Eugenio Paccagnini

Electron-tomographic analysis of intraflagellar transport particle trains in situ

Ultrastructural study of Chlamydomonas cilia shows that anterograde IFT particles form trains that are long and narrow, while retrograde IFT form short, compact particle trains.

Redox Imbalance and Morphological Changes in Skin Fibroblasts in Typical Rett Syndrome

Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (−43.6%) and GSH/GSSG ratio (−3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients.

HIV-1 gp120 increases the permeability of rat brain endothelium cultures by a mechanism involving substance P.

Objective:To analyse whether an HIV-1 envelope protein might play a role in damaging the blood-brain barrier as a fundamental step in the early invasion of the central nervous system by HIV-1. Design:Analysis of permeability of rat brain endothelium cultures to albumin, to assess the functional integrity of the vascular component of the blood-brain barrier. Methods:Rat brain endothelium cultures prepared by cerebral microvessels were exposed to recombinant gp120IIIB on microporous membranes and passage of biotin-labelled albumin was analysed. Scanning electron microscopy was used to analyse cell culture morphology. Some cultures were preincubated with N-nitro-L-arginine methyl ester (L-NAME), a selective inhibitor of nitric oxide synthase, or with spantide, a selective substance P antagonist. Results:HIV-1 gp120 increased the permeability of rat brain endothelial cells to albumin in a dose-dependent manner. Scanning electron microscopy revealed profound gp120-induced alterations in cell morphology accounting for the increased permeability to macromolecules. These alterations were neutralized by anti-gp120 monoclonal antibody but not by isotype control antibody or L-NAME. By contrast, spantide and anti-substance P polyclonal antibody completely blocked the gp120-induced increase in albumin permeability. Control cultures exposed to measles virus nucleoprotein showed an increase in permeability that was not blocked by spantide. Brain endothelial cells, exposed to gp120, displayed cell surface immunoreactivity for substance P, suggesting that substance P is secreted by brain endothelium in response to gp120 stimulation and binds to brain endothelial cells through a receptor-mediated mechanism. Conclusions:These findings suggest a role for substance P in the gp120-induced increase in permeability of brain endothelium.

Substance P antagonist blocks leakage and reduces activation of cytokine-stimulated rat brain endothelium.

We recently demonstrated that substance P mediates increased permeability of brain endothelium exposed to HIV-1 gp120. To test whether substance P is involved in immune processes at the blood-brain barrier (BBB), we stimulated rat brain endothelial cultures prepared from cerebral microvessels with Interferon-gamma (IFN-gamma) and Tumor necrosis factor-alpha (TNF-alpha), two proinflammatory cytokines that alter the BBB and measured permeability to albumin and expression of adhesion molecule ICAM-1 and MHC class II antigen in the presence and absence of spantide, a powerful substance P antagonist. In a dose-dependent manner, spantide completely neutralized increased permeability induced by TNF-alpha and IFN-gamma and expression of MHC class II molecule induced by IFN-gamma and prevented associated cell morphological changes as revealed by scanning electron microscope. Spantide also reduced expression of ICAM-1 induced by TNF-alpha and IFN-gamma by 35% and 30%, respectively. Substance P mRNA was found in unstimulated brain endothelial cells and was upregulated after stimulation with TNF-alpha and IFN-gamma. These in vitro findings demonstrate that substance P plays a major pathogenetic role in damaging and activating the BBB vascular component in the presence of proinflammatory cytokines.

Studies on the biological effects of ozone: 9. Effects of ozone on human platelets

During the course of ozonated autohaemotherapy (O3-AHT) using heparin as an anticoagulant, it was occasionally observed that a few clots were retained in the filter during blood reinfusion. This observation prompted an investigation on the effect of ozone (O3) on human platelets. We have now shown, both by biochemical and morphological criteria, that heparin in the presence of O3 can promote platelet aggregation. In contrast, after Ca(2+) chelation with citrate, platelet aggregation is much reduced. The potential role of the transient formation of hydrogen peroxide (H2O2) in the presence of Ca2+ with the possible expression of adhesion molecules is briefly discussed.

The proapoptotic and antimitogenic protein p66SHC acts as a negative regulator of lymphocyte activation and autoimmunity

The ShcA locus encodes 3 protein isoforms that differ in tissue specificity, subcellular localization, and function. Among these, p66Shc inhibits TCR coupling to the Ras/MAPK pathway and primes T cells to undergo apoptotic death. We have investigated the outcome of p66Shc deficiency on lymphocyte development and homeostasis. We show that p66Shc(-/-) mice develop an age-related lupus-like autoimmune disease characterized by spontaneous peripheral T- and B-cell activation and proliferation, autoantibody production, and immune complex deposition in kidney and skin, resulting in autoimmune glomerulonephritis and alopecia. p66Shc(-/-) lymphocytes display enhanced proliferation in response to antigen receptor engagement in vitro and more robust immune responses both to vaccination and to allergen sensitization in vivo. The data identify p66Shc as a negative regulator of lymphocyte activation and show that loss of this protein results in breaking of immunologic tolerance and development of systemic autoimmunity.

Morphological changes and oxidative damage in Rett Syndrome erythrocytes

BACKGROUND Hypoxemia and increased oxidative stress (OS) have been reported in Rett Syndrome (RTT), a genetical neurodevelopmental disorder. Although OS and hypoxemia can lead to red blood cells (RBCs) shape abnormalities, no information on RBCs morphology in RTT exists. Here, RBCs shape was evaluated in RTT patients and healthy subjects as a function of OS markers, blood oxygenation, pulmonary gas exchange, and cardio-respiratory parameters. METHODS RBCs morphology was evaluated by Scanning Electron Microscopy. Intraerythrocyte and plasma non protein-bound iron (NPBI), esterified F(2)-Isoprostanes (F(2)-IsoPs), 4-HNE protein adducts (4-HNE PAs) were measured. Pulmonary oxygen gradients and PaO(2) were evaluated by gas analyzers and cardiopulmonary variables by pulse oximetry. In RTT patients these parameters were assessed before and after ω-3 polyunsaturated fatty acids (ω-3 PUFAs) administration. RESULTS Altered RBCs shapes (leptocytes) and increased NPBI were present in RTT, together with increased erythrocyte membrane esterified F(2)-IsoPs and 4-HNE PAs. Abnormal erythrocyte shapes were related to OS markers levels, pulmonary gas exchange, PaO(2) and cardio-respiratory variables. After ω-3 PUFAs, a decrease of leptocytes was accompanied by a progressive increase in reversible forms of RBCs. This partial RBCs morphology rescue was related to decreased OS damage markers, improved pulmonary oxygen exchange, and cardiopulmonary physiology. CONCLUSIONS These findings indicate that in RTT 1) RBCs shape is altered; 2) the OS-hypoxia diad is critical in generating altered RBCs shape and membrane damage; 3) ω-3 PUFAs are able to partially rescue RBCs morphology and the OS-derived damage. GENERAL SIGNIFICANCE RBCs morphology is an important biosensor for OS imbalance and chronic hypoxemia.

Two cases of cutaneous phaeohyphomycosis by Alternaria alternata and Alternaria tenuissima

Two cases of cutaneous phaeohyphomycosis, one with a nodular appearance and the other with an erythematous infiltrating patch, are reported in immunocompromised patients. Diagnosis was based on histological examination, which revealed hyphae and round-shaped fungal cells in a granulomatous dermal infiltrate, and on identification of the moulds when biopsy fragments were cultured on Sabouraud-dextrose agar without cycloheximide. The pathogens were Alternaria tenuissima in the first case and A. alternata in the second. The fungi were examined by scanning electron microscopy. The patients were checked for bone and lung involvement and were then treated with surgical excision and itraconazole, and itraconazole only, respectively, with clinical and mycological resolution.

Onychomycosis caused by Alternaria spp. in Tuscany, Italy from 1985 to 1999

Summary. Cutaneous phaeohyphomycosis due to Alternaria spp. is reported with increasing frequency, especially in patients with immune deficiency. Onychomycosis caused by this mould is still rarely observed. Here we report nine cases observed in Tuscany in the period 1985–99; the agent was Alternaria alternata in eight cases and Alternaria chlamidospora in one. Diagnosis was made on the basis of repeated direct microscopic mycological examination and culture, confirmed by scanning electron microscope observation of fragments of colonies. In most cases, the clinical manifestations were dystropy and distal subungual hyperkeratosis of one or two nails of the feet or hands. Seven cases were treated with oral itraconazole, successfully in six cases, as clinical and mycological recovery was confirmed at follow‐up 1 year later.

Amyloidosis, Inflammation, and Oxidative Stress in the Heart of an Alkaptonuric Patient

Background. Alkaptonuria, a rare autosomal recessive metabolic disorder caused by deficiency in homogentisate 1,2-dioxygenase activity, leads to accumulation of oxidised homogentisic acid in cartilage and collagenous structures present in all organs and tissues, especially joints and heart, causing a pigmentation called ochronosis. A secondary amyloidosis is associated with AKU. Here we report a study of an aortic valve from an AKU patient. Results. Congo Red birefringence, Th-T fluorescence, and biochemical assays demonstrated the presence of SAA-amyloid deposits in AKU stenotic aortic valve. Light and electron microscopy assessed the colocalization of ochronotic pigment and SAA-amyloid, the presence of calcified areas in the valve. Immunofluorescence detected lipid peroxidation of the tissue and lymphocyte/macrophage infiltration causing inflammation. High SAA plasma levels and proinflammatory cytokines levels comparable to those from rheumatoid arthritis patients were found in AKU patient. Conclusions. SAA-amyloidosis was present in the aortic valve from an AKU patient and colocalized with ochronotic pigment as well as with tissue calcification, lipid oxidation, macrophages infiltration, cell death, and tissue degeneration. A local HGD expression in human cardiac tissue has also been ascertained suggesting a consequent local production of ochronotic pigment in AKU heart.