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Professore Ordinario
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

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2023 - Aged gastrocnemius muscle of mice positively responds to a late onset adapted physical training [Articolo su rivista]
Cisterna, B.; Lofaro, F. D.; Lacavalla, M. A.; Boschi, F.; Malatesta, M.; Quaglino, D.; Zancanaro, C.; Boraldi, F.

Introduction: A regular physical training is known to contribute to preserve muscle mass and strength, maintaining structure and function of neural and vascular compartments and preventing muscle insulin resistance and inflammation. However, physical activity is progressively reduced during aging causing mobility limitations and poor quality of life. Although physical exercise for rehabilitation purposes (e.g., after fractures or cardiovascular events) or simply aiming to counteract the development of sarcopenia is frequently advised by physicians, nevertheless few data are available on the targets and the global effects on the muscle organ of adapted exercise especially if started at old age.Methods: To contribute answering this question for medical translational purposes, the proteomic profile of the gastrocnemius muscle was analyzed in 24-month-old mice undergoing adapted physical training on a treadmill for 12 weeks or kept under a sedentary lifestyle condition. Proteomic data were implemented by morphological and morphometrical ultrastructural evaluations.Results and Discussion: Data demonstrate that muscles can respond to adapted physical training started at old age, positively modulating their morphology and the proteomic profile fostering protective and saving mechanisms either involving the extracellular compartment as well as muscle cell components and pathways (i.e., mitochondrial processes, cytoplasmic translation pathways, chaperone-dependent protein refolding, regulation of skeletal muscle contraction). Therefore, this study provides important insights on the targets of adapted physical training, which can be regarded as suitable benchmarks for future in vivo studies further exploring the effects of this type of physical activity by functional/metabolic approaches.

2023 - Circulating and Tumor-Associated Neutrophils in the Era of Immune Checkpoint Inhibitors: Dynamics, Phenotypes, Metabolism, and Functions [Articolo su rivista]
Gibellini, L.; Borella, R.; Santacroce, E.; Serattini, E.; Boraldi, F.; Quaglino, D.; Aramini, B.; De Biasi, S.; Cossarizza, A.

Simple Summary Neutrophils are the most abundant leukocytes in the circulation, represent the first line of defense in the immune system and mediate inflammation. Increasing evidence suggests that neutrophils constitute a large population of cells with phenotypic and functional heterogeneity. In this review, we summarize and discuss new findings delineating that both circulating neutrophils and tumor-associated neutrophils have a role in tumor prognosis and resistance to immune checkpoint inhibitors. Neutrophils are the most abundant myeloid cells in the blood and are a considerable immunological component of the tumor microenvironment. However, their functional importance has often been ignored, as they have always been considered a mono-dimensional population of terminally differentiated, short-living cells. During the last decade, the use of cutting-edge, single-cell technologies has revolutionized the classical view of these cells, unmasking their phenotypic and functional heterogeneity. In this review, we summarize the emerging concepts in the field of neutrophils in cancer, by reviewing the recent literature on the heterogeneity of both circulating neutrophils and tumor-associated neutrophils, as well as their possible significance in tumor prognosis and resistance to immune checkpoint inhibitors.

2023 - Validation of airway porcine epithelial cells as an alternative to human in vitro preclinical studies [Articolo su rivista]
Genna, V. G.; Adamo, D.; Galaverni, G.; Lepore, F.; Boraldi, F.; Quaglino, D.; Lococo, F.; Pellegrini, G.

Animal models are currently used in several fields of biomedical research as useful alternatives to human-based studies. However, the obtained results do not always effectively translate into clinical applications, due to interspecies anatomical and physiological differences. Detailed comparability studies are therefore required to verify whether the selected animal species could be a representative model for the disease or for cellular process under investigation. This has proven to be fundamental to obtaining reliable data from preclinical studies. Among the different species, swine is deemed an excellent animal model in many fields of biological research, and has been largely used in respiratory medicine, considering the high homology between human and swine airways. In the context of in vitro studies, the validation of porcine airway epithelial cells as an alternative to human epithelial cells is crucial. In this paper, porcine and human tracheal and bronchial epithelial cells are compared in terms of in vivo tissue architecture and in vitro cell behaviour under standard and airlifted conditions, analyzing the regenerative, proliferative and differentiative potentials of these cells. We report multiple analogies between the two species, validating the employment of porcine airway epithelial cells for most in vitro preclinical studies, although with some limitations due to species-related divergences.

2022 - Inhibition of the DNA Damage Response Attenuates Ectopic Calcification in Pseudoxanthoma Elasticum [Articolo su rivista]
Huang, Jianhe; Ralph, Douglas; Boraldi, Federica; Quaglino, Daniela; Uitto, Jouni; Li, Qiaoli

Pseudoxanthoma elasticum (PXE) is a heritable ectopic calcification disorder with multiorgan clinical manifestations. The gene at default, ABCC6, encodes an efflux transporter, ABCC6, which is a critical player regulating the homeostasis of inorganic pyrophosphate, a potent endogenous anticalcification factor. Previous studies suggested that systemic inorganic pyrophosphate deficiency is the major but not the exclusive cause of ectopic calcification in PXE. In this study, we show that the DNA damage response (DDR) and poly(ADP-ribose) (PAR) pathways are involved locally in PXE at sites of ectopic calcification. Genetic inhibition of PAR polymerase 1 gene PARP1, the predominant PAR-producing enzyme, showed a 54% reduction of calcification in the muzzle skin in Abcc6-/-Parp1-/- mice, compared with that of age-matched Abcc6-/-Parp1+/+ littermates. Subsequently, oral administration of minocycline, an inhibitor of DDR/PAR signaling, resulted in an 86% reduction of calcification in the muzzle skin of Abcc6-/- mice. Minocycline treatment also attenuated the DDR/PAR signaling and reduced the calcification of dermal fibroblasts derived from patients with PXE. The anticalcification effect of DDR/PAR inhibition was not accompanied by alterations in plasma inorganic pyrophosphate concentrations. These results suggest that local DDR/PAR signaling in calcification-prone tissues contributes to PXE pathogenesis and that its inhibition might provide a promising treatment strategy for ectopic calcification in PXE, a currently intractable disease.

2022 - Metabolic reprograming shapes neutrophil functions in severe COVID-19 [Articolo su rivista]
Borella, Rebecca; De Biasi, Sara; Paolini, Annamaria; Boraldi, Federica; Tartaro, Domenico Lo; Mattioli, Marco; Fidanza, Lucia; Neroni, Anita; Caro-Maldonado, Alfredo; Meschiari, Marianna; Franceschini, Erica; Quaglino, Daniela; Guaraldi, Giovanni; Bertoldi, Carlo; Sita, Marco; Busani, Stefano; Girardis, Massimo; Mussini, Cristina; Cossarizza, Andrea; Gibellini, Lara

: To better understand the mechanisms at the basis of neutrophil functions during SARS-CoV-2 we studied patients with severe COVID-19 pneumonia. They had high blood proportion of degranulated neutrophils and elevated plasma levels of myeloperoxidase (MPO), elastase and MPO-DNA complexes, which are typical markers of neutrophil extracellular traps (NET). Their neutrophils display dysfunctional mitochondria, defective oxidative burst, increased glycolysis, glycogen accumulation in the cytoplasm, and increase glycogenolysis. Hypoxia-inducible factor 1α (ΗΙF-1α) is stabilized in such cells, and it controls the level of glycogen phosphorylase L (PYGL), a key enzyme in glycogenolysis. Inhibiting PYGL abolishes the ability of neutrophils to produce NET. Patients displayed significant increases of plasma levels of molecules involved in the regulation of neutrophils' function, including CCL2, CXCL10, CCL20, IL-18, IL-3, IL-6, G-CSF, GM-CSF, IFN-γ. Our data suggest that metabolic remodelling is vital for the formation of NET and for boosting neutrophil inflammatory response, thus suggesting that modulating ΗΙF-1α or PYGL could represent a novel approach for innovative therapies. This article is protected by copyright. All rights reserved.

2022 - The “Elastic Perspective” of SARS-CoV-2 Infection and the Role of Intrinsic and Extrinsic Factors [Articolo su rivista]
Boraldi, F.; Lofaro, F. D.; Cossarizza, A.; Quaglino, D.

Elastin represents the structural component of the extracellular matrix providing elastic recoil to tissues such as skin, blood vessels and lungs. Elastogenic cells secrete soluble tropoelastin monomers into the extracellular space where these monomers associate with other matrix proteins (e.g., microfibrils and glycoproteins) and are crosslinked by lysyl oxidase to form insoluble fibres. Once elastic fibres are formed, they are very stable, highly resistant to degradation and have an almost negligible turnover. However, there are circumstances, mainly related to inflammatory conditions, where increased proteolytic degradation of elastic fibres may lead to consequences of major clinical relevance. In severely affected COVID-19 patients, for instance, the massive recruitment and activation of neutrophils is responsible for the profuse release of elastases and other proteolytic enzymes which cause the irreversible degradation of elastic fibres. Within the lungs, destruction of the elastic network may lead to the permanent impairment of pulmonary function, thus suggesting that elastases can be a promising target to preserve the elastic component in COVID-19 patients. Moreover, intrinsic and extrinsic factors additionally contributing to damaging the elastic component and to increasing the spread and severity of SARS-CoV-2 infection are reviewed.

2022 - The pathogenic c.1171A>G (p.Arg391Gly) and c.2359G>A (p.Val787Ile) ABCC6 variants display incomplete penetrance causing pseudoxanthoma elasticum in a subset of individuals [Articolo su rivista]
Szeri, Flora; Miko, Agnes; Navasiolava, Nastassia; Kaposi, Ambrus; Verschuere, Shana; Molnar, Beatrix; Li, Qiaoli; Terry, Sharon F; Boraldi, Federica; Uitto, Jouni; van de Wetering, Koen; Martin, Ludovic; Quaglino, Daniela; Vanakker, Olivier M; Tory, Kalman; Aranyi, Tamas

ABCC6 promotes ATP efflux from hepatocytes to bloodstream. ATP is metabolized to pyrophosphate, an inhibitor of ectopic calcification. Pathogenic variants of ABCC6 cause pseudoxanthoma elasticum, a highly variable recessive ectopic calcification disorder. Incomplete penetrance may initiate disease heterogeneity, hence symptoms may not, or differently manifest in carriers. Here, we investigated whether incomplete penetrance is a source of heterogeneity in pseudoxanthoma elasticum. By integrating clinical and genetic data of 589 patients, we created the largest European cohort. Based on allele frequency alterations, we identified two incomplete penetrant pathogenic variants, c.2359G>A (p.Val787Ile) and c.1171A>G (p.Arg391Gly), with 6.5% and 2% penetrance, respectively. However, when penetrant, the c.1171A>G (p.Arg391Gly) manifested a clinically unaltered severity. After applying in silico and in vitro characterization, we suggest that incomplete penetrant variants are only deleterious if a yet unknown interacting partner of ABCC6 is mutated simultaneously. The low penetrance of these variants should be contemplated in genetic counseling.

2021 - A case report of pseudoxanthoma elasticum with rare sequence variants in genes related to inherited retinal diseases [Articolo su rivista]
Lofaro, F. D.; Mucciolo, D. P.; Murro, V.; Pavese, L.; Quaglino, D.; Boraldi, F.

A case of a patient with an early and severe visual impairment is described. Due to the occurrence of skin papules a suspect of pseudoxanthoma elasticum (PXE) was posed. PXE is a rare autosomal recessive disease clinically characterized by skin, cardiovascular and ocular manifestations, these last being those that most severely affect patients’ quality of life. A whole exome sequencing approach focusing on 340 genes related to the calcification process and/or to inherited retinal diseases (IRDs) was performed. Rare monoallelic sequence variants in ABCA4, ABCC6, IMPG1, POC1B and RAX2 were found. The presence of calcified elastic fibers was assessed by ultrastructural analysis on a skin biopsy. Diagnosis of PXE was based on clinical, biomolecular and morphological results, although the additional involvement of several IRD genes is important to explain the unexpectedly severe ophthalmological phenotype of the patient also in prognostic and therapeutic perspectives. Data indicate that genetic screening using a wide‐spectrum analysis approach is essential to assist ophthalmologists in improving patient counseling.

2021 - Adaptive Optics Imaging in Patients Affected by Pseudoxanthoma Elasticum [Articolo su rivista]
Murro, V.; Mucciolo, D. P.; Giorgio, D.; Pavese, L.; Boraldi, F.; Quaglino, D.; Finocchio, L.; Sodi, A.; Virgili, G.; Giansanti, F.

Purpose: To describe the retinal findings of patients affected by pseudoxanthoma elasticum (PXE) using a multimodal imaging approach including flood-illumination adaptive optics ophthalmoscopy (AO). Design: Retrospective case series. Materials and Methods: Patients affected by PXE were retrospectively studied. Clinical data, color, infrared and autofluorescence fundus imaging, optical coherence tomographic scans, and AO examinations were collected. Furthermore, the photoreceptor count was assessed. PXE diagnosis was confirmed by a positive skin biopsy and/or genetic testing. Results: Twenty-one eyes of 18 patients (11 females and 7 males) were included in the study. In 3 patients, both eyes were studied. The mean age at examination was 37 ± 16.4 years (range 11-66) and the mean best-corrected visual acuity (BCVA) was 0.1 ± 0.2 logMAR (range 0-1). We identified 3 types of angioid streaks (AS) using AO: “crack,” “band,” and “hypopigmented.” The first 2 were very similar and they differed in size; the third type showed specific clinical features. Comet lesions appeared as hyper-reflective round lesions on AO imaging. In all eyes, the cone mosaic appeared reduced inside the streaks compared to the neighboring areas (13,532.8 ± 1,366.5 cones/mm2 vs 16,817.1 ± 1,263.0 cones/mm2 respectively). Conclusion: Using AO imaging in PXE-related retinopathy, we were able to observe the presence of the photoreceptors within the angioid streaks, differentiate 3 types of angioid streaks, based on size and reflective features, and identify the very small crystalline bodies not identifiable using other retinal imaging techniques.

2021 - Age-related changes in the matrisome of the mouse skeletal muscle [Articolo su rivista]
Lofaro, F. D.; Cisterna, B.; Lacavalla, M. A.; Boschi, F.; Malatesta, M.; Quaglino, D.; Zancanaro, C.; Boraldi, F.

Aging is characterized by a progressive decline of skeletal muscle (SM) mass and strength which may lead to sarcopenia in older persons. To date, a limited number of studies have been performed in the old SM looking at the whole, complex network of the extracellular matrix (i.e., matrisome) and its aging-associated changes. In this study, skeletal muscle proteins were isolated from whole gastrocnemius muscles of adult (12 mo.) and old (24 mo.) mice using three sequential extractions, each one analyzed by liquid chromatography with tandem mass spectrometry. Muscle sections were investigated using fluorescence-and transmission electron microscopy. This study provided the first characterization of the matrisome in the old SM demonstrating several statisti-cally significantly increased matrisome proteins in the old vs. adult SM. Several proteomic findings were confirmed and expanded by morphological data. The current findings shed new light on the mutually cooperative interplay between cells and the extracellular environment in the aging SM. These data open the door for a better understanding of the mechanisms modulating myocellular behavior in aging (e.g., by altering mechano-sensing stimuli as well as signaling pathways) and their contribution to age-dependent muscle dysfunction.

2021 - Apoptosis in the Extraosseous Calcification Process [Articolo su rivista]
Boraldi, F.; Lofaro, F. D.; Quaglino, D.

Extraosseous calcification is a pathologic mineralization process occurring in soft connective tissues (e.g., skin, vessels, tendons, and cartilage). It can take place on a genetic basis or as a consequence of acquired chronic diseases. In this last case, the etiology is multifactorial, including both extra- and intracellular mechanisms, such as the formation of membrane vesicles (e.g., matrix vesicles and apoptotic bodies), mitochondrial alterations, and oxidative stress. This review is an overview of extraosseous calcification mechanisms focusing on the relationships between apoptosis and mineralization in cartilage and vascular tissues, as these are the two tissues mostly affected by a number of age-related diseases having a progressively increased impact in Western Countries.

2021 - Dermal Alterations in Clinically Unaffected Skin of Pseudoxanthoma elasticum Patients [Articolo su rivista]
Boraldi, F; Lofaro, Fd; Losi, L; Quaglino, D

Background: Pseudoxanthoma elasticum (PXE), due to rare sequence variants in the ABCC6 gene, is characterized by calcification of elastic fibers in several tissues/organs; however, the pathomechanisms have not been completely clarified. Although it is a systemic disorder on a genetic basis, it is not known why not all elastic fibers are calcified in the same patient and even in the same tissue. At present, data on soft connective tissue mineralization derive from studies performed on vascular tissues and/or on clinically affected skin, but there is no information on patients' clinically unaffected skin. Methods: Skin biopsies from clinically unaffected and affected areas of the same PXE patient (n = 6) and from healthy subjects were investigated by electron microscopy. Immunohistochemistry was performed to evaluate p-SMAD 1/5/8 and p-SMAD 2/3 expression and localization. Results: In clinically unaffected skin, fragmented elastic fibers were prevalent, whereas calcified fibers were only rarely observed at the ultrastructural level. p-SMAD1/5/8 and p-SMAD2/3 were activated in both affected and unaffected skin. Conclusion: These findings further support the concept that fragmentation/degradation is necessary but not sufficient to cause calcification of elastic fibers and that additional local factors (e.g., matrix composition, mechanical forces and mesenchymal cells) contribute to create the pro-osteogenic environment.

2021 - Domains 12 to 16 of tropoelastin promote cell attachment and spreading through interactions with glycosaminoglycan and integrins alphaV and alpha5beta1 [Articolo su rivista]
Bochicchio, B.; Yeo, G. C.; Lee, P.; Emul, D.; Pepe, A.; Laezza, A.; Ciarfaglia, N.; Quaglino, D.; Weiss, A. S.

Elastin is an extracellular matrix (ECM) component with key structural and biological roles in elastic tissues. Interactions between resident cells and tropoelastin, the monomer of elastin, underpin elastin's regulation of cellular processes. However, the nature of tropoelastin-cell interactions, and the contributions of individual tropoelastin domains to these interactions are only partly elucidated. In this study, we identified and characterized novel cell-adhesive sites in the tropoelastin N-terminal region between domains 12 and 16. We found that this region interacts with αV and α5β1 integrin receptors, which mediate cell attachment and spreading. A peptide sequence from within this region, spanning domains 14 to mid-domain 16, binds heparan sulfate through electrostatic interactions with peptide lysine residues, and induces conformational ordering of the peptide. We propose that domains 14-16 direct initial cell attachment through cell-surface heparan sulfate glycosaminoglycans, followed by αV and α5β1 integrin-promoted attachment and spreading on domains 12-16 of tropoelastin. These findings advance our mechanistic understanding of elastin matrix biology, with the potential to enhance tissue regenerative outcomes of elastin-based materials.

2021 - From Clinical Diagnosis to the Discovery of Multigene Rare Sequence Variants in Pseudoxanthoma elasticum: A Case Report [Articolo su rivista]
Lofaro, F. D.; Mucciolo, D. P.; Murro, V.; Pavese, L.; Quaglino, D.; Boraldi, F.

Pseudoxanthoma elasticum (PXE) is a rare autosomal recessive disease clinically characterised by early cutaneous alterations, and by late clinically relevant ocular, and cardiovascular manifestations. ABCC6 genetic tests are used to confirm clinical PXE diagnosis, but this strategy may be rather challenging when only one ABCC6 pathogenic variant is found. A next-generation sequencing approach focusing on 362 genes related to the calcification process and/or to inherited retinal diseases was performed on a patient with clinical PXE diagnosis (skin papules and laxity, angioid streaks, and atrophy) who was carrier of only one ABCC6 rare sequence variant. Beside ABCC6, several rare sequence variants were detected which can contribute either to the occurrence of calcification (GGCX and SERPINF1 genes) and/or to ophthalmological manifestations (ABCA4, AGBL5, CLUAP1, and KCNV2 genes). This wide-spectrum analysis approach facilitates the identification of rare variants possibly involved in PXE, thus avoiding invasive skin biopsy as well as expensive and time-consuming diagnostic odyssey and allows to broaden and to deepen the knowledge on this complex rare disease and to improve patients' counselling, also with a future perspective of personalised medicine.

2021 - Phenotypic Features and Genetic Findings in a Cohort of Italian Pseudoxanthoma Elasticum Patients and Update of the Ophthalmologic Evaluation Score [Articolo su rivista]
Boraldi, F; Murro, V; Lofaro, Fd; Mucciolo, Dp; Costa, S; Pavese, L; Quaglino, D

Background: Pseudoxanthoma elasticum (PXE) is a rare ectopic calcification genetic disease mainly caused by ABCC6 rare sequence variants. The clinical phenotype is characterized by typical dermatological, ophthalmological and cardiovascular manifestations, whose frequency and severity are differently reported in the literature. Methods: A retrospective study was performed on 377 PXE patients of Italian origin, clinically evaluated according to the Phenodex Index, who underwent ABCC6 biomolecular analyses. Moreover, 53 PXE patients were further characterized by in-depth ophthalmological examinations. Results: A total of 117 different ABCC6 rare sequence variants were detected as being spread through the whole gene. The severity of the clinical phenotype was dependent on age, but it was not influenced by gender or by the type of sequence variants. In-depth ophthalmological examinations focused on the incidences of coquille d'oeuf, comet lesions, pattern dystrophy-like lesions, optic disk drusen and posterior-pole atrophy. Conclusion: Given the large number of patients analyzed, we were able to better evaluate the occurrence of less frequent alterations (e.g., stroke, myocardial infarction, nephrolithiasis). A more detailed description of ophthalmological abnormalities allowed us to stratify patients and better evaluate disease progression, thus suggesting a further update of the PXE score system.

2020 - Altered bioenergetics and mitochondrial dysfunction of monocytes in patients with COVID-19 pneumonia [Articolo su rivista]
Gibellini, L.; De Biasi, S.; Paolini, A.; Borella, R.; Boraldi, F.; Mattioli, M.; Lo Tartaro, D.; Fidanza, L.; Caro-Maldonado, A.; Meschiari, M.; Iadisernia, V.; Bacca, E.; Riva, G.; Cicchetti, L.; Quaglino, D.; Guaraldi, G.; Busani, S.; Girardis, M.; Mussini, C.; Cossarizza, A.

In patients infected by SARS-CoV-2 who experience an exaggerated inflammation leading to pneumonia, monocytes likely play a major role but have received poor attention. Thus, we analyzed peripheral blood monocytes from patients with COVID-19 pneumonia and found that these cells show signs of altered bioenergetics and mitochondrial dysfunction, had a reduced basal and maximal respiration, reduced spare respiratory capacity, and decreased proton leak. Basal extracellular acidification rate was also diminished, suggesting reduced capability to perform aerobic glycolysis. Although COVID-19 monocytes had a reduced ability to perform oxidative burst, they were still capable of producing TNF and IFN-γ in vitro. A significantly high amount of monocytes had depolarized mitochondria and abnormal mitochondrial ultrastructure. A redistribution of monocyte subsets, with a significant expansion of intermediate/pro-inflammatory cells, and high amounts of immature monocytes were found, along with a concomitant compression of classical monocytes, and an increased expression of inhibitory checkpoints like PD-1/PD-L1. High plasma levels of several inflammatory cytokines and chemokines, including GM-CSF, IL-18, CCL2, CXCL10, and osteopontin, finally confirm the importance of monocytes in COVID-19 immunopathogenesis.

2020 - Pattern dystrophy-like changes and coquille d’oeuf atrophy in elderly patients affected by pseudoxanthoma elasticum [Articolo su rivista]
Murro, V.; Mucciolo, D. P.; Giorgio, D.; Sodi, A.; Boraldi, F.; Quaglino, D.; Virgili, G.; Giansanti, F.

Purpose: To evaluate the retinal features of elderly patients affected by pseudoxanthoma elasticum (PXE). Materials and methods: This is a retrospective case series of 62 eyes of 31 elderly PXE patients (age > 50 years). Clinical data, ultra-widefield fundus imaging (color, red-free (RF), infra-red imaging (IR), fundus autofluorescence (FAF)), and OCT examinations were collected. Diagnosis was confirmed by genetic testing or skin biopsy. Results: Thirty-one patients (10 males and 21 females (mean age 61.3 years, range 50–74 years)) were included in our study. Visual acuity ranged from 20/20 Snellen equivalent to 20/200. The mean follow-up was 66.4 ± 20.7 months (range 10–88). Pattern dystrophy-like changes (PD) (52 eyes of 26 patients, 83.8%) and atrophy resembling the “diffuse trickling” pattern described in geographic atrophy were present in the majority of patients. Twenty-three eyes of 12 patients (67.6%) had peripapillary atrophy, 9 eyes of 5 patients (26.4%) macular atrophy, 6 eyes of 3 patients (17.6%) displayed posterior pole atrophy and in 6 eyes of 3 patients (17.6%), atrophy could be detected beyond the vascular arcades (mid-peripheral atrophy). End-stage atrophy covered the entire area indicated as “coquille d’oeuf” (eggshell). Choroidal neovascularization occurred in 49 eyes of 26 patients (94.2%) with PD and in 6 eyes of 3 patients (60%) without PD. Genetic examinations were available for 29 patients (29/31, 93.5%). Conclusions: The elderly PXE patients were characterized by pattern dystrophy-like changes with more or less extensive atrophy, progressive over time, which in some cases affected the whole area of the coquille d’oeuf during the course of the disease.

2020 - Rare Co-occurrence of Beta-Thalassemia and Pseudoxanthoma elasticum: Novel Biomolecular Findings [Articolo su rivista]
Boraldi, F.; Lofaro, F. D.; Costa, S.; Moscarelli, P.; Quaglino, D.

A number of beta-thalassemia patients, independently from the type of beta-thalassemia (β0 or β+) and blood transfusion requirements, may develop, after puberty, dermal, cardiovascular, and ocular complications associated with an ectopic mineralization phenotype similar to that observed in another rare genetic disorder, namely, Pseudoxanthoma elasticum (PXE). To date, the causes of these alterations in beta-thalassemia patients are not known, but it has been suggested that they could be the consequence of oxidative stress-driven epigenetic regulatory mechanisms producing an ABCC6 down-regulation. Since, in the last years, several genes have been associated to the ectopic mineralization phenotype, this study, for the first time, applied, on beta-thalassemia patients with ectopic mineralization phenotype, a multigene testing strategy. Selection of genes to be analyzed was done on the basis of (i) their genetic involvement in calcification diseases or (ii) their role in calcium-phosphate equilibrium. Although, due to the rarity of these conditions, a limited number of patients was analyzed, the detection of pathogenic variants represents the proof of concept that PXE and beta-thalassemia traits co-occur on a genetic basis and that, in addition to causative mutations, functional polymorphisms may further influence connective tissue manifestations. The use of a multigene-based next-generation sequencing represents a useful time- and cost-effective approach, allowing to identify sequence variants that might improve prognostic assessment and better management of these patients, especially in the current era of precision medicine aiming to identify individual optimal care based on a unique personal profile.

2020 - Relationship Between Mitochondrial Structure and Bioenergetics in Pseudoxanthoma elasticum Dermal Fibroblasts [Articolo su rivista]
Lofaro, F. D.; Boraldi, F.; Garcia-Fernandez, M.; Estrella, L.; Valdivielso, P.; Quaglino, D.

Pseudoxanthoma elasticum (PXE) is a genetic disease considered as a paradigm of ectopic mineralization disorders, being characterized by multisystem clinical manifestations due to progressive calcification of skin, eyes, and the cardiovascular system, resembling an age-related phenotype. Although fibroblasts do not express the pathogenic ABCC6 gene, nevertheless these cells are still under investigation because they regulate connective tissue homeostasis, generating the “arena” where cells and extracellular matrix components can promote pathologic calcification and where activation of pro-osteogenic factors can be associated to pathways involving mitochondrial metabolism. The aim of the present study was to integrate structural and bioenergenetic features to deeply investigate mitochondria from control and from PXE fibroblasts cultured in standard conditions and to explore the role of mitochondria in the development of the PXE fibroblasts’ pathologic phenotype. Proteomic, biochemical, and morphological data provide new evidence that in basal culture conditions (1) the protein profile of PXE mitochondria reveals a number of differentially expressed proteins, suggesting changes in redox balance, oxidative phosphorylation, and calcium homeostasis in addition to modified structure and organization, (2) measure of oxygen consumption indicates that the PXE mitochondria have a low ability to cope with a sudden increased need for ATP via oxidative phosphorylation, (3) mitochondrial membranes are highly polarized in PXE fibroblasts, and this condition contributes to increased reactive oxygen species levels, (4) ultrastructural alterations in PXE mitochondria are associated with functional changes, and (5) PXE fibroblasts exhibit a more abundant, branched, and interconnected mitochondrial network compared to control cells, indicating that fusion prevail over fission events. In summary, the present study demonstrates that mitochondria are modified in PXE fibroblasts. Since mitochondria are key players in the development of the aging process, fibroblasts cultured from aged individuals or aged in vitro are more prone to calcify, and in PXE, calcified tissues remind features of premature aging syndromes; it can be hypothesized that mitochondria represent a common link contributing to the development of ectopic calcification in aging and in diseases. Therefore, ameliorating mitochondrial functions and cell metabolism could open new strategies to positively regulate a number of signaling pathways associated to pathologic calcification.

2020 - The biology of vascular calcification [Capitolo/Saggio]
Quaglino, D.; Boraldi, F.; Lofaro, F. D.

Vascular calcification (VC), characterized by different mineral deposits (i.e., carbonate apatite, whitlockite and hydroxyapatite) accumulating in blood vessels and valves, represents a relevant pathological process for the aging population and a life-threatening complication in acquired and in genetic diseases. Similarly to bone remodeling, VC is an actively regulated process in which many cells and molecules play a pivotal role. This review aims at: (i) describing the role of resident and circulating cells, of the extracellular environment and of positive and negative factors in driving the mineralization process; (ii) detailing the types of VC (i.e., intimal, medial and cardiac valve calcification); (iii) analyzing rare genetic diseases underlining the importance of altered pyrophosphate-dependent regulatory mechanisms; (iv) providing therapeutic options and perspectives.

2020 - The mineralization process of insoluble elastin fibrillar structures: Ionic environment vs degradation [Articolo su rivista]
Boraldi, F.; Moscarelli, P.; Lofaro, F. D.; Sabia, C.; Quaglino, D.

Despite its long half-life and physiological role, elastin undergoes irreversible changes (i.e elastolysis and/or calcification) impairing resilience of soft connective tissues. At present, it is still undefined: 1) to which extent elastin fibers have to be fragmented in order to increase their susceptibility to calcify; 2) which is the contribution of ionic environment on elastin mineralization; 3) why, in the same tissue area, mineralized coexist with non-mineralized fibers. The in vitro mineralization process was investigated on insoluble elastin, hydrolyzed or not-hydrolyzed, and incubated in different cell-free ionic environments. Mineral deposition is favored on hydrolyzed fibrillar structures due to exposure of multiple charged sites increasing the adsorption of Ca2+ that can attract phosphate and increase the local ion concentration over the point of supersaturation, representing the minimum requirement for hydroxyapatite nucleation sites. At physiological pH, the degree of elastin mineralization is influenced by hydrolysis and complexity of medium composition, since ionic species, as sodium, potassium, magnesium, in addition to calcium and phosphorus, interfere with the calcification process. These findings broaden the knowledge on the factors controlling hydroxyapatite deposition on insoluble elastin and can also explain why, in vivo, calcified and non-calcified fibers can be observed within the same tissue.

2019 - Coquille d’oeuf in young patients affected with Pseudoxantoma elasticum [Articolo su rivista]
Murro, V.; Mucciolo, D. P.; Giorgio, D.; Sodi, A.; Boraldi, F.; Quaglino, D.; Virgili, G.; Rizzo, S.

Purpose: To evaluate the fundus phenotype of young patients affected with Pseudoxantoma Elasticum (PXE). Materials and Methods: Retrospective case series of five young PXE patients. Clinical data, ultra-widefield imaging (color, red-free (RF), choroidal (Ch) and fundus autofluorescence (FAF)) and OCT examination were collected. Diagnosis was confirmed by the characteristic histopathological abnormalities in skin biopsies and genetic testing results. Results: Five patients, 2 males and 3 females (mean age 16 years, range 12–20 years) were included in our study. The visual acuity was 20/20 in all subjects. Fundus evaluation revealed peau d’orange in all patients: multiple, yellowish/white round lesions, scattered from the posterior pole to the mid-peripheral retina of each eye. Ultra-wide field imaging allows us to capture and describe the entire area of coquille d’oeuf/peau d’orange in a single picture, facilitating their identification and discrimination. Angiod streaks were visible in both eyes of four patients. In one patient optic disc drusen were detected in both eyes. All patients presented comet lesions. Conclusions: PXE-related retinopathy findings: peau d’orange/coquille d’oeuf, angioid streaks, comet lesions and drusen of the optic disc were present early in PXE patients. The early detection of coquille d’oeuf/peau d’orange revealed a preferable area into midperiphery where Bruch’s membrane will be more likely to be affected.

2019 - Endogenous Calcification Inhibitors in the Prevention of Vascular Calcification: A Consensus Statement From the COST Action EuroSoftCalcNet [Articolo su rivista]
Back, M.; Aranyi, T.; Cancela, M. L.; Carracedo, M.; Conceicao, N.; Leftheriotis, G.; Macrae, V.; Martin, L.; Nitschke, Y.; Pasch, A.; Quaglino, D.; Rutsch, F.; Shanahan, C.; Sorribas, V.; Szeri, F.; Valdivielso, P.; Vanakker, O.; Kempf, H.

The physicochemical deposition of calcium-phosphate in the arterial wall is prevented by calcification inhibitors. Studies in cohorts of patients with rare genetic diseases have shed light on the consequences of loss-of-function mutations for different calcification inhibitors, and genetic targeting of these pathways in mice have generated a clearer picture on the mechanisms involved. For example, generalized arterial calcification of infancy (GACI) is caused by mutations in the enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (eNPP1), preventing the hydrolysis of ATP into pyrophosphate (PPi). The importance of PPi for inhibiting arterial calcification has been reinforced by the protective effects of PPi in various mouse models displaying ectopic calcifications. Besides PPi, Matrix Gla Protein (MGP) has been shown to be another potent calcification inhibitor as Keutel patients carrying a mutation in the encoding gene or Mgp-deficient mice develop spontaneous calcification of the arterial media. Whereas PPi and MGP represent locally produced calcification inhibitors, also systemic factors contribute to protection against arterial calcification. One such example is Fetuin-A, which is mainly produced in the liver and which forms calciprotein particles (CPPs), inhibiting growth of calcium-phosphate crystals in the blood and thereby preventing their soft tissue deposition. Other calcification inhibitors with potential importance for arterial calcification include osteoprotegerin, osteopontin, and klotho. The aim of the present review is to outline the latest insights into how different calcification inhibitors prevent arterial calcification both under physiological conditions and in the case of disturbed calcium-phosphate balance, and to provide a consensus statement on their potential therapeutic role for arterial calcification.

2019 - Exome sequencing and bioinformatic approaches reveals rare sequence variants involved in cell signalling and elastic fibre homeostasis: new evidence in the development of ectopic calcification [Articolo su rivista]
Boraldi, Federica; Lofaro, Francesco Demetrio; Romano, Oriana; Grilli, Andrea; Losi, Lorena; Moscarelli, Pasquale; Bicciato, Silvio; Quaglino, Daniela

Elastic fibres undergo aberrant mineralization in genetic as well as in acquired pathologic conditions causing severe impairment of tissue mechanical properties. Despite the number of investigations performed so far, the pathogenesis of these alterations is still elusive, due to both the complexity of the elastin network and the involvement of many genes and/or pro-osteogenic signalling pathways. Whole Exome Sequencing (WES) was performed on DNA from three patients affected by beta-thalassemia exhibiting soft connective tissue calcification. WES data were analysed with a bioinformatic approach, allowing to screen and to select genes carrying rare sequence variants. These genes were matched with those present in Extracellular Matrix DB. This approach enables to shed light on the involvement of the extracellular matrix in the occurrence of ectopic calcification. Results revealed a number of rare sequence variants in genes related to elastic fibre assembly and integrity. For instance, the involvement of fibrillins and collagen type VI in the formation of a modified microfibrillar scaffold may lead to elastic fibres less resilient and more prone to hydroxyapatite deposition. Moreover, data reveal that changes in mitochondrial metabolic pathways are sustained by a genetic background and emphasize that a persistent chronic oxidative stress can further influence extracellular matrix homeostasis and cell signalling through the TGFβ-BMP axis. Eventually, the presence of multiple rare sequence variants in the Solute Carrier Family 25 Member 5 (SLC25A5) gene is suggestive of the role of this gene as a key factor linking mitochondria metabolism, ADP/ATP ratio and oxidative stress thus affecting extracellular matrix homeostasis and activation of pro-osteogenic factors.

2019 - Intraretinal hyperreflective foci in PXE-related retinopathy with acquired vitelliform lesions: a long-term follow-up [Articolo su rivista]
Murro, V.; Mucciolo, D. P.; Giorgio, D.; Sodi, A.; Boraldi, F.; Quaglino, D.; Virgili, G.; Rizzo, S.

Purposes: To describe the long-term follow-up of a patient affected by Pseudoxanthoma Elasticum (PXE) and acquired macular vitelliform lesions in both eyes. Material and methods: Case report Results: We reported the 9-year follow-up of a patient affected by PXE. We described the onset and the resolution of the vitelliform macular lesions which lasted 5 years. The vitelliform lesion appeared almost simultaneously in both eyes with an initial increase in size, even though asymmetrical. We detected the intraretinal migration of hyper-reflective foci in both eyes during the follow-up. Choroidal neovascularization (CNV) occurred in her right eye during the follow-up. Visual acuity decreased from 20/20 to 20/32 in left eye; from 20/20 to 20/100 in her right eye. Conclusions: we reported the natural history of acquired vitelliform lesion in PXE-related retinopathy describing the Intraretinal hyperreflective foci migration.

2019 - Signaling pathways in elastic tissues [Articolo su rivista]
Daamen, W. F.; Quaglino, D.

For many years elastin was considered as the matrix component structurally required to provide tissue elasticity. However, the expanded knowledge on the regulation of connective tissue homeostasis has revealed that elastic fibers also represent a source of elastokines and are the target of a number of signaling pathways mainly involving the TGF-β/BMP axis. A better understanding of these complex regulatory networks may pave the way for targeted therapeutic strategies in a number of genetic as well as acquired diseases and for the development of new functionalized biomaterials.

2018 - Heparan sulfates facilitate harmless amyloidogenic fibril formation interacting with elastin-like peptides [Articolo su rivista]
Boraldi, Federica; Moscarelli, Pasquale; Bochicchio, Brigida; Pepe, Antonietta; Salvi, Anna M.; Quaglino, Daniela

Heparan sulfates (HSs) modulate tissue elasticity in physiopathological conditions by interacting with various matrix constituents as tropoelastin and elastin-derived peptides. HSs bind also to protein moieties accelerating amyloid formation and influencing cytotoxic properties of insoluble fibrils. Interestingly, amyloidogenic polypeptides, despite their supposed pathogenic role, have been recently explored as promising bio-nanomaterials due to their unique and interesting properties. Therefore, we investigated the interactions of HSs, obtained from different sources and exhibiting various degree of sulfation, with synthetic amyloidogenic elastin-like peptides (ELPs), also looking at the effects of these interactions on cell viability and cell behavior using in vitro cultured fibroblasts, as a prototype of mesenchymal cells known to modulate the soft connective tissue environment. Results demonstrate, for the first time, that HSs, with differences depending on their sulfation pattern and chain length, interact with ELPs accelerating aggregation kinetics and amyloid-like fibril formation as well as self-association. Furthermore, these fibrils do not negatively affect fibroblasts' cell growth and parameters of redox balance, and influence cellular adhesion properties. Data provide information for a better understanding of the interactions altering the elastic component in aging and in pathologic conditions and may pave the way for the development of composite matrix-based biomaterials.

2018 - Interactions between elastin-like peptides and an insulating poly(ortho-aminophenol) membrane investigated by AFM and XPS [Articolo su rivista]
Carbone, Maria Elvira; Ciriello, Rosanna; Moscarelli, Pasquale; Boraldi, Federica; Bianco, Giuliana; Guerrieri, Antonio; Bochicchio, Brigida; Pepe, Antonietta; Quaglino, Daniela; Salvi, Anna Maria

This investigation was undertaken to explore the mutual recognition of the pentapeptide (ValGlyGlyValGly)n, a hydrophobic elastin-like peptide (ELP), suspended in deionized water in monomer (n = 1) and trimer (n = 3) forms and the outer surface of a very thin, insulating polymer, poly(ortho-aminophenol) (PoAP), electrochemically grown on a platinum foil by cyclic voltammetry in a neutral medium (phosphate-buffered saline, I = 0.1M) immersed in the suspension. As a prior task, the proved propensity of the ValGlyGlyValGly sequence, at the given minimal length (three or more repeats), to self-assemble into amyloid-like fibrils when solubilized in an aqueous environment was considered within the framework of testing PoAP surfaces for the specific detection of amyloid precursors. From our knowledge of the chemical structure and physical properties of both biomacromolecule families obtained in previous studies, we focused on the efficacy of the binding sites offered to ELP fibrils by PoAP in its as-prepared form or properly modified either by postsynthesis oxidation or by adsorption/entrapping of ELP monomer(s) with or without protecting terminal groups. Consistent with all methods of preparation, the best surfaces, recognizable by the trimer fibrils, are those modified to carry a larger number of carbonyls, particularly by entrapment of ELP monomer(s) during PoAP electrosynthesis using an imprinting-inspired method. The degree of attachment of fibrillar aggregates, detected by atomic force microscopy and X-ray photoelectron spectroscopy, provides unequivocal evidence of the cooperative forces involving PoAP–ELP interactions. The results obtained suggest the prospect of using the proposed Pt/PoAP/ELP systems as biodetectors in Alzheimer disease.

2018 - Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations [Articolo su rivista]
Boraldi, Federica; Burns, Jorge S.; Bartolomeo, Angelica; Dominici, Massimo; Quaglino, Daniela

Background aims: Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported. Methods: To compare mineralization of human mesenchymal cell types known to differ in extracellular matrix calcification potency, bone marrow–derived mesenchymal stromal cells and dermal fibroblasts from neonatal and adult donors, at both low and high passages, were investigated in an ex vivo experimental model by supplementing the osteogenic induction medium with FBS or with hPL. Four commercial hPL preparations were profiled by liquid chromatography/electrospray ionization quadrupole time-of-flight spectrometry, and mineralization was visualized by von Kossa staining and quantified by morphometric evaluations after 9, 14 and 21 days of culture. Results: Data demonstrate that (i) commercial hPL preparations differ according to mass spectra profiles, (ii) hPL variously influences mineral deposition depending on cell line and possibly on platelet product preparation methods, (iii) donor age modifies mineral deposition in the presence of the same hPL and (iv) reduced in vitro proliferative capacity affects osteogenic induction and response to hPL. Conclusion: Despite the standardized procedures applied to obtain commercial hPL, this study highlights the divergent effects of different preparations and emphasizes the importance of cellular ontology, donor age and cell proliferative capacity to optimize the osteogenic induction capabilities of mesenchymal stromal cells and design more effective cell-based therapeutic protocols.

2018 - Peripapillary comet lesions and comet rain in PXE-related retinopathy [Articolo su rivista]
Murro, Vittoria; Mucciolo, Dario Pasquale; Sodi, Andrea; Boraldi, Federica; Quaglino, Daniela; Virgili, Gianni; Rizzo, Stanislao

Purpose: To study peripapillary comet lesions (PCL) in Italian patients affected with pseudoxanthoma elasticum (PXE). Methods: Retrospective review of fundoscopic and swept-source (SS) optical coherence tomography (OCT) images of patients with PXE examined at the Regional Reference Center for Hereditary Retinal Degenerations at the Careggi Teaching Hospital of Florence from 2012 to 2017. Results: From 148 eyes of 74 patients affected with PXE, we identified 24 eyes of 14 patients (11 were female) with a mean age of 39 years (range, 20–58 years) characterized by peripapillary comet lesions. Of these 24 eyes, 15 eyes (of 10 patients) were characterized by comet rain. The smallest comet lesion at the OCT examination appeared as a focal roundish hyper-reflective alteration at the level of the outer retinal segments and RPE-Bruch’s membrane complex; the larger lesions appeared as circular and ovoid structures with hyper-reflective borders in the outer nuclear layer. Conclusion: The comet lesion formation process involves the outer layers of the retina and RPE/Bruch’s membrane complex. It consists of a degenerative/rearrangement process of the photoreceptors which occurs in an area of focal altered RPE/Bruch’s membrane resembling the outer retinal tubulation.

2018 - Segregation analysis revealed hemizygotic causative mutations in a pseudoxanthoma elasticum patient [Articolo su rivista]
Perazzolli, G.; Girolomoni, G.; Colato, C.; Quaglino, D.

2018 - The Effects of Parenteral K1 Administration in Pseudoxanthoma Elasticum Patients Versus Controls. A Pilot Study [Articolo su rivista]
Carrillo-Linares, Juan Luis; García-Fernández, María Inmaculada; Morillo, María José; Sánchez, Purificación; Rioja, José; Barón, Francisco Javier; Ariza, María José; Harrington, Dominic J.; Card, David; Boraldi, Federica; Quaglino, Daniela; Valdivielso, Pedro

Introduction: Pseudoxanthoma elasticum (PXE) is a rare disease caused by mutations in the ABCC6 gene. Vitamin K1 is involved in the posttranslational carboxylation of some proteins related to inhibition of the calcification process. Our aim was to investigate, in patients affected by PXE, baseline levels of vitamin K1-dependent proteins and -metabolites and whether parenteral administration of phytomenadione was effective in modulating their levels. Methods: We included eight PXE patients with typical clinical symptoms (skin, retina, and vascular calcification) and two ABCC6 causative mutations; 13 clinically unaffected first-degree patients' relatives (9 carrying one ABCC6 mutation and 4 non-carriers). We assessed urinary vitamin K1 metabolites and serum Glu- and Gla-OC, Gas6 and undercaboxylated prothrombin (PIVKA-II), at baseline and after 1 and 6 weeks after a single intramuscular injection of 10 mg vitamin K1. Results: Comparison of PXE patients, heterozygous, and non-carriers revealed differences in baseline levels of serum MK-4 and of urinary vitamin K metabolites. The response to phytomenadione administration on vitamin K-dependent proteins was similar in all groups. Conclusion: The physiological axis between vitamin K1 and vitamin K-dependent proteins is preserved; however, differences in the concentration of vitamin K metabolites and of MK-4 suggest that vitamin K1 metabolism/catabolism could be altered in PXE patients.

2017 - Pigment epithelial-derived factor: a new player in dermal elastic fibre calcification? [Articolo su rivista]
Boraldi, Federica; Losi, Lorena; Quaglino, Daniela

Pigment epithelium-derived factor (PEDF) is an endogenously produced glycoprotein expressed in several organs during developmental stages and adulthood mainly acting on cell differentiation.(1) In vitro and in vivo studies have demonstrated that PEDF has neurotrophic and antioxidant activities as well as the ability to counteract angiogenesis, tumorigenesis, atherosclerosis, thrombosis and inflammation.(1,2) In addition, PEDF has been also related to bone metabolism, increasing alkaline phosphatase (ALP) expression and promoting osteoblast differentiation.(3) This article is protected by copyright. All rights reserved.

2017 - Regeneration of the entire human epidermis using transgenic stem cells [Articolo su rivista]
Hirsch, Tobias; Rothoeft, Tobias; Teig, Norbert; Bauer, Johann W.; Pellegrini, Graziella; De Rosa, Laura; Scaglione, Davide; Reichelt, Julia; Klausegger, Alfred; Kneisz, Daniela; Romano, Oriana; SECONE SECONETTI, Alessia; Contin, Roberta; Enzo, Elena; Jurman, Irena; Carulli, Sonia; Jacobsen, Frank; Luecke, Thomas; Lehnhardt, Marcus; Fischer, Meike; Kueckelhaus, Maximilian; Quaglino, Daniela; Morgante, Michele; Bicciato, Silvio; Bondanza, Sergio; De Luca, Michele

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, lifethreatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

2016 - Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction [Articolo su rivista]
Boraldi, Federica; Bartolomeo, Angelica; De Biasi, Sara; Orlando, Stefania; Costa, Sonia; Cossarizza, Andrea; Quaglino, Daniela

Introduction Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascu- lar manifestations. A number of studies highlighted the role of these cells in age-related dis- eases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination. Methods Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentia- tion markers in healthy subjects and in patients affected by peripheral vascular manifesta- tions associated with ectopic calcification. Results This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accu- rately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vas- cular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification. Conclusion Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context.

2016 - Magnesium modifies the structural features of enzymatically mineralized collagen gels affecting the retraction capabilities of human dermal fibroblasts embedded within this 3D system [Articolo su rivista]
Boraldi, Federica; Bartolomeo, Angelica; Annovi, Giulia; Debret, Romain; Quaglino, Daniela

Mineralized collagen gels have been developed as in vitro models to better understand the mechanisms regulating the calcification process and the behavior of a variety of cell types. The vast majority of data are related to stem cells and to osteoblast-like cells, whereas little information is available for dermal fibroblasts, although these cells have been associated with ectopic calcification and consequently to a number of pathological conditions. Therefore, we developed and characterized an enzymatically mineralized collagen gel in which fibroblasts were encapsulated within the 3D structure. MgCl2 was also added during gel polymerization, given its role as (i) modulator of ectopic calcification; (ii) component of biomaterials used for bone replacement; and (iii) constituent of pathological mineral deposits. Results demonstrate that, in a short time, an enzymatically mineralized collagen gel can be prepared in which mineral deposits and viable cells are homogeneously distributed. MgCl2 is present in mineral deposits and significantly affects collagen fibril assembly and organization. Consequently, cell shape and the ability of fibroblasts to retract collagen gels were modified. The development of three-dimensional (3D) mineralized collagen matrices with both different structural features and mineral composition together with the use of fibroblasts, as a prototype of soft connective tissue mesenchymal cells, may pave new ways for the study of ectopic calcification.

2015 - Donor's age and replicative senescence favour the in-vitro mineralization potential of human fibroblasts [Articolo su rivista]
Boraldi, Federica; Bartolomeo, Angelica; Di Bari, Caterina; Cocconi, Andrea; Quaglino, Daniela

Aberrant mineralization of soft connective tissues (ectopic calcification) may occur as a frequent age-related complication. Still, it remains unclear the role of mesenchymal cell donor's age and of replicative senescence on ectopic calcification. Therefore, the ability of cells to deposit in-vitro hydroxyapatite crystals and the expression of progressive ankylosis protein homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), tissue non specific alkaline phosphatase (TNAP) and osteopontin (OPN) have been evaluated in human dermal fibroblasts derived from neonatal (nHDF) and adult (aHDF) donors (ex-vivo ageing model) or at low and high cumulative population doublings (CPD) up to replicative senescence (in-vitro ageing model). This study demonstrates that: 1) replicative senescence favours hydroxyapatite formation in cultured fibroblasts; 2) donor's age acts as a major modulator of the mineralizing potential of HDF, since nHDF are less prone than aHDF to induce calcification; 3) donor's age and replicative senescence play in concert synergistically increasing the calcification process; 4) the ANKH+ENPP1/TNAP ratio, being crucial for pyrophosphate/inorganic phosphate balance, is greatly influenced by donor's age, as well as by replicative senescence, and regulates mineral deposition; 5) OPN is only modulated by replicative senescence.

2015 - Histology-directed and imaging mass spectrometry: An emerging technology in ectopic calcification [Articolo su rivista]
Taverna, Domenico; Boraldi, Federica; De Santis, Giorgio; Caprioli, Richard M.; Quaglino, Daniela

The present study was designed to demonstrate the potential of an optimized histology directed protein identification combined with imaging mass spectrometry technology to reveal and identify molecules associated to ectopic calcification in human tissue. As a proof of concept, mineralized and non-mineralized areas were compared within the same dermal tissue obtained from a patient affected by Pseudoxanthoma elasticum, a genetic disorder characterized by calcification only at specific sites of soft connective tissues. Data have been technically validated on a contralateral dermal tissue from the same subject and compared with those from control healthy skin. Results demonstrate that this approach 1) significantly reduces the effects generated by techniques that, disrupting tissue organization, blend data from affected and unaffected areas; 2) demonstrates that, abolishing differences due to inter-individual variability, mineralized and non-mineralized areas within the same sample have a specific protein profile and have a different distribution of molecules; and 3) avoiding the bias of focusing on already known molecules, reveals a number of proteins that have been never related to the disease nor to the calcification process, thus paving the way for the selection of new molecules to be validated as pathogenic or as potential pharmacological targets.

2015 - In vitro differentiation of human amniotic epithelial cells into insulin-producing 3D spheroids [Articolo su rivista]
Okere, Bernard; Alviano, Francesco; Costa, Roberta; Quaglino, Daniela; Ricci, Francesca; Dominici, Massimo; Paolucci, Paolo; Bonsi, Laura; Iughetti, Lorenzo

Regenerative medicine and stem cell therapy may represent the solution for the treatment of non-curable human diseases such as type 1 diabetes. In this context of growing demand for functional and safe stem cells, human amniotic epithelial cells (hAECs) from term placenta have attracted increasing interest for their wide availability, stem cell properties, and differentiation plasticity, which make them a promising tool for stem cell-based therapeutic applications. We initially assayed the stemness characteristics of hAECs in serum-free conditions. Subsequently we developed a culture procedure on extracellular matrix for the formation of three-dimensional (3D) spheroids. Finally, we tested the immunomodulation and differentiation potential of hAEC spheroids: the presence of pancreatic endocrine hormones was revealed with transmission electron microscopy and immunofluorescence analyses; the release of C-peptide in hyperglycemic conditions was assayed with ELISA.The serum-free culture conditions we applied proved to maintain the basic stemness characteristics of hAECs. We also demonstrated that 3D spheroids formed by hAECs in extracellular matrix can be induced to differentiate into insulin-producing cells. Finally, we proved that control and induced cells equally inhibit the proliferation of activated mononuclear cells.The results of this study highlight the properties of amnion derived epithelial cells as promising and abundant source for cell-based therapies. In particular we are the first group to show the in vitro pancreatic induction of hAECs cultured on extracellular matrix in a 3D fashion. We accordingly propose the outcomes of this study as a novel contribution to the development of future cell replacement therapies involving placenta-derived cells.

2014 - Can APOE and MTHFR polymorphisms have an influence on the severity of cardiovascular manifestations in Italian Pseudoxanthoma elasticum affected patients? [Articolo su rivista]
Boraldi, Federica; Costa, Sonia; Rabacchi, Claudio; Ciani, Miriam; Vanakker, Olivier; Quaglino, Daniela

Background: The clinical phenotype of Pseudoxanthoma elasticum (PXE) affected patients, although progressive with age, is very heterogeneous, even in the presence of identical ABCC6 mutations, thus suggesting the occur- rence of modifier genes. Beside typical skin manifestations, the cardiovascular (CV) system, and especially the peripheral vasculature, is frequently and prematurely compromised. Methods and results: A cohort of 119 Italian PXE patients has been characterized for apolipoprotein E (APOE) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms by PCR. The severity of the clinical phe- notype has been quantified according to the Phenodex PXE International score system. Statistical analysis (chi2 test, odd ratio, regression analysis, analysis of variance) were done by GraphPad. Data demonstrate that the fre- quency of APOE alleles is similar in PXE patients and in healthy subjects and that the allelic variant E2 confers a protection against the age-related increase of CV manifestations. By contrast, PXE patients are characterized by high frequency of the MTHFR-T677T polymorphism. With age, CV manifestations in T677T, but also in C677T, pa- tients are more severe than those associated with the C677C genotype. Interestingly, compound heterozygosity for C677T and A1298C polymorphisms is present in 70% of PXE patients. Conclusions: PXE patients may be screened for these polymorphisms in order to support clinicians for a better management of disease-associated CV complications.

2014 - Changes in dermal fibroblasts from Abcc6-/- mice are present before and after the onset of ectopic tissue mineralization [Articolo su rivista]
Boraldi, Federica; Bartolomeo, Angelica; Li, Q.; Uitto, J.; Quaglino, Daniela

Pseudoxanthoma elasticum (PXE), a rare genetic disease caused by mutations in the ABCC6 gene, is characterized by progressive calcification of elastic fibers in the skin, eyes, and the cardiovascular system. The pathomechanism of the mineralization is still obscure. Several hypotheses have been proposed, one of them suggesting a role for fibroblasts in controlling the amount and the quality of the calcified extracellular matrix. This hypothesis raises the question whether changes in mesenchymal cells are the cause and/or the consequences of the calcification process. In this study, fibroblasts were isolated and cultured from Abcc6+/+ and Abcc6-/- mice of different ages to investigate parameters known to be associated with the phenotype of fibroblasts from PXE patients. Results demonstrate that a few changes (Ank and Opn downregulation) are already present before the occurrence of calcification.By contrast, a modification of other parameters (intracellularO2- content, Tnap activity, and Bmp2 upregulation) can be observed inAbcc6-/- mice after the onset of tissue mineralization.These data suggest that in the Abcc6 -/- genotype, dermal fibroblasts actively contribute to changes that promote matrix calcification and that these cells can be further modulated with time by the calcified environment, thus contributing to the age-dependent progression of the disease.

2014 - Fibroblasts from patients affected by Pseudoxanthoma elasticum exhibit an altered PPi metabolism and are more responsive to pro-calcifying stimuli [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; Bartolomeo, Angelica; Quaglino, Daniela

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a genetic disorder characterized by progressive calcification of soft connective tissues. The pathogenesis is still hard to pin down. In PXE dermal fibroblasts, in addition to impaired carboxylation of the vitamin K-dependent inhibitor matrix Gla protein (MGP), we have also demonstrated an up-regulation of alkaline phosphatase activity. In the light of these data we have suggested that both calcium and phosphate metabolism might be locally altered, both pathways acting in synergy on the occurrence of matrix calcification. OBJECTIVE: This study aims to better explore if cultured PXE fibroblasts, compared to control cells, exhibit a modified inorganic pyrophosphate (PPi) metabolism and are more responsive to pro-calcifying stimuli. METHODS: Primary human dermal fibroblasts isolated from healthy individuals and from PXE patients were cultured for different time points in standard and in pro-calcifying media. The expression of ANKH/ANKH, ENPP1/PC1, ALPL/TNAP, SPP1/OPN was evaluated by qRT-PCR and Western blot, respectively. TNAP activity was measured by spectrophotometric analyses, whereas calcification was investigated by light and electron microscopy as well as by micro-analytical techniques. RESULTS: In the presence of pro-calcifying stimuli, dermal fibroblasts alter their phenotype favouring matrix mineralization. In particular, ENPP1/PC1 and SPP1/OPN expression, as well as TNAP activity, was differently expressed in control and in PXE fibroblasts. Moreover, in pathologic cells the ratio between factors favouring and reducing PPi availability exhibits a more pronounced shift towards a pro-calcifying balance. CONCLUSION: PXE fibroblasts are more susceptible to pro-calcifying stimuli and in these cells an altered PPi metabolism contributes to matrix calcification.

2014 - Impact of Candida albicans hyphal wall protein 1 (HWP1) genotype on biofilm production and fungal susceptibility to microglial cells [Articolo su rivista]
Orsi, Carlotta Francesca; Borghi, Elisa; Colombari, Bruna; Neglia, Rachele Giovanna; Quaglino, Daniela; Ardizzoni, Andrea; Morace, Giulia; Blasi, Elisabetta

The hyphal wall protein 1 (HWP1) gene of Candida albicans encodes for a fungal cell wall protein, required for hyphal development and yeast adhesion to epithelial cells; yet, its role in pathogenesis remains largely unknown. In the present study, we analyzed two C. albicans laboratory strains, the DAY286 (HWP1/HWP1) and the null mutant FJS24 (hwp1/hwp1) and six clinical isolates [3 harbouring the homozygous HWP1 gene (HWP1/HWP1) and 3 the heterologous gene (HWP1/hwp1)]. Biofilm production, fungal HWP1 mRNA levels and ultrastructural morphology were investigated; also, the susceptibility of these strains to microglial cells was evaluated, in terms of fungal damage and immune cell-mediated secretory response. When comparing the two laboratory strains, biofilm was produced to a similar extent independently on the genetic background, while the susceptibility to microglial cell-mediated damage was higher in the hwp1/hwp1 mutant than in the HWP1/HWP1 counterpart. Also, transmission electron microscopy revealed differences between the two in terms of abundance in surface adhesin-like structures, fungal cell wall shape and intracellular granules. When comparing the clinical isolates grouped according to their HWP1 genotype, reduced biofilm production and increased susceptibility to microglial cell-mediated damage occurred in the HWP1/hwp1 isolates with respect to the HWP1/HWP1 counterparts; furthermore, upon exposure to microglial cells, the HWP1/HWP1 isolates, but not the HWP1/hwp1 counterpart, showed enhanced HWP1 mRNA levels. Finally, both laboratory and clinical isolates exhibited reduced ability to stimulate TNFα and nitric oxide production by microglial cells in the case of heterozygous or null mutant HWP1 genotype. Overall, these data indicate that C. albicans HWP1 genotype influences pathogen morphological structure as well as its interaction with microglial cells, while fungal biofilm production results unaffected, thus arguing on its role as virulence factor that directly affects host mediated defences.

2014 - In Vitro derivation of insulin-producing cells from 3D spheroids of human amniotic epithelial cells [Abstract in Rivista]
Okere, B; Alviano, F; Patianna, V; Costa, R; Predieri, Barbara; Quaglino, Daniela; Ricci, F; Dominici, Massimo; Paolucci, P; Bonsi, L; Iughetti, Lorenzo

Objective: Regenerative medicine and stem cell therapy represent a promising tool for the treatment of non-curable human diseases such as type 1 diabetes. Human amniotic epithelial cells (hAECs) from term placenta have attracted growing interest for their immunological properties, plasticity and availability which make them a promising tool for stem cell-based therapeutic applications. The aim of our study was to culture hAECs in serum-free condition preserving their phenotypic and genetic traits, evaluating their pancreatic differentiative potential in a 3D fashion. Methods: hAECs were isolated and cultured in standard serum-rich medium and serum-free optimized media. Flow Cytometry analysis was performed to evaluate stemness and specific epithelial cells markers. qPCR assessed stem cell and proliferation markers. We established a 3D culture procedure on basement membrane extracts to obtain spheroids mimicking the in vivo morphology and spatial organization of pancreatic islets. Results: The serum free protocol we developed proved to maintain hAECs stemness characteristics and confirmed their immunomodulatory activity on PHA stimulated PBMCs as revealed by proliferation assays. Immunofluorescence revealed the presence of pancreatic endocrine hormones and transmission electron microscopy (TEM) analysis showed a clear membrane-associated organization of secretory granules, consistent with beta cell ultrastructure in vivo. Conclusion: We accordingly propose the outcomes of this study as a novel contribution to the development of a future cell replacement therapy for type 1 diabetes.

2014 - Long-term stability and safety of transgenic cultured epidermal stem cells in gene therapy of junctional epidermolysis bullosa. [Articolo su rivista]
DE ROSA, Laura; Carulli, S; Cocchiarella, Fabienne; Quaglino, Daniela; Enzo, Elena; Franchini, Eleonora; Giannetti, A; DE SANTIS, Giorgio; Recchia, Alessandra; Pellegrini, Graziella; DE LUCA, Michele

We report a long-term follow-up (6.5 years) of a phase I/II clinical trial envisaging the use of autologous genetically modified cultured epidermal stem cells for gene therapy of junctional epidermolysis bullosa, a devastating genetic skin disease. The critical goals of the trial were to evaluate the safety and long-term persistence of genetically modified epidermis. A normal epidermal-dermal junction was restored and the regenerated transgenic epidermis was found to be fully functional and virtually indistinguishable from a normal control. The epidermis was sustained by a discrete number of long-lasting, self-renewing transgenic epidermal stem cells that maintained the memory of the donor site, whereas the vast majority of transduced transit-amplifying progenitors were lost within the first few months after grafting. These data pave the way for the safe use of epidermal stem cells in combined cell and gene therapy for genetic skin diseases.

2014 - Retinitis pigmentosa, cutis laxa, and pseudoxanthoma elasticum-like skin manifestations associated with GGCX mutations [Articolo su rivista]
Kariminejad, Ariana; Bozorgmehr, Bita; Najafi, Abdolhamid; Khoshaeen, Atefeh; Ghalandari, Maryam; Najmabadi, Hossein; Kariminejad, Mohamad H.; Vanakker, Olivier M.; Hosen, Mohammad J.; Malfait, Fransiska; Quaglino, Daniela; Florijn, Ralph J.; Bergen, Arthur A. B.; Hennekam, Raoul C.

Gamma-glutamyl carboxylase (GGCX) mutations have been reported in patients with a pseudoxanthoma elasticum (PXE)-like phenotype, loose redundant skin, and multiple vitamin K-dependent coagulation factor deficiencies. We report on the clinical findings and molecular results in 13 affected members of two families who had a uniform phenotype consisting of (PXE)-like skin manifestations in the neck and trunk, loose sagging skin of the trunk and upper limbs, and retinitis pigmentosa confirmed by electroretinographies in 10 affected individuals. There were no coagulation abnormalities. Molecular investigations of the ATP-binding cassette subfamily C member 6 did not yield causative mutations. All 13 affected family members were found to be homozygous for the splice-site mutation c.373+3G>T in the GGCX gene. All tested parents were heterozygous for the mutation, and healthy siblings were either heterozygous or had the wild type. We suggest that the present patients represent a hitherto unreported phenotype associated with GGCX mutations. Digenic inheritance has been suggested to explain the variability in phenotype in GGCX mutation carriers. Consequently, the present phenotype may not be explained only by the GGCX mutations only but may be influenced by variants in other genes or epigenetic and environmental factors.

2014 - Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells. [Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Boraldi, Federica; Giorgio, Valentina; Bernardi, Paolo; Bartolomeo, Regina; Nasi, Milena; DE BIASI, Sara; Missiroli, Sonia; Carnevale, Gianluca; Losi, Lorena; Tesei, Anna; Pinton, Paolo; Quaglino, Daniela; Cossarizza, Andrea

Lon is a nuclear-encoded, mitochondrial protease that assists protein folding, degrades oxidized/damaged proteins, and participates in maintaining mtDNA levels. Here we show that Lon is up-regulated in several human cancers and that its silencing in RKO colon cancer cells causes profound alterations of mitochondrial proteome and function, and cell death. We silenced Lon in RKO cells by constitutive or inducible expression of Lon shRNA. Lon-silenced cells displayed altered levels of 39 mitochondrial proteins (26% related to stress response, 14.8% to ribosome assembly, 12.7% to oxidative phosphorylation, 8.5% to Krebs cycle, 6.3% to β-oxidation, and 14.7% to crista integrity, ketone body catabolism, and mtDNA maintenance), low levels of mtDNA transcripts, and reduced levels of oxidative phosphorylation complexes (with >90% reduction of complex I). Oxygen consumption rate decreased 7.5-fold in basal conditions, and ATP synthesis dropped from 0.25 ± 0.04 to 0.03 ± 0.001 nmol/mg proteins, in the presence of 2-deoxy-d-glucose. Hydrogen peroxide and mitochondrial superoxide anion levels increased by 3- and 1.3-fold, respectively. Mitochondria appeared fragmented, heterogeneous in size and shape, with dilated cristae, vacuoles, and electrondense inclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid, a Lon inhibitor, partially mimics Lon silencing. In summary, Lon is essential for maintaining mitochondrial shape and function, and for survival of RKO cells.-Gibellini, L., Pinti, M., Boraldi, F., Giorgio, V., Bernardi, P., Bartolomeo, R., Nasi, M., De Biasi, S., Missiroli, S., Carnevale, G., Losi, L., Tesei, A., Pinton, P., Quaglino, D., Cossarizza, A. Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells.

2014 - Structural characterization and biological properties of the amyloidogenic elastin-like peptide (VGGVG)3 [Articolo su rivista]
Moscarelli, P.; Boraldi, Federica; Bochicchio, B.; Pepe, A.; Salvi, A. M.; Quaglino, Daniela

The peculiar and unique properties of elastin are due to the abundance of hydrophobic residues and of repetitive sequences as XGGZG (X, Z = V, L or A). Unexpectedly, these sequences not only provide elasticity to the whole protein, but are also able to form amyloid-like fibrils. Even though amyloid fibrils have been associated for a long time to the development of serious disorders as Alzheimer's disease, recent evidence suggests that toxicity may be related to oligomeric species or to pre-fibrillar intermediates, rather than to mature fibrils. In addition, a number of studies highlighted the potential of “bio-inspired” materials based on amyloid-like nanostructures. The present study has been undertaken with the aim to characterize a chemically synthesized elastin-like peptide (VGGVG)3. Structural and biological features were compared with those of peptides as poly(VGGVG) and VGGVG that, having the same amino acid sequence, but different length and supramolecular structure have been previously investigated for their amyloidogenic properties. Results demonstrate that a minimum sequence of 15 amino acids is sufficient to aggregate into short amyloid-like fibrils, whose formation is however strictly dependent on the specific VGGVG repeated sequence. Moreover, in the attempt to elucidate the relationship among aggregation properties, fibers morphology and biocompatibility, 3T3 fibroblasts were grown in the presence of VGGVG-containing elastin-like peptides (ELPs) and analyzed for their ability to proliferate, attach and spread on ELPs-coated surfaces. Data clearly show that amyloid-like fibrils made of (VGGVG)3 are not cytotoxic at least up to the concentration of 100 μg/ml, even after several days of culture, and are a good support for cell attachment and spreading.

2014 - Thymic maturation and programmed cell death [Capitolo/Saggio]
Quaglino, Daniela; Accorsi, Alice; Boraldi, Federica; Ottaviani, Enzo

The thymus plays a crucial role in the development and maintenance of the immune system, being the main site of T cell differentiation and maturation throughout life. Associated to dramatic structural changes, its function seems to markedly diminish with time, never the less, there are several data indicating that, despite organ atrophy, at least part of the thymus remains active throughout one's lifetime. In the last decades, several studies, aiming to understand the significance of age-dependent changes in thymic structure and function, highlighted the concept that developmental and maturational stages strongly depend on the balanced and coordinated occurrence of life and death options. In particular, programmed cell death represents a fundamental requirement in order to assure a proper functionality of the immune response and to avoid the formation of uncontrolled and potentially self-damaging lymphocytic clones. By contrast, the time-dependent thymic atrophy is due to progressive replacing of lymphoid with adipose tissue. In the light of the increased knowledge on the factors/mechanisms controlling the process of adipogenesis, it could be suggested that fat accumulation in the thymic stroma might not be considered a passive, deleterious consequence of aging, but instead a potential source of molecules with various biological functions. Therefore, thymus represents a very interesting model in terms of energy expenditure and trade off, tissue homeostasis, immune defence and disease escape. The implications of changes in thymic structure, in the ratio of proliferation and programmed cell death as well as the occurrence of fat involution still represent an open question and will be discussed in the present chapter.

2013 - bis-Dehydroxy-Curcumin Triggers Mitochondrial-Associated Cell Death in Human Colon Cancer Cells through ER-Stress Induced Autophagy. [Articolo su rivista]
Basile, Valentina; Belluti, Silvia; Ferrari, Erika; Gozzoli, C; Ganassi, Sonia; Quaglino, Daniela; Saladini, Monica; Imbriano, Carol

Background: The activation of autophagy has been extensively described as a pro-survival strategy, which helps to keep cells alive following deprivation of nutrients/growth factors and other stressful cellular conditions. In addition to cytoprotective effects, autophagy can accompany cell death. Autophagic vacuoles can be observed before or during cell death, but the role of autophagy in the death process is still controversial. A complex interplay between autophagy and apoptosis has come to light, taking into account that numerous genes, such as p53 and Bcl-2 family members, are shared between these two pathways. Methodology/Principal Findings: In this study we showed a potent and irreversible cytotoxic activity of the stable Curcumin derivative bis-DeHydroxyCurcumin (bDHC) on human colon cancer cells, but not on human normal cells. Autophagy is elicited by bDHC before cell death as demonstrated by increased autophagosome formation -measured by electron microscopy, fluorescent LC3 puncta and LC3 lipidation- and autophagic flux -measured by interfering LC3-II turnover. The accumulation of poly-ubiquitinated proteins and ER-stress occurred upstream of autophagy induction and resulted in cell death. Cell cycle and Western blot analyses highlighted the activation of a mitochondrial-dependent apoptosis, which involves caspase 7, 8, 9 and Cytochrome C release. Using pharmacological inhibitions and RNAi experiments, we showed that ER-stress induced autophagy has a major role in triggering bDHC-cell death. Conclusion/Significance: Our findings describe the mechanism through which bDHC promotes tumor selective inhibition of proliferation, providing unequivocal evidence of the role of autophagy in contrasting the proliferation of colon cancer cells.

Bartolomeo, Angelica; Boraldi, Federica; Uitto, J.; Quaglino, Daniela

Progressive calcification of elastic fibers is typical of Pseudoxanthoma elasticum (PXE), a rare genetic disease due to mutations in the ABCC6 gene.The pathogenesis of mineral- ization is only partially known.1 Previous studies on dermal fibroblasts from PXE patients demonstrated that the calcifica- tion process is associated to impaired carboxylation of matrix- gla-protein that, in its active form, binds to calcium, therefore inhibiting mineralization.2 However, the recent observation that PXE fibroblasts exhibit also a significant upregulation of alka- line phosphatase (TNAP) activity suggested that an abnormal phosphate metabolism may take place within soft connective tis- sues, thus contributing to ectopic calcification.3 To improve the understanding of PXE it was developed a transgenic mouse model by specifically inactivating the Abcc6 gene.4 Consistently, Abcc6-/- mice recapitulate several histopathological findings typ- ical of PXE, including the slow progression of the disease.5 Aim of the present study was to isolate fibroblasts from Abcc6+/+ and Abcc6-/- mice of different ages (i.e. before and after the devel- opment of ectopic calcification) and to investigate proteins con- trolling phosphate levels in the extracellular matrix. Results demonstrate a down-regulation of pyrophosphatase/phosphodi- esterase 1, of progressive ankylosis protein and of osteopontin, whereas bone morphogenetic protein 2 and TNAP activity were up-regulated in fibroblasts from Abcc6-/- animals. These data support the hypothesis that in PXE the unbalanced ratio between factors locally controlling both calcium and phosphate homeostasis are crucial in triggering tissue calcification.

2013 - Ectopic calcification in β-thalassemia patients is associated with increased oxidative stress and lower MGP carboxylation [Articolo su rivista]
Boraldi, Federica; Garcia Fernandez, M.; PAOLINELLI DEVINCENZI, Chiara; Annovi, Giulia; Schurgers, L.; Vermeer, C.; Cianciulli, P.; Ronchetti, I.; Quaglino, Daniela

A number of beta-thalassemia (β-thal) patients in the course of the disease exhibit ectopic calcification affecting skin, eyes and the cardiovascular system. Clinical and histopathological features have been described similar to those in pseudoxanthoma elasticum (PXE), although different genes are affected in the two diseases. Cultured dermal fibroblasts from β-thal patients with and without PXE-like clinical manifestations have been compared for parameters of redox balance and for the expression of proteins, which have been already associated with the pathologic mineralisation of soft connective tissues. Even though oxidative stress is a well-known condition of β-thal patients, our results indicate that the occurrence of mineralized elastin is associated with a more pronounced redox disequilibrium, as demonstrated by the intracellular increase of anion superoxide and of oxidized proteins and lipids. Moreover, fibroblasts from β-thal PXE-like patients are characterized by decreased availability of carboxylated matrix Gla protein (MGP), as well as by altered expression of proteins involved in the vitamin K-dependent carboxylation process. Results demonstrate that elastic fibre calcification is promoted when redox balance threshold levels are exceeded and the vitamin K-dependent carboxylation process is affected decreasing the activity of MGP, a well-known inhibitor of ectopic calcification. Furthermore, independently from the primary gene defect, these pathways are similarly involved in fibroblasts from PXE and from β-thal PXE-like patients as well as in other diseases leading to ectopic calcification, thus suggesting that can be used as markers of pathologic mineralisation.

2013 - Fibroblast involvement in soft connective tissue calcification [Articolo su rivista]
Ronchetti, I.; Boraldi, Federica; Annovi, Giulia; Cianciulli, P.; Quaglino, Daniela

Soft connective tissue calcification is not a passive process, but the consequence of metabolic changes of local mesenchymal cells that, depending on both genetic and environmental factors, alter the balance between pro- and anti-calcifying pathways. While the role of smooth muscle cells and pericytes in ectopic calcifications has been widely investigated, the involvement of fibroblasts is still elusive. Fibroblasts isolated from the dermis of pseudoxanthoma elasticum (PXE) patients and of patients exhibiting PXE-like clinical and histopathological findings offer an attractive model to investigate the mechanisms leading to the precipitation of mineral deposits within elastic fibers and to explore the influence of the genetic background and of the extracellular environment on fibroblast-associated calcifications, thus improving the knowledge on the role of mesenchymal cells on pathologic mineralization

Orsi, Carlotta Francesca; Borghi, Elisa; Colombari, Bruna; Neglia, Rachele Giovanna; Quaglino, Daniela; Morace, Giulia; Blasi, Elisabetta

Introduzione. Il gene HWP1 di Candida albicans (Ca) codifica per una proteina della parete cellulare fungina coinvolta nella crescita ifale e nell’adesione del lievito alle cellule epiteliali; il ruolo di questa proteina nella patogenesi della candidosi non è ancora completamente chiarito. Il presente studio, mediante l’impiego di ceppi d’isolamento clinico di Ca e ceppi di laboratorio isogenici per HWP1, intende valutare il ruolo del genotipo HWP1 nella formazione del biofilm e nell’interazione in vitro tra Ca e le cellule microgliali (immunoeffettore cerebrale). Materiali e metodi. Sei ceppi clinici di Ca (3 eterozigoti e 3 omozigoti per il gene HWP1, indicati rispettivamente con gli acronimi H/h e H/H) e 2 ceppi di laboratorio (DAY286, wildtype per HWP1, H/H-wt, e FJS24, suo mutante isogenico HWP1-deleto, H/H-KO), sono stati studiati in vitro per valutare: a) la capacità di formare biofilm, b) i livelli di espressione del gene HWP1, c) l’ultrastruttura e d) la suscettibilità alle cellule microgliali, sia in termini di inibizione della formazione del biofilm che di induzione di una risposta secretoria. Risultati. Confrontando tra loro i ceppi clinici, i genotipi H/h hanno dimostrato una ridotta capacità di formare biofilm e una maggiore suscettibilità al danno mediato dalla cellula microgliale; a differenza della controparte H/H, i genotipi H/h hanno anche esibito una ridotta capacità a stimolare la produzione di ossido nitrico e di TNFα da parte delle cellule microgliali. A seguito dell’esposizione alle cellule microgliali, i ceppi H/H, ma non quelli H/h, hanno mostrato aumentati livelli di mRNA HWP1 specifico. Confrontando i 2 ceppi isogenici per HWP1, il genotipo H/H-KO ha mostrato una aumentata suscettibilità al danno mediato dalla microglia rispetto al ceppo H/H-wt, mentre il biofilm è stato prodotto in misura comparabile da parte di entrambi i ceppi. Inoltre, la microscopia elettronica a trasmissione ha rivelato differenze tra i due ceppi in termini di spessore della parete fungina, presenza di strutture superficiali adesine-simili e granuli intracitoplasmatici; tali differenze sono mantenute anche in seguito all'esposizione alle cellule microgliali. Conclusioni. Questi dati indicano che il genotipo HWP1 di Ca influenza la suscettibilità delle cellule fungine alla microglia, ma non ha effetto sulla capacità del fungo di formare biofilm.

2013 - Matrix gla protein and alkaline phosphatase are differently modulated in human dermal fibroblasts from PXE patients and controls [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; Vermeer, C.; Schurgers, L. J.; Trenti, T.; Tiozzo, Roberta; Guerra, Deanna; Quaglino, Daniela

Mineralization of elastic fibers in pseudoxanthoma elasticum (PXE) has been associated with low levels of carboxylated matrix gla protein (MGP), most likely as a consequence of reduced vitamin K (vit K) availability. Unexpectedly, vit K supplementation does not exert beneficial effects on soft connective tissue mineralization in the PXE animal model. To understand the effects of vit K supplementation and in the attempt to interfere with pathways leading to the accumulation of calcium and phosphate within PXE-mineralized soft connective tissues, we have conducted in vitro studies on dermal fibroblasts isolated from control subjects and from PXE patients. Cells were cultured in standard conditions and in calcifying medium (CM) in the presence of vit K1 and K2, or levamisole, an alkaline phosphatase (ALP) inhibitor. Control and PXE fibroblasts were characterized by a similar dose-dependent uptake of both vit K1 and vit K2, thus promoting a significant increase of total protein carboxylation in all cell lines. Nevertheless, MGP carboxylation remained much less in PXE fibroblasts. Interestingly, PXE fibroblasts exhibited a significantly higher ALP activity. Consistently, the mineralization process induced in vitro by a long-term culture in CM appeared unaffected by vit K, whereas it was abolished by levamisole.

2013 - Mixed type I and type II collagen scaffold for cartilage repair: ultrastructural study of synovial membrane response and healing potential versus microfractures (a pilot study) [Articolo su rivista]
Enea, D; Guerra, Deanna; Roggiani, Jessika; Cecconi, S; Manzotti, S; Quaglino, Daniela; Pasquali Ronchetti, I; Gigante, A.

The association between microfracture of the subchondral plate and a coverage scaffold has emerged as a promising strategy to treat cartilage lesions in a one-step procedure. Between different types of scaffolds (e.g. collagen, hyaluronic acid, polyglycolic acid) currently studied, type I collagen scaffold is the most used for this purpose, and is currently adopted for humans. The aim of this study was to test a novel scaffold made of mixed type I and II collagen (I-IICS) in order to define the immunological reaction of the synovial tissue and the repair capabilities induced by the collagen membrane when associated with microfracture. Eight New Zealand White rabbits, aged 180 days, were operated on bilaterally on the medial femoral condyle. A circular cartilage lesion was performed up to the calcified layer of the medial femoral condyle, and the centre of the lesion was microfractured. Randomly, one of the two lesions was covered with the I-IICS (treated), and the other was left uncovered (control). The synovial membrane reaction and the quality of the cartilage tissue repair were investigated at 2, 90, 180 and 270 days macroscopically, histomorphologically and ultrastructurally. Expression of tumor necrosis factor-alpha (TNF-alpha) in synovial tissue by immunocytochemistry analyses was also investigated. In the control group, at 2 days gold particles were localized mainly on synoviocyte type A, less on synoviocytes type B and on collagen bundles; in the treated group the reaction is more intense in cells in the matrix, but at 180 days controls and treated joints were very similar. The synovial membranes of the joints receiving the I-IICS did not reveal significant changes compared to the age-matched controls. Signs of inflammation were present at the 90-day time-point, and became less evident at afterwards. The degradation of the scaffolds was already evident at the 90-day time-point. The quality of the cartilage repair of the rabbits treated with the I-IICS was slightly better in 5 cases out of 6 in comparison to the controls. However, a statistically significant difference was not detected (p=0.06). Scaffolds made of mixed type I and II collagen exhibited good biocompatibility properties in vivo and favoured cartilage restoration when associated with microfracture, as shown in this pilot study.

2013 - Oxidative stress is a key regulator of ectopic calcifications in beta-Thalassemia patients [Abstract in Rivista]
Quaglino, Daniela; Boraldi, Federica; Annovi, Giulia; Cianciulli, Paolo

A surprisingly high percentage of clinical complications affecting beta-thalassemia (beta-thal) patients is due to ectopic calcification. In these patients, elastic fiber calcification is associated with accumulation of anion superoxide and to increased levels of oxidized proteins and lipids. As a consequence, carboxylation of the calcification inhibitor Matrix Gla Protein (MGP) might be impaired. Independently from the gene defect, common pathogenic pathways are associated with ectopic calcification in PXE and in a number of beta-thal patients.

2012 - Heparan sulfate affects elastin deposition in fibroblasts cultured from donors of different ages [Articolo su rivista]
Annovi, Giulia; Boraldi, Federica; Moscarelli, P.; Guerra, Deanna; Tiozzo, Roberta; Parma, B.; Sommer, P.; Quaglino, Daniela

Abstract Heparan sulfate (HS), due to its presence on the cell surface and in the extracellular milieu and its ability to modulate cell signaling, has a fundamental role in both physiological and pathological conditions. For decades we have demonstrated the occurrence of interactions between glycosaminoglycans (GAGs) and elastic fibers. In particular, we have recently shown that HS is present inside elastic fibers and plays a role in the assembly and stability of elastin coacervates. Elastin represents, within the extracellular matrix, the component most severely affected during aging, and changes in the synthesis and posttranslational modifications of HS have been described, possibly influencing cellular behavior and protein interactions. Thus, the present study has investigated, in two different in vitro experimental models, the role of HS on elastin deposition and assembly. Results demonstrate that: (1) Biological effects of HS are partly dependent on the physicochemical characteristics of the GAGs; (2) HS does not affect attachment, viability, and growth of human dermal fibroblasts; (3) HS does not modify elastin gene expression nor elastin synthesis, but favors α-elastin aggregation and, independently from the age of donors, elastin assembly; (4) HS significantly increases the expression of fibulin 5, and these effects are especially evident in fibroblasts isolated from aging donors. These data provide a better understanding of the biological role of HS and offer new perspectives regarding the possibility of restoring and/or preserving the elastic component with aging

2012 - Patologia della matrice extracellulare. [Capitolo/Saggio]
I., Ronchetti; Quaglino, Daniela

Il capitolo illustra le caratteristiche delle principali componenti della matrice extracellulare ed il loro ruolo in una ampia serie di patologie sia genetiche che acquisite. Inoltre viene analizzato, sia da un punto di vista morfologiche che biomolecolare, il coinvolgimento della matrice nell'invechiamento, durante la riparazione tessutale, nonchè durante l'insorgenza e la progressione tumorale.

Boraldi, Federica; Annovi, Giulia; Tiozzo, Roberta; Quaglino, Daniela

Soft connective tissues calcifications (i.e. ectopic calcifications) represent a deleterious consequence of diabetes, renal disorders and aging, being a key determinant of cardiovascular morbidity and mortality. Although the molecular pathways leading to this undesired mineralization have been largely investigated in smooth muscle cell cultures (SMC), to date no effective treatments are available. In order to further investigate the process of ectopic calcifications, an in vitro calcification assay has been established by isolating dermal fibroblasts (DF) from healthy adult individuals and from patients affected by Pseudoxanthoma elasticum, a disease characterized by progressive calcification of elastic fibres. Cells were grown up to 30 days in standard or in a calcifying medium. The degree of mineralization was evaluated after Von Kossa staining, whereas markers of calcification (ALP, ANKH, BMP2, ENPP1, MGP, SPP1) were assessed by RT-PCR and Western Blot. Results demonstrate that: 1) in contrast to SMC, DF do not develop a calcifying signature, 2) changes in the expression of some osteogenic markers are more related to the aging of cell cultures, 3) the development of a calcified matrix is tightly dependent on the characteristics of the extracellular environment, 4) increased ALP activity is necessary but not sufficient to have mineral deposit formation; 5) the complex balance between pro- and anti-calcifying factors, including circulating factors as MGP and fetuin, plays a significant role in the occurrence of ectopic calcifications in vivo.

Boraldi, Federica; Annovi, Giulia; Schurgers, L.; Vermeer, C.; Quaglino, Daniela

Pseudoxanthoma elasticum (PXE) is a genetic disease characterized by progressive mineralization of the elastic component, which has been related to a reduced expression of the active form of matrix gla protein (MGP), a strong inhibitor of ectopic calcifications. To be fully effective, MGP has to be carboxylated (c-MGP) through a vitamin K-depend- ent pathway. The observation that in PXE, patients have lower levels of circulating vitamin K, arose the question whether elastic fiber mineralization is due to an insufficient amount of the vitamin reaching peripheral connective tissues, or to the inability of PXE mesenchymal cells to utilize the vitamin. Unexpectedly, vitamin K supplementation does not exert bene- ficial effects on soft connective tissue mineralization in the PXE animal model. For a better understanding of the role of vitamin K-dependent carboxylation of MGP in PXE patho- genesis, aim of the present study was to investigate the effects of vitamin K1 (phylloquinone) and vitamin K2 (menaquinone- 4, MK-4) supplementation on control and on PXE dermal fibroblasts cultured in standard conditions and in calcifying medium. Results demonstrate that vitamin K1 and K2 can be taken up and accumulated at similar levels by control and PXE fibroblasts, that the carboxylation process can be conse- quently similarly up-regulated, that both vitamins K1 and K2, independently from concentration, similarly down-regulate the expression of CALU in all cell strains, whereas changes are negligible in the case of PDI, indicating that the effect of vita- min K supplementation on the expression of ER-proteins involved in vitamin K cycle is not pathway-specific. Surprisingly, MGP cannot be adequately carboxylated, even at increased levels of vitamin K. It can be therefore excluded that PXE fibroblasts are not capable to utilize the vitamin, thus suggesting that altered MGP characteristics/properties could contribute to defective carboxylation. Moreover, the observa- tion that in an in vitro calcification model, both vitamin K1 and K2 are ineffective in inhibiting the mineralization process, also in control fibroblasts, i.e. in cells that do not exhibit reduced cMGP, may underline the importance and the com- plexity of the extracellular environment in mineral deposit for- mation and in regulating cell behavior.

Boraldi, Federica; Annovi, Giulia; Tiozzo, Roberta; Quaglino, Daniela

In order to investigate the calcification process in both physiological or pathological conditions, in vitro osteogenic assays are generally performed using bone-derived cells, bone-marrow-derived mesenchymal stromal cells or vascular smooth muscle cells. In normal healthy individuals, mineral formation is limited to specialized tissues as skeletal bone and teeth, however, there are many disorders ( i.e. diabetes, kidney diseases,atherosclerosis as well as genetic conditions) in which soft connective tissues undergo mineralization. In the present study a calcification assay has been established by isolating dermal fibroblasts from adult individuals and by growing these cells in a calcifying medium in which DMEM has been supplemented with 10mM β-glycerophosphate, 50μg/ml ascorbic acid and 10 nM dexametasone. After different periods of culture, up to 40 days, fibroblast cell cultures were stained with the Von Kossa method and the activity of alkaline phosphatase (ALP) measured by a spectrophometric assay. Results indicate that in-vitro human dermal fibroblasts, which are characterized by a limited life span in culture, are capable to mineralize their secreted extracellular matrix, when grown in the presence of an osteogenic medium. Moreover, the process of mineralization appeared to progresses with time, since areas of calcifications become visible after two weeks of culture. Consistently with the activation of the osteogenic phenotype, fibroblasts exhibited also an upregulation of ALP activity. However, we have observed a remarkable heterogeneity among cells from different individuals, supporting the hypothesis that ALP is not a unique marker of calcification and that the mineralization process is the result of a fine regulation of many inhibitors and stimulatory factors.

2011 - Elastic fiber abnormalities in a case of Beals Sindrome [Abstract in Atti di Convegno]
Guerra, Deanna; Costa, Sonia; Quaglino, Daniela

Abnormalieties in dermal and vascular elastic fibers in a patient affected by Beal Syndrome are described at the ultrastrutural level. The tissue-specific involvement of GAGs are also discussed.

2011 - Fibrillin-1 genetic deficiency leads to pathological ageing of arteries in mice [Articolo su rivista]
B., Mariko; M., Pezet; B., Escoubet; S., Bouillot; J. P., Andrieu; B., Starcher; QUAGLINO, Daniela; M. P., Jacob; P., Huber; F., Ramirez; G., Faury

Fibrillin-1, the major component of extracellular microfibrils that associate with insoluble elastin in elastic fibres, is mainly synthesized during development and postnatal growth and is believed to guide elastogenesis. Mutations in the fibrillin-1 gene cause Marfan syndrome, a multisystem disorder characterized by aortic aneurysms and dissections. The recent finding that early deficiency of elastin modifies vascular ageing has raised the possibility that fibrillin-1 deficiency could also contribute to late-onset pathology of vascular remodelling. To address this question, we examined cardiovascular function in 3-week-old, 6-month-old, and 24-month-old mice that are heterozygous for a hypomorphic structural mutation of fibrillin-1 (Fbn1$^{+/{\rm{mg}}\Delta}$ mice). Our results indicate that Fbn1$^{+/{\rm{mg}}\Delta}$ mice, particularly those that are 24 months old, are slightly more hypotensive than wild-type littermates. Additionally, aneurysm and aortic insufficiency were more frequently observed in ageing Fbn1$^{+/{\rm{mg}}\Delta}$ mice than in the wild-type counterparts. We also noted substantial fragmentation and decreased number of elastic lamellae in the aortic wall of Fbn1$^{+/{\rm{mg}}\Delta}$ mice, which were correlated with an increase in aortic stiffness, a decrease in vasoreactivity, altered expression of elastic fibre-related genes, including fibrillin-1 and elastin, and a decrease in the relative ratio between tissue elastin and collagen. Collectively, our findings suggest that the heterozygous mgΔ mutation accelerates some aspects of vascular ageing and eventually leads to aortic manifestations resembling those of Marfan syndrome. Importantly, our data also indicate that vascular abnormalities in Fbn1$^{+/{\rm{mg}}\Delta}$ mice are opposite to those induced by elastin haploinsufficiency during ageing that affect blood pressure, vascular dimensions, and number of elastic lamellae

2011 - Flavivirus NS4A-induced autophagy protects cells against death and enhances virus replication [Articolo su rivista]
J. E., Mclean; A., Wudzinska; E., Datan; Quaglino, Daniela; Z., Zakeri

Flaviviruses include the most prevalent and medically challenging viruses. Persistent infection with flaviviruses of epithelial cells and hepatocytes that do not undergo cell death is common. Here we report that, in epithelial cells, upregulation of autophagy following flavivirus infection markedly enhances virus replication, and that one flavivirus gene, NS4A, uniquely determines the upregulation of autophagy. Dengue-2 and Modoc (a murine flavivirus) kill primary murine macrophages but protect epithelial cells and fibroblasts against death provoked by several insults. The flavivirus-induced protection derives from upregulation of autophagy, as upregulation of autophagy by starvation or inactivation of mTOR also protects the cells against insult, while inhibition of autophagy via inactivation of PI3K nullifies the protection conferred by flavivirus. Inhibition of autophagy also limits replication of both Dengue-2 and Modoc virus in epithelial cells. Expression of flavivirus NS4A is sufficient to induce PI3K-dependent autophagy and to protect cells against death; expression of other viral genes including NS2A and NS4B fails to protect cells against several stressors. Flavivirus NS4A protein induces autophagy in epithelial cells and thus protects them from death during infection. As autophagy is vital to flavivirus replication in these cells, NS4A is therefore also identified as a critical determinant of flavivirus replication.

2011 - Rearrangements of the ABCC6 gene in Italian patients with PXE [Abstract in Rivista]
Guerra, Deanna; Costa, Sonia; J., Roggiani; Rabacchi, Claudio; CALANDRA BUONAURA, Sebastiano; I., PASQUALI RONCHETTI; Quaglino, Daniela

The objective of this study was to search for the deletion of exon 24 and exons 24-27 that had been previously reported in Italian patients affected by pseudoxanthoma elasticum, anb autosomal recessive disorder characterized by calcification and fragmentation of elastic fibers as a conseguence of mutations in the ABCC6 gene. To detect deletions in the ABCC6 gene the development and use of long range PCR procedure using appropriately designed primers are described.

2011 - The multifaceted complexity of genetic diseases: a lesson from pseudoxanthoma elasticum. [Capitolo/Saggio]
Quaglino, Daniela; Boraldi, Federica; Annovi, Giulia; I., Ronchetti

Pseudoxanthoma elasticum (PXE) is a rare genetic disorder characterized by mineralization of elastic fibers within all connective tissue, although the most important clinical manifestation affect skin, eyes and the cardiovascular system. Despite the dramatic involvement of the extracellular matrix, the first attempts made by researchers to find out the gene defect among those coding for matrix molecules failed and in 2000 three groups, independently, demonstrated that PXE is due to mutation in the ABCC6 gene belonging to the ABC family of membrane transporters. Today the physiological substrate of this transporter is not know and still elusive are the pathogenetic mechanisms linking a defective cellular transporter mainly expressed in liver and kidney to ectopic calcification of connective tissues. This disease may therefore represent a very interesting example of the complexity that regulate molecular pathways, on the influence of metabolism on several other organs/systems. Moreover, there are also evidence that similar endpoints (i.e. clinical and histological alterations) can be observed in some patients starting from different gene defects (Pseudoxanthoma, Beta-thalassemia, vitamin-k dependent coagulation deficiency). These data support the importance of using wide-spread technologies as transcriptomic or proteomic analysis to have a broader view of the cellular pathways that may be involved. Moreover recent findings in the literature highlights the role of polymorphisms in other genes that could be responsible for phenotypic changes and for a different severity of clinical manifestation in this monogenic disorder.

2010 - Comparison of ex vivo and in vitro human fibroblast ageing models [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; Tiozzo, Roberta; Sommer, P.; Quaglino, Daniela

Several studies have analyzed modulation of gene expression during physiological ageing with interesting, but often contradictory results, depending on the model used. In the present report we compare age-related metabolic and synthetic parameters in human dermal fibroblasts (HDF) isolated from young and old subjects (ex vivo ageing model) and cultured from early up to late cumulative population doublings (CPD) (in vitro ageing model) in order to distinguish changes induced in vivo by the aged environment and maintained in vitro, from those associated with cell senescence and progressive CPD. Results demonstrate that fibroblasts from aged donors, already at early CPD, exhibit an impaired redox balance, highlighting the importance of this parameter during ageing, even in the presence of standard environmental conditions, which are considered optimal for cell growth. By contrast, several proteins, as those related to heat shock response, or involved in endoplasmic reticulum and membrane trafficking, appeared differentially expressed only during in vitro ageing, suggesting that, at high CPD, the whole cell machinery becomes permanently altered. Finally, given the importance of the elastic component for a long-lasting connective tissue structural and functional compliance, this study focuses also on elastin and fibulin-5 synthesis and deposition, demonstrating a close relationship between fibulin-5 and ageing.

2010 - Low serum vitamin K in PXE results in defective carboxylation of mineralization inhibitors similar to the GGCX mutations in the PXE-like syndrome. [Articolo su rivista]
O., Vanakker; L., Martin; L., Schurgers; Quaglino, Daniela; C., Vermeer; Ronchetti, Ivonne; P., Coucke; A., De Paepe

Soft-tissue mineralization is a tightly regulated process relying on the activity of systemic and tissue-specific inhibitors and promoters of calcium precipitation. Many of these, such as matrix gla protein (MGP) and osteocalcin (OC), need to undergo carboxylation to become active. This post-translational modification is catalyzed by the gammaglutamyl carboxylase GGCX and requires vitamin K (VK) as an essential co-factor. Recently, we described a novel phenotype characterized by aberrant mineralization of the elastic fibers resulting from mutations in GGCX. Because of the resemblance with pseudoxanthoma elasticum (PXE), a prototype disorder of elastic fiber mineralization, it was coined the PXE-like syndrome. As mutations in GGCX negatively affect protein carboxylation, it is likely that inactive inhibitors of calcification contribute to ectopic mineralization in PXE-like syndrome. Because of the remarkable similarities with PXE, we performed a comparative study of various forms of VK-dependent proteins in serum, plasma (using ELISA), and dermal tissues (using immunohistochemistry) of PXE-like and PXE patients using innovative, conformation-specific antibodies. Furthermore, we measured VK serum concentrations (using HPLC) in PXE-like and PXE samples to evaluate the VK status. In PXE-like patients, we noted an accumulation of uncarboxylated Gla proteins, MGP, and OC in plasma, serum, and in the dermis. Serum levels of VK were normal in these patients. In PXE patients, we found similar, although not identical results for the Gla proteins in the circulation and dermal tissue. However, the VK serum concentration in PXE patients was significantly decreased compared with controls. Our findings allow us to conclude that ectopic mineralization in the PXE-like syndrome and in PXE results from a deficient protein carboxylation of VK-dependent inhibitors of calcification. Although in PXE-like patients this is due to mutations in the GGCX gene, a deficiency of the carboxylation co-factor VK is at the basis of the decreased activity of calcification inhibitors in PXE.

2010 - Pseudoxanthoma elasticum and familial hypercholesterolemia: A deleterious combination of cardiovascular risk factors [Articolo su rivista]
Pisciotta, L; Tarugi, Patrizia Maria; Borrini, C; Bellocchio, A; Fresa, R; Guerra, Deanna; Quaglino, Daniela; Ronchetti, Ivonne; Calandra Buonaura, Sebastiano; Bertolini, S.

Pseudoxanthoma Elasticum (PXE), an autosomal recessive disease due to mutations in ABCC6 gene, is characterised by fragmentation of elastic fibres with involvement of the cardiovascular system. We investigated a 60-year-old female with angina pectoris found to have PXE, associated with elevated plasma LDL-C suspected to be due to autosomal-co-dominant hypercholesterolemia. METHODS: ABCC6, LDLR, PCSK9 and exon 26 of APOB genes were re-sequenced. Cardiovascular involvement was assessed by coronary angiography, single-photon emission computed tomography (SPECT) and ultrasound examination. RESULTS AND CONCLUSIONS: The patient was a compound heterozygous for two ABCC6 mutations (p.S317R and p.R1141X) and heterozygous for a novel LDLR mutation (p.R574H). She had severe coronary stenosis and calcification of the arteries of the lower limbs. Treatment with ezetimibe/simvastatin 10/60mg/day, maintained over a 4.5-year period, reduced of LDL-C and the myocardial ischemic area. In PXE patients LDL-lowering treatment might contribute to delay macrovascular complications.

2009 - A morphological Approach to monitor nanoparticle-cell interaction [Articolo su rivista]
Gatti, Antonietta; Quaglino, Daniela; Sighinolfi, G. L.

Nanotechnologies may change to the better many sectors of industry, but considerable concern is arising about their side effects and possible risks to human life. The potential toxicity of nanoparticles (NPs) versus cells has to be much more clearly investigated than it has been done to date to define their future role in biological, medical and environmental applications.The present study performed in-vitro standardized cytotoxicity tests using Hematite, Magnetite and Valentinite nanoparticles with 3T3 cells. Biological (XTT and Brd-U assays), morphological (ESEM and TEM) and physical (EDS and x-ray diffraction) investigations were performed to evaluate cell-nanoparticle interaction and physical state after interaction. The results identified a threshold toxic concentration for all NPs, beyond which no cytotoxic effects were detectable by standardized tests. Notwithstanding these results, cytoplasmatic and nuclear translocation was observed and verified also during mitotic phase. The limits of the standardized tests are analyzed and discussed.

Aruta, CLAUDIA GAETANA; Croce, Maria Antonietta; Quaglino, Daniela; Guerra, Deanna; Tiozzo, Roberta

We have carried out an in vitro study on the interactions of human macrophages (J111) with two different membranes made of collagen type I and II, isolated from horse tendon and from horse articular and trachea cartilagene in order to obtain data on their biocompatibility. We have described the morphology of cell seeded on collagen films, and we have evaluated their proliferation as well as cytokine production as indicator of macrophage activation. The inflammatory response may in fact induce the destruction of collagen membranes and may interfere with cell and tissue behaviour. Results might be relevant for in vivo application such ad “tissue engeneering” and /or specialized cells implantation.

2009 - Connective tissue and diseases: from morphology to proteomics towards the development of new therapeutic approaches. [Articolo su rivista]
Quaglino, Daniela; Boraldi, Federica; Annovi, Giulia; Guerra, Deanna; Ronchetti, Ivonne

Connective tissue consists of cells separated by the extracellular matrix, whose composition and amount vary according to age, to functional requirements, and to the presence of pathologic conditions. Within this non-random macromolecular assembly, collagens, elastin, proteoglycans and structural glycoproteins are mutually interdependent and modifications of one component, by extrinsic (environmental) and/or intrinsic (systemic, genetic, age-related) factors, may have consequences on the tissue as a whole. Since decades, different microscopical techniques have been applied mainly for diagnostic purposes and for detailed descriptions of changes occurring in cells and in matrix components. More recently, in order to dissect the molecular complexity of the matrix network, to analyse the interactions between cells and matrix and to look for modulators of cell phenotype, histomorphologic investigations have been implemented with proteomic studies that allow to identify possible diagnostic markers, and to better understand patho-mechanisms enabling the design of novel therapeutic strategies. Therefore, the progressively expanding, although incomplete, knowledge on connective tissue biology, sheds new light on the pathogenesis of diseases affecting single molecules (i.e. collagenopathies, mucopolysaccharidoses, elastinopathies) and discloses the importance of matrix components as fundamental regulators of cell phenotype, in relation, for instance, to the aging process and/or to cancer development and progression. Few examples will be presented demonstrating the promises of proteomics as a technique leading to the discovery of new therapies and possibly to the development of individualized treatments for a better patient care.

2009 - Elastin and elastin-based polymers [Capitolo/Saggio]
Quaglino, Daniela; Guerra, Deanna; Ronchetti, Ivonne

Tropoelastin, elastin and elastin-derived peptides are very interesting molecules for the production of nanodevices with different purposes. Because of its peculiar structure and physical chemical characteristics, due to alternate hydrophobic and hydrophilic domains, tropoelastin can be fragmented and each fragment used for specific functions, such as to bring elasticity, self assembling properties, interactions with cells, with artificial polymers and with other matrix constituents, activation of metabolic pathways. Rather interestingly, hydrogels can be also produced for cell delivery of DNA and of drugs, so becoming a promising tool in tumor chemotherapy.

2009 - Fibroblast protein profile analysis highlights the role of oxidative stress and vitamin K recycling in the pathogenesis of pseudoxanthoma elasticum [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; Guerra, Deanna; Paolinelli Devincenzi, Chiara; Maria Inmaculada Garcia, Fernandez; Panico, Fulvio; De Santis, Giorgio; Tiozzo, Roberta; Ronchetti, Ivonne; Quaglino, Daniela

Pseudoxanthoma elasticum (PXE) is a genetic disorder associated to mutations in the ABCC6 gene, however the pathogenetic mechanisms leading to elastic fibre calcification and to clinical manifestations are still unknown. Dermal fibroblasts, directly involved in the production of the extracellular milieu, have been isolated from healthy subjects and from patients affected by PXE, cultured in vitro and characterized for their ability to produce reactive oxygen species, for structural and functional properties of their cell membranes, for changes in their protein profile. Data demonstrate that oxidative stress has profound and endurable consequences on PXE fibroblast phenotype being responsible for: reduced levels of global DNA methylation, increased amount of carbonylated proteins and of lipid peroxidation products, altered structural properties of cell membranes, modified protein expression. In conclusion, these data shed new light on pathogenetic pathways in PXE, by identifying a network of proteins affecting elastic fibre calcification through inefficient vitamin K recycling, and highlighting the role of several differentially expressed proteins that could be regarded as targets for validating the efficacy of future therapeutic strategies aiming to delay and/or revert the pathologic phenotype of PXE fibroblasts. Moreover, these data open new perspectives for investigating PXE-like phenotypes in the absence of ABCC6 mutations.

2009 - New insights into autophagic cell death in the gypsy moth Lymantria dispar: a proteomic approach [Articolo su rivista]
Malagoli, Davide; Boraldi, Federica; Annovi, Giulia; Quaglino, Daniela; Ottaviani, Enzo

Autophagy is an evolutionary ancient process based on the activity of genes conserved from yeast to metazoan taxa. Whereas its role as a mechanism to provide energy during cell starvation is commonly accepted, debate continues about the occurrence of autophagy as a means specifically activated to achieve cell death. The IPLB-LdFB insect cell line, derived from the larval fat body of the lepidoptera Lymantria dispar, represents a suitable model to address this question, as both autophagic and apoptotic cell death can be induced by various stimuli. Using morphological and functional approaches, we have observed that the culture medium conditioned by IPLB-LdFB cells committed to death by the ATPase inhibitor oligomycin A stimulates autophagic cell death in untreated IPLB-LdFB cells. Moreover, proteomic analysis of the conditioned media suggests that, in IPLB-LdFB cells, oligomycin A promotes a shift towards lipid metabolism, increases oxidative stress and specifically directs the cells towards autophagic activity.

2008 - A long-term study on female mice fed on a genetically modified soybean: effects on liver ageing. [Articolo su rivista]
M., Malatesta; Boraldi, Federica; Annovi, Giulia; B., Baldelli; S., Battistelli; M., Biggiogera; Quaglino, Daniela

Liver represents a suitable model for monitoring the effects of a diet, due to its key role in controlling the whole metabolism. Although no direct evidence has been reported so far that genetically modified (GM) food may affect health, previous studies on hepatocytes from young female mice fed on GM soybean demonstrated nuclear modifications involving transcription and splicing pathways. In this study, the effects of this diet were studied on liver of old female mice in order to elucidate possible interference with ageing. The morpho-functional characteristics of the liver of 24-month-old mice, fed from weaning on control or GM soybean, were investigated by combining a proteomic approach with ultrastructural, morphometrical and immunoelectron microscopical analyses. Several proteins belonging to hepatocyte metabolism, stress response, calcium signalling and mitochondria were differentially expressed in GM-fed mice, indicating a more marked expression of senescence markers in comparison to controls. Moreover, hepatocytes of GM-fed mice showed mitochondrial and nuclear modifications indicative of reduced metabolic rate. This study demonstrates that GM soybean intake can influence some liver features during ageing and, although the mechanisms remain unknown, underlines the importance to investigate the long-term consequences of GM-diets and the potential synergistic effects with ageing, xenobiotics and/or stress conditions.

2008 - A proteomic approach for investigating the aging process; the human fibroblast model. [Relazione in Atti di Convegno]
Quaglino, Daniela; Boraldi, Federica; Annovi, Giulia

Proteomic analysis of changes in protein expession of fibroblasts aged in vitro or isolated from aging donors highlights some characteristics of the aging process.

2008 - Comparison of Fibroblasts from Patients Affected by Pseudoxanthoma elasticum (PXE) or by β-Thalassemia with (β-thal-PXE+) and without PXE-Like Clinical Manifestations (β-thal-PXE-) [Abstract in Rivista]
Boraldi, Federica; PAOLINELLI DEVINCENZI, Chiara; M. I., GARCIA FERNANDEZ; Annovi, Giulia; Quaglino, Daniela; Tiozzo, Roberta; P., Cianciulli; Ronchetti, Ivonne

The role of altered redox balance in the pathogenesis of PXE-like clinical manifestations in patients affected by beta-thalassemia is discussed.

2008 - Elastin haploinsufficiency induces alternative aging processes in the aorta [Articolo su rivista]
M., Pezet; M. P., Jacob; B., Escoubet; Gheduzzi, Dealba; E., Tillet; P., Perret; P., Huber; Quaglino, Daniela; R., Vranckx; D. Y., Li; B., Starcher; W. A., Boyle; R. P., Mecham; G., Faury

Elastin, the main component of elastic fibers, is synthesized only in early life and provides the blood vessels with their elastic properties. With aging, elastin is progressively degraded, leading to arterial enlargement, stiffening, and dysfunction. Also, elastin is a key regulator of vascular smooth muscle cell proliferation and migration during development since heterozygous mutations in its gene (Eln) are responsible for a severe obstructive vascular disease, supravalvular aortic stenosis, isolated or associated to Williams syndrome. Here, we have studied whether early elastin synthesis could also influence the aging processes, by comparing the structure and function of ascending aorta from 6- and 24-month-old Eln+/- and Eln+/+ mice. Eln+/- animals have high blood pressure and arteries with smaller diameters and more rigid walls containing additional although thinner elastic lamellas. Nevertheless, longevity of these animals is unaffected. In young adult Eln+/- mice, some features resemble vascular aging of wild-type animals: cardiac hypertrophy, loss of elasticity of the arterial wall through enhanced fragmentation of the elastic fibers, and extracellular matrix accumulation in the aortic wall, in particular in the intima. In Eln+/- animals, we also observed an age-dependent alteration of endothelial vasorelaxant function. On the contrary, Eln+/- mice were protected from several classical consequences of aging visible in aged Eln+/+ mice, such as arterial wall thickening and alteration of alpha(1)-adrenoceptor-mediated vasoconstriction. Our results suggest that early elastin expression and organization modify arterial aging through their impact on both vascular cell physiology and structure and mechanics of blood vessels.

2008 - Exon 26-coded polypeptide: an isolated hydrophobic domain of human tropoelastin able to self-assemble in vitro. [Articolo su rivista]
A., PEPE A; R., Flamis; Guerra, Deanna; Quaglino, Daniela; B., Bochicchio; Ronchetti, Ivonne; A. M., Tamburro

Hydrophobic domains of human tropoelastin are able to aggregate in a variegated manner. Some aggregates have typical features of the whole protein while others show peculiar self-assembling profiles. Among these hydrophobic domains, an important role in the self-assembling properties of tropoelastin in vitro could be assigned to the peptide encoded by exon 26 of the human tropoelastin gene, that, although unstructured in solution, has great tendency to self-assemble in an ordered manner. The present report describes the aggregation properties of this hydrophobic domain of human tropoelastin analysed by different ultra-structural approaches. Transmission electron microscopy shows that the peptide is able to form different aggregation entities from short rods to very long and flexible fibers, depending on the temperature and on the incubation time. At a microm scale, very long fibers as well as fractal aggregation patterns were observed. Data show that the isolated domain encoded by exon 26 of the tropoelastin gene is able to aggregate in a manner very similar to the whole tropoelastin protein. The aggregation properties are due to the peculiar sequence of EX26, and not to its amino acid composition, as evidenced by the supramolecular analysis of a scrambled sequence of exon 26-coded domain of human tropoelastin, showing a quite different aggregation patterns. These findings confirm that specific sequences can play a driving role in the aggregation process of tropoelastin molecule, at least in vitro, and indicate exon 26-encoded domain among these sequences.

2008 - New insights into Pseudoxanthoma elasticum pathogenesis by proteome analysis. [Abstract in Rivista]
Quaglino, Daniela; Boraldi, Federica; Annovi, Giulia; Guerra, Deanna; Panico, Fulvio; Spaggiari, Antonio; DE SANTIS, Giorgio; Tiozzo, Roberta; Ronchetti, Ivonne

Proteome analysis on fibroblasts from PXE patients allowed to investigate on pathways involved in the pathogenesis of the disorder, thus highlighting the importance of oxidative stress and vitamin K recycling.

2008 - Parameters of oxidative stress are present in the circulation of PXE patients [Articolo su rivista]
M. I., Garcia Fernandez; Gheduzzi, Dealba; Boraldi, Federica; PAOLINELLI DEVINCENZI, Chiara; P., Sanchez; P., Valdivielso; M. J., Morilla; Quaglino, Daniela; Guerra, Deanna; Casolari, Sara; L., Bercovitch; Ronchetti, Ivonne

Pseudoxanthoma elasticum (PXE) is an inherited disorder characterized by calcification of elastic fibres leading to dermatological and vascular alterations associated to premature aged features and to life threatening clinical manifestations. The severity of the disease is independent from the type of mutation in the ABCC6 gene, and it has been suggested that local and/or systemic factors may contribute to the occurrence of clinical phenotype. The redox balance in the circulation of 27 PXE patients and of 50 healthy subjects of comparable age was evaluated by measuring the advanced oxidation protein products (AOPP), the lipid peroxidation derivatives (LOOH), the circulating total antioxidant status (TAS), the thiol content and the extracellular superoxide dismutase activity (EC-SOD). Patients were diagnosed by clinical, ultrastructural and molecular findings. Compared to control subjects, PXE patients exhibited significantly lower antioxidant potential, namely circulating TAS and free thiol groups, and higher levels of parameters of oxidative damage, as LOOH and of AOPP, and of circulating EC-SOD activity. Interestingly, the ratio between oxidant and antioxidant parameters was significantly altered in PXE patients and related to various score indices. This study demonstrates, for the first time, that several parameters of oxidative stress are modified in the blood of PXE patients and that the redox balance is significantly altered compared to control subjects of comparable age. Therefore, in PXE patients the circulating impaired redox balance may contribute to the occurrence of several clinical manifestations in PXE patients, and/or to the severity of disease, thus opening new perspectives for their management.

2008 - Resistance of mtDNA-depleted cells to apoptosis [Articolo su rivista]
Ferraresi, Roberta; Troiano, Leonarda; Pinti, Marcello; Roat, Erika; Lugli, Enrico; Quaglino, Daniela; D., Taverna; D., Bellizzi; G., Passarino; Cossarizza, Andrea

Cells lacking mitochondrial genome (defined as rho(0)) are useful models in studies on cancer, aging, mitochondrial diseases and apoptosis, but several of their functional aspects have been poorly characterized. Using different clones of rho(0) cells derived from the human osteosarcoma line 143B, we have tested the effects of different apoptogenic molecules such as staurosporine (STS), doxorubicin, daunomycin and quercetin, and have analyzed apoptosis, mitochondrial membrane potential (MMP), levels of oxygen free radicals, reduced glutathione (GSH) content, and expression of P-glycoprotein (P-gp). When compared to parental cells, rho(0) cells resulted much less sensitive to apoptosis. MMP was well maintained in rho(0) cells, and remained unchanged after adding apoptogenic agents, and did not change after treatment with molecules able to depolarize mitochondria such as valinomycin. After adding STS, the production of reactive oxygen species was similar in both cell types, but rho(0) cells maintained higher levels of GSH. In rho(0) cells, P-gp was strongly over-expressed both at mRNA and protein level, and its functionality was higher. The resistance to apoptosis of rho(0) cells could be not only due to an increased scavenger capacity of GSH, but also due to a selection of multidrug resistant cells that hyperexpress P-gp.

2008 - The effect of serum withdrawal on the protein profile of quiescent human dermal fibroblasts in primary cell culture [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; PAOLINELLI DEVINCENZI, Chiara; Tiozzo, Roberta; Quaglino, Daniela

The effect of serum deprivation on proliferating cells is well known, in contrast its role on primary cell cultures, at confluence, has not been deeply investigated. Therefore, in order to explore the response of quiescent cells to serum deprivation, ubiquitous mesenchymal cells, as normal human dermal fibroblasts, were grown, for 48 h after confluence, in the presence or absence of 10% FBS. Fibroblast behaviour (i.e. cell morphology, cell viability, ROS production and elastin synthesis) was evaluated morphologically and biochemically. Moreover, the protein profile was investigated by 2-DE and differentially expressed proteins were identified by MS. Serum withdrawal caused cell shrinkage but did not significantly modify the total cell number. ROS production, as evaluated by the dihydroethidium (DH2) probe, was increased after serum deprivation, whereas elastin synthesis, measured by a colorimetric method, was markedly reduced in the absence of serum. By proteome analysis, 41 proteins appeared to significantly change their expression, the great majority of protein changes were related to the cytoskeleton, the stress response and the glycolytic pathway. Data indicate that human dermal fibroblasts in primary cell culture can adapt themselves to environmental changes, without significantly altering cell viability, at least after a few days of treatment, even though serum withdrawal represents a stress condition capable to increase ROS production, to influence cell metabolism and to interfere with cell behaviour, favouring the expression of several age-related features.

2007 - Antioxidant status in the circulation of patients with Pseudoxanthoma elasticum (PXE). [Abstract in Rivista]
Boraldi, Federica; M. I., GARCIA FERNANDEZ; Gheduzzi, Dealba; PAOLINELLI DEVINCENZI, Chiara; P., Sanchez; P., Valdivielso; M. J., Morilla; Guerra, Deanna; Casolari, Sara; Quaglino, Daniela; Ronchetti, Ivonne

Several markers of redox balance have been measured in the plasma of PXE patients. Data demonstrate the presence of an altered redox balance not only at a local level (connective tissue fibroblasts), but also in the circulation. A correlation with clinical manifestations is provided.

2007 - Eosinophils, but not neutrophils, exibit an efficient DNA repair machinary and high nucleolar activity [Articolo su rivista]
Salati, Simona; Bianchi, Elisa; Zini, Roberta; Tenedini, Elena; Quaglino, Daniela; Manfredini, Rossella; Ferrari, Sergio

BACKGROUND AND OBJECTIVES: Traditionally eosinophils have been considered terminally differentiated cells that play a role in host protection against parasites. However, there is some evidence showing that eosinophils are, in fact, multifunctional leukocytes involved in inflammatory responses, as well as in tissue homeostasis. We characterized the transcriptome profile of human eosinophils, and, for the purpose of comparison, the transcriptome profile of neutrophils, monocytes and hematopoietic progenitor cells. Moreover, we studied the activation of selected cellular processes for which a significant differential expression was demonstrated. DESIGN AND METHODS: We profiled gene expression using Affymetrix GeneChips. DNA repair capacity was tested using the comet assay. Nucleoli and their activity were characterized by transmission electron microscopy analysis, silver staining of nucleolus regions (AgNOR) and RNA staining. RESULTS: Gene expression profiling showed that eosinophils appear hierarchically closer to monocytes than to neutrophils. Gene ontology mapping of differentially expressed genes revealed that eosinophils express categories very similar to those expressed by monocytes, related to DNA repair and nucleolar functions. Moreover, our data show that eosinophils and monocytes maintain the ability to repair both double and single strand DNA breaks, whereas neutrophils lack this capacity. Furthermore, eosinophils exhibit nucleolar activity, which is lacking in neutrophils, but resembles that in monocytes. INTERPRETATION AND CONCLUSIONS: The presence of large, active nucleoli in eosinophils, coupled to marked activity of DNA repair systems, suggests that eosinophils are not terminally differentiated cells. Indeed, their transcriptome profile and functional properties are more similar to those of non-terminally differentiated cells such as monocytes, rather than to neutrophils.

2007 - Fibroblasts from patients with beta-Thalassemia and Pseudoxanthoma elasticum-like clinical manifestations. [Abstract in Rivista]
PAOLINELLI DEVINCENZI, Chiara; Boraldi, Federica; M. I., GARCIA FERNANDEZ; Annovi, Giulia; Quaglino, Daniela; Tiozzo, Roberta; Ceccarelli, Daniela; P., Cianciulli; P. L., Forni; Ronchetti, Ivonne

In vitro studies on the phenotype of dermal fibroblasts from patients with beta-thalassemia and PXE-like clinical manifestations are described and the the role of altered redox balance is discussed

2007 - Hypoxia influences the cellular cross-talk of human dermal fibroblasts. A proteomic approach [Articolo su rivista]
Boraldi, Federica; Annovi, Giulia; Carraro, F.; Naldini, A.; Tiozzo, Roberta; Sommer, P.; Quaglino, Daniela

The ability of cells to respond to changes in oxygen availability is critical for many physiological and pathological processes (i.e. development, aging, wound healing, hypertension, cancer). Changes in the protein profile of normal human dermal fibroblasts were investigated in vitro after 96 h in 5% CO(2) and 21% O(2) (pO(2) = 140 mm Hg) or 2% O(2) (pO(2) = 14 mm Hg), these parameters representing a mild chronic hypoxic exposure which fibroblasts may undergo in vivo. The proliferation rate and the protein content were not significantly modified by hypoxia, whereas proteome analysis demonstrated changes in the expression of 56 proteins. Protein identification was performed by mass spectrometry. Data demonstrate that human fibroblasts respond to mild hypoxia increasing the expression of hypoxia inducible factor (HIF1a) and of the 150-kDa oxygen-regulated protein. Other differentially expressed proteins appeared to be related to stress response, transcriptional control, metabolism, cytoskeleton, matrix remodelling and angiogenesis. Furthermore, some of them, like galectin 1, 40S ribosomal protein SA, N-myc-downstream regulated gene-1 protein, that have been described in the literature as possible cancer markers, significantly changed their expression also in normal hypoxic fibroblasts. Interestingly, a bovine fetuin was also identified that appeared significantly less internalised by hypoxic fibroblasts. In conclusion, results indicate that human dermal fibroblasts respond to an in vitro mild chronic hypoxic exposure by modifying a number of multifunctional proteins. Furthermore, data highlight the importance of stromal cells in modulating the intercellular cross-talk occurring in physiological and in pathologic conditions.

2007 - Is matrix GLA Protein (MGP) a key regulator of elastic fiber calcification? [Abstract in Rivista]
Annovi, Giulia; Gheduzzi, Dealba; Boraldi, Federica; L. J., Schurgers; Tiozzo, Roberta; Quaglino, Daniela; Ronchetti, Ivonne

The role of vitamin k dependent carboxylation of Matrix Gla protein in PXE patogenesis is described and discussed.

2007 - Matrix Gla Protein involved in elastic fiber calcification in the dermis of pseudoxanthoma elasticum patients. [Articolo su rivista]
Gheduzzi, Dealba; Boraldi, Federica; Annovi, Giulia; Paolinelli Devincenzi, Chiara; L., Schurgers; C., Vermeer; Quaglino, Daniela; Ronchetti, Ivonne

Mature MGP (Matrix gamma-carboxyglutamic acid protein) is known to inhibit soft connective tissues calcification. We investigated its possible involvement in pseudoxanthoma elasticum (PXE), a genetic disorder whose clinical manifestations are due to mineralization of elastic fibers. PXE patients have lower serum concentration of total MGP compared to controls (P<0.001). Antibodies specific for the noncarboxylated (Glu-MGP) and for the gamma-carboxylated (Gla-MGP) forms of MGP were assayed on ultrathin sections of dermis from controls and PXE patients. Normal elastic fibers in controls and patients were slightly positive for both forms of MGP, whereas Gla-MGP was more abundant within control's than within patient's elastic fibers (P<0.001). In patients' calcified elastic fibers, Glu-MGP intensively colocalized with mineral precipitates, whereas Gla-MGP precisely localized at the mineralization front. Data suggest that MGP is present within elastic fibers and is associated with calcification of dermal elastic fibers in PXE. To investigate whether local cells produce MGP, dermal fibroblasts were cultured in vitro and MGP was assayed at mRNA and protein levels. In spite of very similar MGP mRNA expression, cells from PXE patients produced 30% less of Gla-MGP compared to controls. Data were confirmed by immunocytochemistry on ultrathin sections. Normal fibroblasts in vitro were positive for both forms of MGP. PXE fibroblasts were positive for Glu-MGP and only barely positive for Gla-MGP (P<0.001). In conclusion, MGP is involved in elastic fiber calcification in PXE. The lower ratio of Gla-MGP over Glu-MGP in pathological fibroblasts compared to controls suggests these cells may play an important role in the ectopic calcification in PXE.

2007 - New insights on the pathogenesis of Pseudoxanthoma elasticum by proteome analysis. [Abstract in Rivista]
Boraldi, Federica; Annovi, Giulia; PAOLINELLI DEVINCENZI, Chiara; Gheduzzi, Dealba; Tiozzo, Roberta; Ronchetti, Ivonne; Quaglino, Daniela

Data are presented on the protein profile of dermal fibroblasts cultured in vitro from PXE patients, with the aim to elucidate molecular pathways involved in the pathogenesis of the disease

2007 - Response of confluent human dermal fibroblasts to serum deprivation investigated by proteome analysis. [Abstract in Rivista]
Boraldi, Federica; Annovi, Giulia; PAOLINELLI DEVINCENZI, Chiara; Guidetti, Rita; Tiozzo, Roberta; Quaglino, Daniela

A preliminary characterization of the protein profile of human dermal fibroblstats cultured in vitro after confluence, in the absence of serum factors, is provided.

2007 - Scanning electron microscopy of thinned specimens: From multilayers to biological samples [Articolo su rivista]
V., Morandi; P. G., Merli; Quaglino, Daniela

The rules governing image formation of thin specimens in scanning and scanning transmission electron microscopy at low energy, deduced from the observation of semiconductor multilayers, were validated on specimens defined by a much more complex structure as the biological ones. It is shown that for a suitable specimen thickness it is possible to have, at the same time, backscattered electron images and scanning transmission electron images with a comparable resolution. Moreover, the nonconductive biological samples can be observed without charging effects if they are thin enough to ensure that a significant fraction of the electron beam crosses the specimen.

2007 - Tropoelastin deposition is affected by addition of heparan sulphate. [Abstract in Rivista]
Guidetti, Rita; Croce, Maria Antonietta; Gheduzzi, Dealba; B., Parma; Ronchetti, Ivonne; Tiozzo, Roberta; Quaglino, Daniela

The interactions between tropoelastin and heparan sulphate are described and their role in elastic fiber formation are discussed

2006 - Oxidative stress in fibroblasts from patients with pseudoxanthoma elasticum: possible role in the pathogenesis of clinical manifestations [Articolo su rivista]
Ronchetti, Ivonne; MI Garcia, Fernandez; Boraldi, Federica; Quaglino, Daniela; Gheduzzi, Dealba; De Vincenzi Paolinelli, C.; Tiozzo, Roberta; Bergamini, Stefania; D., Ceccarelli; U., Muscatello

Pseudoxanthoma elasticum (PXE) is a genetic disease characterized by calcification and fragmentation of elastic fibres of the skin, cardiovascular system and eye, caused by mutations of the ABCC6 gene, which encodes the membrane transporter MRP6. The pathogenesis of the lesions is unknown. Based on studies of similar clinical and histopathological damage present in haemolytic disorders, our working hypothesis is that PXE lesions may result from chronic oxidative stress occurring in PXE cells as a consequence of MRP6 deficiency. Our results show that PXE fibroblasts suffer from mild chronic oxidative stress due to the imbalance between production and degradation of oxidant species. The findings also show that this imbalance results, at least in part, from the loss of mitochondrial membrane potential (Delta Psi(m)) with overproduction of H2O2. Whether mitochondrial dysfunction is the main factor responsible for the oxidative stress in PXE cells remains to be elucidated. However, mild chronic generalized oxidative stress could explain the great majority of structural and biochemical alterations already reported in PXE.

2006 - The lack of Pneumococcal surface protein C (PspC) increases the susceptibility of Streptococcus pneumoniae to the killing by microglial cells [Articolo su rivista]
Peppoloni, Samuele; Colombari, Bruna; Neglia, Rachele Giovanna; Quaglino, Daniela; F., Iannelli; Mr, Oggioni; G., Pozzi; Blasi, Elisabetta

Microglial cells, the resident phagocytes in the brain, share many phenotypical and functional characteristics with peripheral macrophages, suggesting that they may participate in an innate immune response against microorganisms invading the central nervous system (CNS). In this study, we demonstrate that the microglial cells constitutively exhibit antibacterial activity in vitro against Streptococcus pneumoniae. By using a Pneumococcal surface protein C (PspC)-deleted strain and its wild-type counterpart, we found that the extent of such an activity is significantly influenced by the presence of a PspC molecule on the bacterial surface. The PspC- mutant FP20 is indeed more susceptible than the PspC+ strain HB565 to microglial killing. Interestingly, this phenomenon is observed when using a medium supplemented with heat-inactivated foetal bovine serum (FBS). Electron microscopy studies indicate that the microglial cells interact more efficiently with PspC- than with PspC+ pneumococci. Moreover, upon infection with the PspC- mutant, microglial cells produce levels of TNF-alpha, MIP-2, IL-10 and nitric oxide, significantly higher than those observed with PspC+ bacteria. These findings indicate that the lack of PspC significantly enhances the susceptibility of S. pneumoniae to both bactericidal activity and secretory response by the microglial cells, suggesting that this molecule may play an important role in the invasion of CNS by pneumococcus.

2006 - 1956-2006 Cinquanta anni di microscopia in Italia tra storia, progresso ed innovazione [Curatela]
Quaglino, Daniela; Falcieri, E; Catalano, M; Diaspro, A; Montone, A; Mengucci, P; Pelicciari, C.

This volume celebrates the 50th anniversary of SIME/SISM foundation by describing the major advances in the different fields of microscopy and some of their applications in biology, material science, medicine, botanics, environmental science.

2005 - ABCC6 gene analysis in Italian subjects affected by Pseudoxanthoma elasticum. [Abstract in Rivista]
Gheduzzi, Dealba; E., Giacobazzi; M., Rebecchi; Guidetti, Rita; Quaglino, Daniela; Ronchetti, Ivonne

Techniques used to screen PXE patients for causative mutations in the ABCC6 gene are described and compared in order to reach a better mutation detection rate.

2005 - Ageing of connective tissues [Relazione in Atti di Convegno]
Quaglino, Daniela; Gheduzzi, Dealba; Boraldi, Federica; Ronchetti, Ivonne

Aging, considered as a chronological and/or biological event, is an extremely complex and multifactorial process, which may represent the consequences of environmental noxae interfering with genetic and developmental programs.The extracellular matrix plays a crucial role in age-related degenerations, and the so-called "premature aging syndromes" give further evidence for the complexity of the relationships between connective tissue, age and diseases. Several reports already focused on the correlations between age and phenotypic expression of mesenchimal cells in vitro; moreover, it was also pointed out that morphological and functional alterations of connective tissues, at some extent, might be related in vivo to increasing age. Correlation between morphologic data and proteomic analyses sustains the hypothesis that senescence is the result of both genetic and epigenetic factors.

2005 - Dermal fibroblasts from pseudoxanthoma elasticum patients have raised MMP-2 degradative potential [Articolo su rivista]
Quaglino, Daniela; Sartor, L; Garbisa, S; Boraldi, Federica; Croce, Maria Antonietta; Passi, A; De Luca, G; Tiozzo, Roberta; Ronchetti, Ivonne

Cultured fibroblasts from the dermis of normal subjects and of Pseudoxanthoma elasticum (PXE) patients were analysed for enzyme activity, protein and mRNA expression of metalloproteases (MMP-2, MMP-3, MMP-9, MT1-MMP) and of their specific inhibitors (TIMP-1, TIMP-2 and TIMP-3). MMP-3, MMP-9 and TIMP-3 mRNAs and proteins failed to be detected in both the medium and the cell layer of both controls and PXE patients. MMP-2 mRNA was significantly more expressed in PXE than in control cell lines, whereas MT1-MMP, TIMP-1 and TIMP-2 mRNAs appeared unchanged. MMP-2 was significantly higher in the cell extracts from PXE fibroblasts than in control cells, whereas differences were negligible in the cell medium. Data suggest that PXE fibroblasts have an increased proteolytic potential, and that MMP-2 may actively contribute to connective tissue alterations in this genetic disorder.

2005 - Dissection of human tropoelastin: Supramolecular organization of polypeptide sequences coded by particular exons [Articolo su rivista]
A., Pepe; Guerra, Deanna; B., Bochicchio; Quaglino, Daniela; Gheduzzi, Dealba; Ronchetti, Ivonne; A. M., Tamburro

Polypeptide sequences encoded by some exons of the human tropoelastin gene (EDP, elastin-derived peptide) have been analysed for their ability to coacervate and to self-assembly. The great majority of them were shown to form organized structures, but only a few were indeed able to coacervate. Negative staining and rotary shadowing transmission electron microscopy showed the polypeptides to adopt a variety of supramolecular organization, from filaments, as those typical of tropoelastin, to amyloid-like fibers. The results obtained gave significant insight to the possible roles played by specific polypeptide sequences of tropoelastin.

2005 - Heparan sulphate interacts with tropoelastin, with some tropoelastin peptides and is present in human dermis elastic fibers [Articolo su rivista]
Gheduzzi, Dealba; Guerra, Deanna; B., Bochicchio; A., Pepe; Am, Tamburro; Quaglino, Daniela; S., Mithieux; Ronchetti, Ivonne; As, Weiss

A number of reports point to the presence of proteoglycans and/or glycosaminoglycans within elastic fibers in normal and in pathological conditions. We present data that heparan sulphate (HS)-containing proteoglycans are associated with normal elastic fibers in human dermis and that isolated HS chains interact in vitro with recombinant tropoelastin and with peptides encoded by distinct exons of the human tropoelastin gene (EDPs). By immunocytochemistry, HS chains were identified as associated with the amorphous elastin component in the human dermis and remained associated with the residual elastin in the partially degenerated fibers of old subjects. HS appeared particularly concentrated in the mineralization front of elastic fibers in the dermis of patients affected by pseudoxanthoma elasticum (PXE). In in vitro experiments, HS induced substantial changes in the coacervation temperature and in the aggregation properties of recombinant tropoelastin and of synthetic peptides (EDPs) corresponding to sequences encoded by exons 18, 20, 24 and 30 of the human tropoelastin gene. In particular, HS modified the coacervation temperature and favoured the aggregation into ordered structures of tropoelastin molecules and of EDPs 18, 20 and 24, but not of EDP30. These data strongly indicate that HS-elastin interactions may play a role in tissue elastin fibrogenesis as well as modulating elastin stability with time and in diseases.

2005 - Identification of mineralized elastic fibers on wet samples by SEM [Articolo su rivista]
Boraldi, Federica; Tonelli, Massimo; Gheduzzi, Dealba; Ronchetti, Ivonne; Quaglino, Daniela

A method is described that could be of potential use for the rapid ultrastructural identification of abnormal and fragmented elastic fibers in very small wet samples of dermal biopsies from patients affected by Pseudoxanthoma elasticum (PXE). Moreover, the method, which consists of the use of sealed capsules transparent to electrons, allows the rapid and accurate localization and detection of mineralized areas in PXE patients and of their ion composition by X-ray microanalysis. This methodology could be of great help in any tissue disorder, especially in connective tissue disorders, characterized by structural alterations associated with ion precipitation.

2005 - New insights on the pathogenesis of clinical manifestations in Pseudoxathoma elasticum. [Abstract in Rivista]
Ronchetti, Ivonne; M. I., GARCIA FERNANDEZ; Boraldi, Federica; Quaglino, Daniela; Gheduzzi, Dealba; PAOLINELLI DEVINCENZI, Chiara; Tiozzo, Roberta; Bergamini, Stefania; Ceccarelli, Daniela; Muscatello, Umberto

Data are presented on the presence of oxidative stress in fibroblasts from PXE patients and the potential role in PXE pathogenesis is discussed.

2005 - On the pathogenesis of PXE-like clinical manifestations in Beta-thalassemic patients [Abstract in Rivista]
Boraldi, Federica; PAOLINELLI DEVINCENZI, Chiara; M. I., Garcia Fernandez; Quaglino, Daniela; Tiozzo, Roberta; Croce, Maria Antonietta; P., Cianciulli; F., Sorrentino; G. L., Forni; Ronchetti, Ivonne

Altered fibroblast phenotype and membrane transport properties are described in patients affected by beta-thalassemia with elastic fiber mineralization and PXE-like clinical manifestations.

2005 - Ruolo della proteina pneumococcica di superficie PspC nel l’interazione in vitro di Streptococcus pneumoniae con le cellule microgliali [Abstract in Atti di Convegno]
Peppoloni, Samuele; Colombari, Bruna; Neglia, Rachele Giovanna; Quaglino, Daniela; Martino, Antonio; F., Iannelli; M. R., Oggioni; G., Pozzi; Blasi, Elisabetta

Ruolo della proteina pneumococcica di superficie PspC nel l’interazione in vitro di Streptococcus pneumoniae con le cellule microglialiIntroduzione: Le cellule microgliali, i principali fagociti residenti del distretto cerebrale, possiedono caratteristiche fenotipiche e funzionali tipiche dei macrofagi e perciò si pensa possano svolgere un ruolo importante nell’immunità innata contro le infezioni cerebrali. Streptococcus pneumoniae è responsabile di gravi malattie, quali polmonite, batteremia e meningite. Tra i suoi fattori di virulenza c’è PspC, una molecola di adesione presente nel 75% circa dei pneumococchi, che oltre a svolgere un ruolo fondamentale nel processo di colonizzazione, interferisce con i processi di attivazione del complemento, in quanto lega il fattore H e previene così l’opsonofagocitosi. In questo studio, abbiamo valutato il ruolo di PspC nell’interazione di S. pneumoniae con la cellula microgliale. Materiali e Metodi: mediante un modello di infezione in vitro della linea microgliale BV2 con i ceppi batterici HB565 ed il suo mutante isogenico PspC- F20, abbiamo valutato la morfologia, l’attività antipneumococcica e secretoria della microglia. Risultati: le cellule microgliali BV2 mostrano una marcata attività antibatterica nei confronti di entrambi i ceppi; tuttavia, esse legano ed uccidono in maniera più efficiente i batteri mutanti PspC- rispetto a quelli del ceppo parentale HB565, anche in assenza di complemento. Infine, cellule BV2 stimolate con il ceppo PspC- producono livelli di citochine, quali TNF-α, MIP-2, IL-10 e ossido nitrico più elevati di quelli osservati in seguito a stimolazione con il ceppo selvaggio.Conclusioni: l’assenza di PspC aumenta sia l’attività battericida che la risposta secretoria delle cellule microgliali, suggerendo che questa molecola possa giocare un ruolo importante nell’insorgenza della meningite pneumococcica.

2005 - Supramolecular amyloid-like assembly of the polypeptide sequence coded by exon 30 of human tropoelastin [Articolo su rivista]
Am, Tamburro; A., Pepe; B., Bochicchio; Quaglino, Daniela; Ronchetti, Ivonne

Elastin is known to self-aggregate in twisted-rope filaments. However, an ultrastructural organization different from the fibrils typical of elastin, but rather similar to those shown by amyloid networks, is shown by the polypeptide sequence encoded by exon 30 of human tropoelastin. To better understand the molecular properties of this sequence to give amyloid fibers, we used CD, NMR, and FTIR (Fourier transform infrared spectroscopy) to identify the structural characteristics of the peptide. In this study, we have demonstrated, by FTIR, that antiparallel beta-sheet conformation is predominant in the exon 30 fibers. These physical-chemical studies were combined with transmission electron microscopy and atomic force microscopy to analyze the supramolecular structure of the self-assembled aggregate. These studies show the presence of fibrils that interact side-by-side probably originating from an extensive self-interaction of elemental cross beta-structures. Similar sequences, of the general type XGGZG (X, Z = V, L, A, 1), are widely found in many proteins such as collagens IV and XVII, major prion protein precursor, amyloid beta A4 precursor protein-binding family, etc., thus suggesting that this sequence could be involved in contributing to the self-assembly of amyloid fibers even in other proteins.

2005 - The Pneumococcal surface protein C (PspC) influences the susceptibility of Streptococcus pneumoniae to microglia [Abstract in Atti di Convegno]
Peppoloni, Samuele; Colombari, Bruna; Quaglino, Daniela; Neglia, Rachele Giovanna; Martino, Antonio; F., Iannelli; M. R., Oggioni; S., Ricci; G., Pozzi; Blasi, Elisabetta

The Pneumococcal surface protein C (PspC) influences the susceptibility of Streptococcus pneumoniae to microglia

2005 - The role of hypoxic condition on the protein profile profile of human fibroblasts [Abstract in Rivista]
Boraldi, Federica; Annovi, Giulia; Carraro, F; Ronchetti, Ivonne; Tiozzo, Roberta; Quaglino, Daniela

Proteome analysis of normal human dermal fibroblasts culture in normoxic and hypoxic conditions

2004 - ABCC6 mutations in Italian families affected by pseudoxanthoma elasticum(PXE). [Articolo su rivista]
Gheduzzi, Dealba; Guidetti, Rita; Anzivino, Claudia; Tarugi, Patrizia Maria; DI LEO, E; Quaglino, Daniela; Ronchetti, Ivonne

Pseudoxanthoma elasticum (PXE) is a genetic disorder, characterized by cutaneous, ocular and cardiovascular clinical symptoms, caused by mutations in a gene (ABCC6) that encodes for MRP6 (Multidrug Resistance associated Protein 6), an ATP-binding cassette membrane transporter. The ABCC6 gene was sequenced in 38 unrelated PXE Italian families. The mutation detection rate was 82.9%. Mutant alleles occurred in homozygous, compound heterozygous and heterozygous forms, however the great majority of patients were compound heterozygotes. Twenty-three different mutations were identified, among which 11 were new. Fourteen were missense (61%); five were nonsense (22%); two were frameshift (8.5%) and two were putative splice site mutations (8.5%). The great majority of mutations were located from exon 24 to 30, exon 24 being the most affected. Among the others, exons 9 and 12 were particularly involved. Almost all mutations were located in the intracellular site of MRP6. A positive correlation was observed between patient's age and severity of the disorder, especially for eye alterations. The relevant heterogeneity in clinical manifestations between patients with identical ABCC6 mutations, even within the same family, seems to indicate that, apart from PXE causative mutations, other genes and/or metabolic pathways might influence the clinical expression of the disorder

2004 - Effects of metabolites and analogs of amiodarone on alveolar macrophages: structure-activity relationship [Articolo su rivista]
Quaglino, Daniela; Ha, Hr; Duner, E; Bruttomesso, D; Bigler, L; Follath, F; Realdi, G; Pettenazzo, A; Baritussio, A.

Amiodarone, an antiarrhythmic drug toxic toward the lung, is metabolized through sequential modifications of the diethylaminoethoxy group to mono-N-desethylamiodarone (MDEA), di-N-desethylamiodarone (DDEA), and amiodarone-EtOH (B2-O-EtOH), whose effects on lung cells are unclear. To clarify this, we exposed rabbit alveolar macrophages to analogs with different modifications of the diethylaminoethoxy group and then searched for biochemical signs of cell damage, formation of vacuoles and inclusion bodies, and interference with the degradation of surfactant protein A, used as a tracer of the endocytic pathway. The substances studied included MDEA, DDEA, and B2-O-EtOH, analogs with different modifications of the diethylaminoethoxy group, fragments of the amiodarone molecule, and the antiarrhythmic agents dronedarone (SR-33589) and KB-130015. We found the following: 1) MDEA, DDEA, and B2-O-EtOH rank in order of decreasing toxicity toward alveolar macrophages, indicating that dealkylation and deamination of the diethylaminoethoxy group represent important mechanisms of detoxification; 2) dronedarone has greater, and KB-130015 has smaller, toxicity than amiodarone toward alveolar macrophages; and 3) the benzofuran moiety, which is toxic to liver cells, is not directly toxic toward alveolar macrophages.

2004 - Functional genomics: the PXE model [Abstract in Atti di Convegno]
Ronchetti, Ivonne; Quaglino, Daniela; Gheduzzi, Dealba; Croce, Maria Antonietta; Boraldi, Federica; Tiozzo, Roberta

Pseudoxanthoma elasticum represent a disease model for investigating the relationships bettween altered fibroblast phenotype and elastic fiber degeneration/calcification.

2004 - Heparan sulphate-elastin interactions in vivo and in vitro [Abstract in Rivista]
Gheduzzi, Dealba; Guerra, Deanna; Pepe, A; Tamburro, A; Bochicchio, B; Mithieux, S; Weiss, A; Quaglino, Daniela; Ronchetti, Ivonne

Investigation on the role of heparan sulfate-tropoelastin interactions in elastin fibrogenesis.

2004 - La proteina di superficie PspC di pneumococco conferisce resistenza al killing in vitro di Streptococcus pneumoniae da parte di cellule microgliali in maniera indipendente dal complemento [Abstract in Atti di Convegno]
Peppoloni, Samuele; Colombari, Bruna; Quaglino, Daniela; Neglia, Rachele Giovanna; Martino, Antonio; F., Iannelli; S., Ricci; M. R., Oggioni; G., Pozzi; Blasi, Elisabetta

La proteina di superficie PspC di pneumococco conferisce resistenza al killing in vitro di Streptococcus pneumoniae da parte di cellule microgliali in maniera indipendente dal complemento. In assenza di complemento le cellule microgliali BV2 uccidono efficientemente S. pneumoniae in vitro e la presenza di PspC ne riduce la capacità battericida, così come quella secretoria. PcpC svolge quindi un ruolo patogenetico importante nell'interazione diretta tra il pneumococco e la microglia

2004 - Post-infectious acute ataxia and facial diplegia associated with anti-GD1a IgG antibody [Articolo su rivista]
Galassi, G; Susuki, K; Quaglino, Daniela; Yuki, N.

The use of anti-GD1a IgG antibody, as a diagnostic marker of GBS in generalized forms, is described in a patient with with acute facial diplegia and hyperreflexia and the usefulness of anti-ganglioside antibody assays is highlighted. Tests for IgG and IgM antibodies against GM2, GM1, GM1b, GalNAc-GD1a, GD1b, GT1a and GQ1b were negative whereas thin-layer chromatography with immunostaining confirmed the presence of anti-GD1a antibody.An enzyme-linked immunosorbent assay revealed elevated anti-GD1a IgG antibody titer of 2000 (normal <500) in serum obtained on day 6. On day 180, there were no residual neurological abnormalities and seventeen months after onset, titer of anti-GD1a IgG antibody was 500.

2004 - Protease activities in pseudoxanthoma elasticum [Abstract in Rivista]
Quaglino, Daniela; Annovazzi, L; Sartor, L; Garbisa, S; Iadarola, P; Passi, A; Tiozzo, Roberta; Ronchetti, Ivonne

On the role of metalloprotease (MMP) expression and activity in cultured fibroblasts from PXE patients

2004 - Supramolecular, amyloid-like organization of the polypeptide sequence coded by exon 30 of human tropoelastin [Abstract in Rivista]
Pepe, A; Bochicchio, B; Quaglino, Daniela; Ronchetti, Ivonne; Tamburro, Am

Structural investigation on amyloid-like organization in elastin.

2004 - The effect on rat thymocytes of the simultaneous invivo exposure to 50-Hz electric and magnetic field and to continuous light. [Articolo su rivista]
Quaglino, Daniela; Capri, M; Zecca, L; Franceschi, C; Ronchetti, Ivonne

Thymus plays an important role in the immune system and can be modulated by numerous environmental factors, including electromagnetic fields (EMF). The present study has been undertaken with the aim to investigate the role of long-term exposure to extremely low frequency electric and magnetic fields (ELF-EMF) on thymocytes of rats housed in a regular dark/light cycle or under continuous light. Male Sprague-Dawley rats, 2 months old, were exposed or sham exposed for 8 months to 50-Hz sinusoidal EMF at two levels of field strength (1 kV/m, 5 microT and 5 kV/m, 100 microT, respectively). Thymus from adult animals exhibits signs of gradual atrophy mainly due to collagen deposition and fat substitution. This physiological involution may be accelerated by continuous light exposure that induces a massive death of thymocytes. The concurrent exposure to continuous light and to ELF-EMF did not change significantly the rate of mitoses compared to sham-exposed rats, whereas the amount of cell death was significantly increased, also in comparison with animals exposed to EMF in a 12-h dark-light cycle. In conclusion, long-term exposure to ELF-EMF, in animals housed under continuous light, may reinforce the alterations due to a photic stress, suggesting that, in vivo, stress and ELF-EMF exposure can act in synergy determining a more rapid involution of the thymus and might be responsible for an increased susceptibility to the potentially hazardous effects of ELF-EMF

2004 - The protein profile of fibroblasts: the role of proteomics. [Articolo su rivista]
Quaglino, Daniela; Boraldi, Federica; L., Bini; Volpi, Nicola

Fibroblasts represent one of the most widely used cell types to investigate the biology of connective tissues in normal and pathologic conditions. Aim of the present review is to emphasize, in the light of the current literature, the importance of fibroblast proteomics as a powerful resource for functional genomics in health and disease. Only very recently, proteomic techniques has been applied to characterise human dermal fibroblasts, but few data are available concerning fibroblasts of various animal origins or derived from different tissues. Functional proteomic methods have been successfully used in order i) to investigate changes in protein synthesis resulting from stimulation of fibroblasts with exogenous and endogenous factors and in the presence of conditioned media; ii) to identify the underlying mechanisms that modulate fibroblast protein profile during senescence; iii) to obtain increased knowledge about the pathogenesis of diseases such as peribronchial fibrosis; iv) to better understand the molecular basis of biocompatibility. In addition, comparison of data obtained by proteome analysis, on in vitro aged human embryo fibroblasts and on in vitro cultured human fibroblasts from subjects of different ages, allowed differences and similarities of the aging process in different models to be highlighted. Although the number of proteomic studies has exponentially increased during the past couple of years, several proteins are still under-represented in most proteome maps, i.e. membrane, low abundant and basic proteins. Since a comprehensive proteomic approach must use a technology platform that is not biased against any protein class and is able to resolve co- and post-translationally modified forms of proteins, we exemplify here the major technical improvements in protein separation and identification. Moreover, glycosylation is the most common type of post-translational protein modification, and a special emphasis is therefore placed on the expanding role of glycomics.

2004 - The skin equivalent model: developments and perspectives. [Articolo su rivista]
Croce, Maria Antonietta; Sammarco, Rita; PAOLINELLI DEVINCENZI, Chiara; Boraldi, Federica; Gheduzzi, Dealba; Damour, O.; Sommer, P.; Ronchetti, Ivonne; Tiozzo, Roberta; Quaglino, Daniela

Three-dimensional culture systems have been developed to mimic natural interactions among cells and between cells and the extracellular matrix. The “skin equivalent ” is a 3-D co-culture system of fibroblasts and keratinocytes used as a covering surface in extended wounds, and as a model for studying the influence of each cell type on the synthesis of the extracellular matrix. In the present study, a three-dimensional scaffold made of chitosan, glycosaminoglycans and collagen has been colonised by keratinocytes from newborn foreskin an by fibroblasts isolated from donors of different ages. In the skin equivalent, the neo-synthesized connective tissue is characterized by the presence of amorphous elastic fibers, strengthening the usefulness of this three-dimensional model as a powerful tool for investigating connective tissue metabolism in different physiological (i.e. aging) and pathological conditions. Interestingly, when fibroblasts isolated from aged donors were cultured in vitro, even though in the presence of abundant nutrients and growth factors, their old phenotype appeared not to be completely overcome, indicating that aging is the result of a continuous remodelling controlled by epigenetic as well as genetic factors. Finally, the aged phenotype of fibroblasts seems to exert a stronger influence on keratinocytes compared to that of keratinocytes on fibroblasts.

2003 - Apoptosis-resistant phenotype in HL-60-derived cells HCW-2 is related to changes in expression of stress-induced proteins that impact on redox status and mitochondrial metabolism [Articolo su rivista]
S., Salvioli; G., Storci; Pinti, Marcello; Quaglino, Daniela; L., Moretti; M., Merlo Pich; G., Lenaz; S., Filosa; A., Fico; M., Bonafe; D., Monti; Troiano, Leonarda; Nasi, Milena; Cossarizza, Andrea; C., Franceschi

The onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapic drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.

2003 - Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan [Articolo su rivista]
Boraldi, Federica; Croce, Maria Antonietta; Quaglino, Daniela; Sammarco, Rita; Carnevali, Elena; Tiozzo, Roberta; Ronchetti, Ivonne

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell-matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5 x 10(5) to 2.7 x 10(6) Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell-substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type 1-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast-collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling. (C) 2003 Elsevier Science Ltd. All rights reserved.

2003 - Extracutaneous ultrastructural alterations in pseudoxanthoma elasticum [Articolo su rivista]
Gheduzzi, Dealba; Sammarco, Rita; Quaglino, Daniela; L., Bercovitch; S., Terry; W., Taylor; Ronchetti, Ivonne

Pseudoxanthoma elasticum (PXE) is caused by mutations in the ABCC6 gene, encoding for the membrane transporter MRP6, whose physiological role is still unknown. PXE is characterized by skin, eye, and cardiovascular alterations mainly due to mineralization of elastic fibers. The ultrastructural alterations of a large number of tissues obtained at autopsy from 2 PXE patients were analyzed and compared to clarify the involvement of the various organs in PXE and to identify cell types responsible for clinical manifestations. Ultrastructural alterations typical of PXE were present in all organs examined and consisted mostly of fragmentation and mineralization of a number of elastic fibers, abnormalities of collagen fibril shape and size, and, less frequently, deposition of aggregates of matrix constituents in the extracellular space. The severity of alterations was more pronounced in the organs affected by the clinical manifestations of PXE. Interestingly, veins and arteries were similarly damaged, the adventitia and the perivascular connective tissue being the most affected areas. Therefore, alterations in PXE are systemic and affect all soft connective tissues, even in the absence of specific clinical manifestations. The localization of alterations suggests that fibroblasts and/or smooth muscle cells are very likely involved in the pathogenesis of the disorder. These findings may help in the diagnosis of PXE when clinical manifestations affect internal organs.

2003 - Hyaluronan uptake by adult human skin fibroblasts in vitro [Articolo su rivista]
Croce, M. A.; Boraldi, F.; Quaglino, D.; Tiozzo, R.; Pasquali-Ronchetti, I.

Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism.

2003 - Multidrug resistance protein-6 (MRP6) in human dermal fibroblasts. Comparison between cells from normal subjects and from Pseudoxanthoma elasticum patients [Articolo su rivista]
Boraldi, Federica; Quaglino, Daniela; Croce, Maria Antonietta; M. I. G., Fernandez; Tiozzo, Roberta; Gheduzzi, Dealba; Bacchelli, Barbara; Ronchetti, Ivonne

Multidrug resistance protein-6 (MRP6) is a membrane transporter whose deficiency leads to the connective tissue disorder Pseudoxanthoma elasticum (PXE). In vitro dermal fibroblasts from normal and PXE subjects, homozygous for the R1141X mutation, were compared for their ability to accumulate and to release fluorescent calcein, in the absence and in the presence of inhibitors and competitors of the MDR-multidrug resistance protein (MRP) systems, such as 3-(3-(2-(7-choro-2 quinolinyl) ethenyl)phenyl ((3-dimethyl amino-3-oxo-propyl)thio) methyl) propanoic acid (MK571), verapamil (VPL), vinblastine (VBL), chlorambucil (CHB), benzbromarone (BNZ) and indomethacin (IDM). In the absence of chemicals, calcein accumulation was significantly higher and the release significantly slower in PXE cells compared to controls. VBL and CHB reduced calcein release in both cell strains, without affecting the differences between PXE and control fibroblasts. VPL, BNZ and IDM consistently delayed calcein release from both control and PXE cells; moreover, they abolished the differences between normal and MRP6-deficient fibroblasts observed in the absence of chemicals. These findings suggest that VPL, BNZ and IDM interfere with MRP6-dependent calcein extrusion in in vitro human normal fibroblasts. Interestingly, MK571 almost completely abolished calcein release from PXE cells, whereas it induced a strong but less complete inhibition in control fibroblasts, suggesting that MRP6 is not inhibited by MK571. Data show that MRP6 is active in human fibroblasts, and that its sensitivity to inhibitors and competitors of MDR-MRPs' membrane transporters is different from that of other translocators, namely, MRP1. It could be suggested that MRP1 and MRP6 transport different physiological substances and that MRP6 deficiency cannot be overcome by other membrane transporters, at least in fibroblasts. These data further support the hypothesis that MRP6 deficiency may be relevant for fibroblast metabolism and responsible for the metabolic alterations of these cells at the basis of connective tissue clinical manifestations of PXE. (C) 2003 Elsevier B.V./Intemational Society of Matrix Biology. All rights reserved.

2003 - Normal human dermal fibroblasts: Proteomic analysis of cell layer and culture medium [Articolo su rivista]
Boraldi, Federica; L., Bini; S., Liberatori; A., Armini; V., Pallini; Tiozzo, Roberta; Ronchetti, Ivonne; Quaglino, Daniela

Proteins present within the cell layer and those released in the cell medium from in vitro cultured normal human dermal fibroblasts were separated and characterized in terms of their isoelectric point and molecular weight, by two-dimensional (2-D) gel electrophoresis. All spots in the synthetic gel were firstly analyzed by the Melanie 3 software and compared with those of breast cancer cells, colorectal epithelial cells, HL60, lymphoma cells, and platelets, already available on-line. From the identification of 144 spots from both the cell layer and the medium, we were able to recognize 89 different proteins, since a certain number of spots represented different isoforms of the same molecule. Identifications were performed by matching with on-line 2-D databases, and by matrix assisted laser-desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), in order to confirm the identification by matching, or to identify new proteins. The procedure we used allows (i) to design a highly reproducible reference map of the proteome of adult human normal fibroblasts in culture, (ii) to evaluate protein species produced in the cell layer as well as those released in the culture medium, and (iii) to compare data from gel matching with those obtained by MS. This work represents an essential step for a better knowledge of mesenchymal cells, given the widespread use of this cell type in both clinical and experimental investigations.

2003 - Proteome analysis of dermal fibroblasts cultured in vitro from human healthy subjects of different ages [Articolo su rivista]
Boraldi, Federica; Bini, L.; Liberatori, S.; Armini, A.; Pallini, V.; Tiozzo, Roberta; Ronchetti, Ivonne; Quaglino, Daniela

Aging is a complex multifactorial process still far from being completely understood. The aim of the present study was to compare the proteome of in vitro cultured dermal fibroblasts from healthy subjects of different ages (i.e. 15 +/- 2, 41 +/- 4 and 82 +/- 3 years old). Proteins of the cell layer were separated by two-dimensional electrophoresis and protein identification was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry; moreover, synthetic gels were qualitatively and quantitatively analyzed by Melanie 3 software. Our study did not reveal any protein typical of any one age group. On the other hand, we observed 38 proteins exhibiting more than three-fold reproducible variations with aging, some (45%) being reduced such as F-actin capping protein alpha1, proteasome subunit alpha type 3, heat shock protein 27, ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial thioredoxin-dependent peroxide reductase, cathepsin B, glutathione S-transferase P, cyclophilin A and calgizzarin. In contrast, T-complex protein 1, probable protein disulfide isomerase ER60, phosphoglycerate kinase 1, Ran-specific GTPase-activating protein, proteasome subunit alpha type 5, triosephosphate isomerase and superoxide dismutase (Mn) increased with age. Furthermore, annexin 1, elongation factor 1beta, proteasome activator complex subunit 1, phosphoglycerate mutase, superoxide dismutase (Cu-Zn) and cofilin, exhibited the highest levels in adult cells; whereas, septin 2 homolog, RNA-binding protein regulatory subunit and ATP synthase D chain revealed the lowest values in adults. The present investigation, underlining the complexity of the aging process, highlights the role of synthetic and degradative pathways in modulating the whole cell machinery and emphasizes that metabolic impairment with age could depend partly on different expression of a number of genes and leading to an imbalance among functional proteins.

2002 - Cyclin dependent kinase 5 and its interacting proteins in cell death induced in vivo by cyclophosphamide in developing mouse embryos [Articolo su rivista]
Y., Zhu; L., Lin; S., Kim; Quaglino, Daniela; Ra, Lockshin; Z., Zakeri

Activation or inactivation of members of the cyclin-dependent kinase family is important during cell cycle progression. However, Cdk5, a member of this family that was originally identified because of its high structural homology to Cdc2, is activated during cell differentiation and cell death but not during cell cycle progression. We previously demonstrated a correlation between the up-regulation of Cdk5 protein and kinase activity and cell death during development and pathogenesis. We report here that cyclophosphamide (CP) induces massive apoptotic cell death in mouse embryos and that Cdk5 is expressed in apoptotic cells displaying fragmented DNA. During CP-induced cell death, Cdk5 protein expression is substantially increased as detected by immunohistochemistry but not by Western blot, while its mRNA level remains the same as control, and its kinase activity is markedly elevated. The up-regulation of Cdk5 during CP-induced cell death is not due to de novo protein synthesis. We also examined p35, a regulatory protein of Cdk5 in neuronal differentiation. Using a yeast two-hybrid system, we isolated p35, a neuronal differentiation specific protein, as a protein that interacts with Cdk5 in CP-treated embryos, p35 mRNA level does not change, but the protein expression of p25, a truncated form of p35, is elevated during cell death in vivo, as established here, as well as during cell death in vitro. Our results suggest a role for Cdk5 and its regulatory proteins during CP induced cell death. These results further support the view that Cdk5 and its regulation may be key players in the execution of cell death regardless of how the cell dies, whether through biological mechanisms, disease states such as Alzheimer's disease, or induction by CP.

2002 - The Cardiovascular system [Capitolo/Saggio]
Quaglino, Daniela; Ronchetti, Ivonne

The organization of the extracellular matrix components within the cardiovascular system are descrided with special emphasis to their role in development, aging and disease.

2001 - Amiodarone inhibits lung degradation of SP-A and perturbs the distribution of lysosomal enzymes [Articolo su rivista]
Baritussio, A; Marzini, S; Agostini, M; Alberti, A; Cimenti, C; Bruttomesso, D; Manzato, E; Quaglino, Daniela; Pettenazzo, A.

Amiodarone may induce lung damage by direct toxicity or indirectly through inflammation. To clarify the mechanism of direct toxicity, we briefly exposed rabbit alveolar macrophages to amiodarone and analyzed their morphology, synthesis, and degradation of dipalmitoylphosphatidylcholine (DPPC); distribution of lysosomal enzymes; and uptake of diphtheria toxin and surfactant protein (SP) A used as tracers of the endocytic pathway. Furthermore, in newborn rabbits, we studied the clearance of DPPC and SP-A instilled into the trachea together with increasing amounts of amiodarone. We found that in vitro amiodarone decreases the surface density of mitochondria and lysosomes while increasing the surface density of inclusion bodies, increases the incorporation of choline into DPPC, modifies the distribution of lysosomal enzymes, and does not affect the uptake and processing of diphtheria toxin but inhibits the degradation of SP-A. In vivo amiodarone inhibits the degradation of SP-A but not of DPPC. We conclude that the acute exposure to amiodarone perturbs the endocytic pathway acting after the early endosomes, alters the traffic of lysosomal enzymes, and interferes with the turnover of SP-A.

2001 - Cell death in the rat thymus: a minireview [Articolo su rivista]
Quaglino, Daniela; Ronchetti, Ivonne

During the last decades, the literature has clearly established the fundamental role of the thymus in the development of an effective immune system. During thymocyte development and maturation, potentially autoreactive thymocytes are eliminated by a process known as apoptosis or programmed cell death responsible for the negative selection occurring within the thymus. This process is in sharp contrast to other types of cell death referred to as necrosis. Actually, three different types of cell death have been recently observed morphologically in the rat thymus, i.e. necrosis, apoptosis and clustered cell death. Moreover, among the numerous factors influencing thymocyte cell death, particular attention has been paid to hormones, chemicals, biological compounds and physical agents that may influence the type and/or the extent of cell death. Finally, a brief overview has been devoted to the contribution of mitochondria, nitric oxide, glutathione and intracellular levels of cations in addition to the activity of genes as cdk2, p53, Fas and members' of the Bcl2 family in modulating rat thymus cell death.

2001 - Characterization of pseudoxanthoma elasticum-like lesions in the skin of patients with beta-thalassemia [Articolo su rivista]
Contri, Miranda; Bacchelli, Barbara; Boraldi, Federica; Quaglino, Daniela; Ronchetti, Ivonne; Taparelli, Francesca; Carnevali, Elena; Seidenari, Stefania; A., Francomano; V., Bettoli; V., De Sanctis

Background: Pseudoxanthoma elasticum (PXE), an inherited disorder of unknown pathogenesis, is characterized by elastic fiber mineralization, collagen fibril alterations, and accumulation of thread material in the extracellular space. PXE-like clinical lesions have been described in patients with beta -thalassemia. Objective and Methods: Dermal lesions in these two genetic disorders were compared by light and electron microscopy and by immunocytochemistry. Results: In both disorders, elastic fiber polymorphism, fragmentation, and mineralization were structurally identical. Elastic fiber mineralization in beta -thalassemia was associated with vitronectin, bone sialoprotein, and alkaline phosphatase, similar to what was observed in inherited PXE. Furthermore, abnormalities of collagen fibrils and filament aggregates were identical in both disorders. In both inherited and beta -thalassemia-associated PXE, unrelated gene defects seem to induce cell metabolic abnormalities that lead to identical clinical and structural phenotypes. Conclusion: Data indicate that patients with beta -thalassemia may undergo important alterations of connective tissues, a better understanding of which may help in preventing clinical complications.

2001 - DL-penicillamine induced alteration of elastic fibers of periosteum-perichondrium and associated growth inhibition: an experimental study [Articolo su rivista]
A., Gigante; C., Chillemi; Quaglino, Daniela; M., Miselli; Ronchetti, Ivonne

Perichondrium-periosteum, being of collagen and elastic fiber, is regarded as a bone growth regulating factor. The aim of the present study was to investigate the role of collagen and elastic fibers on bone growth, by interfering with the fiber assembly in growing chicks upon administration of DL-penicillamine (DL-PNA). Our findings demonstrated that DL-PNA determined relevant modifications in the perichondrium-periosteum, as shown by histochemical. histomorphometrical,biochemical and ultrastructural analysis. This chemical has been shown to inhibit the formation of desmosine cross-links in elastin and to induce an increase of elastin associated microfibrils. On the contrary, the collagen network and the biochemical collagen markers were not affected. These changes resulted in a dramatically reduced growth of long bones in comparison with control. Perichondrial-periosteal regulation of bone growth may be mediated by mechanical and biological factors. This study demonstrates a microstructural change in the perichondrium-periosteum with decreased elastin and increased elastic microfibrils content in penicillamine treated chicks. The mechanism linking changes in the perichondrium-periosteum with altered growth still needs to be elucidated. (C) 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

2001 - Hyaluronan affects protein and collagen synthesis by in vitro human skin fibroblasts [Articolo su rivista]
Croce, Maria Antonietta; K., Dyne; Boraldi, Federica; Quaglino, Daniela; G., Cetta; Tiozzo, Roberta; Ronchetti, Ivonne

Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1 muM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P < 0.05), and particularly in collagen (- 40%; P < 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.

2001 - The placenta in pseudoxanthoma elasticum: clinical, structural and immunochemical study. [Articolo su rivista]
Gheduzzi, Dealba; Taparelli, Francesca; Quaglino, Daniela; C., Di Rico; L., Bercovitch; S., Terry; D. B., Singer; Ronchetti, Ivonne

Pseudoxanthoma elasticum (PXE) is a rare genetic disorder clinically characterized by skin, cardiovascular and eve manifestations, mainly due to calcification and fragmentation of elastic fibres. although infrequent, complications during pregnancy in nomen affected by PXE have been reported. The aim of the present stud! was to compare structural features of placentae at term from 14 control and 15 PXE-affected nomen, in order to better understand if and hen: abnormal mineral and/or matrix accumulation might affect placental function in PSE. In all cases, pregnancy, fetus growth and delivery were normal. Both gross and light microscopy examination did not reveal dramatic differences between placentae of PSE patients and controls, with regard to weight, dimensions, infarcts, thrombi, inflammatory lesions or vessels. However, necrotic changes and mineralization appealed statistically more pronounced in PXE. By electron microscopy the most remarkable differences between PXE and control placentae were observed in the localization and morphology of mineral precipitates; a significant higher deposition of mineral precipitates was observed associated with the ´matrix´-type fibrinoid and among collagen fibrils, especially on the maternal side. Immunocytochemistry revealed the presence of vitronectin and fibronectin associated with the PXE-specific mineralizations and the absence of mineralization on the small and scarce elastic fibres in either controls or in PXE.

2000 - A cytofluorimetric study of T lymphocyte subsets in rat lymphoid tissues (thymus, lymph nodes) and peripheral blood: a continuous remodelling during the first year of life [Articolo su rivista]
M., Capri; Quaglino, Daniela; G., Verzella; D., Monti; M., Bonafe; Cossarizza, Andrea; Troiano, Leonarda; L., Zecca; Ronchetti, Ivonne; C., Franceschi

We previously demonstrated that the rat thymus undergoes a progressive remodelling long before the appearance of typical signs of involution [Quaglino, D., Capri, M., Bergamini, G., Euclidi, E., Zecca, L., Franceschi, C., Pasquali Ronchetti, I., 1998. Age-dependent remodelling of rat thymus. Morphological and cytofluorimetric analysis from birth up to one year of age. Eur. J. Cell. Biol. 76, 156-166]. To focus better on the complex remodelling that occurs in the rat immune system during the first year of life, we analysed the phenotype profile of thymocytes, and T lymphocytes from mesenteric lymph nodes and peripheral blood of the same animals by flow cytometry. Two experimental sets were performed simultaneously using the same animal strain, but starting and ending the study at different ages (15 days up to 300 days in the first experimental set, and 90 days up to 360 days of life in the second). In the rat these ages appear to be crucial not only for developmental, maturative and early involutional processes of the thymus, but also of the entire immune system. The main findings were the following: (1) in the thymus, CD8(-)CD3(-) cells increased, CD5(+) alpha beta TCR- and CD8(+)CD4(+) thymocytes decreased, while the most mature cell subset appeared well preserved with ageing; (2) in the lymph nodes, T helper and T cytotoxic lymphocytes decreased in the most aged animals. Memory/activated CD4(+)CD45RC(-) T cells decreased, while naive/resting CD4(+)CD45RC(+) cells increased in the youngest animals and decreased in the oldest. CD8(+)CD45RC(-) and CD8(+)CD45RC(+) lymphocytes showed a complex age-dependent trend, and (3) in peripheral blood, minor modifications were evident, such as an age-dependent increase in the alpha beta TCR(+)CD25(+) cell subset. Some of these changes were related to the developmental process, while others could likely be interpreted as early signs of immunosenescence. The role of these modifications in immune system is discussed within the framework of the remodelling hypothesis of immunosenescence. The age-dependent changes in these three lymphoid compartments, however, appear to be different and only partially overlapping, thus suggesting that the maturational, developmental and ageing processes have distinct characteristics in the central and peripheral lymphoid organs. (C) 2000 Elsevier Science Inc. All rights reserved.

2000 - Abnormal phenotype of in vitro dermal fibroblasts from patients with pseudoxanthoma elasticum (PXE) [Articolo su rivista]
Quaglino, Daniela; Boraldi, Federica; Barbieri, D; Croce, Maria Antonietta; Tiozzo, Roberta; Ronchetti, Ivonne

Pseudoxanthoma elasticum (PXE) is a genetic connective tissue disease, whose gene and pathogenesis are still unknown. Dermal fibroblasts from patients affected by PXE have been compared in vitro with fibroblasts taken from sex and age-matched normal individuals. Cells were grown and investigated in monolayer, into three-dimensional collagen gels and in suspension. Compared with normal cells, PXE fibroblasts cultured in monolayer entered more rapidly within the S phase and exhibited an increased proliferation index; on the contrary, similarly to normal fibroblasts, PXE cells did not grow in suspension. Furthermore, compared with normal fibroblasts, PXE cells exhibited lower efficiency in retracting collagen type I lattices and lower adhesion properties to collagen type I and to plasma fibronectin. This behavior was associated with higher expression of integrin subunits alpha 2, alpha 5, alpha v, whereas beta 1 subunit as well as alpha 2 beta 1 and alpha 5 beta 1 integrin expression was lower than in controls. Compared to controls, PXE fibroblasts had higher CAM protein expression in accordance with their high tendency to form cellular aggregates, when kept in suspension. The demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities, is consistent with the hypothesis that the gene responsible for PXE might have a broad regulatory role on the cellular machinery. (C) 2000 Elsevier Science B.V. All rights reserved.

2000 - Citochine. [Monografia/Trattato scientifico]
Quaglino, Daniela; J. M., Davidson

Sono prese in considerazioni le proprietà biologiche delle principali citochine ed il loro ruolo in condizioni fisiologiche e patologiche

2000 - Modulation of cell death in the rat thymus - Light and electron microscopic investigations [Articolo su rivista]
Quaglino, Daniela; M., Capri; Ronchetti, Ivonne

In mammals, the thymus is the primary central organ of the lymphoid system; after birth, it progressively diminishes in size, undergoing gradual atrophy. Physiological maturation and/or involution of the thymus may be accelerated by endogenous or exogenous factors. Exposure to extremely low frequency EMF seems to interfere with thymic cell death. Data suggest that, in the rat model, a prolonged exposure to 50 Hz electric and magnetic fields, independently from field strength, seems to affect thymic cell death and possibly thymic physiology, since alterations in the balance of cell death and other parameters such as mitoses might interfere with the positive and negative selection of thymocytes and with the immunosurveillance properties of the thymus.

2000 - Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum [Articolo su rivista]
O., Le Saux; Z., Urban; C., Tschuch; K., Csiszar; B., Bacchelli; Quaglino, Daniela; Ronchetti, Ivonne; F. M., Pope; A., Richards; S., Terry; L., Bercovitch; A., de Paepe; C. D., Boyd

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degefieration(1-5). PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed(6). Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families(7,8). Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16013.1 has been reported (refs 8,9). We have refined this locus to an 820-kb region containing 6 candidate genes(10). Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6).

1999 - Hyaluronic acid by atomic force microscopy [Articolo su rivista]
I., Jacoboni; U., Valdrè; Mori, Giuseppe; Quaglino, Daniela; Ronchetti, Ivonne

Hyaluronic acid (HA) of different molecular weights has been examined by atomic force microscopy (AFM) in air. This technique allows 3-D surface images of soft samples without any pretreatment, such as shadowing or staining. In the present study we examined the supermolecular organization of HA chains when deposited on mica and graphite, to better understand the interchain and intrachain interactions of HA molecules in solution. The concentration of the solution deposited varied from 0.001 to 1 mg/ml. On both substrates, and independent of the concentration, high-molecular-mass HA formed networks in which molecules ran parallel for hundreds of nanometers, giving rise to hat sheets and tubular structures that separate and rejoin into similar neighboring aggregates. Accurate measurements of the thickness of the thinnest sheets were consistent with a monolayer of HA molecules, 0.3 nm thick, strongly indicating lateral aggregation forces between chains as well as rather strong hydrophilic interactions between mica and HA The results agree with an existing model of HA tertiary structure in solution in which the network is stabilized by both hydrophilic and hydrophobic interactions. Our images support this model and indicate that hydrophobic interactions between chains may exert a pivotal role in aqueous solution.

1999 - Identification of heterozygote carriers in families with a recessive form of pseudoxanthoma elasticum (PXE) [Articolo su rivista]
Bacchelli, Barbara; Quaglino, Daniela; Gheduzzi, Dealba; Taparelli, Francesca; Boraldi, Federica; B., Trolli; O., Le Saux; Cd, Boyd; Ronchetti, Ivonne

Skin biopsies of 18 healthy relatives of patients with pseudoxanthoma elasticum (PXE), belonging to six different recessive families, have been examined by optical and electron microscopy in order to determine morphologic alterations potentially useful for the identification of carriers of this genetic disorder. These morphologic features have been compared with those observed in the same tissue areas of eight PXE patients belonging to the same families, with six normal subjects, and to the carrier status of these apparently unaffected relatives as determined by haplotype analysis using informative markers surrounding the locus of the PXE gene on chromosome 16p. The dermis of all the relatives of PXE patients, established by haplotype analysis to be heterozygote carriers of a mutation in the PXE gene, exhibited several alterations very similar, although less severe, to those typical in PXE patients. Alterations were present in the reticular dermis and consisted of irregular-sized collagen bundles and elastic fibers; elastic fibers fragmented, cribriform, and mineralized; numerous fibroblasts, larger than normal, and subendothelial elastin in small vessels. Strikingly, none of these dermal changes were noted in an unaffected relative in one family who was identified as a noncarrier by haplotype analysis. Although many of these alterations are not specific for PXE, the presence of these morphologic changes in unaffected relatives of PXE patients indicates alterations in skin that could be diagnostic for carriers of a subclinical phenotype of PXE.

1999 - The effect of caloric restriction on the aortic tissue of aging rats [Articolo su rivista]
Fornieri, Claudio; Taparelli, Francesca; Quaglino, Daniela; Contri, Miranda; Jm, Davidson; S., Algeri; Ronchetti, Ivonne

Connective tissue shows peculiar and complex age-related modifications, which can be, at least in part, responsible for altered functions and increased susceptibility to diseases. Food restriction has long been known to prolong life in rodents, having antiaging effects on a variety of physiologic and pathologic processes. Therefore, the aorta has been investigated in rats fed normal or hypocaloric diet, from weaning to senescence. Compared with controls, caloric-restricted animals showed less pronounced age-dependent alterations such as elastic fiber degradation, collagen accumulation and cellular modifications. Immunocytochemical analyses revealed that elastic fibers were positively labelled for biglycan, decorin, ApoB100 (LDL), ApoAl (HDL) and elastase and that the intensity of the reactions was time- and diet-dependent, With age, the major changes affecting aortic elastic fibers were increased positivity for decorin, LDL and elastase, Compared with age-matched normal fed rats, caloric restricted animals revealed lower content of LDL, decorin and elastase and higher positivity for HDL. These data suggest that a caloric restricted diet might influence the aging process of the arterial wall in rats, delaying the appearance of age-related degenerative features, such as structural alterations of cells and matrix and modified interactions of elastin with cells and with other extracellular matrix molecules.

1998 - Age-dependent remodeling of rat thymus. Morphological and cytofluorimetric analysis from birth up to one year of age [Articolo su rivista]
Quaglino, Daniela; Capri, M; Bergamini, G; Euclidi, E; Zecca, L; Franceschi, C; Ronchetti, Ivonne

Structural and phenotypic modifications of rat thymocytes from birth up to one year of age, i.e. during maturation and at the beginning of the involutive process of the thymus are described. Since the biological significance and the mechanisms of thymic involution are still a matter of debate, this study aims at clarifying the complexity of the compensatory events occurring during this relatively neglected period of time. Thymuses from Sprague-Dawley rats were analyzed morphologically and morphometrically by light and electron microscopy, At the same time, thymocyte subsets, isolated from the same animals, were characterized by flow cytometry according to physical parameters and phenotypic markers. Results indicate that major changes occur during the first month from birth and from six months onward. In particular, already during the first weeks after birth, thymocytes undergo a slight reduction of mitoses associated with an increased number of apoptoses, Moreover, during the same period of time, flow cytometry revealed an expansion of small thymocytes and changes in thymocyte subsets such as increase of CD4+CD8+ and CD5+alpha beta TCR- and a decrease of CD4-CD8-, CD4-CD8+ cells. The thymus of adult rats was characterized by time-dependent decrease of both mitoses and apoptoses, progressive physical disconnection among cells, increase of necrotic areas and fibrosis. Around one year of age tissue changes were associated with a dramatic reduction of the population of large thymocytes and the rise of numerous small thymocytes that were unexpectedly negative for all tested markers. By contrast, medium-size thymocytes exhibited a marked decrease of CD4+CD8+ and CD5+alpha beta TCR- subsets. In conclusion, our data indicate that thymus undergoes, with time, a complex remodeling and suggest that thymic involution is not only a simple shrinkage of the organ but rather the result of a series of compensatory mechanisms among different cell populations in a setting of progressive involution.

1998 - In chyloptysis, SP-A affects the clearance of serum lipoproteins entering the airways [Articolo su rivista]
Alberti, A; Ravenna, F; Quaglino, Daniela; Luisetti, M; Muraca, M; Previato, L; Enzi, Gb; Bruni, R; Baritussio, A.

Serum lipoproteins may enter the airways and appear in sputum (chyloptysis) when the lymphatic circulation is impaired by inflammation, neoplasia, or an abnormal proliferation of smooth muscle cells. While analyzing the bronchoalveolar lavage fluid of a patient with chyloptysis, we noticed that surfactant could not be separated from contaminating serum lipoproteins and speculated that lipoproteins might interact with surfactant components. To clarify this point we immobilized surfactant protein (SP) A on microtiter wells and incubated it with I-125-labeled very low density lipoproteins (VLDLs), low-density lipoproteins, and high-density lipoproteins. We found that SP-A binds lipoproteins. Studying in greater detail the interaction of SP-A with VLDLs, we found that the binding is time and concentration dependent; is inhibited by unlabeled lipoproteins, phospholipids, and antibodies to SP-A; is increased by Ca2+; and is unaffected by methyl alpha-D-mannopyranoside. Whole surfactant is a potent inhibitor of binding. Furthermore, we found that SP-A increases the degradation of VLDLs by alveolar macrophages and favors the association of VLDLs with alveolar surfactant. We conclude that SP-A influences the disposal of serum lipoproteins entering the airways and speculate that binding to alveolar surfactant might represent an important step in the interaction between exogenous substances and the lung.

1998 - N-1,N-12-bis(ethyl)spermine effect on growth of cis-diamminedichloroplatinum(II)-sensitive and -resistant human ovarian-carcinoma cell lines [Articolo su rivista]
Marverti, Gaetano; Piccinini, Giorgio; S., Ghiaroni; D., Barbieri; Quaglino, Daniela; Moruzzi, Maria Stella

The results presented here demonstrate that a cis-diamminedichloroplatinum(II) (DDP)-resistant human ovarian-carcinoma cell line is also cross-resistant to the spermine analogue N-1,N-12-bis(ethyl)spermine (BESPM). We report that C13* cells, which are approximately IO-fold resistant to DDP, similarly showed 7-fold resistance to BESPM by colony-forming assay with an IC50 value of 24.6 +/- 2 mu M vs. 3.4 +/- 0.8 mu M of 2008 cells. Resistance appears to be the result of many effects, such as different morphological and functional modifications of mitochondria Furthermore, although BESPM accumulation was almost identical in sensitive and resistant cells, the intracellular polyamine pool of the 2 cell lines was differentially affected by this polyamine analogue. In fact, when spermidine (SPD) was still detectable in C13* cells, in 2008 cells it was not, and the spermine (SPM) content was always more markedly reduced in sensitive cells than in the resistant variant. The lower polyamine content of 2008 cells could be related to a higher degree of induction of sperdimine/spermine N-1-acetyltransferase (SSAT) activity by BESPM in sensitive cells than in their resistant counterpart. Despite the observed cross-resistance, the combination of the 2 drugs resulted in supra-additive and synergistic effects in both cell lines, depending on concentration, as assessed by median-effect analysis of the survival data. The effectiveness of this combination was also confirmed by the increased accumulation of cells in the G(2)/M phase of the cell cycle in both cell lines. Taken together, these data suggest that BESPM effect on cell growth of DDP-sensitive and DDP-resistant cells involves multiple mechanisms that are differently modulated by the DDP-resistant phenotype. Int J. Cancer 78:33-40, 1998.

1998 - Study of elastic fiber organization by scanning force microscopy [Articolo su rivista]
Ronchetti, Ivonne; Alessandrini, Andrea; Contri, Miranda; Fornieri, Claudio; Mori, Giuseppe; Quaglino, Daniela; U., Valdré

Elastic fibers of beef ligamentum nuchae were observed by atomic force microscopy and data compared with those obtained by conventional and freeze-fracture electron microscopy. Fresh isolated elastin fibers as well as thin sections of ligament fragments, which were fixed and embedded either in relaxed or in stretched, conditions, were analysed. The results confirm that, at least in beef ligamentum nuchae, elastic fibers consist of beaded filaments which can be oriented by stretching in the direction of the force applied. Moreover, atomic force microscopy revealed that these beaded filaments are laterally connected by periodical bridges which become more pronounced upon stretching. The data clearly show that elastin molecules are organized in a rather ordered array, at least at the super-molecular level, and a depiction of the elastin organization in beef ligament um nuchae is attempted.

1997 - Ascorbate differentially regulates elastin and collagen biosynthesis in vascular smooth muscle cells and skin fibroblasts by pretranslational mechanisms [Articolo su rivista]
Jm, Davidson; Pa, Luvalle; O., Zoia; Quaglino, Daniela; Mg, Giro

Ascorbate contributes to several metabolic processes including efficient hydroxylation of hydroxyproline in elastin, collagen, and proteins with collagenous domains, yet hydroxyproline in elastin has no known function. Prolyl hydroxylation is essential for efficient collagen production; in contrast, ascorbate has been shown to decrease elastin accumulation in vitro and to alter morphology of elastic tissues in vivo, Ascorbate doses that maximally stimulated collagen production (10-200 mu M) antagonized elastin biosynthesis in vascular smooth muscle cells and skin fibroblasts, depending on a com bination of dose and exposure time. Diminished elastin production paralleled reduced elastin mRNA levels, while collagen I and III mRNAs levels increased. We compared the stability of mRNAs for elastin and collagen I with a constitutive gene after ascorbate supplementation or withdrawal. Ascorbate decreased elastin mRNA stability, while collagen I mRNA was stabilized to a much greater extent. Ascorbate withdrawal decreased collagen I mRNA stability markedly (4.9-fold), while elastin mRNA became more stable. Transcription of elastin was reduced 72% by ascorbate exposure. Differential effects of ascorbic acid on collagen I and elastin mRNA abundance result from the combined, marked stabilization of collagen mRNA, the lesser stability of elastin mRNA, and the significant repression of elastin gene transcription.

1997 - Cell behaviour and cell-matrix interactions of human palmar aponeurotic cells in vitro [Articolo su rivista]
Quaglino, Daniela; G., Bergamini; Croce, Maria Antonietta; Boraldi, Federica; D., Barbieri; Caroli, Alessandro; A., Marcuzzi; Tiozzo, Roberta; Ronchetti, Ivonne

The present investigation has been performed to better characterize, in vitro, normal aponeurotic cells in comparison with dermal fibroblasts and with cells derived from Dupuytren´s affected aponeuroses. Cells were cultured in monolayer and/or into three-dimensional collagen gels. Cell structure, adhesion, and spreading capability on different substrates, as well as integrin expression were investigated by light and electron microscopy and by flow cytometry. Cell-matrix interactions were also analyzed by gel retraction experiments in the presence, or absence, of RGD peptides and anti-integrin antibodies. Normal aponeurotic cells, compared with dermal fibroblasts, exhibited in vitro peculiar structural features, which were substantially maintained in Dupuytren´s aponeurotic cells, irrespective of the substrate they were grown on. By contrast, the aponeurotic cell behavior was different in normal and diseased cells, these latter approaching that of dermal fibroblasts. Normal aponeurotic cells, in fact, were characterized by low efficiency in retracting the collagen gel, low alpha(2), alpha(1) and alpha(5) integrin subunit expression and low adhesion properties onto collagen and fibronectin, whereas cells isolated from the aponeuroses of Dupuytren´s patients exhibited higher capability of retracting the collagen gel, increased adhesion properties toward collagen and fibronectin, and higher levels of integrin expression. No differences were observed between dermal fibroblasts from Dupuytren´s patients or from normal subjects. These in vitro results are consistent with those previously obtained in situ, suggesting that palmar aponeurotic cells have a peculiar phenotype and that changes in cell-matrix interactions occur in Dupuytren´s contracture. Moreover, by comparing data obtained from the retracted fibrotic cords and the still clinically unaffected aponeuroses of the same patients, it may be noted that Dupuytren´s disease is not only confined to the clinically involved branches, but includes the whole aponeurosis of the affected hand.

1997 - Hyaluronan-phospholipid interactions [Articolo su rivista]
Ronchetti, Ivonne; Quaglino, Daniela; Bacchelli, Barbara; Mori, Giuseppe; P., Ghosh

Hyaluronan-phospholipid interactions have been studied in vitro by negative staining and rotary shadowing electron microscopy. Hyaluronan (HA) molecules of different molecular weights (around 170 000; 740 000, and 1.9 x 10(6) Ha) were added to phospholipid suspensions (DPPC or egg lecithin) that were in the form of either unilamellar particles or multilamellar vesicles. Suspensions were then gently stirred and incubated at different temperatures from 24 hr up to 7 days. After 24 hr, at temperatures just above the melting point of the phospholipid used, both unilamellar particles and multilamellar vesicles were already shown to change their organization in the presence of HA, giving rise to the formation of (1) huge perforated membrane-like structures lying on the substrate; (2) 12-nm-thick ´´cylinders´´ (rollers) with a tendency to aggregate and to form sheets. These structures were seen only in the presence of high-molecular-weight HA, whereas low-molecular-weight HA (170 kDa) induced fragmentation of liposomes and formation of a few short rollers. These data show,that phospholipids and HA interact and suggest they may also do so in vivo within the joint cavity, where both chemical species are present, giving rise to complexes which might exhibit peculiar lubricating and protective properties. It is also proposed that such interactions may not be as efficient in arthritic joints, where HA is degraded to low-molecular-weight fragments.

1997 - Protein synthesis, DNA degradation, and morphological changes during programmed cell death in labial glands of Manduca sexta [Articolo su rivista]
Jochova, J; Quaglino, Daniela; Zakeri, Z; Woo, K; Sikorska, M; Weaver, V; Lockshin, Ra

Labial glands of the tobacco hornworm Manduca sexta (Lepidoptera: Sphingiidae), homologues of Drosophila salivary glands, undergo programmed cell death (PCD) in a 4-day period during larva-to-pupa metamorphosis. The programmed death of the labial gland was examined by electron microscopy and measurement of protein synthesis as well as measurement of DNA synthesis, end-labeling of single strand breaks, and pulsed-field gel electrophoresis. One of the earliest changes observed is a sharp drop in synthesis of most proteins, coup ed with synthesis of a glycine-rich protein, reminiscent of silk-like proteins. From a morphological standpoint, during the earliest phases the most prominent changes are the formation of small autophagic vacuoles containing ribosomes and an apparent focal dissolution of the membranes of the endoplasmic reticulum, whereas later changes include differing destruction at the lumenal and basal surfaces of the cell and erosion of the basement membrane. By the fourth day of metamorphosis, individual cells become rapidly vacuolated in a cell-independent manner. In the vacuolated cells on day 3, chromatin begins to coalesce. it is at this period that unequivocal nucleosomal ladders are seen and end-labeling in situ or electrophoretic techniques document single or double-strand breaks, respectively DNA synthesis ceases shortly after the molt to the fifth instar, as detected by incorporation of tritiated thymidine and weak TUNEL labeling. Large size fragments of DNA are seen shortly after DNA synthesis ceases and thence throughout the instar, raising the possibility of potential limitations built into the cells before their final collapse.

1997 - Reversal of the wound healing deficit in diabetic rats by combined basic fibroblast growth factor and transforming growth factor-β1 therapy [Articolo su rivista]
Davidson, J. M.; Broadley, K. N.; Quaglino, D.

Wound healing is impaired in the diabetic state because of, at least in part, low expression of growth factors. Individual growth factors can partially activate healing, yet the actual wound environment presents a dynamic continuum of multiple cellular signals. Complex interactions among growth factors and target cells can have synergistic effects, and several examples of combinatorial, in vivo activity are evident in the literature. In this study, the implantation of a combination of basic fibroblast growth factor and transforming growth factor-β in rats induced fivefold to sevenfold increases in granulation tissue formation in comparison with implantation of each growth factor alone. Diabetes was induced in adult, male Sprague-Dawley rats with streptozotocin. Incisional wounds and sponge granulation tissue were produced in separate groups and then treated with an injection of 2 μg transforming growth factor-β1 combined with 10 μg basic fibroblast growth factor on day 3. DNA, collagen, and protein were analyzed in granulation tissue on days 7 and 9, and biomechanical properties of incisions were tested on days 7, 14, and 21. The combination of transforming growth factor-β1 and basic fibroblast growth factor had marked, positive effects on biochemical parameters of wound healing and reversed the tensile strength deficit of diabetic wounds. Nonradioactive in situ hybridization showed increased expression of messenger RNA for type I and III procollagen and transforming growth factor-β1 in normal and diabetic wounds, whereas ultrastructural examination showed a marked reorganization of collagen fibrils. These findings reinforce the concept that appropriate mixtures of cytokines rather than individual cytokines may more adequately stimulate tissues in cases of impaired wound healing.

1997 - Surfactant protein A (SP-A) interacts with serum lipoproteins entering the airways [Articolo su rivista]
Alberti, A.; Ravenna, F.; Quaglino, D.; Bruni, R.; Baritussio, A.

Serum lipoproteins may enter the airways and appear in the sputum (chylophtysis) when the lymphatic circulation is impaired by inflammation, neoplasm, or by an abnormal proliferation of smooth muscle cells. While analyzing the bronchoalveolar lavage fluid of a patient with chylophtysis, we noticed that surfactant could not be separated from contaminating lipoproteins, and we speculated that surfactant components may interact with lipoproteins entering the airways. To clarify this point, we immobilized SP-A on microtiter wells and incubated it with 125I-VLDL. We found that SP-A binds VLDL. The binding is time and concentration dependent, is inhibited by excess VLDL and by liposomes (dipalmitoyl phosphatidylcholine: egg phosphatidylcholine: phosphatidylglycerol: cholesterol, 50:25:15:10), is increased by 5 mM Ca++, is unaffected by surfactant proteins B and C, or by methyl mannoside, and is greatly decreased if SP-A is alkylated and reduced before immobilization. Furthermore, we found that soluble SP-A increases the degradation of VLDL by alveolar macrophages collected from normal subjects, and favors the binding of VLDL to surfactant membranes. We conclude that SP-A affects the catabolism of serum lipoproteins entering the airways.

1996 - Ascorbic acid and connective tissue [Capitolo/Saggio]
Ronchetti, I. P.; Quaglino, D.; Bergamini, G.

1996 - Connective tissue in skin biopsies from patients suffering systemic sclerosis [Articolo su rivista]
Quaglino, Daniela; Bergamini, G; Boraldi, Federica; Manzini, E; Davidson, Jm; Ronchetti, Ivonne

Little information is available on elastin during systemic sclerosis since biochemical and morphological data have primarily focused on collagen and glycosaminoglycan alterations of connective tissues in this pathological process. We performed ultrastructural, morphometric, biochemical and in situ hybridisation analyses on skin biopsies from patients affected by scleroderma and from site and age-matched control subjects. Affected skin revealed alterations in the distribution and organisation of collagen bundles and fibrils together with zonal increase of the microfibrillar component. Elastic fibres were significantly more numerous than in control shin, were more frequently vacuolated and characterised by electron-dense inclusions; moreover, morphometric analysis provided evidence for a significant increase of the percentage of both collagen bundles and elastin fibres in the measured tissue, compared to normal skin. Biochemical analysis seemed to confirm the increased elastin content per unit of dried weight tissue in sclerodermic skin. Differences observed among patients were only partially associated with disease duration and/or to severity of clinical manifestations. The abnormal amount of elastic fibres observed in skin biopsies from patients, and data from in situ hybridisation suggest the presence of a deregulation of the whole extracellular matrix that might be related to the role of cytokines such as TGF-beta, which has already been suggested to be involved in systemic sclerosis and in enhanced collagen and elastin production.

1996 - Earthworm coelomocytes in vitro: Cellular features and ''granuloma'' formation during cytotoxic activity against the mammalian tumor cell target K562 [Articolo su rivista]
Quaglino, Daniela; Cooper, El; Salvioli, S; Capri, M; Suzuki, Mm; Ronchetti, Ivonne; Franceschi, C; Cossarizza, Andrea

Earthworms possess specific, adaptive, cellular immunodefense as well as non-specific responses found in other complex metazoans. Here we characterized coelomocytes from the earthworm Eisenia foetida by electron microscopy and cytofluorimetric analyses, and investigated structural changes that occur when effector coelomocytes and target K562 erythromyeloid human tumor cells interact during cytotoxic activity. In in vitro cultures 1) the two earthworm cell types (i.e. small and large coelomocytes) retained their morphological features; 2) their DNA content was significantly less than that of human lymphocytes and the erythromyeloid human tumor cell line K562; 3) significant percentages of coelomocytes were found to be in S or G2/M phases of the cell cycle. When cultivated alone for up to 3 h, coelomocytes formed no aggregates. However, when mixed with K562, coelomocytes spontaneously killed tumor cells, and cytotoxic reactivity was accompanied by the formation of multiple aggregates similar to granulomas. These results are the first to describe this type of earthworm non-specific ''inflammatory'' response in vitro against tumor cells.

1996 - Earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines [Articolo su rivista]
Cossarizza, Andrea; Cooper, El; Suzuki, Mm; Salvioli, S; Capri, M; Gri, G; Quaglino, Daniela; Franceschi, C.

Earthworm coelomocytes (leukocytes) effect cytotoxicity at significantly high levels against the NK-sensitive, human tumor cell line, K562, and the NK-resistant targets (U937, BSM, GEM). By cytofluorimetric analyses using mouse anti-human monoclonal antibodies and by morphological evaluations, two types of coelomocytes were identified: (1) small (8-11 mu m) electron-dense cells (SC): CD11a+, CD45RA+, CD45RO+, CDw49b+, CD54+, beta(2)-m+, and Thy-1+; (2) large (12-15 mu m) electron-lucent cells (LC) that are negative for these markers. Both cell types were negative for other CD and MHC class I and class II markers. SC were active during recognition, rapidly binding to targets; LC were phagocytic. Release of Cr-51 revealed rapid, significant, and equal levels of killing at 4 degrees, 20 degrees, and 37 degrees C. We propose that primitive NK-like activity appeared early in evolution.

1996 - Programmed cell death in the tobacco hornworm, Manduca sexta: Alteration in protein synthesis [Articolo su rivista]
Zakeri, Z; Quaglino, Daniela; Latham, T; Woo, K; Lockshin, Ra

The metamorphic death of the labial glands of the tobacco hornworm, Manduca sexta, occurs during a 4 day period during larva-to-pupa metamorphosis. The earliest changes marking the death of the cell, all occurring on the first day, are a sharp drop in protein synthesis, coupled with the selective survival or upregulation of a few messages. An early rearrangement of the rough endoplasmic reticulum is presumably related to the generalized decrease in protein synthesis. Lysosomal acid phosphatase also begins to increase very early, and ultimately the bulk of the cytoplasm is destroyed in autophagic vacuoles, but activation of lysosomes does not account for the decreased rate of synthesis. The mechanism by which most protein synthesis is depressed remains under investigation.

1996 - Ultrastructural and morphometrical evaluations on normal human dermal connective tissue - The influence of age, sex and body region [Articolo su rivista]
Quaglino, Daniela; G., Bergamini; Boraldi, Federica; Ronchetti, Ivonne

In order to give detailed structural and quantitative evaluations for some of the most important dermal constituents such as collagen, elastic fibres and mesenchymal cells, and for the nonstructured extracellular matrix, we performed ultrastructural investigations on dermal biopsies from 50 healthy caucasian subjects aged from 6 fetal months to 83 years. Striking changes were observed, mainly in the perinatal period, for collagen, elastin and mesenchymal cells and, after 50 years of age, for collagen and elastin. Only slight or negligible differences were noted between males and females and in skin specimens taken from different parts of the body but similarly exposed to environmental factors (i.e. UV radiation). Modifications of the non-structured extracellular matrix appeared to be the consequence of changes affecting the other components. The results, therefore, emphasize the importance of the ageing factor in connective tissue metabolism and give further information on both qualitative and quantitative characteristics of normal human dermis.

Cooper, El; Cossarizza, Andrea; Suzuki, Mm; Salvioli, S; Capri, M; Quaglino, Daniela; Franceschi, C.

Earthworm coelomocytes have been used as effector cells against the human tumor target, K562. To first assess the viability of effecters, incorporation of [H-3]-thymidine was tested and was higher in autogeneic (A double left right arrow A, self) than in allogeneic (A double left right arrow B, nonself) coelomocytes, A double left right arrow A showed significantly greater numbers in S, G2, or M phases than A double left right arrow B coelomocytes. When A double left right arrow A or A double left right arrow B were cultured, no significant cell killing occurred in either, as measured in a 4-hr Cr-51 release assay, A double left right arrow A but not A double left right arrow B killed K562 target cells, Cytotoxicity was dependent upon membrane binding between small, electron-dense coelomocytes and targets; it was enhanced by adding PHA. The heat labile supernatant from A double left right arrow A but not from A double left right arrow B killed K562 targets after cultivation for 10 min at 22 degrees C, but not immediately after washing, Recognition of, binding to, and killing of foreign cells in a natural killer cell-like reaction may reflect natural immunity in earthworms.

1995 - Duodenal ferritin synthesis in genetic hemochromatosis [Articolo su rivista]
Pietrangelo, Antonello; Casalgrandi, Giovanna; Quaglino, Daniela; R., Gualdi; D., Conte; S., Milani; Montosi, Giuliana; Ventura, Ezio; L., Cesarini; G., Cairo

Background/Aims: The molecular defect of genetic hemochromatosis (GH) is unknown. It is believed that low expression of duodenal ferritin in GH is caused by tissue or cell specific defect of ferritin synthesis. Our study was designed to ascertain whether the control of duodenal ferritin synthesis in GH was defective. Methods: Expression at the single cell level of H and L ferritin messenger RNAs and protein and activity of the iron regulatory factor, which controls the translation of ferritin messenger RNA, were assessed in 43 duodenal biopsy specimens from individuals with GH, secondary hemochromatosis (SH), anemia, or normal iron balance. Results: Signal for ferritin H and L subunit messenger RNAs was detected in both absorptive and nonabsorptive cells by in situ hybridization, but in 10 of 14 patients with untreated GH, the signal was lower than in patients with SH or normal subjects. However, immunostaining for ferritin protein documented a diffuse/cytoplasmic pattern, whereas a supranuclear/granular staining was found in normal subjects or patients with SH. The spontaneous activity of duodenal iron regulatory factor was consistently higher in patients with GH than in normal subjects or subjects with anemia or SH. Conclusions: In patients with GH, ferritin gene transcription is preserved in both absorptive and nonabsorptive intestinal cells. Low accumulation of ferritin is not caused by a defective control of ferritin synthesis but by low expression of ferritin messenger RNA and sustained activity of iron regulatory factor.

1995 - Erratum: Duodenal ferritin synthesis in genetic hemochromatosis (Gastroenterology (1995) 108 (208-207)) [Articolo su rivista]
Pietrangelo, A.; Casalgrand, G.; Quaglino, D.; Gualdi, R.; Conte, D.; Milani, S.; Montosi, G.; Cesarini, L.; Ventura, E.; Cairo, G.

Cossarizza, Andrea; Cooper, El; Quaglino, Daniela; Salvioli, S; Kalachnikova, G; Franceschi, C.

Earthworm coelomocytes exist in two forms, i.e., small (SC) and large (LC) cells, as demonstrated by velocity sedimentation, electron microscopy, and FCM. However, we know little concerning the functional activities of various, important organelles, such as mitochondria. In comparison with SC, LC from Eisenia foetida have a higher number of mitochondria, and, accordingly, showed a greater fluorescence intensity when mitochondrial mass was measured by nonyl acridine orange and FCM. To measure MMP we used both the lipophilic cationic probe JC-1 and Rh123. The intracellular localization of JC-1 in SC and LC was observed by fluorescence microscopy. Using JC-1, MMP was analyzed separately on SC and LC by FCM, and significant percentages of coelomocytes (>95% of SC and about 90% of LC) displayed a high MMP. Adding 0.1 mu M VAL caused most SC to depolarize, while this occurred in only a few LC. Rh123 gave different results: no effects of VAL were observed either in SC or in LC. In coelomocytes there may be several energy-independent Rh123-binding sites whose role must still be elucidated. On the whole, these data indicate that it is possible to analyze mitochondrial parameters by FCM in intact invertebrate coelomocytes, and that the type of cell and the probe used have a critical importance.

1995 - Transforming growth factor-beta reverses a posttranscriptional defect in elastin synthesis in a Cutis Laxa skin fibroblast strain [Articolo su rivista]
Mc, Zhang; Mg, Giro; Quaglino, Daniela; Jm, Davidson

Skin fibroblasts from two cases of autosomal recessive cutis laxa (CL), having insignificant elastin production and mRNA levels, were challenged with transforming growth factor beta-1 (TGF-beta 1). Elastin production was brought from undetectable values to amounts typical of normal human skin fibroblasts in a dose-dependent fashion. Basic fibroblast growth factor (100 ng/ml) alone or in combination with TGF-beta 1 reduced elastin production and mRNA expression in CL skin fibroblasts more extensively than in normal cells. In situ hybridization showed that these effects were at the transcript level. One of the CL strains was examined in detail. Transcription rates for elastin were similar in normal and CL and unchanged by TGF-beta 1 or TGF-beta 2 (10 ng/ml), while in CL elastin mRNA half-life was increased > 10-fold by TGF-beta 2 and reduced 6-fold after TGF-beta 2 withdrawal, as compared with a control strain. Cycloheximide partially reversed elastin mRNA instability. These data are consistent with a defect in elastin mRNA stability that requires synthesis of labile factors or intact translational machinery, resulting in an extremely low steady state level of mRNA present in this strain of CL. Furthermore, TGF-beta can relieve elastin mRNA instability in at least one CL strain and elastin production defects in both CL strains.

1995 - Ultrastructure of elastin [Capitolo/Saggio]
Ronchetti, Ip.; Fornieri, C.; Contri, Mb.; Quaglino, D.

Almost all structural studies on elastin have been done in higher vertebrates, in which it is organized as an extracellular network of branched fibres which vary from fractions f microns to several microns in diameter. By conventional electron microscopy, elastin appears amorphous. By both freeze-fracture and negative staining on cryosections, it can be resolved as beaded filaments 5 nm in diameter forming a 3D meshwork that, upon stretching, becomes oriented in the direction of the force applied. This filamentous aggregation of elastin molecules is confirmed in vitro by the observation that its soluble precursor, tropoelastin, shows a strong tendency to associate into short 5 nm-thick filaments that, with time, become longer and aggregate into bundles of various dimensions. If chemically fixed and embedded, these aggregates appear amorphous and identical to natural elastin fibres. The tendency of tropoelastin to aggregate into 4-5 nm-thick beaded filaments, which then associate into 12 nm-thick filaments forming a 3D network, has been observed by atomic force microscopy for recombinant human tropoelastin. Therefore, the amorphous structure of elastin seems to be a technical artefact. Apart from elastin-associated microfibrils, which are always present at the periphery of growing elastic fibres and probably have a role more complex than being a scaffold for tropoelastin aggregation in vivo, the elastic fibres seem to be composed of several matrix constituents, which are different in different organs and change with age and in pathological conditions. This is demonstrated by immunocytochemical studies on ultrathin sections.

Zakeri, Z; Quaglino, Daniela; Ahuja, Hs

We examined the importance of cell death in producing the limb deformity phenotype of the Hammertoe mouse. In order to make this evaluation, we first combined several techniques to verify the reliability of cell death markers in the normal mouse limb. In the normal limb, cell death occurs between Days 10.5 and 15.5 in the anterior, interdigital, and posterior zones. Morphological examination and staining for acid phosphatase or macrophages all reveal regions of cell death by detecting phagocytosis, while in. situ end labeling of DNA defines nuclear degeneration as apoptotic. The phagocytes display surface markers characteristic of macrophages. The Hammertoe mutant shows strongly reduced cell death only in the interdigital region. This result is consistent with the phenotype and suggests that the mutation influences the patterning or control of cell death rather than its mechanism.

1994 - Elastin production and degradation in Cutis Laxa acquisita [Articolo su rivista]
Fornieri, Claudio; Quaglino, Daniela; G., Lungarella; E., Cavarra; Tiozzo, Roberta; Mg, Giro; M., Canciani; Jm, Davidson; Ronchetti, Ivonne

A case of cutis laxa acquisita was studied with the aim of defining the molecular defects involved and comparing them with those of an inherited form of cutis laxa. In the acquisita form of cutis laxa ultrastructural and biochemical observations confirmed a dramatic reduction of dermal elastin, whereas collagen content was normal. Elastin mRNA expression as well as tropoelastin production by dermal fibroblasts, in vitro, were normal compared with control cells, as revealed by in situ hybridization and enzyme-linked immunosorbent assay, respectively. Lysyl oxidase activity, measured on cultured fibroblasts, was reduced to 60% compared with age-matched control subjects. Unlike control skin fibroblasts or fibroblasts from inherited cutis laxa, the affected skin cells from cutis laxa acquisita predominantly expressed an elastolytic activity identified as cathepsin G. Patient serum also has reduced elastase inhibitory capacity and reduced levels of alpha 1-antiproteinase inhibitor (alpha 1-antitrypsin). Although cutis laxa acquisita is a heterogeneous group of disorders, findings in this patient were consistent with excessive loss of cutaneous elastin due to the combined effects of several factors, such as low lysyl oxidase activity together with high levels of cathepsin G and reduction of circulating proteinase inhibitor(s).

1994 - G2.1 Earthworm leukocytes (CD11a+, CD45RA+, ß2-m+, Thy-1+) can kill human K562 tumor cell targets [Relazione in Atti di Convegno]
Cooper, E. L.; Suzuki, M. M.; Cossarizza, A.; Salvioli, S.; Capri, M.; Quaglino, D.; Pasquali-Ronchetti, I.; Franceschi, C.

M., Marinelli; Quaglino, Daniela; S., Bettuzzi; P., Strocchi; Davalli, Pierpaola; Corti, Arnaldo

In the ventral prostate of the intact rat, clusterin mRNA is expressed in a small population of cuboidal epithelial cells undergoing apoptosis, but not in the columnar cells comprising the vast majority of the glandular epithelium. Upon castration, clusterin mRNA expression and apoptotic activity are turned off in the cuboidal cells and turned on in the columnar ones. We show here that the progressive enhancements in the abundance of clusterin mRNA, occurring in the rat ventral prostate upon aging, are based on increases of the cuboidal cells at the expense of the columnar ones. DNA fragmentation, a typical sign of apoptosis, assayed both by agarose gel electrophoresis and in situ staining, was undetectable in 3-month-old rats but was evident among the cuboidal cells of 24-month-old animals and columnar cells of 1-day castrates. The DNA content of the ventral prostate did not change significatively between young and old rats, indicating that no increase in the rate of cell death occurs within the age interval examined. It is concluded that the enhancement in cuboidal cell population, the consequent augmented accumulation of clusterin mRNA, and the increased frequency of DNA fragmentation that we have detected in the aging rat ventral prostate are not directly related to the rate of apoptosis.

Baritussio, A; Alberti, A; Quaglino, Daniela; Pettenazzo, A; Dalzoppo, D; Sartori, L; Pasqualironchetti, I.

Transformations of surfactant after secretion are incompletely understood. To clarify them, we lavaged lungs in fetuses and in newborn rabbits, fractionated the lavage fluid by differential and density gradient centrifugation, and analyzed the distribution of surfactant protein (SP) phospholipids, SP-A, SP-B, and SP-C. Furthermore, we administered into trachea of newborn rabbits labeled surfactant and compared the alveolar clearance of SP-A, SP-B, SP-C and saturated phosphatidylcholine. We found that, in the fetus, secreted lamellar bodies contain all components of surfactant, except a small amount of SP-A. As breathing starts and new surfactant subtypes are generated, the proteins are mostly associated with dense subtypes, but SP-B and SP-C are especially concentrated in dense materials that contain minor amounts of phospholipids and SP-A. Furthermore, we found that, during breathing, alveolar surfactant is degraded into more than one type of remnant, that the lavage fluid contains a pool of SP-A not associated with membranes, and that SP-A, SP-B, and SP-C are all turned over at a faster rate than saturated phosphatidylcholine.

Zakeri, Zf; Quaglino, Daniela; Latham, T; Lockshin, Ra

DNA fragmentation was evaluated in three instances of programmed cell death, interdigital cell death in embryonic mouse limbs, and metamorphic death of both the labial glands and intersegmental muscle in the tobacco hornworm Manduca sexta. In the mouse, we evaluated both developmental cell death and expanded-range cell death induced by retinoic acid. The status of DNA was examined in several ways. Nuclei were examined by electron microscopy and Feulgen staining. Quantitative assessment of total DNA content in Feulgen-stained degenerating nuclei was made for the gland. In the labial gland, DNA content does not drop during the early phases of cell death; nor is an endonucleolytic ladder seen when DNA was examined by ethidium bromide staining or prelabeling with [H-3]thymidine. Only by using end labeling of DNA could we detect DNA fragmentation at a very late stage in cell death, day 4 of the collapse of the gland. In contrast, WEHI 7.1 lymphoma cells display an early and extensive ladder after treatment with glucocorticoids. In mouse limb, for which cell death follows a more classic apoptotic morphology, a ladder is likewise not seen. We conclude that activation of an endonuclease is neither a trigger nor a necessary or defining component of the early phases of developmental programmed cell death, and that reported failure by others to find such a ladder may depend on limitations in the system that is under investigation.

1993 - Extracellular Matrix Modifications in Rat Tissues of Different Ages: Correlations between elastin and collagen type I mRNA expression and Iysyl-oxidase activity [Articolo su rivista]
Quaglino, Daniela; Fornieri, C.; Nanney, L. B.; Davidson, J. M.

We used a rat model to correlate age, matrix gene expression and lysyl oxidase activity in three connective tissues, skin, aorta and lung. By in situ hybridization, we showed that intense collagen type I and elastin mRNA expression were limited to a brief postnatal period. Although there were some organ-specific differences, the mRNA abundance for these two scleroproteins drastically diminished with time. Thus, the majority of mesenchymal cells in young (60 days) and old (720 days) animals, appeared to be in a quiescent state, consistent with the slow turnover of these two scleroproteins. We also measured the activity of lysyl oxidase, an enzyme which plays a crucial role in the formation of crosslinks in both procollagen and tropoelastin molecules. In all the organs investigated, we observed a tissue-dependent pattern of activity. Moreover in this study we focused on the importance of gene matrix expression in evaluating lysyl oxidase activity of aging tissues.

I., Pasqualironchetti; Contri, Miranda; Fornieri, Claudio; G., Mori; Quaglino, Daniela

Elastin fibres are present in all connective tissues of vertebrates, and are particularly abundant in those undergoing physiological mechanical stress. Elastin, the main component of the fibres, is secreted as soluble tropoelastin molecules, that are crosslinked into insoluble aggregates in the extracellular space. Tropoelastin is highly hydrophobic and is encoded by a single copy gene, localized on chromosome 7 in humans; the primary transcript undergoes alternate splicing giving rise to different tropoelastin molecules, whose distribution and physiological significance are still under investigation. Elastin shows a transient developmental expression, with barely detectable turnover during adult life. In thin sections, elastin fibres appear rather compact and consist of an amorphous material (true elastin) and of longitudinal strips, probably made of various components. By freeze-fracture and negative staining, the elastin fibre has been shown to be made of a loose irregular network of beaded filaments, which can be oriented by stretching. Cytochemical and immunocytochemical approaches have shown that elastin fibres contain proteoglycans as well as sites recognized by antibodies to lysyl oxidase and microfibrillar components. It has been suggested that microfibrils and proteoglycans might have a role in elastin fibrogenesis. Elastin fibres undergo irreversible alterations during aging and in pathological conditions. These alterations consist mainly of accumulation of ions and of abnormal matrix constituents, and/or swelling and disaggregation of the fibre itself.

1992 - Enhanced hepatic collagen gene expression in a rodent model of haemochromatosis [Capitolo/Saggio]
Pietrangelo, Antonello; Quaglino, Daniela; Gualdi, Rossana; G., Casalgrandi; G., Cairo; Ventura, Ezio


Fornieri, Claudio; Quaglino, Daniela; Mori, G.

Connective tissues such as blood vessels are known to be greatly affected by age because of impaired functional properties and increased susceptibility to diseases. With the aim of providing further information on the role of the extracellular matrix in age-related modifications, we investigated the aorta in the rat model from birth to senescence by means of morphological and morphometric observations and by evaluation of lysyl oxidase activity. Results focused on the dramatic vascular rearrangements due to progressive fibrosis of the extracellular matrix and on prominent elastin modifications. The presence of lysyl oxidase activity, even in the oldest animals, might be at least partly responsible for the increased stiffness of the aging extracellular matrix. The striking age-related remodeling of the aortic architecture and the alterations of the interactions between cellular and extracellular compartments might greatly influence the functional properties of the arterial wall in senescence, at least contributing to the consequences of some apparently age-related vascular disorders.

Quaglino, Daniela; Fornieri, Claudio; Botti, B; Davidson, Jm; Pasqualironchetti, I.

Ascorbic acid plays an important role in connective tissue metabolism, where, among other effects, it acts as a reducing factor in the reactions catalyzed by prolyl and lysyl hydroxylases. In vitro, ascorbic acid has been shown to have a positive influence on collagen synthesis at pre- and/or post-translational levels and a negative effect on elastin production. In the present work, the effects of vitamin C on extracellular matrix deposition have been studied in vivo. Stereological analysis on electron micrographs showed, compared to age-matched controls, a 50 to 60% increase of collagen deposition in the media and in the adventitia of the aorta of rats treated for 30 days from the 18th day of life with 10% ascorbate in their drinking water. By contrast, elastin volume density was significantly reduced by the treatment at all ages examined. These morphological data were supported by in situ hybridization observations showing enhanced collagen type I mRNA and reduced elastin mRNA expression upon treatment. Although vitamin C did not inhibit lysyl oxidase activity in vivo, being only slightly higher than in controls, enzyme activity was significantly reduced, when high doses of ascorbate were added in vitro. Lysyl oxidase activity may be a function of enhanced collagen metabolism rather than a direct effect of the vitamin on the enzyme activity. These data indicate that ascorbate exerts opposite effects on the deposition of two major components of the extracellular matrix in vivo, at least during periods of rapid growth.

1991 - Transforming growth factor-beta stimulates wound healing and modulates extracellular matrix gene expression in pig skin: Incisional wound model [Articolo su rivista]
Quaglino, D.; Nanney, L. B.; Ditesheim, J. A.; Davidson, J. M.

Enhanced wound healing is elicited by exogenous administration of transforming growth factor-β1 (TGF-β1) in splitthickness, excisional wounds in the pig (Quaglino, Lab Invest 63:307-319, 1990). A study was designed to investigate if the selective and localized effects of TGF-β1 found in the previous model were dependent upon the type of wound or could be considered a more general effect of the cytokine. Transdermal, sutured incisions in the pig were evaluated by conventional histology and by in situ hybridization to reveal locally affected gene expression of collagen, elastin, fibronectin, stromelysin, TGF-β1, and basic fibroblast growth factor. Granulation tissue formation was markedly enhanced at 6 d by a single injection of recombinant human TGFβ1 at the time of wound closure. Although granulation tissue was confined within the margins of the incisional wound, prominent differences in hybridization signals were observed between control and treated wounds. The stimulatory effect of TGF-β1 on granulation tissue formation was accompanied by a distinct enhancement in cells expressing mRNA for several different extracellular matrix proteins including collagens type I and III and elastin, whereas a single injection of human recombinant TGFβ1 (4 μg) at the wound site diminished the expression of the neutral metalloprotease, stromelysin, and enhanced the frequency and intensity of cells expressing TGF-β1. The data reinforce the concept that TGF-β1 can act as a potent, auto-inductive modulator of connective tissue remodeling during the repair process. © 1991.

Ronchetti, Ivonne; Quaglino, Daniela; Dyne, Km; Zanaboni, G; Cetta, G.

Ultrastructural analyses were performed on clinically normal skin from forearm and/or femoral regions of five subjects, all excreting high levels of gly-pro dipeptides into the urine and exhibiting very low prolidase activity on hemolyzed erythrocytes. In both regions, the overall organization of the dermis was normal. Stereological analysis, however, showed that collagen volume density was reduced when compared to that of age-matched controls. Collagen fibrils did not show ultrastructural alterations, but they were distributed into a higher number of small bundles, and their diameters shifted towards lower values compared to age-matched controls. The elastin volume density was slightly reduced in the patients, especially in the femoral areas. In both forearm and femoral dermis, elastin fibers were significantly more numerous and smaller than in controls. Furthermore, elastin fibers were apparently normal in the forearm dermis, whereas appeared polymorphic and cribriform in the femoral skin. The results focused on the importance of efficient proline re-utilization for normal collagen and elastin synthesis and deposition. The differences between femoral and forearm skin regions from both clinical and ultrastructural points of view, may depend on mechanisms that regulate circulation and, possibly, on other factors which modulate the phenotypic expression of mesenchymal cells.

Baccaranicontri, M; Vincenzi, D; Quaglino, Daniela; Mori, G; Pasqualironchetti, I.

Polyclonal antibodies to human placenta lysyl oxidase (Kuivaniemi et al., 1984) were used to localize the enzyme at ultrastructural level in human placenta, skin and aorta, by using the indirect immunogold method. The antibodies were tested on thin sections of tissues fixed and embedded in various experimental conditions. With all methods employed, the immunoreaction was always positive on collagen fibers in all tissues examined, independently of the age of the subjects. In placenta, the reaction was also slightly positive on matrix microfilaments and cells. In dermis, fibroblasts and elastin were scarcely positive in a normal 5 day-old child, in a child with skin hyperelasticity, and in two babies with osteogenesis imperfecta type II; whereas they were negative in two 16 and 40 year-old normal subjects. In aorta, the immunoreaction was always positive on collagen, scarcely positive on cells and on elastin of a 24 week-old fetus, of a normal child, and of two babies who died of complications associated with O.I. type II; on the contrary, the reaction was negative on cells and elastin fibers of a 16 week-old fetus, and of a normal 19 year-old girl. When present on elastin, gold particles were localized mostly inside the fibers. Contrary to what was observed by Kagan and coworkers on bovine aorta by using antibodies against aortic lysyl oxidase (Kagan et al., 1986), no specific localization of gold particles could be observed on or adjacent to the elastin/associated microfibrils. The results indicate that antibodies raised against placenta lysyl oxidase recognize collagen-associated as well as elastin-associated epitopes

1990 - Localized effects of transforming growth factor beta on extracellular matrix gene expression during wound healing. Excisional wound model. [Articolo su rivista]
Quaglino, Daniela; Nanney, L. B.; Kennedy, R.; Davidson, Jm

The effect of transforming growth factor-beta 1 (TGF-beta 1) on matrix gene expression has been investigated during the process of wound repair, where the formation of new connective tissue represents a critical step in restoring tissue integrity. Split-thickness excisional wounds in the pig were studied by in situ hybridization in order to obtain subjective findings on the activity and location of cells involved in matrix gene expression after the administration of recombinant TGF-beta 1. Data focus on the stimulatory role of this growth factor in granulation tissue formation, on the enhanced mRNA content of collagen types I and III, fibronectin, TGF-beta 1 itself, and on the reduction in stromelysin mRNA, suggesting that increased matrix formation measured after treatment with TGF-beta 1 is due to fibroplasia regulated by the abundance of mRNAs for several different structural, matrix proteins as well as inhibition of proteolytic phenomena elicited by metalloproteinases. These studies reveal elastin mRNA early in the repair process, and elastin mRNA expression is enhanced by administration of TGF-beta 1. Moreover, we show that TGF-beta 1 was auto-stimulating in wounds, accounting, at least in part, for the persistent effects of single doses of this multipotential cytokine.

Conforti, G; Zanetti, A; Pasqualironchetti, I; Quaglino, Daniela; Neyroz, P; Dejana, E.

The vitronectin (Vn) receptor belongs to the integrin family of proteins and although its biochemical structure is fully characterized little is known about its binding affinity and specificity. We report here that Vn receptor binding to different matrix proteins is influenced by the surrounding lipid composition of the membrane. Human placenta affinity purified Vn receptor was inserted into liposomes of different composition: (i) phosphatidylcholine (PC); (ii) PC+phosphatidylethanolamine (PE); (iii) PC+PE+phosphatidylserine (PS) + phosphatidylinositol (PI) + cholesterol (chol). The amount of purified material that could be incorporated into the three lipid vesicle preparations was proportional to the efficiency of the vesicle formation that increased from PC (38%) to PC+PE and PC+PE+PS+PI+chol (about 50%) vesicles. Electron microscopy analysis showed that the homogeneity and size of the three liposome preparations were comparable (20-nm diameter) but their binding capacity to a series of substrates differed widely. Vn receptor inserted in PC liposomes bound only Vn, but when it was inserted in PC+PE and PC+PE+PS+PI+chol liposomes it also attached to von Willebrand factor (vWF) and fibronectin (Fn). Vn receptor had higher binding capacity for substrates when it was inserted in PC+PE+PS+PI+chol than PC+PE liposomes. Antibodies to Vn receptor blocked Vn receptor liposome binding to Vn, vWF, and Fn. The intrinsic emission fluorescence spectrum of the Vn receptor reconstituted in PC+PE+PS+PI+chol liposomes was blue-shifted in relation to PC liposomes, suggesting a conformational change of the receptor in the membranes. These data provide direct evidence that the Vn receptor is "promiscuous" and can associate with Vn, vWF and Fn. The nature of the membrane lipid composition surrounding the receptor could thus influence its binding affinity, possibly by changing its conformation or exposure or both.

1990 - Synovial tissue expression of connective matrix molecule genes [Relazione in Atti di Convegno]
Quaglino, D.; Fava, R.; Davidson, J. M.; Pasquali Ronchetti, I.

1989 - Correlations between age and rat dermis modifications. Ultrastructural- morphometric evaluations and lysyl oxidase activity [Articolo su rivista]
Fornieri, C.; Quaglino, D.; Mori, G.

The extracellular matrix is a complex, integrated macromolecular system which plays a crucial role in the economy of each organ. In this study we focused our attention on the correlations between age and rat skin dermis. The latter was chosen as a model of the connective tissue, and was analyzed by means of electron microscopy and by measurement of the activity of lysyl oxidase, the enzyme involved in collagen and elastin crosslink formation. Ultra-structural and morphometric evaluations associated to body weight growth, showed a progressive increase in the amounts of extracellular components and a progressive reduction in the cell density. Skin from adult animals appeared characterized by a well organized matrix; by contrast, in old rats, we observed several degenerative features such as the disorganization of collagen bundles, the vacuolization of elastic fibers, and the atrophy of the mesenchimal cells. Morphometric evaluations in old animals showed a slight but significant reduction in the percentage of the total collagen measured, a fair stability in the area occupied by the elastin fibers, and an increase of the apparently non-structured matrix. The fact that lysyl oxidase activity was diminished in old rats does not corroborate the observation by several authors that increased collagen insolubility is a consequence of higher intra-and intermolecular crosslinking. This would suggest that other chemical modifications, such as crosslink oxidation or non enzymatic glycosylation, might be involved during the aging of connective tissue. The qualitative and quantitative modifications observed at all ages illustrate the correlation between connective tissue modifications and structural and/or functional properties of the skin. © 1989, Editrice Kurtis s.r.l.. All rights reserved.

1989 - Dermal alterations in patients with Wilson's disease treated with D-penicillamine [Articolo su rivista]
Pasquali Ronchetti, I.; Quaglino, D.; Baccarani Contri, M.; Hayek, J.; Galassi, G.

1989 - Dermal elastin and collagen in systemic sclerosis. Effect of D-penicillamine treatment [Articolo su rivista]
Ronchetti, Ip.; Guerra, D.; Quaglino, D.; Vincenzi, D.; Manzini, E.; Canossi, B.; Manzini, C. U.

Skin biopsies from 4 systemic sclerosis (SSc) patients, 6 SSc patients treated with D-penicillamine (from 8 to 60 months) and 4 normal subjects were analyzed by light and electron microscopy. By light microscopy, collagen bundles of SSc dermis were thicker and more compact than in age-matched controls; D-penicillamine treatment did not significantly modify their organization. On the contrary, a stereological analysis showed that the elastin volume density was higher in patients than in controls, and increased again after D-penicillamine treatment: moreover, the number of elastin fibers per unit area was significantly higher in the dermis of patients compared to controls, and became even higher after D-penicillamine treatment. The phenomena were evident in all strata of the dermis; however, the most significant increase of elastin in SSc patients compared to controls was in the superficial dermis, whereas after D-penicillamine treatment, all the strata of the dermis showed a significant increase in the percentage of elastin and in the number of elastin fibers per unit area compared to untreated patients and to controls. There were no relationships between elastin increase and time from the onset of SSc or time and dose of D-penicillamine treatment. At the ultrastructural level, collagen fibrils had rather heterogeneous diameters and formed more compact fibers, especially in the reticular and in the deep dermis of SSc patients compared to controls. After D-penicillamine, collagen fibril diameters in three of 5 patients examined were statistically wider and more heterogeneous than in controls and in untreated patients, whereas in the other 2 subjects both the mean diameter and the distribution of the class diameter were similar to both controls and untreated patients. This could suggest a specific individual reaction to the drug. Elastin fibers were smaller, more numerous and polymorphous in all patients compared to controls. After D-penicillamine, elastin fibers became even more numerous and smaller than in untreated patients. There was no correlation between the number and size of the elastin fibers and the time or dose of D-penicillamine. The internal organization of the elastin fibers was normal. These data indicate that the amount and distribution of collagen and elastin are altered in the dermis of SSc patients, and that D-penicillamine interferes with the deposition of both fibrous proteins in the dermal extracellular space.

1989 - Terminal cisternae of denervated rabbit skeletal muscle: Alterations of functional properties of Ca2+ release channels [Articolo su rivista]
Zorzato, F.; Volpe, P.; Damiani, E.; Quaglino, D.; Margreth, A.

Terminal cisternae (TC) of skeletal muscle represent the specialized compartment from which Ca2+ is released into the myoplasm after a propagated action potential. In this study we have investigated the morphology, protein composition, and Ca2+ release properties of TC isolated from rabbit gastrocnemius muscle 2 wk after nerve sectioning. Thin-section electron microscopy showed that TC vesicles from denervated muscle were enriched in calsequestrin (CS) and contained a larger fraction of the junctional sarcoplasmic reticulum (SR), as judged by membrane profiles with morphologically intact feet structures. Accordingly, the yield of junctional SR from denervated muscle was twice that of control muscle, and the protein pattern of TC vesicles exhibited an increase in junctional protein components, e.g., CS and the 350-kDa protein. The larger content of the 350-kDa protein, or ryanodine receptor (F.A. Lai, H. Erickson, E. Rousseau, Q.-Y. Liu, and G. Meissner. Nature Lond. 331: 315-319, 1988; T. Imagawa, J.S. Smith, R. Coronado, and K. P. Campbell. J. Biol. Chem. 262: 10636-10643, 1987; L. Hymel, M. Inui, S. Fleischer, and H. Schindler. Proc. Natl. Acad. Sci. USA 85: 441-445, 1988) was paralleled by an increased binding site density (B(max)) for ryanodine binding in denervated muscle TC. The effects of ruthenium red, a Ca2+ release blocker, on Ca2+ loading rate and Ca2+-ATPase activity suggested that TC from denervated muscle were less permeable to Ca2+. After active Ca2+ loading, both doxorubicin and caffeine induced Ca2+ release from isolated TC, yet Ca2+ release rates were reduced in denervated muscle TC. This study provides evidence that denervation causes 1) proliferation of the junctional SR and 2) significant modifications of the functional properties of the Ca2+ release channels of isolated TC.

1988 - Denervation‐induced proliferative changes of triads in rabbit skeletal muscle [Articolo su rivista]
Salvotri, S.; Damiani, E.; Zorzato, F.; Volpe, P.; Pierobon, S.; Quaglino, D.; Salviati, G.; Margreth, A.

Protein compositional and functional differences exist between longitudinal and junctional sarcoplasmic reticulum (SR) in relation to Ca transport and to Ca release. In light of this knowledge, we have reinvestigated the effects of denervation on SR of rabbit gastrocnemius, a predominantly fast muscle. Electron microscopy of 2‐weeks denervated muscle showed proliferation of transverse tubules (TT), forming junctional contacts with SR terminal cisternae (TC). At coincident periods, the yield of muscle microsomes was increased, and their fractionation by sucrose‐density centrifugation demonstrated a relative increase of heavy vesicles. Thin‐section electron microscopy of heavy SR from denervated muscle showed an increased number of vesicles containing calsequestrin (CS) as compared with control muscle. Electrophoretic analysis confirmed the relative decrease of Ca‐ATPase protein and the striking increase of CS both in total microsomes and in heavy SR vesicles. Calcium loading and Ca‐ATPase activity as well as the density of Ca‐ATPase protein were decreased to a similar extent (20–30%) in denervated muscle microsomes. Stimulation of Ca‐ATPase activity by Ca‐ionophore A23187 showed that the vesicles were tightly sealed. When probed by competitive ELISA with antibody to SR Ca‐ATPase from pure fast muscle, the Ca‐ATPase of denervated microsomes was found to be highly cross reactive. Cleveland's peptide maps of the Ca‐ATPase protein after partial digestion with S. aureus V8 protease also showed no significant change after denervation. Changes in cholesterol content and in the ratio of Mg‐ATPase to Ca‐ATPase activity of denervated muscle microsomes indicated a 4‐fold increase of TT protein, i.e., from about 3% to not more than 12% of total protein, at 2 weeks after denervation. All these changes were totally reversed upon reinnervation of muscle fibers, and the consequent muscle recovery, as obtained by nerve crushing instead of nerve sectioning. From these results, we conclude that denervated adult fast muscle, similarly to immature fast muscle, contains more junctional SR. However, the molecular and catalytic properties of the Ca‐ATPase are unaffected by denervation. Copyright © 1988 John Wiley &amp; Sons, Inc.

1988 - Mitochondrial oculoskeletal myopathy: case report [Articolo su rivista]
Colombo, A.; Merelli, E.; Sola, P.; Panzetti, P.; Quaglino, D.; Fornieri, C.

We report a case of oculoskeletal myopathy with abnormal mitochondria in which the chief clinical feature was ophthalmoplegia. Muscle weakness was mild and there were no retinal or cerebellar abnormalities, no deafness and no cardiac defects. The muscle biopsy specimen revealed subsarcolemmal mitochondrial aggregates and ragged red fibers. Electronmicroscopy showed that the aggregates were made up of mitochondria of variable size with structural abnormalities of the cristae and crystalloid inclusions. We believe that this oculoskeletal myopathy is distinct from Kearn-Sayre syndrome. © 1988 Masson Italia Periodici.

1988 - Postnatal transformations of alveolar surfactant in the rabbit: changes in pool size, pool morphology and isoforms of the 32-38 kDa apolipoprotein [Articolo su rivista]
Bruni, R.; Baritussio, A.; Quaglino, D.; Gabelli, C.; Benevento, M.; Ronchetti, Ip.

To clarify perinatal transformations of surfactant we performed lung lavage in term fetuses and in 0-24-h-old newborn rabbits. Lavage fluid was separated into three pools, namely lavage pellet, lavage supernatant and cells. We found that at birth the pellet contains 94.1 ± 1.4% (S.E.) saturated phosphatidylcholine, while the supernatant and cells contain traces of it. At birth the pellet contains secreted lamellar bodies while the supernatant lacks any recognizable structure. After birth, the alveolar saturated phosphatidylcholine level increases 5.1-times in 24 h, the proportions between pools reaching adult values in 90 min (pellet = 75.9 + 4.8%, supernatant = 22.7 ± 4.9%), and small vesicles appear in the supernatant, probably originating from the turnover of alveolar surfactant during breathing. The saturated phosphatidylcholine associated with cells remains unchanged. At birth, the 32-38 kDa surfactant apolipoprotein appears to be less extensively sialylated than in adult life. © 1988.

1987 - Familial Ehlers‐Danlos Syndrome Type II: Abnormal Fibrillogenesis of Dermal Collagen [Articolo su rivista]
Rizzo, R.; Contri, M. B.; Micafi, G.; Quaglino, D.; Pavone, L.; Ronchetti, I. P.

Abstract: We examined a father and son affected by Ehlers‐Danlos syndrome type II. Both patients had micrognathia together with ligament and skin hy‐perlaxity. The son exhibited complete cleft palate. Ultrastructural studies revealed abnormal collagen fibrils in the dermis of both patients. In the child the most striking alterations consisted of lateral fusion of an enormous number of collagen fibrils giving rise to huge polymorphic collagen masses. In the father's dermis the great majority of collagen fibrils appesired normal; however, lateral fusion of fibrils together with local abnormal collagen aggregation were occasionally seen. In both patients the dermal elastic network was well developed and elastic fibers appeared normal. Copyright © 1987, Wiley Blackwell. All rights reserved

1987 - Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas [Articolo su rivista]
Fornieri, C.; Contri, Mb.; Quaglino, D.; Ronchetti, Ip.

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G.M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the exent of the enzyme inhibition. When inhibition was &gt;65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

1986 - Aortic Elastin Abnormalities in Osteogenesis Imperfecta Type II [Articolo su rivista]
Ronchetti, Ip.; Quaglino, D.; Contri, Mb.; Tenconi, R.; Bressan, G. M.; Volpin, D.

Skin and aortic samples from two patients who died by lethal perinatal Osteogenesis Imperfecta (O. I.) were studied by optical and electron microscopy and compared with similar samples from two normal human fetuses and one newborn child. No significant abnormalities were observed in the dermis of O. I. patients apart from small differences in the diameter of reticular collagen fibrils. On the contrary, in the aortas of both patients collagen fibrils were significantly smaller than in the controls; moreover, elastin lamellae were deeply altered and consisted of roundish aggregates of elastin, massively permeated by cytochemically recognizable glycosaminoglycans. As identical features were described in experimental lathyrism by using inhibitors of the enzyme lysyl oxidase (Pasquali Ronchetti et al., 1984), the conclusion is reached that in the two cases of lethal perinatal O. I. examined, a severe lysyl oxidase deficiency could account for the observed ultrastructural abnormalities of elastin and that, besides defects of collagen type I, additional alterations of cellular metabolism might be responsible for the clinical heterogeneity of the diesease. © 1986, Gustav Fischer Verlag · Stuttgart · New York. All rights reserved.

1986 - Effect of DL-penicillamine on the aorta of growing chickens. Ultrastructural and biochemical studies [Articolo su rivista]
Ronchetti, Ip.; Fornieri, C.; Contri, Mb.; Quaglino, D.; Caselgrandi, E.

The effect of DL-penicillamine on the architecture of the aortic wall of growing chickens was studied, with particular attention to elastin and collagen. Penicillamine was added to the diet (0.2% and 0.4%, in the presence or not of 10 mg/kg CuSO4 and 100 mg/kg vitamin B6) from hatching, for periods from 7 days up to 2 months. The same regions of the thoracic aortas were examined and compared in all the different experimental conditions. The results showed that penicillamine induced relevant modifications in the process of elastin fibrogenesis. The alterations consisted of an increase of elastin in the extracellular space, associated with an increase in the number of elastin fibers per unit area, and a decrease of the mean profile area of the fibers. Interestingly, penicillamine induced the formation of numerous bundles of microfibrils associated or not with elastin fibers. After prolonged treatment, elastin tended to diminish and the fibers tended to fuse into polymorphic syncytia. Collagen fibrils were larger, showed more heterogeneous cross diameters, were less numerous, and were more spread out within the tissue. All the other components of the aortic wall appeared not to be altered by the chemical. Penicillamine did not modify the copper content of chick aortas, whereas it induced a 40-50% reduction of the activity of both salt and 4 M urea-soluble peptidyl lysyl oxidases in the same tissue. These data may help in understanding some of the pathologic manifestations in human beings during penicillamine treatment.

1985 - Alterations of elastin fibrogenesis by inhibition of the formation of desmosine crosslinks. Comparison between the effect of beta-aminopropionitrile (βAPN) and penicillamine [Articolo su rivista]
Ronchetti, I. P.; Contri, M. B.; Fornieri, C.; Quaglino, D.; Mori, G.

Experimental lathyrism was induced by feeding newborn chicks a diet containing 0.2 and 0.4% DL-Penicillamine, with or without CuSO4 (10 mg/Kg diet) and Vitamin B6 (100 mg/Kg diet), or 0.015 and 0.1% beta-aminopropionitrile fumarate (βAPN). After 7, 15, 25 and 55 days of treatment the animals were killed, the aortas removed and processed for electron microscopy in the presence of markers for proteoglycans, and the elastic fibers were carefully examined. Penicillamine, which prevents the formation of desmosine crosslinks by binding to precursors, induced the production of numerous new elastin fibers which appeared normal from the ultrastructural point of view. It seems, therefore, that at least in chick aortas, desmosine crosslinks are not necessary for the aggregation of tropoelastin molecules into structurally normal fibers. On the contrary, βAPN, a classical inhibitor of lysyl oxidase, induced the tropoelastin molecules to aggregate into abnormal protuberances on the old fibers. Moreover, the elastin deposited during βAPN treatment was always permeated by cytochemically revealed proteoglycans, which were never observed after penicillamine treatment. It is speculated that, at least in the system under study, the e-amino groups of tropoelastin molecules may offer the binding sites for matrix proteoglycans until they are removed by lysyl oxidase, and that matrix proteoglycans might play a role in elastin fibrogenesis by preventing spontaneous tropoelastin aggregation in areas far from growing elastin fibers. © 1985 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1985 - Biology and pathology of the elastin fiber: ultrastructural and biochemical approach [Articolo su rivista]
Ronchetti, Ip.; Fornieri, C.; Contri, Mb.; Quaglino, D.