. Proteins peculiar of the adults appeared in saliva with different time courses during human development. Acidic proline-rich proteins encoded by PRH2 locus and glycosylated basic proline-rich proteins encoded by PRB3 locus appeared following 180 days of postconceptional age, followed at 7 months (±2 weeks) by histatin 1, statherin, and P-B peptide. The other histatins and acidic proline-rich proteins encoded by PRH1 locus appeared in whole saliva of babies from 1 to 3 weeks after the normal term of delivery, S-type cystatins appeared at 1 year (±3 months), and basic proline-rich proteins appeared at 4 years (±1 year) of age. All of the proteinases involved in the maturation of salivary proteins were more active in preterm than in at-term newborns, on the basis of the truncated forms detected. The activity of the Fam20C kinase, involved in the phosphorylation of various proteins, started around 180 days of postconceptional age, slowly increased reaching values comparable to adults at about 2 years (±6 months) of age. Instead, MAPK14 involved in the phosphorylation of S100A9 was fully active since birth also in preterm newborns.

Vestibulo-Ocular Reflex Modification after Virtual Environment Exposure

Immersion in an illusory world is possible by means of virtual reality (VR), where environmental perception is modified by artificial sensorial stimulation. The application of VR for the assessment and rehabilitation of pathologies affecting the vestibular system, in terms of both diagnosis and care, could represent an interesting new line of research. Our perception of reality is in fact based on static and dynamic spatial information perceived by our senses. During head movements in a virtual environment the images on the display and the labyrinthine information relative to the head angular accelerations differ and therefore a visuo-vestibular conflict is present. It is known that mismatches between visual and labyrinthine information may modify the vestibulo-oculomotor reflex (VOR) gain. We studied the post-immersion modifications in 20 healthy subjects (mean age 25 years) exposed to a virtual environment for 20 min by wearing a head-mounted display. VOR gain and phase were measured by means of harmonic sinusoidal stimulation in the dark before, at the end of and 30 min after VR exposure. A VOR gain reduction was observed in all subjects at the end of VR exposure which disappeared after 30 min. Our data show that exposure to a virtual environment can induce a temporary modification of the VOR gain. This finding can be employed to enable an artificial, instrumental modification of the VOR gain and therefore opens up new perspectives in the assessment and rehabilitation of vestibular diseases.Immersion in an illusory world is possible by means of virtual reality (VR), where environmental perception is modified by artificial sensorial stimulation. The application of VR for the assessment and rehabilitation of pathologies affecting the vestibular system, in terms of both diagnosis and care, could represent an interesting new line of research. Our perception of reality is in fact based on static and dynamic spatial information perceived by our senses. During head movements in a virtual environment the images on the display and the labyrinthine information relative to the head angular accelerations differ and therefore a visuo-vestibular conflict is present. It is known that mismatches between visual and labyrinthine information may modify the vestibulo-oculomotor reflex (VOR) gain. We studied the post-immersion modifications in 20 healthy subjects (mean age 25 years) exposed to a virtual environment for 20 min by wearing a head-mounted display. VOR gain and phase were measured by means of harmonic sinusoidal stimulation in the dark before, at the end of and 30 min after VR exposure. A VOR gain reduction was observed in all subjects at the end of VR exposure which disappeared after 30 min. Our data show that exposure to a virtual environment can induce a temporary modification of the VOR gain. This finding can be employed to enable an artificial, instrumental modification of the VOR gain and therefore opens up new perspectives in the assessment and rehabilitation of vestibular diseases.

Age-related Histopathological Changes of the Stria Vascularis: An Experimental Model: Cambios histopatólogicos relacionados con la edad en la estría vascular: Un modelo experimental

The stria vascularis (SV) of 3-, 8- and 18-month-old C57 mice was examined with a fluorescence microscope in order to evaluate the capillary density and vessel diameter. The capillary density was significantly reduced in 18-month-old mice compared to 3-month-old (P<0.001) and to 8-month-old (.P<0.001) mice. The difference between 3- and 18-month-old mice was significant for the basal, middle and apical cochlear turns, while the difference between 8- and 18-month-old mice was significant only for the middle and basal turns. Vessel diameter within the whole SV was significantly reduced in all turns in 18-month-old mice (P<0.01) versus 3-month-old animals, while vessel diameter was reduced only in the basal turn in 8-month-old versus 3-month-old mice. The results suggest that previously described histopathological changes in the organ of Corti of C57 mice may be related to modifications of strial capillaries.

Expression of vascular endothelial growth factor and its receptors in the cochlea of various experimental animals

Conclusion. The results of this study indicate that vascular endothelial growth factor (VEGF) may be an important regulator of the vascular network of the inner ear and suggest that the VEGF signalling pathway may play a role in pathophysiologic conditions. Objective. In order to clarify the role of vascular growth factor in the modulation of the vascular network of the cochlea, we studied the expression of VEGF and its receptors—fms-like tyrosine kinase (Flt-1) and foetal liver kinase (Flk-1)—in the inner ear of 3-month-old rodents of different species: C57BL/6J mice, Wistar albino rats and Hartley albino guinea pigs. Material and methods. Qualitative immunohistochemical studies were performed by using specific antibodies to VEGF and its receptors on paraffin sections of the cochlea. The expression levels of VEGF and its receptors were quantified by means of Western blot analysis of cochlea protein extracts. Results. We demonstrated that VEGF and its receptors are expressed in the cochlea and described their distribution in the inner ear. In particular, VEGF and Flt-1 are present at the level of the modiolus, spiral ganglion, spiral ligament, basilar membrane, supporting cells, outer and inner hair cells and stria vascularis. Flk-1 was less strongly expressed in the cochlea and was not detected in the organ of Corti.

Can Chronic Nasal Obstruction Cause Dysfunction of the Paratubal Muscles and Otitis Media? An Experimental Study in Developing Wistar Rats

Objective—To quantitatively analyze modifications of the paratubal muscles in developing Wistar rats following nasal obstruction. Material and Methods—Twenty-four Wistar rats were used. Twelve were examined at 6, 8 and 12 weeks after birth and were considered normal controls. The nostrils of the remaining 12 rats were bilaterally obstructed by means of a synthetic resin 28 days after birth. The animals were sacrificed at either 2, 4 or 8 weeks after nostril occlusion. Serial sections were made in the dorsoventral plane and stained with hematoxylin–eosin. Four 5×5 μm2 areas, selected within the paratubal muscles, were histologically analyzed and the number of muscular fibers was counted manually. Results—The number of tensor veli palatini muscle fibers progressively decreased in the obstructed rats compared with age-matched normal controls and in those that had been obstructed for 4 and 8 weeks these reductions were statistically significant. Conclusion—The correct development of the paratubal muscles seems to be linked to physiological nasal breathing and is negatively affected by oral breathing.