14 results on '"Iorio E"'
Search Results
2. Reconstruction of a human hemicornea through natural scaffolds compatible with the growth of corneal epithelial stem cells and stromal keratocytes
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Barbaro, V., Ferrari, S., Fasolo, A., diego ponzin, and Di Iorio, E.
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Indoles ,Staining and Labeling ,Tissue Engineering ,Tissue Scaffolds ,Stem Cells ,Biocompatible Materials ,Epithelial Cells ,Cell Separation ,Limbus Corneae ,Biomechanical Phenomena ,Extracellular Matrix ,Colony-Forming Units Assay ,Cornea ,Corneal Transplantation ,Mice ,Animals ,Humans ,sense organs ,Stromal Cells ,Biomarkers ,Research Article ,Cell Proliferation - Abstract
To reconstruct a human hemicornea in vitro by means of limbal stem cells cultured onto human keratoplasty lenticules (HKLs) and to obtain a natural corneal graft for clinical applications.Limbal stem cells were seeded onto HKLs with or without the presence of feeder layers of lethally irradiated 3T3-J2 cells and compared with the current "gold standard" scaffold, i.e., the fibrin glue. The effects of the scaffold on the preservation of stemness and/or induction of differentiation pathways were investigated through analysis of a variety of markers, including p63 and DeltaNp63alpha for stemness, 14-3-3sigma for early differentiation, keratins 3, 14, 12, and 19 to determine cell phenotype, and alpha6, beta1, and beta4 integrins to evaluate interactions with the stroma. Integrity of the stroma was assessed through analysis of keratan sulfate, CD-34 and aldehyde dehydrogenase 3A1 (ALDH3A1) (for keratocytes), visual system homeobox 1 (VSX1), and alpha-smooth muscle actin (alpha-SMA) (for fibroblasts and myofibroblasts). The structural properties of the reconstructed "hemicornea" were investigated through scanning electron microscopy. To evaluate the preservation of the stemness potential, cells were trypsinized from each scaffold and clonogenic/proliferative characteristics analyzed.Limbal stem cells expanded onto HKLs gave rise to a stratified squamous keratinized epithelium morphologically similar to that of normal corneas. The resulting corneal epithelium was characterized by basal expression of p63 and DeltaNp63alpha, while expression of 14-3-3sigma, keratin 3, and keratin 12 was found in the upper cell layers. The basal cuboidal epithelial cells were anchored to the basement membrane and expressed keratin 14 and alpha6, beta1, and beta4 integrins. In the stroma of HKLs, keratocytes maintained the biosynthetic and phenotypic appearances typical of resting/quiescent cells and expressed keratan sulfate, CD-34, and ALDH3A1. Fibroblastic transformation was observed with the appearance of VSX1 and alpha-SMA. Scanning electron microscopy analysis showed that HKLs maintained their native conformation with collagen fibrils interconnected to the network and parallel to the corneal surface. HKLs did not alter the clonogenic/proliferative capacity of limbal stem cells. No differences were seen when HKL was compared to fibrin glue, one of the scaffolds currently used for limbal stem cell transplantation.Our findings demonstrate that HKL could be a suitable scaffold for corneal epithelial stem cells as they were shown to proliferate, express differentiation markers, and bind to the underlying stroma with no alterations in clonogenic potential. HKLs have some advantages over currently used scaffolds, such as the possibility to allow cell growth with no feeder layers, to be freeze dried, and to preserve the integrity and viability of stromal keratocytes. The development of a tissue-engineered "hemicornea" might offer new therapeutic perspectives to patients affected by total limbal stem cell deficiency with stromal scarring.
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- 2009
3. A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe
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Parekh, M., Stefano Ferrari, Di Iorio, E., Barbaro, V., Camposampiero, D., Karali, M., Ponzin, D., Salvalaio, G., Parekh, M, Ferrari, S, Di Iorio, E, Barbaro, V, Camposampiero, D, Karali, M, Ponzin, D, and Salvalaio, G
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Cornea ,genetic structures ,Humans ,Medicine ,sense organs ,eye diseases ,Eye Evisceration ,Retina - Abstract
Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly. The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision(4,5,6). The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina(5,6). The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve(5,6). The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.
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- 2012
4. Oxidative species and S-glutathionyl conjugates in the apoptosis induction by allyl thiosulfate
- Author
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Nepravishta, R, Sabelli, R, Iorio, E, Micheli, L, Paci, M, and Melino, Sm
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Magnetic Resonance Spectroscopy ,Blotting, Western ,Cell Cycle ,ROS ,Apoptosis ,Sulfuric Acid Esters ,Lymphoma, T-Cell ,Glutathione ,Fluorescence ,Allyl Compounds ,garlic ,thiyl radicals ,glutathione ,organ sulfur compounds ,Tumor Cells, Cultured ,Humans ,Settore BIO/10 ,Oxidation-Reduction ,Cell Proliferation - Abstract
Natural allyl sulfur compounds show antiproliferative effects on tumor cells, but the biochemical mechanisms underlying the antitumorigenic properties of the organ sulfur compounds are not yet fully understood. Sodium 2-propenyl-thiosulfate is a garlic water-soluble organo-sulfane sulfur compound able to promote apoptosis in cancer cells, affecting the 'managing' of the redox state in the cell. Our studies show that sodium 2-propenyl-thiosulfate reacts spontaneously with reduced glutathione at physiological pH, leading to the formation of S-allyl-mercapto-glutathione, radicals and peroxyl species, which are able to induce inhibition of enzymes with cysteine in the catalytic site, such as sulfurtransferases. S-Allyl-mercapto-glutathione was purified and characterized by NMR and MS, and its cytotoxic effect at 500 μm on HuT 78 cells was analyzed, showing activation of the p38-MAPK pathway. Many allyl sulfur compounds are also able to promote chemoprevention by induction of xenobiotic-metabolizing enzymes, inducing down-activation or detoxification of the carcinogens. Thus, the effects of the S-allyl-mercapto-glutathione on proteins involved in the cellular detoxification system, such as glutathione S-transferase, have been evaluated both in vitro and in HuT 78 cells. Although the antitumor properties of water-soluble sulfur compounds may arise from several mechanisms and it is likely that more cellular events occur simultaneously, a relevant role is played by the formation of both reduced glutathione conjugates and radical species that affect the activity of the thiol-proteins involved in fundamental cellular processes.
- Published
- 2011
5. Oral absorption of bendazac lysine salt in man after repeated administrations
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Angeli D. Galli, G. Barillari, Catanese B, and Iorio E
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Adult ,Male ,Pharmacology ,medicine.medical_specialty ,Clinical tests ,Indazoles ,Time Factors ,business.industry ,Administration, Oral ,Absorption (skin) ,Plasma levels ,Kinetics ,Endocrinology ,Bendazac lysine salt ,Intestinal Absorption ,Internal medicine ,Bendazac ,medicine ,Humans ,Pyrazoles ,Female ,business ,Blood Chemical Analysis ,medicine.drug - Abstract
Summary The plasma levels of the lysine salt of bendazac were studied in man after repeated administrations. The substance was administered in 500 mg tablets three times daily for 21 days. Concentrations of bendazac ranging between 15 and 30 μg/ml were found in the plasma within 10 hours after the first administration. No significant differences were observed between the first and following days of treatment. Common laboratory and clinical tests did not show any marked change.
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- 1983
6. Oxidative stress in chronic lymphocytic leukemia: still a matter of debate
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Eugenio Luigi Iorio, Francesco La Rocca, Vitalba Ruggieri, Mario Capunzo, Ilaria Migliaccio, Agostino Festa, Giovanni D'Arena, Elisa Seneca, Pellegrino Musto, Michele Caraglia, Aldo Giudice, Gioacchino Calapai, Vincenzo De Feo, D'Arena, G., Seneca, E., Migliaccio, I., De Feo, V., Giudice, A., La Rocca, F., Capunzo, M., Calapai, G., Festa, A., Caraglia, M., Musto, P., Iorio, E. L., and Ruggieri, V.
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Chronic lymphocytic leukemia ,oxidative stress ,prognostication ,Hematology ,Oncology ,Cancer Research ,Energy metabolism ,Mitochondrion ,medicine.disease_cause ,03 medical and health sciences ,0302 clinical medicine ,hemic and lymphatic diseases ,Biomarkers, Tumor ,medicine ,Humans ,chemistry.chemical_classification ,oxidative stre ,Reactive oxygen species ,Chemistry ,Prognosis ,medicine.disease ,Leukemia, Lymphocytic, Chronic, B-Cell ,Mitochondria ,Oxidative Stress ,030220 oncology & carcinogenesis ,Cancer research ,Energy Metabolism ,Reactive Oxygen Species ,Carcinogenesis ,Oxidative stress ,030215 immunology - Abstract
There is a large body of evidence showing a strong correlation between carcinogenesis of several types of human tumors, including chronic lymphocytic leukemia (CLL), and oxidative stress (OS). The mechanisms by which OS may promote cancer pathogenesis have not been completely deciphered yet and, in CLL, as in other neoplasms, whether OS is a primary cause or simply a downstream effect of the disease is still an open question. It has been demonstrated that, in CLL, OS concomitantly results from increased reactive oxygen species (ROS) production, mainly ascribable to CLL cells mitochondrial activity, and impaired antioxidant defenses. Interestingly, OS evaluation in CLL patients, at diagnosis, seems to have a prognostic significance, thus getting new insights in the biological comprehension of the disease with potential therapeutic implications.
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- 2018
7. Prognostic relevance of oxidative stress measurement in chronic lymphocytic leukaemia
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Marta Coscia, Carlo Visco, Vitalba Ruggieri, Gioacchino Calapai, Luca Laurenti, Candida Vitale, Nicola Matteo Dario Di Minno, Silvia Deaglio, Eugenio Luigi Iorio, Omar Perbellini, Vincenzo Pizza, Pellegrino Musto, Giovanni Di Minno, Giovanni D'Arena, Idanna Innocenti, Francesco La Rocca, Aldo Giudice, D'Arena, G., Vitale, C., Perbellini, O., Coscia, M., La Rocca, F., Ruggieri, V., Visco, C., Di Minno, N. M. D., Innocenti, I., Pizza, V., Deaglio, S., Di Minno, G., Giudice, A., Calapai, G., Musto, P., Laurenti, L., and Iorio, E. L.
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Male ,0301 basic medicine ,Antioxidant ,medicine.medical_treatment ,Chronic lymphocytic leukemia ,d-ROMs ,medicine.disease_cause ,CD49d ,Gastroenterology ,Photometry ,0302 clinical medicine ,80 and over ,oxidative stress ,BAP test ,d-ROM ,Chronic ,Stage (cooking) ,Aged, 80 and over ,Leukemia ,Hematology ,General Medicine ,Middle Aged ,Oxidants ,Lymphocytic ,chronic lymphocytic leukaemia ,prognosis ,030220 oncology & carcinogenesis ,Female ,Human ,Oxidant ,Adult ,medicine.medical_specialty ,Aged ,Biomarkers ,Humans ,Karyotyping ,Leukemia, Lymphocytic, Chronic, B-Cell ,Neoplasm Staging ,Prognosis ,Oxidative Stress ,Prognosi ,03 medical and health sciences ,Internal medicine ,medicine ,In patient ,oxidative stre ,Lymphocytic leukaemia ,business.industry ,BAP test, Chronic lymphocytic leukaemia, d-ROMs ,Oxidative stress, Prognosis, Hematology ,B-Cell ,Cytogenetics ,Biomarker ,medicine.disease ,Settore MED/15 - MALATTIE DEL SANGUE ,030104 developmental biology ,Immunology ,business ,Oxidative stress - Abstract
Objective To evaluate the prognostic significance of oxidative stress (OS) and antioxidant defense status measurement in patients with chronic lymphocytic leukemia (CLL). Method d-ROMs test and BAP test were evaluated at diagnosis of 165 patients with CLL and correlated with clinical-biological features and prognosis. Results An increased oxidative damage (d-ROMs test) and a reduced antioxidant potential (BAP test), were found in CLL patients than normal controls (p
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- 2017
8. Limbal Stem Cell Deficiency and Ocular Phenotype in Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome Caused by p63 Mutations
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Giuseppe Castaldo, Vanessa Barbaro, John A. McGrath, Diego Ponzin, Elisabetta Böhm, Paola Nardiello, Enzo Di Iorio, Stephen B. Kaye, Colin E. Willoughby, Stefano Ferrari, Di Iorio, E, Kaye, Sb, Ponzin, D, Barbaro, V, Ferrari, S, Böhm, E, Nardiello, P, Castaldo, Giuseppe, Mcgrath, Ja, and Willoughby, Ce
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Male ,Pathology ,Ectodermal dysplasia ,Ectrodactyly ,genetic structures ,DNA Mutational Analysis ,Visual Acuity ,Meibomian gland ,Polymerase Chain Reaction ,Corneal Diseases ,Ectodermal Dysplasia ,Missense mutation ,Limbal stem cell ,Child ,Fluorescent Antibody Technique, Indirect ,Stem Cells ,Epithelium, Corneal ,Middle Aged ,Cleft Palate ,limbal stem cell deficiency LSCD ,Phenotype ,medicine.anatomical_structure ,Child, Preschool ,Immunohistochemistry ,Adult ,medicine.medical_specialty ,Adolescent ,Cleft Lip ,Mutation, Missense ,Vision Disorders ,p63 sequence variants ,Limbus Corneae ,Ophthalmology ,medicine ,Humans ,Retrospective Studies ,ectrodactyly ectodermal dysplasia clefting EEC syndrome ,business.industry ,Tumor Suppressor Proteins ,Epithelial Cells ,Retrospective cohort study ,medicine.disease ,eye diseases ,stomatognathic diseases ,Histopathology ,sense organs ,business ,Keratoplasty, Penetrating ,Transcription Factors - Abstract
Objective To describe the ocular phenotype in patients with ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome (MIM#604292) and to determine the pathogenic basis of visual morbidity. Design Retrospective case series. Participants Nineteen families (23 patients) affected by EEC syndrome from the United Kingdom, Ireland, and Italy. Methods General medical examination to fulfill the diagnostic criteria for EEC syndrome and determine the phenotypic severity. Mutational analysis of p63 was performed by polymerase chain reaction–based bidirectional Sanger sequencing. All patients with EEC syndrome underwent a complete ophthalmic examination and ocular surface assessment. Limbal stem cell deficiency (LSCD) was diagnosed clinically on the basis of corneal conjunctivalization and anatomy of the limbal palisades of Vogt. Impression cytology using immunofluorescent antibodies was performed in 1 individual. Histologic and immunohistochemical analyses were performed on a corneal button and corneal pannus from 2 EEC patients. Main Outcome Measures The EEC syndrome phenotypic severity (EEC score), best-corrected Snellen visual acuity (decimal fraction), slit-lamp biomicroscopy, tear function index, tear breakup time, LSCD, p63 DNA sequence variants, impression cytology, and corneal histopathology. Results Eleven heterozygous missense mutations in the DNA binding domain of p63 were identified in all patients with EEC syndrome. All patients had ocular involvement and the commonest was an anomaly of the meibomian glands and lacrimal drainage system defects. The major cause of visual morbidity was progressive LSCD, which was detected in 61% (14/23). Limbal stem cell deficiency was related to advancing age and caused a progressive keratopathy, resulting in a dense vascularized corneal pannus, and eventually leading to visual impairment. Histologic analysis and impression cytology confirmed LSCD. Conclusions Heterozygous p63 mutations cause the EEC syndrome and result in visual impairment owing to progressive LSCD. There was no relationship of limbal stem cell failure with the severity of EEC syndrome, as classified by the EEC score, or the underlying molecular defect in p63 . Financial Disclosure(s) The authors have no proprietary or commercial interest in any of the materials discussed in this article.
- Published
- 2012
9. A new locus (DFNA47) for autosomal dominant non-syndromic inherited hearing loss maps to 9p21-22 in a large Italian family
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Pio D'Adamo, Salvatore Melchionda, Angela D'Eustacchio, Francesca Donaudy, Paolo Gasparini, Enzo Di Iorio, D'Adamo, ADAMO PIO, Donaudy, F, D'Eustacchio, A, DI IORIO, E, Melchionda, S, and Gasparini, Paolo
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Adult ,Male ,Adolescent ,Hearing loss ,Locus (genetics) ,Biology ,Genome ,Genetic determinism ,Degenerative disease ,Genetic linkage ,otorhinolaryngologic diseases ,Genetics ,medicine ,Humans ,Hearing Loss ,Gene ,Progressive hearing impairment ,Genetics (clinical) ,Genes, Dominant ,Chromosome Mapping ,Middle Aged ,medicine.disease ,Pedigree ,Italy ,Female ,Lod Score ,medicine.symptom ,Chromosomes, Human, Pair 9 ,Microsatellite Repeats - Abstract
Hearing loss is the most common sensory disorder in humans, and genetic factors are a major cause. Approximately 15-20% of genetic cases exhibit an autosomal dominant pattern of transmission. So far, 41 autosomal dominant loci have been mapped and 17 genes have been identified. Here we report the mapping of a novel locus for autosomal dominant non-syndromic hearing loss, DFNA47, to chromosome 9p21-22 in a large multigenerational Italian family with progressive hearing impairment. Most affected individuals noticed hearing impairment after their teens with subsequent gradual progression to a moderate-severe loss. There were no obvious vestibular dysfunction and other associated abnormalities. A maximum lod score of 3.14 was obtained with marker D9S157 (at theta=0) after a genome wide search. The study of additional markers allowed us to confirm this region with positive lod scores of 3.58 (at theta=0 from D9S285) and of 3.67 (at theta=0 from D9S162). Recombinants define a region of approximately 9 cM flanked by markers D9S268 and D9S942. Multipoint linkage analysis showed a Lod score of 4.26. Few known genes map to the region, and those possibly related by function to hearing are being screened for disease-causing mutations.
- Published
- 2003
10. A novel de novo missense mutation in TP63 underlying germline mosaicism in AEC syndrome: Implications for recurrence risk and prenatal diagnosis
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Felice Amato, Stefano Ferrari, Mohit Parekh, Giuseppe Castaldo, Diego Ponzin, Paola Nardiello, Cristina Parolin, E. Bonifazi, Vanessa Barbaro, Arianna Calistri, Enzo Di Iorio, Colin E. Willoughby, Barbaro, V, Nardiello, P, Castaldo, Giuseppe, Willoughby, Ce, Ferrari, S, Ponzin, D, Amato, Felice, Bonifazi, E, Parekh, M, Calistri, A, Parolin, C, and Di Iorio, E.
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Male ,Ectodermal dysplasia ,TP63 mutation ,Cleft Lip ,Genetic counseling ,Molecular Sequence Data ,Mutation, Missense ,Prenatal diagnosis ,Germline mosaicism ,C%22">c.1568T>C ,Biology ,medicine.disease_cause ,Germline mutation ,Prenatal Diagnosis ,TP63 ,Genetics ,medicine ,Humans ,ankyloblepheron-ectodermal defects- cleft lip/palate ,Missense mutation ,sporadic mutation ,Amino Acid Sequence ,Genetics (clinical) ,Mutation ,germline mosaicism ,Sequence Homology, Amino Acid ,Mosaicism ,Tumor Suppressor Proteins ,Syndrome ,medicine.disease ,p.L523P ,Pedigree ,Cleft Palate ,Germ Cells ,Female ,Ankyloblepharon ectodermal defects cleft lip palate syndrome ,Transcription Factors - Abstract
Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome is a rare autosomal dominant ectodermal dysplasia syndrome. It is caused by heterozygous mutations in TP63, encoding a transcriptional factor of the p53 family. Mutations in TP63, mainly missense in exons 13 and 14 encoding the sterile alpha motif (SAM) and the transactivation inhibitory (TI) domains, account for 99% of mutations in individuals with AEC syndrome. Of these, ≥70% are de novo mutations, present in the affected patient, but not in parents nor in healthy siblings. However, when a mutation appears de novo, it is not possible to differentiate between a sporadic mutation, or germline mosaicism in the parents. In this latter case, there is a risk of having additional affected offspring. We describe two sisters with AEC syndrome, whose parents were unaffected. Both patients carried the heterozygous c.1568T>C substitution in exon 13 of TP63, resulting in a p.L523P change in the SAM domain of the protein. Analyses of DNA from parental blood cells, seminal fluid (from the father) and maternal cells (buccal, vaginal, and cervical) did not reveal the mutation, suggesting that the mosaicism may involve a very low percentage of cells (very low grade somatic mosaicism) or, more likely, maternal gonadal mosaicism. Mosaicism must be considered for the assessment of recurrence risk during genetic counseling in AEC syndrome, and pre-implantation/prenatal genetic diagnosis should be offered to all couples, even when the mutation is apparently de novo.
- Published
- 2012
11. Outer-membrane porins from Gram-negative bacteria stimulate platelet-activating-factor biosynthesis by cultured human endothelial cells
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Luigi Silvestro, Ciro Capasso, Maria Antonietta Tufano, Luigi Biancone, Fabio Rossano, Eugenio L. Iorio, Giovanni Camussi, Antonella De Martino, Adone Baroni, Tufano, M. A., Biancone, L., Rossano, Fabio, Capasso, C., Baroni, A., DE MARTINO, A., Iorio, E. L., Silvestri, L., and Camussi, G.
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Salmonella typhimurium ,biosynthesis of platelet-activating factor ,Umbilical Veins ,Gram-negative bacteria ,Porins ,Biology ,Biochemistry ,Phospholipases A ,Calcium in biology ,chemistry.chemical_compound ,Phospholipase A2 ,Acetyltransferases ,Gram-Negative Bacteria ,Extracellular ,Humans ,Platelet Activating Factor ,Cells, Cultured ,Platelet-activating factor ,biology.organism_classification ,endothelial cells ,Cell biology ,Phospholipases A2 ,Cytosol ,chemistry ,Porin ,biology.protein ,Calcium ,lipids (amino acids, peptides, and proteins) ,Endothelium, Vascular ,Bacterial outer membrane ,Bacterial Outer Membrane Proteins - Abstract
Porins are a family of hydrophobic proteins located in the outer membrane of the cell wall in Gram-negative bacteria. The effect of porins on the biosynthesis of platelet-activating factor (PAF) by cultured human umbilical-cord-vein-derived endothelial cells (HUVEC) was investigated. The results demonstrate that porins were able to induce a dose-dependent synthesis of PAF in HUVEC. PAF, synthesized after stimulation with porins, was mainly cell associated and the synthesis peaked at 15 min, decreasing rapidly thereafter. Experiments with radiolabeled precursors demonstrated that PAF, a 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, was synthesized via the remodeling pathway involving the acetylation of 1-O-alkyl-2-lyso-sn-glyceryl-3-phosphorylcholine (2-lysoPAF) generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase-A2 activity. The activation of phospholipase A2 in HUVEC stimulated by porins was detected by observing the mobilization of [14C]arachidonic acid. In addition, the activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphorylcholine 2-O-acetyltransferase was transiently increased in porin-stimulated HUVEC and, after incubation with [3H]CoASAc or [3H]acetate, the [3H]acetyl group was incorporated into newly synthesized PAF. Porins, by forming transmembrane channels, induced a sustained influx of extracellular 45Ca2+ into the cytosol. The activation of PAF synthesis by porins depended on this influx rather than on intracellular calcium mobilization, since PAF synthesis did not occur in the absence of extracellular Ca2+.
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- 1993
12. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells
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Chiara Bonini, Giuliana Ferrari, Alberto Giannetti, Elena Provasi, Fulvio Mavilio, Sergio Capurro, Graziella Pellegrini, Andrea Conti, Enzo Di Iorio, Stefano Ferrari, Alessandra Recchia, Giulietta Maruggi, Francesca Di Nunzio, Cristina Magnoni, Michele De Luca, Mavilio, F, Pellegrini, G, Ferrari, S, Di Nunzio, F, Di Iorio, E, Recchia, A, Maruggi, G, Ferrari, Giuliana, Provasi, E, Bonini, MARIA CHIARA, Capurro, S, Conti, A, Magnoni, C, Giannetti, A, and De Luca, M.
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Adult ,Male ,Pathology ,medicine.medical_specialty ,Genetic Vectors ,Junctional/*therapy Feasibility Studies Gene Therapy/*methods Genetic Vectors Humans Male Mice Retroviridae *Stem Cell Transplantation Tissue Engineering/methods ,Junctional epidermolysis bullosa (medicine) ,General Biochemistry, Genetics and Molecular Biology ,Cultured Epidermis/*cytology Epidermolysis Bullosa ,Mice ,Laminin ,medicine ,Animals ,Humans ,Cells, Cultured ,Basement membrane ,Tissue Engineering ,integumentary system ,biology ,Epidermis (botany) ,3T3 Cells Adult Animals Cell Adhesion Molecules/genetics Cells ,3T3 Cells ,Genetic Therapy ,General Medicine ,medicine.disease ,Transplantation ,Retroviridae ,medicine.anatomical_structure ,Epidermal Cells ,Immunology ,biology.protein ,Feasibility Studies ,Epidermolysis bullosa ,Stem cell ,Epidermolysis Bullosa, Junctional ,Keratinocyte ,Cell Adhesion Molecules ,Stem Cell Transplantation - Abstract
The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles(1,2). Cultured keratinocyte stem cells, known as holoclones(3-6), generate sheets of epithelium used to restore severe skin, mucosal and corneal defects(7-9). Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder(10). Epidermal stem cells from an adult patient affected by LAM5-beta 3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA ( encoding LAM5-beta 3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up ( 1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease. The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.
- Published
- 2006
13. Hearing loss: frequency and functional studies of the most common connexin26 alleles
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Salvatore Melchionda, Leopoldo Zelante, Paolo Gasparini, Paola D'Andrea, Massimiliano Bicego, Roberto Bruzzone, Enzo Di Iorio, Valentina Veronesi, D'Andrea, Paola, Veronesi, V, Bicego, M, Melchionda, S, Zelante, L, DI IORIO, E, Bruzzone, R, and Gasparini, Paolo
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Hearing loss ,Protein Conformation ,Population ,Mutant ,Biophysics ,Mutation, Missense ,Connexin ,Biology ,Deafness ,medicine.disease_cause ,Biochemistry ,Connexins ,Gene Frequency ,otorhinolaryngologic diseases ,medicine ,Missense mutation ,Humans ,Allele ,education ,Molecular Biology ,Allele frequency ,Alleles ,Genetics ,Mutation ,education.field_of_study ,Cell Biology ,Connexin 26 ,Italy ,medicine.symptom ,HeLa Cells - Abstract
Mutations in the GJB2 gene, encoding the gap-junction channel protein connexin 26, account for the majority of recessive forms and some of the dominant cases of deafness. Here, we report the frequency of GJB2 alleles in the Italian population affected by hearing loss and the functional analysis of six missense mutations. Genetic studies indicate that, apart from the common 35delG, only few additional mutations can be detected with a significant frequency in our population. Transfection of communication-incompetent HeLa cells with Cx26 missense mutations revealed three distinct classes of functional deficits in terms of protein expression, subcellular localisation and/or functional activity. Moreover, the M34T mutant acted as a dominant inhibitor of wild-type Cx26 channel activity when the two proteins were co-expressed in a manner mimicking a heterozygous genotype. These data support the hypothesis of a functional role for M34T as a dominant allele and represent a further step towards a complete understanding of the role of GJB2 in causing hearing loss.
- Published
- 2002
14. Increased blood levels of platelet-activating factor in insulin-dependent diabetic patients with microalbuminuria
- Author
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Eugenio L. Iorio, C Olivetti, Antonella Martino, Daniela Furlani, Lucro Quagliuolo, Giovanni Camussi, Maurizio Cassader, Guiseppe Montrucchio, Marcia Rosario Boccellino, Gabriella Gruden, Luigi Servillo, Enrico Lupia, Paolo Cavallo-Perin, CAVALLO PERIN, P, Lupia, E, Gruden, G, Olivetti, C, DE MARTINO, A, Cassader, M, Furlani, D, Servillo, Luigi, Quagliuolo, Lucio, Iorio, E, Boccellino, Mr, Montrucchio, G, and Camussi, G.
- Subjects
Adult ,Male ,medicine.medical_specialty ,Endothelium ,medicine.medical_treatment ,Diabetes ,Exercise ,Microalbuminuria ,PAF ,1-Alkyl-2-acetylglycerophosphocholine Esterase ,Albuminuria ,Diabetes Mellitus, Type 1 ,Female ,Humans ,Phospholipases A ,Platelet Activating Factor ,Reference Values ,Angiopathy ,Diabetic nephropathy ,chemistry.chemical_compound ,Internal medicine ,medicine ,Diabetes Mellitus ,Endothelial dysfunction ,Transplantation ,Platelet-activating factor ,business.industry ,Insulin ,Glomerular basement membrane ,medicine.disease ,medicine.anatomical_structure ,Endocrinology ,chemistry ,Nephrology ,business ,Type 1 - Abstract
angiopathy [1]. In particular, the increased albumin Background. Platelet-activating factor (PAF ), a phos- excretion rate (AER) is an early predictor for the pholipid mediator of inflammation, may induce an development of overt diabetic nephropathy [2,3]. enhanced size- and charge-dependent glomerular per- Although the exact mechanism of microalbuminuria meability in experimental animals. Studies on the role is still unknown, several factors that may contribute of PAF in enhanced glomerular permeability in the to its development have been identified [4]. early phase of diabetic nephropathy are still lacking. Intraglomerular haemodynamic alterations [4] and Methods. We evaluated the intravascular levels of PAF changes in size- and charge-dependent permeability and its main catabolic enzyme, the PAF-specific plasma selectivity of the glomerular basement membrane acetyl-hydrolase (PAF-AH ), in basal conditions and (GBM ) [5,6 ] have been implicated in the pathogenesis after exercise, in normo- or micro-albuminuric insulin- of the enhanced albumin excretion. Recently, it has dependent diabetic (IDD) patients and in normal been suggested that endothelial dysfunction precedes subjects. the development of microalbuminuria [7]. Endothelial Results. The results obtained indicate that the concen- dysfunctions include an increase in vascular resistance tration of PAF in whole blood was significantly due to reduced synthesis of nitric oxide [8] and enhanced in basal conditions, during and after exercise enhanced production of endothelin by the endothelium in all microalbuminuric IDD patients, but not in [9], a decrease in endothelial anticoagulant and profibnormoalbuminuric IDD or in control subjects. The rinolytic properties [10], and an altered barrier funcincreased concentration of PAF did not correlate with tion of the endothelium [1]. Several mechanisms of changes in the activity of PAF-AH, suggesting an inflammation have been studied as potential candidates enhanced production rather than a decreased cata- for the endothelial alterations [8–10] and the changes bolism of PAF. in permaselectivity of GBM [5,6 ]. Due to its potent Conclusions. These results indicate an association biological actions on endothelial cells, leukocytes and between increased production of PAF and enhanced platelets (PLTs), platelet-activating factor (PAF ) is a glomerular permeability in microalbuminuric IDD potential candidate for the enhanced vascular and patients. glomerular permeability observed in diabetes [11]. Previous studies on experimental animals have shown
- Published
- 2000
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