29 research outputs found
Immunohistochemical analysis of axillary skin biopsies for the detection of adrenergic innervation of sweat glands in normal subjects and Parkinson’s disease patients
Beside the typical motor symptoms Parkinson’s disease (PD) is characterized, with varying severity, by autonomic dysfunction. Several studies have shed light on the anatomical and molecular changes that underlie the peripheral neuronal degeneration associated with PD and other Lewy body (LB) diseases (LBDs). By using skin biopsies from LBDs patients it was possible to detect misfolded phospho-α-synuclein (p-syn) deposits within dermal nerve fibers and correlate them with a reduced density of small nerve fibers. (1, 2). The skin biopsy approach is an inexpensive and minimally invasive technique. To date, there is not a standardized procedure for sampling site, tissue processing and nerve fibre assessment, so the goal of a diagnostic instrument for an early diagnosis of (LBDs) still remains a challenge. We have carried out a retrospective study setting up a novel protocol based on 10 µm thick serial sections from FFPE axillary skin biopsies. This choice take advantage from the presence of apocrine glands in the axillary region, as they receive a dense sympathetic adrenergic innervation, exploitable for a clear nervous fibers tracking. The biopsies were taken from 14 individuals who had been, in the first instance, diagnosed with various traits of motor and neurological dysfunction and two control subjects. Serial tissue sections were analysed by IHC (DAB chromogen) and by immunofluorescent labelling, using anti-p-α-synuclein (S129), anti- α -synuclein, anti-PGP9.5 and anti-tyrosine hydroxylase antibodies. This particular setting has proven useful to well highlight the adrenergic fibers surrounding the apocrine sweat glands and to visualize the fibers α -synuclein deposition. Our results enabled us to support the first diagnosis in various cases with probable PD but gave a negative p-Syn-S129 immunoreactivity results for samples from vascular Parkinson, multiple system atrophy, essential tremor and frontotemporal dementia. Our methodological setting is able to detect the adrenergic innervation of sweat apocrine glands and both the presence of Lewy bodies and Lewy neurites in axillary skin biopsies
Glutamate signaling in human melanoma cell line SK-MEL-28
Glutamate, recognized as the major excitatory neurotransmitter in the mammalian central nervous system, has been shown to regulate proliferation, migration and survival of immature and mature neurons. In addition, glutamate exerts regulatory roles in the physiology of non-neuronal cells, as confirmed by its expression in peripheral tissues [1]. Recently, the involvement of both ionotropic and metabotropic glutamate receptors in the pathophysiology of various human malignancies such as melanoma has been proposed [2]. In this study, we investigate the role of ionotropic NMDA and AMPA receptors in human melanoma cell line SK-MEL-28, via the evaluation of gene expression profile of markers for neural crest (Slug, Snail, Twist), mesenchymal stem cell (Vimentin) and embryonic stem cell (Nanog and Oct4). We reported significant alterations in neural crest and embryonic stem cell markers expression in SK-MEL-28 following stimulation with 10 or 100 mM AMPA or NMDA. In addition, modulatory actions of glutamate receptors on cell proliferation and migration were also demonstrated via in vitro proliferation assay and wound healing assay. Overall, our results enhance the knowledge of glutamate signaling in human melanoma
Expression and Function of Gonadotropin-releasing Hormone (GnRH) Receptor in Human Olfactory GnRH-secreting Neurons AN AUTOCRINE GnRH LOOP UNDERLIES NEURONAL MIGRATION
Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmann's syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time- and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dose-dependent increase in migration (up to 3-4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons
Effects of culture system and hypoxia on long-term expansion and differentiation of mesenchymal stem cells derived from periodontal ligament
Periodontal ligament stem cells (PDLSCs), located in the perivascular space of the periodontium were able to differentiate into periodontal cell types in vitro [1]. In this study, we investigated the effect of three different culture media and of low oxygen tension (1%) on the immunophenotype, proliferation rate and osteogenic potential of PDLSCs. This study was the first report to compare the PDLSCs from the same person in different culture systems. PDLSCs were harvested from three healthy third molars and the single-cells suspensions were cultured in the culture media a-MEM, DMEM and a new medium formulation (Enriched Ham’s F12 Medium, EHFM), respectively. PDLSCs were subcultured (4 x 103/cm2) until passage 7. The characterization of PDLSCs included FACS, immunofluorescence analysis and cell proliferation assay in both normoxia and hypoxia (1%). After culture in osteogenic medium for 7, 14 and 21 days, osteoblastic differentiation was evaluated by alkaline phosphatase activity, mineralization (alizarin red staining) and gene expression of osteogenic markers. Osteoblastic differentiation was also evaluated under hypoxic conditions. PDLSCs cultured in EHFM showed increased proliferation rate and CD73 overexpression compared to cells maintained in a-MEM and DMEM. On the other hand, PDLSCs grown in a-MEM and DMEM showed higher osteogenic differentiation potential compared to EHFM. Hypoxia affected both proliferation rate and osteogenic potential. On the basis of these results, we propose a two stages protocol for the osteogenic induction of PDLSCs, in which the early expansion stage could be performed in EHFM without loss of cell stemness. Furthermore, the results obtained in the different conditions (normoxia and hypoxia) suggest that oxygen tension plays a critical role in PDLSCs physiology
Hypoxia induces Galectin-3 and Bcl-2 over-expression in human umbilical vein endothelial cells (HUVECs)
Angiogenesis, the growth of new blood vessels from pre-existing endothelium, is a critical phenomenon occurring during development and tissue regeneration. In pathological conditions such as inflammation and malignancies, hypoxia represent one of the most important driver of angiogenesis, mainly via the release of nitric oxide [1]. Here we investigate the behavior of human umbilical vein endothelial cells (HUVECs) treated with 100 mM CoCl2 for 24 hours, a condition mimicking hypoxia by the stabilization of HIF-1α and HIF-2α [2,3]. MTT and wound healing assays were performed to evaluate cell migration and proliferation, respectively, while Bcl-2 and Galectin-3 expression levels were analyzed by western blotting. We showed that hypoxic condition resulted in reduced proliferation and migration with increased expression of Galectin-3 and Bcl-2. These preliminary results provide new insights in the characterization of Galectin-3/Bcl-2 interplay in endothelial cell survival under hypoxic condition, and will contribute to a better understanding of hypoxia influences on tumor angiogenesis
Overstimulation of glutamate signaling in hamster hippocampal neurons: what’s new?
It is known that ischemic complications arise from neuronal and glial dysfunctions occurring in almost all brain areas. Within some neuronal networks, an early excitatory/inhibitory circuit imbalance tends to account for premature neuronal damages especially during the initial stages of perinatal development. Interestingly, cellular conditions reported in ischemia were also detected during the different phases of hibernation cycle and above all arousal state. Hibernating animals are able to survive under these conditions without neurological damage, so their neuronal circuits present an opportunity to investigate molecular strategies involved in mammalian cell survival under unfavorable conditions. We reported a contextual alterations of both ionotropic and metabotropic Glutamatergic systems in perinatal hippocampal neurons in response to ischemic-like condition, according to their early activation during neuronal development (Giusi et al., 2009; Di Vito et a.l, 2012). In addition, an altered expression was also reported for specific PSD scaffold proteins, which regulate Glutamate receptors targeting (Al-Hallaq et al., 2007). From our preliminary results, we can suggest that specific alterations of glutamatergic receptors, which differ significantly from those reported in other rodent, could play a major role toward the correction of neuronal development aberrations linked to clinical disorders
Surprising autopsy diagnosis in unclear initial situation. A case of intravascular B cell lymphoma
Intravascular large B cell lymphoma (IVLBCL) is a rare extranodal non-Hodgkin lymphoma characterized by proliferation of malignant cells within the lumen of small vessels, with a predilection for the central nervous system and the skin [1]. IVLBCL clinical course is highly aggressive, clinical signs and symptoms are not specific and may consist in neurological deficits, fever of unknown origin, cutaneous lesions, lacking of a typical neuroimaging pattern [1]. For all these reasons the diagnosis is frequently missed and the exitus is frequent, therefore post-mortem evaluation is necessary to clarify the clinical history. We present a case of IVLBCL in a 62-year-old female with unusual symptomatology, mimicking a vascular multinfarctual cerebropathy; post-mortem autopsy was diriment to define the nature of the disease. Immu- nohistochemical analysis for anti-CD20 revealed the ubiquitary presence of malignant lymphoid B-cells into the vessels of all organs analyzed, allowing the definitive diag- nosis. Although the diagnostic procedure for such pathology is still a matter for fur- ther studies, adequate interpretation of neuro-imaging and morphological findings, as well as of systemic symptoms can provide a right diagnostic hypothesis, suggest- ing focused biopsy in vivo
The Case of Medication-Related Osteonecrosis of the Jaw Addressed from a Pathogenic Point of View. Innovative Therapeutic Strategies: Focus on the Most Recent Discoveries on Oral Mesenchymal Stem Cell-Derived Exosomes
Bisphosphonates-related osteonecrosis of the jaw (BRONJ) was firstly reported by Marx in 2003. Since 2014, the term medication-related osteonecrosis of the jaw (MRONJ) is recommended by the American Association of Oral and Maxillofacial Surgeons (AAOMS). Development of MRONJ has been associated to the assumption of bisphosphonates but many MRONJ-promoting factors have been identified. A strong involvement of immunity components has been suggested. Therapeutic intervention includes surgical and non-surgical treatments, as well as regenerative medicine procedures for the replacement of the lost tissues. The literature confirms that the combination of mesenchymal stem cells (MSCs), biomaterials and local biomolecules can support the regeneration/repair of different structures. In this review, we report the major open topics in the pathogenesis of MRONJ. Then, we introduce the oral tissues recognized as sources of MSCs, summing up in functional terms what is known about the exosomes release in physiological and pathological conditions