in english and italian Morphophysiological Remarks E xperimental M edicine R eviews вЂњplumeliaвЂќ edizioni Edited by Aldo Gerbino Giovanni Zummo Giuseppe Crescimanno Di.Me.S. вЂњplumeliaвЂќ ricerca in english and italian Morphophysiological Remarks E xperimental M edicine R eviews вЂњplumeliaвЂќ edizioni Edited by Aldo Gerbino Giovanni Zummo Giuseppe Crescimanno Di.Me.S. Dipartimento di Medicina Sperimentale UniversitГ degli Studi di Palermo FacoltГ di Medicina e Chirurgia UniversitГ degli Studi di Palermo Centro Interdipartimentale di Tecnologie della Conoscenza Di.Me.S. Dipartimento di Medicina Sperimentale Sezioni: Anatomia Umana вЂњEmerico LunaвЂќ (via del Vespro, 129) Istologia ed Embriologia вЂњArcangelo Pasqualino di MarineoвЂќ (via del Vespro, 129) Fisiologia Umana вЂњGiuseppe PaganoвЂќ (corso Tukory, 129) В© Copyright Experimental Medicine Reviews Morphophysiological Remarks in English and Italian Palermo, Italy - vol 2/3, 2008/2009 Edited by Aldo Gerbino, Full Professor of Histology and Embryology, Histology Section Giovanni Zummo, Full Professor of Human Anatomy, Human Anatomy Section Giuseppe Crescimanno, Full Professor of Physiology, Physiology Section Scientific editorial office Francesco Cappello, Laura Uzzo, Maurizio Casarrubea Editing Simona Corrao, Annamaria Mauro Graphic design Rosario Notaro Plumelia edizioni Collana вЂњRicercaвЂќ ISBN 978-88-89876-15-2 This volume can be found on line at the following address: www.unipa.it/dimes Printed by Officine Tipografiche Aiello & Provenzano, Bagheria (Palermo) Note The heterogeneity of scientific papers, a member of different schools, is divided into trasversal methodology that today spreads through way of research. This makes, on value editorial uniformity of single capitols, some problems about harmonization. It is necessary and perhaps useful that rise some differences about impostation, that not seem damage editingвЂ™s quality. The Editors 5 Editorial board Everly Conway de Macario University of Maryland Biotechnology Institute, Baltimore, MD, U.S.A. В Felipe Cortes de Bonavides University of Sevilla, Sevilla, Spain В Donna E. Davies University of Southampton, Southampton, U.K. В Regina M. Day Uniformed Services University of the Health Sciences, Bethesda, MD, U.S.A. В JosГ© M. Icardo University of Cantabria, Santander, Spain В Alberto J. L. Macario University of Maryland Biotechnology Institute, Baltimore, MD, U.S.A. Abdo Jurjus Faculty of Medicine, American University of Beirut, Beirut, Lebanon Tomasz Konopka Department of Oral Pathology, Wroclaw Medical University, Poland 6 Guest contribution Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 THE CHAPERONING SYSTEM: PHYSIOLOGY AND PATHOLOGY [Il sistema delle chaperonine: fisiologia e patologia] Alberto J. L. Macario and Everly Conway de Macario University of Maryland Biotechnology Institute, Center of Marine Biotechnology, Columbus Center, Baltimore, USA Key words: Chaperoning system, Molecular chaperones, Heat Shock Proteins, Chaperone subpopulations, Chaperonology, Chaperonopathies, Chaperonotherapy Parole chiave: Sistema delle Chaperonine, Chaperoni molecolari, Proteine da Shock Termico, Chaperonopatie, Chaperonoterapie Abstract. The concept of chaperoning system is presented here for the first time. The system encompasses all the molecular chaperones and their anatomically and functionally related molecules and higher order structures (multimolecular assemblies, cells, tissues). This ensemble of functionally related molecules and structures is viewed as a physiological system whose central role is the control of protein homeostasis in what concerns maintenance of a complete set of proteins in all fluids, cells, and tissues with the correct, native, functional conformation. The system ensures proper protein folding, re-folding, degradation, and translocation, and participates in a wide range of other molecular and cellular processes from antigen presentation to hormone receptor assembly and ligand binding, and others, that require stabilization of structure and facilitation of intermolecular interaction. The science that studies this system is called chaperonology, which also includes the study of disease caused by system malfunction and defective chaperones, i.e., the chaperonopathies, and their prevention and treatment using chaperone genes or proteins, i.e., chaperonotherapy. Riassunto. Viene presentato, per la prima volta, il concetto dellвЂ™esistenza di un sistema degli chaperoni molecolari. Questo sistema comprende tutti gli chaperoni molecolari e le molecole strutturalmente e funzionalmente correlate, tanto quanto strutture di livello superiore (complessi multimolecolari in cellule e tessuti). Questo insieme di molecole e strutture funzionalmente correlate tra di loro viene descritto come un вЂњsistema fisiologicoвЂќ il cui ruolo centrale ГЁ il controllo dellвЂ™omeostasi proteica, specie per ciГІ che concerne il mantenimento della corretta (nativa) conformazione funzionale di proteineВ presenti in tutti i fluidi biologici, cosГ¬ come nelle cellule e nei tessuti. Questo sistema, quindi, assicura il corretto folding delle proteine, cosГ¬ come il loro re-folding, ma anche la degradazione e la traslocazione; inoltre partecipa ad un elevato numero di altre attivitГ cellulari e molecolari, che vanno dalla presentazione antigenica allвЂ™assemblamento dei recettori ormonali, dalla formazione di complessi con ligandi ad altre azioni che richiedono la stabilizzazione delle strutture proteiche e la facilitazione dellвЂ™interazione tra molecole. La scienza che studia questo sistema ГЁ detta chaperonologia, e questa include anche lo studio delle malattie determinate dal suo malfunzionamento (c.d. chaperonopatie), come ad esempio per la presenza di chaperoni вЂњdifettosiвЂќ, nonchГ©В ilВ loro utilizzoВ in ambito preventivo e terapeutico (c.d. chaperonoterapia), attraverso lвЂ™espressione genica o lвЂ™utilizzo delle stesse proteine. Note. This article contains most of the lecture by the authors entitled вЂњMolecular chaperones and chaperonopathies in oncopathologyвЂќ presented at the Symposium вЂњNew molecular targets in diagnostic oncopathology,вЂќ on the occasion of the opening of the Advanced Oncopathology Laboratory at the A.R.N.A.S. Ospedale Civico-University of Palermo, Palermo, Italy, December 13, 2008. 9 Alberto J. L. Macario, Everly Conway de Macario Introduction Since 1962 the response to stress has been studied at various levels [1]. The stressor often tested was, and is still today, a temperature elevation, heat shock. However, there are many other stressors of various natures, chemical, physical, mechanical, biological, psychological, social, occupational, etc., which have also been investigated [2,3]. One consequence of stress that was examined from the beginning was the response of certain genes to heat shock which resulted in the production proteins called heat-shock proteins (Hsp). The functions of Hsp were then studied in various ways and some were found to assist protein folding and were called molecular chaperones [1]. Work on Hsp and chaperones has been performed over many years in many laboratories around the world and the results have been published in a broad range of printed and electronic publications, and made available also in databases. Thus, a huge amount of data and ideas about stress, stress response, Hsp, chaperones, and anti-stress mechanisms is disperse in a wide variety of sources. A few years ago we started the unification of the field into a coherent rational structure, a new scientific discipline we called chaperonology [4,5]. This new discipline includes subspecialties such as chaperonomics, chaperonotherapy, and the study of chaperonopathies [4-6]. In this article, we propose the unification of the molecular chaperones and Hsp molecules and the higher order structures anatomically and functionally related to these molecules into a single physiological system. Physiology. Definitions Chaperoning system The chaperoning system is a newly identified physiological set of molecules and molecular teams, and pertinent cells and tissues, key to maintaining protein homeostasis and other cellular functions. The molecular components of the system have been known for sometime but they were not considered as members of a single physiological system until now. Likewise, the cells containing chaperones and/or producing the chaperones were not considered before as part of a specialized physiological system centered on chaperones. Terminology The ensemble of chaperones and closely related molecules in terms of interaction and function and pertinent supramolecular structures (multimolecular machines, cells, tissues) could be considered a system or an apparatus. System was chosen over apparatus because of its connotation of interconnected functions and pathways made of chaperones and related molecules and cells and tissues, whereas apparatus has a less open-ended feel as 10 The Chaperoning system: physiology and pathology it keeps the focus on the вЂњhardwareвЂќ molecules. The system could be named in various ways, for instance the chaperone system, the chaperonic system, or the chaperoning system. We have chosen at this time the term chaperoning system because chaperone system may be taken to: a) designate the system pertaining to a single chaperone from among the many that exist; b) indicate that the chaperonesвЂ™ only function is to assist (to chaperone) nascent not yet folded polypeptides and unfolded proteins to gain or regain a folded, native conformation when, in fact, chaperones have other functions, too; and c) exclude components of the system that are not typical chaperones but interact with them in a critical way, or that are not molecules, such as the cells that contain and/or produce the chaperones. The term chaperonic system was not preferred either because the spoken change of emphasis, from the soft вЂњвЂ¦oneвЂќ of chaperone to the hard вЂњвЂ¦onicвЂќ of chaperonic is both unattractive to the ear and non-intuitive. Furthermore, chaperonic does not reflect as well as chaperoning does the functions and interaction of the components of the system. Chaperonology The identification of the central components of the chaperoning system, the molecular chaperones, has originated the novel field of science named chaperonology. Chaperonology deals with the chaperoning system and with its components, molecules and higher order structures (molecular assemblies, cells, tissues), and their abnormalities and pathologies. The roles of chaperones The classic concept is that chaperones assist other proteins to fold and re-fold, and usher defective proteins toward degradation. Thus, chaperoning proteins to fold or refold and guiding proteins toward degradation can be considered typical functions of chaperones. However, many chaperones play roles that are not typically related to protein folding but are seemingly quite different, for example the role of Hsp70 in tumor-antigen presentation and tumor immunity [7,8]. These could be considered atypical functions of typical chaperones. Conversely, molecules that are not typical chaperones may have functions similar to the atypical functions of typical chaperones. For example, Hsp32 is the product of a heat shock-inducible gene that plays a significant role in protection against oxidative stress but has no known function in protein folding or refolding [9]. Thus, Hsp32 is a typical heat-shock protein with a role in anti-stress mechanisms like many chaperones but it is not a typical chaperone. 11 Alberto J. L. Macario, Everly Conway de Macario Chaperones Autochthonous Imported Sessile Mobile Sedentary Nomadic Single Team Network Complex Fig. 1 Subpopulations of chaperones: autochthonous vs. imported (with regard to any given cell); sessile vs. mobile (the latter can be sedentary or nomadic); single or member of a chaperoning team with other chaperones, co-chaperones, and co-factors, also named chaperoning machine. A chaperoning team or machine can be a member of a chaperoning network, which is formed by various chaperoning teams and, possibly, other molecules or molecular assemblies. A chaperone can also form a complex with another molecule or structure (tumor antigen, cell-surface receptor, cytosolic glucocorticoid-hormone receptor, chemical compound), but in this case the complex is not a chaperoning machine; it has other functions more or less unrelated to chaperoning. Examples: 1) Hsp70 forms complexes with tumor antigens (peptides) and cell-surface receptors; 2) Hsp90 binds glucocorticoidhormone receptor (a protein which is a transcription factor); 3) Hsp90 binds some anti-tumor compounds like the antibiotic geldanamycin. See text for references. Key. Circled C, molecular chaperone; 1, mobile chaperone in the cytosol; 2, chaperone inside an organelle, such as the nucleus or mitochondrion; 3, sessile chaperone anchored to a particle (e.g., ribosome) in the cytosol; 4 and 5, sessile chaperone anchored to the cell membrane on the cytosolic side (4) or on the outside in the extracellular space (5); 6, mobile chaperones in the intercellular space; 7, mobile chaperone in circulation inside a vessel (blood or lymph); 8, sessile chaperone anchored to the vessel wall on the inside; 9, mobile chaperone in the cytosol like that shown in 1, but imported from another cell. Molecular chaperones can be found also in other locations such as cerebrospinal fluid and secretions (e.g., saliva and urine), not shown in this figure (see Table 3) 12 The Chaperoning system: physiology and pathology Molecules, cells and tissues of the chaperoning system Chaperones are made in cells for work in the same cells in which they are made or for export (Fig. 1). Chaperones for export are made in a cell and then travel to other locations, inside cells or in extracellular sites, in which they will take residence and work. It can be predicted that there are cells, and tissues or defined zones within certain tissues or organs, specialized in the production of chaperones for export. Chaperones are present in tissues and fluids and, as explained earlier, we call the entire chaperone population of an organism the chaperoning system (Fig. 1). Each cell has its own set of chaperones or subsystem that typically includes more than one chaperoning complex or team, and teams interact forming networks inside the cell. Pertinent timely questions The words molecular chaperones and chaperoning system immediately suggest a number of issues and questions (Table 1). Answers to some of these questions can be found in this article and in the bibliography. Table 1: Questions What are the components of the chaperoning system? Where and when are the molecular chaperones made? Where do the chaperones reside and work? What do the chaperones do? What are the diseases caused by defective chaperones? What types of chaperonopathies are known? How would one diagnose a chaperonopathy? When would treatment with chaperones, i.e., chaperonotherapy, be indicated? What types of chaperonotherapies are available? What to do next? Classification of molecular chaperones A) Types of chaperones according to their size (molecular weight) Heat-shock proteins (Hsp) senso stricto are the product of genes induced by a temperature elevation (heat shock), but the name is also applied to proteins from genes inducible by any other stressor. Many Hsp are chaperones (for example the heat-inducible form of Hsp70 in humans) but not all chaperones are Hsp. An example of the latter is the AlphaHemoglobin Stabilizing Protein (AHSP), which is a dedicated chaperone whose substrate is the alpha hemoglobin chain and is encoded in a gene not known to be inducible by heat or any other stressor [10]. Conversely, many Hsp are not chaperones, for example Hsp32 or 13 Alberto J. L. Macario, Everly Conway de Macario heme oxygenase-1 (HO-1). This enzyme is the inducible isoform of heme oxygenase that catalyzes the NADPH, O2 and cytochrome P450-reductase dependent oxidation of heme to carbon monoxide, iron, and biliverdin, which is immediately reduced to bilirubin [9]. Thus Hsp32 is associated with the generation of biliverdin and bilirubin, potent antioxidants, and therefore it has to be considered part of the anti-stress mechanisms of which many molecular chaperones are important components. Although many Hsp do not act as chaperones and, vice versa, many chaperones are the product of genes not inducible by any stressor, the terms Hsp and chaperone have been used as synonyms for years in a huge number of printed and electronic publications, and in databases. It is virtually impossible at this time in the history of chaperonology to correct these abuses in nomenclature usage. Therefore, we use here both terms as synonyms. Hsp-chaperones can be grouped according to their size in a classification that has practical utility in research and, particularly in pathology and clinics (Table 2). Table 2: Classification of Hsp-chaperones according to molecular weight1 Chaperone subpopulation Name Other names Heavy High MW; Hsp100 Hsp90 Hsp70 Chaperones; DnaK Hsp60 Chaperonins; Cpn60 Hsp40 DnaJ Small Hsp sHsp; alpha-crystallins Proteases; isomerases; AAA+ proteins (e.g., Other paraplegin, spastin]), etc. 1 MW (kDa) 100 or higher 81-99 65-80 55-64 35-54 34 or less Various From references [2,20] B) Types of chaperones according to their origin with regard to their place of residence and work Chaperones are in fixed locations inside the various cell compartments as well as in biological fluids moving around (Fig. 1; Table 3). Note: The following two terms refer to chaperones inside any given cell without taking into account the cellвЂ™s organelles or membranes. Autochthonous: a chaperone originated in the cell in which it resides. The cell of origin and of residence is one and the same, i.e., an autochthonous chaperone resides and works in the same cell in which it was produced, disregarding in which cell compartment the chaperone resides and works. Imported: the place of residence (a given cell) of an imported 14 The Chaperoning system: physiology and pathology chaperone is not the same as that of its origin but another cell. Table 3: Places at which chaperones reside and work Location Compartment Cellular Nucleus Cytosol Mitochondria Endoplasmic reticulum Lysosomes Vesicles Membrane on the inside Chloroplasts Pericellular Membrane on the outside Extracellular Intercellular space Blood (plasma, serum) Cerebrospinal fluid Secretions (e.g., saliva) C) Types of chaperones according to their ability to move and change residence Chaperones can be classified according to their mobility (Fig. 1). Sessile: fixed, anchored to another structure (e.g., cell membrane). Mobile: not fixed, capable of moving inside a cell (e.g., from cytosol to nucleus), or outside cells (e.g., in the blood) and change place of residence and work. Mobile chaperones can be of two subtypes: i) Sedentary, reside always in the same cell or cell compartment but are not fixed to any structure, so they can move within the confines of their вЂњhomeвЂќ or cellular realm; and ii) Nomadic, change residence, i.e., the chaperone takes residence for a while in one location (e.g., a cell or cell compartment), then in another, and so forth. Nomadic chaperones travel and work in various successive places. Hsp60 is an example of mobile, nomadic chaperone: it is produced in the cytosol and then translocated to the mitochondria from which it can exit and go back to the cytosol, and even exit the cell and appear in the extracellular space [11,12]. D) Types of chaperones according to their relation with other chaperones or other molecules Chaperones exercise their functions alone or in associations with other molecules (Fig. 1), and they can be considered Single, i.e., a chaperone molecule that performs its role not associated with any other chaperone, or Social. The latter form part of a Team (more precisely, Chaperoning Team), namely, a specific association of chaperones to build a chaperone machine. A Chaperoning Team includes chaperones senso stricto (e.g., Hsp70[DnaK]), co-chaperones (e.g., Hsp40[DnaJ]), and co-factors (e.g., Nucleotide Exchange Factor [NEF] 15 Alberto J. L. Macario, Everly Conway de Macario such as GrpE); all these molecules assemble into a multimolecular machine that performs the chaperoning work. Other associations are: Network (more precisely, Chaperoning Network), which is a specific interaction between chaperone machines (e.g., Hsp70-Hsp40NEF, and Hsp60-Hsp10, and Prefoldin), or between a chaperoning team and a single chaperone. Complex designates the association of a chaperone with another molecule which is not a chaperone, for example Hsp70 with tumor antigen [7,8], Hsp90 with glucocorticoid receptor [13] or with geldanamycin and other inhibitors [14-16], and Hsp60 with procaspase-3 [17]. E) Other classifications of chaperones Chaperones can be classified according to gene type into house-keeping vs. anti-stress (constitutive vs. stress-inducible genes, respectively); and according to substrate range into generic vs. dedicated (with many, almost any, client polypeptide vs. with a single, specific client polypeptide or with a very restricted, specific set of client polypeptides, respectively). Table 4: Chaperonopathies1 Type Genetic Mutation hereditary Mutation sporadic Polymorphism Protein coding region Non-coding region: promoter region (5вЂ™ UTR) Acquired Early onset Late onset By mistake Tumors Other Examples AlphaA-crystallin gene, cataracts. AlphaB-crystallin gene, desmin-related myopathy. Hsp27 (HSPB1) dHMN/CMT22 Hsp27 (HSPB1) dHMN/CMT2 References [22-24] [24] HSP70-1 (HspA1A); HSP70-2 (HspA1B); HSP70-Hom (HspA1L) Affect longevity [25] HSP70-1 (HspA1A); HSP70-2 (Hsp70A1B) Affect longevity [25] Alpha-crystallin PTM (cataracts in diabetes) Alpha-crystallin PTM (cataracts of senescence) [26] [27,28] Increased: Hsp60 & Hsp10, colon, exocervix, prostate; Hsp90, breast; HYOU-1, breast, brain Decreased: Hsp10 and Hsp60, lung Hsp70 necessary: breast Prefoldin generates highly toxic Abeta oligomers rather than the less toxic fibrils (ref. AlzheimerвЂ™s disease) [29-35] [33,36] [37] [38] Other examples of chaperonopathies can be found in references [4,5,20,21]. Abbreviations: dHMN/CMT2, distal Hereditary Motor Neuropathy/Charcot-Marie-Tooth disease type 2; UTR, untranslated region; PTM, post-translational modification; Hsp, heat-shock protein. 1 2 16 The Chaperoning system: physiology and pathology Pathology. Disorders of protein homeostasis Identification of chaperones and their functions made clear that the chaperoning system is a major component of anti-stress mechanisms and also plays a central role in protein homeostasis, particularly in what concerns the maintenance of a complete set of proteins with a native, functional conformation throughout the cell and the organism. The realization that chaperones normally play such important roles lead to the idea that malfunctional, defective chaperones must have a role in pathogenesis [2,18,19]. Pathological disorders in which abnormal (sick) chaperones play a pathogenetic role were called chaperonopathies [18-21]. Protein precipitates. Here we consider protein precipitates a key indicator of protein homeostasis abnormality or failure. As when hyperglycemia is detected one thinks of problems with insulin and the pancreas, protein precipitates ought to direct attention to chaperones, i.e., to a defect in the chaperoning system or chaperonopathy. Chaperonopathies. The abnormalities and pathologies of the chaperones are grouped under the newly coined name of chaperonopathies. The most common chaperonopathies are manifestations of ageing [18]. A classification of chaperonopathies is displayed in Table 4. Chaperonopathies by mistake or collaborationism. Pathologic conditions in which normal chaperones are involved in pathogenetic pathways and contribute to the development of disease rather than the contrary are called chaperonopathies by mistake or collaborationism. Significant examples of chaperonopathies by mistake are some types of cancers (Table 4). Treatment Chaperonotherapy. Identification of chaperonopathies and other diseases (e.g., proteinmisfolding diseases) in which normal chaperones could play a beneficial role has lead to the idea of using these molecules for treatment, namely chaperonotherapy [4,5]. Chaperonotherapy refers to the use of chaperones (genes and proteins) for treatment of chaperonopathies, for example replacement of a structurally defective chaperone with a normal version of it, or supplementation of a quantitative deficiency of a chaperone by administering the pertinent chaperone as gene or protein. Chaperonotherapy can also be used for treatment of conditions that are not chaperonopathies at all, or are not primarily chaperonopathies (e.g., some neurodegenerative diseases) but are due primarily to abnormalities in proteins (proteinopathies) that are not chaperones. Treatment of chaperonopathies by mistake, such as certain forms of cancer, requires antichaperone agents. When chaperones are involved in pathogenetic pathways and contribute to the development of disease rather than the contrary they must be inhibited or eliminated. Development of antichaperone agents is one of the most promising lines of research in the fight against cancer. 17 Alberto J. L. Macario, Everly Conway de Macario Conclusions A new physiological system is presented that plays a central role in controlling protein homeostasis at the level of molecular conformation. Proteins are major cellular and organismal components with a broad range of functions. For a cell or organism to function correctly all its proteins in all its compartments must be at the physiological concentrations and possess the right primary, secondary, tertiary and, when pertinent, quaternary structures. The chief function of the chaperoning system is to ensure that all proteins reach after synthesis the mature, correct functional structure beyond the primary one, i.e., the native conformation. The chaperoning system also monitors the native conformation of mature proteins and restores it if these proteins tend to lose it due to stress or any other proteindenaturing factor. Furthermore, the components of the system play a variety of other roles, many of which are not those typically assigned to chaperones (e.g., assisting nascent proteins to fold correctly or to refold if partially denatured, helping protein translocation across membranes, or ushering damaged or unnecessary proteins toward degradation) but are diverse, including in many instances facilitation of intermolecular association and interaction and stabilization of multimolecular assemblies. The system encompasses molecules, cells and tissues but the central components are the molecular chaperones. System malfunction causes disturbances in protein homeostasis that lead to aggregation and formation of protein precipitates. Failure of chaperones due to qualitative or quantitative abnormalities cause pathological conditions named chaperonopathies. Treatment of these conditions can be done using chaperone genes or proteins, a form of therapy called chaperonotherapy. Perspectives The concept of chaperoning system offers a new standpoint to look at molecular chaperones and their associated molecules and higher order structures and to study these molecules and structures using fresh approaches, including those that will take into consideration interactions and interconnections between system components far apart in the cell and in the organism. Likewise, the new concept will be instrumental to unveil aspects not yet completely understood of known diseases. This unveiling of new features will, in turn, provide clues on the strategies and methods that should be used for elucidating them. Also, the chaperoning system concept, in showing new angles of pathologic disorders still poorly characterized, will allow the investigation of these disorders using novel strategies that should uncover details as yet hidden. Acknowledgements. We thank Dr. Francesco Cappello for his encouragement and constructive comments during preparation of the material for this article, and Dr. Adriana Verschoor for her critical discussion of terminology. This is Publication No. 09-200 from the Center of Marine Biotechnology. 18 The Chaperoning system: physiology and pathology References [1] Haak, J. and Kregel, K.C. 1962-2077: a cell stress odyssey. Novartis Foundation Symposium 291 (London, UK, June 2007] 3-15, 2008. Discussion 15-22, 137-40. [2] Macario, A.J.L. Heat-shock proteins and molecular chaperones: Implications for pathogenesis, diagnostics, and therapeutics. Intl J Clin Lab Res 1995; 25: 59-70. [3] Macario, A.J.L. and Conway de Macario, E. 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FEBS J 2008; 275: 5982-5993. 21 Airway cells, epidermis, signaling Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 TGFb signaling: Roles of Smads [La segnalazione del TGFОІ: ruoli degli Smads] Francesca Magno*В§1, Giampiero La RoccaВ§1, Rita Anzalone1, Simona Corrao1, Tiziana Loria1, Melania Lo Iacono1, Gaetana Spagnolo1, Antonino Di Stefano2, Bruno Balbi2 and Giovanni Zummo1 Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana, UniversitГ degli Studi di Palermo, Italy; 2Fondazione S. Maugeri, IRCCS, Veruno, NO (IT) - *Corresponding author - В§The two authors contributed equally to this work 1 Key words: TGFОІ, Smads, Chronic obstructive Pulmonary Disease, Extracellular matrix Parole chiave: TGFОІ, Smads, Broncopneumopatia Cronica Ostruttiva, Matrice extracellulare Abstract. Transforming Growth Factor ОІ (TGFОІ) is involved in regulation of several biological and physiological processes such as stimulation and regulation of lung extracellular matrix (ECM) components. Binding of this cytokine to specific membrane receptors induces a transduction signal mediated mainly by Smad proteins. In the last decade many studies increased the information about the structural characteristics of these molecules, together with their main molecular interactions. In this review we briefly summarized what is know about these proteins, focusing on the knowledge of the key steps of TGFОІ transduction pathway. Riassunto. Il fattore di crescita trasformante ОІ (TGFОІ) ГЁ coinvolto nella regolazione di diversi processi fisiologici, come la stimolazione e regolazione dei componenti della matrice extracellulare (ECM) polmonare. Il legame di questa citochina a specifici recettori di membrana induce una trasduzione del segnale mediata principalmente dalle proteine Smad. NellвЂ™ultimo decennio molti studi hanno contribuito ad aumentare le conoscenze circa le caratteristiche strutturali di queste molecole, insieme alle loro principali interazioni molecolari. In questa review, noi riassumiamo brevemente le informazioni circa queste proteine, focalizzando lвЂ™attenzione sulla conoscenza dei punti chiave del processo di trasduzione del segnale mediato dal TGFОІ. 1. Introduction COPD (Chronic Obstructive Pulmonary Disease) is characterised by a slow progressive and partially reversible airflow limitation that is associated with an abnormal inflammatory response of the lungs to noxious particles or gases. The pathological hallmarks of COPD are typical of the main lung diseases such as chronic bronchitis, emphysema, chronic asthma tissue damage and fibrosis [1]. All of these pathologies are characterized by an inflammatory process with an increase of inflammatory cells and production of a large variety of cytokines. The transforming growth factor ОІ appears to be the most important cytokine involved on stimulation and regulation of lung extracellular matrix (ECM) components. TGF-ОІ superfamily consist of secreted growth factors involved on regulation of different cellular 25 Magno Francesca, La Rocca Giampiero, Anzalone Rita, Corrao Simona, Loria Tiziana, Lo Iacono Melania, Spagnolo G, Di Stefano A, Balbi B, Zummo Giovanni processes, such as cell growth, development, differentiation, proliferation, motility, adhesion and apoptosis [2,3]. The mammalian TGFОІ family comprises three members: TGFОІ1, TGFОІ2 and TGFОІ3. Each of them is encoded by a distinct gene and is expressed in both a tissuespecific and developmentally-regulated fashion. Specifically, TGFОІ1 is expressed in endothelial, hematopoietic, and connective tissue cells; both TGFОІ1 and TGFОІ3 are expressed in structures undergoing morphogenesis and mesenchymal cells, while TGFОІ2 alone is expressed in differentiating and mature epithelium [4,5]. These peptides play an important role in the pathogenesis of different diseases, such as cancer, fibrotic and parasitic diseases [5]. Members of this family are secreted as latent ligands by binding to their propeptide, or in trapped form by binding to occluding factors. The active form of a TGF-ОІ cytokine is a dimer stabilized by hydrophobic interactions, which are further strengthened by an inter-subunit disulfide bridge in most cases. TGFОІ signal is initiated by binding to a specific pair of membrane serine/threonine kinase receptors (type I and type II receptors) and is transduced to the nucleus by Mitogen Activate Protein Kinase (MAPKs) and, principally, by Smad proteins. 2. Basic features of Smad proteins Eight Smad proteins are encoded in the human and mouse genome, four in Drosophila and three in C. Elegans [6]. Only five of the mammalian Smads (R-Smads, namely Smad1, Smad2, Smad3, Smad5 and Smad8 act as substrates for the TGFОІ familyвЂ™s receptors; indeed Smads1, 5, and 8 act mainly as substrates for the BMP (Bone Morphogenetic Protein) and anti-Muellerian receptors, while Smads2 and 3 for the TGFОІ, activin, and nodal receptors. Co-Smad Smad4 serves as a common partner for all R-Smads while Smad6 and 7 are inhibitory proteins. The name вЂњSmadвЂќ was coined in reference to its sequence similarity to the Sma and Mad proteins. Functional studies, together with the X-ray crystal structure analysis, showed that these ~500 amino acids proteins consist of two conserved globular domains (MH1 and MH2 domains) coupled by a flexible linker region rich of binding sites for Smurf (Smad ubiquitination-related factors) ubiquitin ligase, of phosphorylation sites for several classes of protein kinases and, in Smad4, a nuclear export signal (NES) involve on nucleus-cytoplasmatic translocation [7]. The MH1 domain is conserved in all R-Smads and in Co-Smad but not in inhibitory Smads; it functions as DNA-binding site containing a ОІ-harpin structure and is stabilized by a tightly bound zinc atom [8,9]. C-terminal domain, or MH2 domain, is conserved in both Smad proteins and is involved on Smad-Smad interaction and on R-Smad activation/phosphorylation. At limited distance a basic pocket interacts with the phosphorylated region of the activated receptor type I (in R-Smad), or with the phosphorylated tail of R-Smad (in case of Smad4); in the other site, a set of contiguous hydrophobic patches, the вЂњHydrophobic corridorвЂќ, are involved in interaction with nucleoporins, and cytoplasmatic retention proteins [10]. 26 TGFb signaling: Roles of Smads 3. Receptor internalitation Binding of TGFОІ to the type I and type II receptors, induces formation of multimeric receptors. The serine/threonine kinase receptor family in the human genome comprises 12 members, 7 type I and 5 type II receptors, all involved in TGFОІ signaling; both types of receptors consist of about 500 amino acids, organized sequentially into an N-terminal extracellular ligand binding domain, a transmembrane region, and a C-terminal serine/ threonine kinase domain. In contrast with BMPs, TGFОІ and Activin display a high affinity for the type II receptors and do not interact with the isolated type I receptors [6]. TGFОІ binds tightly to the ectodomain of the type II receptor first; this binding allows the subsequent dimerization with the type I receptor, forming a large ligand-receptor complex involving a ligand dimer and four receptor molecules. Lin suggested that once type II receptors phosphorylate type I receptors, they could interact at the cell surface with the cytoplasmatic form of promyelocytic leukemia protein (cPML) that acts as a bridging factor between a Smad Anchor for Receptor Activation (SARA) and Smad2/3. cPML promotes the transfer of the complex formed into early endosomes, where cPML could dissociate from the complex, allowing Smad2/3 to interact with SARA and to be phosphorylated [11]. SARA is an anchor protein which acts by presenting R-Smads to the activated type I receptor and thereby regulating TGFОІ transduction signal. It is a multidomain protein that consist of 80 amino acids Smad-Binding Domain (SBD) and a FYVE phosholipid-binding domain that avidly binds to phosphatidyl inositol 3-phosphate on endosomal membranes and targets SARA preferentially to early endosomes [12]. As revealed by the X-ray crystal structure of the Smad2вЂ“SBD complex, the SBD of SARA makes contact with the three consecutive hydrophobic patches on the MH2 domain surface that constitute the hydrophobic corridor [13], blocking translocation of Smad2/3 into the nucleus and formation of transcriptional complexes; the activated TGFОІ receptor complex therefore undergoes endocytosis via two distinct routes: by internalization via coated vesicles to early endosomes and via caveolae to caveolin-positive vesicles for degradation [12]. However other adaptor proteins were found to cooperate with SARA on Smad signaling, such as FYVE-containing protein, HGS [14] Disabled-2 [15], Axin [16], and the ELF ОІ-spectrin [17]. 4. Smad phosphorylation In the basal state, Smads form homoligomers and remain in an inactive conformation through a hydrophobic interaction between the MH1 and MH2 domains. In solution, the unphosphorylated Smad2 MH2 domain is a monomer but, once phosphorylated, both R-Smads and Smad4 form homotrimic complexes [9,18]. As revealed by X-ray crystallographic studies, the binding of R-Smad to Smad4 forms oligomers, heterodimers (RSmadвЂ“Smad4) or heterotrimers (two R-Smads molecules plus one Smad4 molecule); 27 Magno Francesca, La Rocca Giampiero, Anzalone Rita, Corrao Simona, Loria Tiziana, Lo Iacono Melania, Spagnolo G, Di Stefano A, Balbi B, Zummo Giovanni these oligomers are stabilized by interactions within an extensive proteinвЂ“protein interface between MH2 domains plus the binding of the pSerвЂ“XвЂ“pSer motif of one MH2 domain into the di-phosphoserine binding pocket on the adjacent MH2 domain [19]. Phosphorylation can occur in two different sites: in the linker region sites by several kinases, including MAPKs and CDK [20,21] and mainly on the C-terminal conserved Ser-Val-Ser (Ser-MetSer in Smad2) motif of Smad3 by the activated receptor [7,10]. Actually the cytoplasmatic region of the type I receptor contains a canonical protein kinase domain preceded by a regulatory region or GS domain to which the inhibitor FKBP12 binds to enforce the inactive basal state. Phosphorylation of the GS domain by the type II receptor creates a repeated pS-X-pS motif that serves as a docking site for both R-Smads [7]; as a result, R-Smads decrease their affinity for SARA and the activated oligomer complex is translocated into the nucleus where it can bind directly with DNA promoters or indirectly binding with transcription factor or co-factors. [22, 23]. However TGFОІ-independent signals can involve target sites rich of multiple serine and threonine, both on linker region and on N-terminal domain; indeed MAPKs bind these consensus sites and are responsable of Smad3/2 phosphorylation or Smad4 activation [24]. In vitro, ERK 1 and 2 are able to activate Smad1, 2, and 3 by phosphorylating the linker region; moreover ERK-mediated phosphorylation of Smads 1, 2, and 3 was shown to attenuate the nuclear accumulation and signaling activity of these proteins in response to agonists [25]. The linker region of R-Smads are subject to phosphorylation by Cam Kinase II (Ca++- and Calmodulin-dependent kinase II) [26], while the N-terminal and the linker region of Smad3 can be phosphorylated by CDKs (Cyclin-dependent kinases) [27] or by GRK-2 (G protein-coupled Receptor Kinase 2) that phosphorylates Ser197 in Smad2 [28]. 5. Smad dephosphorylation Recently phosphatases have been suggested as regulatory molecules of the TGFОІ pathway; the interest for phosphatases started from a study in which was demonstrated that in a cell-free system Smad2 was exported from the nucleus in a dephosphorylated state [29]. Protein phosphatases control cellular function through cleavage of phosphate from phosphorylated serine/threonine (pS/Ts) and tyrosine (pY) residues in proteins. The human genome encodes about 146 phosphatases, of these. 40 are able to act on serine/ threonine residues (PS/TP). An important role on Smads activity regulation was shown for members of the PPM subfamily namely PPM1A/PP2C-О± and PPM1B/PP2CОІ. Both PPM phosphatases directly dephosphorylate R-Smads at the C-terminal SXS motif, but not the phospho Ser/Thr residues in the linker region; PPM1A, is exclusively present in the nucleus, and its activity enhances the disassembly of the activated Smad complex and facilitates nuclear export of Smad2/3, unlike PPMB [11,30]. Elevated levels of PPM1A mitigated tran28 TGFb signaling: Roles of Smads scriptional response to TGF-ОІ, and inhibited the anti-proliferation function of TGF-ОІ. However dephosphorylation seems to act on Smads after they end their transcriptional function on DNA, and thereby not interfering directly with TGFОІ signaling. Recently, Wrighton and colleagues discovered another phosphatase able to attenuate Smad signalling, the SCP1 or C-terminal domain phosphatase 1. It is a specific phosphatase that dephosphorylates the linker region and the N-terminal domain, specifically at Thr-220 and Ser-245/250/255 in Smad2 and Thr-179 and Ser-204/Ser-S208/Ser-213 in Smad3 [31], resetting them to their basal unphosphorylated state. The biological mechanism by which they can act on Smad is unclear. 6. Nuclear import and export mechanism of Smads The mechanisms of Smad nuclear import and export have been extensively studied over the last few years, particularly for Smads 2, 3, and 4. Two recent studies showed that Smads do not reside statically in the cytoplasm in absence of ligands or in the nucleus upon TGFОІ stimulation; there is a spontaneous bi-directional shuttling of the R-Smads/ Smad4 complex across the nuclear envelope without TGFОІ stimulation [23] even if the nuclear import of the oligomer complex is offset by export forces, so they cannot reach a high concentration in the nucleus [32]. In the basal state, R-Smads are predominantly localized in the cytoplasm, I-Smads tend to be nuclear, while Smad4 is distributed in both the cytoplasm and the nucleus. The process of nucleocytoplasmatic shuttling can occur with or without nuclear transport factors [23]. Some Smad proteins can undergo nuclear import via importins; importin-О± binds importin-ОІ which directly interacts with the nuclear pore components, namely the nucleoporins CAN/Nup214 and Nup153 [23] to negotiate the passage of the importin-ОІ/importin-О±-cargo complex into the nucleus. However, the nuclear translocation of Smad proteins can occur independently of importins because they can directly interact with nucleoporins [23]. Smad2, 3, and 4 undergo nuclear import by direct interactions with nucleoporins, this process may be aided by importin-ОІ in the case of Smad3 and 4. The intrinsic asymmetry of the nuclear pore complex and the distribution of different nucleoporins along the span of the pore are thought to allow proteins docking on one side of the pore to move unidirectionally to the other side and vice versa [33]. Indeed, the direct interaction of Smad2 and 3 with nucleoporins has been shown to enable nuclear export as well as import [23]. 7. Post-translational regulation of Smads Smad activation and transcriptional function can be regulated in both cytoplasm and nucleus. Several types of mechanisms were involved to limit their activity: one type includes transcriptional co-repressors, such as the homeodomain protein TGIF. Binding of 29 Magno Francesca, La Rocca Giampiero, Anzalone Rita, Corrao Simona, Loria Tiziana, Lo Iacono Melania, Spagnolo G, Di Stefano A, Balbi B, Zummo Giovanni TGIF prevents the interaction of Smads with nuclear co-activators and interferes with activation of TGFОІ-target genes expression. The inhibitory Smads; Smad, 6 and 7, serve as decoys interfering with SmadвЂ“receptor or SmadвЂ“Smad interactions; Smad7 inhibits TGFОІ/ activin and BMP signaling, while Smad6 inhibits primarily the BMP pathway. Smad6 competes with Smad4 for interaction with the receptor-activated Smad1, such that inactive Smad1вЂ“Smad6 complexes are formed [34]. Smad7 acts as a competitive inhibitor able to bind the activated TGFОІ and BMP receptors, competing with R-Smads and prevending or blocking their activation [35], it is involved in regulation of TGF receptor ubiquination by SMURF ubiquitin ligase [36]. Smurf1 and Smurf2 are HECT-domain containing E3 ubiquitin ligases that recognize R-Smads as substrates through a PPXY motif. Smurf2 can bind to Smad7, without immediate ubiquitination and degradation, but favouring export of Smad7 to the cytoplasm. The Smurf-Smad7 complex is able to bind to the TGFОІ receptor complex and promotes its ubiquitination, thereby down-regulating signaling; however the activity of Smad7 is regulated at many levels: it is induced by TGFОІ, activin and BMP signaling and by ERK [37] or inflammatory signals such as interferon-Оі, TNF-О± and interleukin-1ОІ [38]. Activated R-Smads undergo ubiquitination and subsequent degradation. Increasing evidence indicates that ubiquitin modification is important for regulating components of TGFОІ signaling and can occur in both the basal and the activated state: for example, phosphorylated Smad2 is eliminated by the fast action of phosphatases and also by the slower action of ubiquitin-dependent proteosome degradation. The susceptibility to ubiquitination may be controlled by acetylation of the same lysine residues. Following phosphorylation, Smad2 and 3 are translocated to the nucleus where they interact with co-activators and/or co-repressors. Smads transcriptional activity is significantly enhanced by recruitment and direct interaction with p300/CBP (CREB-binding protein). It is a co-activator with intrinsic acetyltransferase activity able to transfer the acetyl group from acetyl coenzyme A to the lysine residues in histones allowing remodeling of chromatin to a more relaxed conformation to allow transcription. Smad2, but not Smad3, can be acetylated by p300/CBP in a ligand-dependent manner, this event involves at least three lysine residues, Lys19, Lys20, and Lys39, that are required for efficient acetylation of Smad2. An acetylation event is required for Smad2 to mediate activin and TGFОІ signaling. Mutations of the three key lysine residues did not alter the stability of Smad2 or its ability to form a complex with Smad4 on promoter DNA, but prevent nuclear accumulation of Smad2 and subsequent TGFОІ and activin responses [39]. Nuclear Smad7 can be also acetylated by p300 at two N-terminal lysines, which are also the sites of ubiquitin attachment. Acetylation prevented receptorinduced ubiquitination of Smad7 by Smurf, without affecting the subcellular distribution of Smad7. Another Smads regulatory process is Sumoylation. In higher eukaryotes there are three SUMO (Small-Ubiquitin-like Modifier) proteins, SUMO-1, SUMO-2 and SUMO-3, 30 TGFb signaling: Roles of Smads that alter proteins function by creating a composite interaction domain [40] and regulating protein stability and subcellular localization. Two sumoylation consensus motifs were observed in Smad4; one in MH1 domain and one in the linker region [40]. As consequence of this regulation, however, two opposite effects were observed, an increase and a decrease of transcriptional activity [41,42], probably as a result of potential competition between ubiquitination and sumoylation of the same lysine residue in Smad4. Ubiquitination of RSmad/Smad4 complex can be a target of proteosomes and as a result, gene expression is reduced. By sumoylation, ubiquitination effects are blocked, half life of the activated complex is prolonged and gene expression is increased. SUMO modification also represses transcriptional activity of a number of transcription factors, such as Sp3, c-Jun, c-Myb, AP2, nuclear receptors and Elk-1, and the general co-activator p300, regulating their biological role [41]. 8. Conclusions In the last ten years the attention of scientists for Smad proteins seem to be increased. Each piece of information contributes to better understand their possible role in maintenance and development of pathological diseases and helps scientists to understand the molecular network of TGFОІ1-induced pathways. In this review we summarized what is known about these molecules. 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Daxx mediates the small ubiquitin-like modifier-dependent transcriptional repression of Smad4. J Biol Chem 2005; 280: 10164-10173. 33 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role [Le Cellule di Langerhans e il loro possibile ruolo tollerogenico] Marco La Manna1, Viviana Bazan2, Aldo Gerbino3 and Alfredo Salerno1 Dipartimento di Biopatologia e Metologie Biomediche - Sezione di Patologia Generale, 2 Dipartimento di Discipline Chirurgiche ed Oncologiche, 3 Di.Me.S. - Sezione di Istologia ed Embriologia. UniversitГ degli Studi di Palermo (IT) 1 Key words: Langerhans cells, Role Tolerogenic Parole chiave: Cellule di Langerhans, Tollerogenesi Abstract. Langerhans cells are the prototype of antigen presenting cell, their role is to work as sentinel in the epidermis. Like every APC Langerhans cells act as bridge between innate and acquired immunity recognizing antigens into the epidermis and bringing them to drying lymph node, their work is well described by the Langerhans cell paradigm. Recently many works designed a new and amazing role of Langerhans cells in fact they often showed tolerogenic capacity, while in many cases, they seems not necessary to promote activation of acquired immunity. Anyway Langerhans cells remaining an interesting target for new vaccine strategies because of their localization in the epidermis and the ability to capture antigens trough different recognition pathways. Also interesting is the possible use of langerhans cells for their tolerogenic capacity, in many experimental models these cells opportunely addressed with appropriated cytokines appears able to down-regulate immune system response in autoimmune pathology. Riassunto. Le cellule di Langerhans sono il prototipo delle APC (antigen presenting cells) professionali, il loro ruolo ГЁ quello di agire da sentinella nellвЂ™epidermide. Come ogni APC le cellule di Langerhans agiscono come ponte tra lвЂ™immunitГ innata e quella acquisita catturando antigeni nellвЂ™epidermide e trasportandoli nel linfonodo drenante, il loro ruolo nellвЂ™ambito del sistema immunologico ГЁ ben descritto dal: вЂњparadigma delle cellule di LangerhansвЂќ. Recentemente molti lavori ha definito un nuovo interessante ruolo per le cellule di Langerhans mostrando una loro importante attivitГ tollerogenica, mentre in molti casi sembra che queste cellule, pur avendo una grande capacitГ come APC, non siano necessarie per promuovere lвЂ™attivazione della immunitГ adattativa. Per la loro attivitГ di APC le cellule di Langerhans restano un interessante bersaglio per la messa a punto di nuovi vaccini grazie alla localizzazione nellвЂ™epidermide ed alla loro capacitГ di catturare diversi antigeni attraverso vari pattern di riconoscimento. Г€ inoltre di notevole interesse un loro utilizzo che sfrutti la loro attivitГ tollerogenica, poichГ© in molti modelli sperimentali queste cellule opportunamente indirizzate con le appropriate citochine sembrano essere in grado di sopprimere la risposta del sistema immune nel contesto di patologie autoimmuni della cute. Le cellule di Langerhans (LC), cellule dendritiche della linea mieloide presenti nellвЂ™epidermide, costituiscono una importante parte del sistema immune della pelle. Sebbene scoperte da Poul Langerhans nel 1868, ci 35 Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno sono voluti piГ№ di 100 anni prima che queste cellule fossero associate al sistema immune e riconosciute come APC (antigen presenting cells). In particolare il gruppo di ricerca di Ralph Steiman scoprГ¬ le caratteristiche funzionali delle LC partendo dal dato che le LC presenti nellвЂ™epidermide murina, in vitro, maturavano in cellule dendritiche con forti capacitГ immunostimolatorie. I lavori che seguirono sullo studio delle LC; disegnarono il concetto di cellule dendritiche (DC) come APC professionali capaci di indurre una risposta immune mediata dai linfociti T successivamente Wilson e Villadangos [4] idearono il paradigma delle LC che assegna tre funzioni chiave alle a queste cellule. Langerhans cells вЂњparadigmвЂќ Langerhans cells (LC) a constituent part of the skin immune system. were discovered by Paul Langerhans in 1868 [1], but it took more than 100 years before they were first associated with the immune system and recognized as antigen presenting cells [2]. In particular, Ralph SteinmanвЂ™s group [3] discovered the functional characteristics of LC starting from the finding that murine epidermal LC mature into potent immunostimulatory dendritic cells (DC) in vitro. The work that followed shaped the concept of DC as professional antigen presenting cells (APC) and inducers of T-cell immune responses largely by studying LC, later the term вЂ�LC paradigmвЂ™ was coined by Wilson and Villadangos [4]. This concept assigned three key functions to LC/DC [5]. First, immature LC that reside in the periphery in the steady state are highly specialized in antigen uptake. LC represent approximately 3% of epidermal cells and form a dense network in the epidermis where they constantly scan the environment and are ideally positioned to detect any pathogen breaching the skin barrier [6]. Second, LC transport antigen to skin draining lymph nodes (LN), which is essential to ensure interaction with rare antigen-specific naive T cells. Stimulation by a number of pathogen products in addition to pro-inflammatory cytokines induces LC activation [7вЂ“9]. Third, during migration the LC undergo a process of maturation in which they process antigen acquired in the skin and present it in the context of MHC class I/II. In addition, they dramatically upregulate their surface expression of co-stimulatory molecules and start to produce cytokines required for proper Th1 or Th2 instruction. Thus, by the time LC arrive in the LN, they have acquired the surface phenotype of a вЂ�functionallyвЂ™ mature DC capable of activating naive T cells and initiating an adaptive immune response specifically designed to fight off the invading cutaneous pathogen [5]. The LC paradigm has been expanded to include LC that migrate to skin-draining LN in the steady state in the absence of activating stimuli where they present self-peptides to T cells [10вЂ“14]. This steady-state presentation of self-antigens has been proposed to eliminate self-reactive T cells and provide a mechanism of peripheral tolerance [15, 16]. Recently the role of LC in peripheral tolerance was deeply investigated, resulting in a 36 New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role pivotal activity perhaps more important than usually attributed APC role Here we wont to analyze the morphological and functional aspects of LC from their origin to migration into lymph node to understand which mechanisms make LCs able to accomplish their role into the immune system. Phenotypical aspects of Langerhans cells As immature cells, LC primary function is constantly scan the environment by extending and retracting their dendrites to sense the environment for danger signals and capture antigens, LC express the C-type lectin CD207 (Langerin), which represent usually the hallmark of LC [17вЂ“19]. Langerin is the major constituent of Birbeck Granules (BG), that are a unique cytoplasmic organelle and constitute a subdomain of the endosomal recycling compartment, perhaps being involved in antigen loading processing. LC also express a characteristic set of cell-surface molecules, such as cutaneous lymphocyte-associated antigen (CLA), E-cadherin, the CC chemokine receptor 6 (CCR6) [2024], the invariant MHCI like CD1a receptor and epithelial cell adhesion molecule (EpCAM) [25] which is expressed in LC, but not in other DC subsets [26], whereas langerin is also expressed in a subset of dermal Dendritic cells (dDC) and in some CD8a1 DC in LN [25, 27, 28]. Langerin and Birbeck Granule. Although BGs have been first observed in 1961 [29], there is still a large part of mystery around their organization and function in the Langerhans cells. Nevertheless, BGs were described as organelles allowing a nonclassical routing for an antigen processing pathway [30, 31]. These distinctive rod-shaped compartments BGs may therefore correspond to a specialized membrane domain of the ERC, devoted to the loading of CD1a with glycolipids internalized and routed to the BGs by Langerin [32]. The characterization of Langerin has provided some insight into the biogenesis of Birbeck granules. Langerin is a transmembrane type II Ca2-dependent lectin with a single carbohydrate recognition domain displaying mannose-binding specificity and an intracellular proline-rich motif [33]. This is consistent with the observation that Ca2+ removal can cause unzipping of BGs to various extents with inner periodical pattern disintegration [34, 35]. In freshly isolated Langerhans cells, recycling Langerin is dynamically retained in the endosomal recycling compartment and that Birbeck granules form where Langerin accumulates. Langerin, necessary for the formation of BG is expressed on the plasma membrane and 37 Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno constitutively internalized into BGs, the internal membranes where it accumulates, before returning to the plasma membrane [32, 33]. A direct role of langerin in BG formation was demonstrated from observation of BG induction in murine fibroblasts as well as in human melanoma cell line upon langerin gene transfection [33, 36] Langerin extracellular region, and more precisely its carbohydrate recognition domain (CRD), was suggested to be a key element in the BG architecture [37]. BGs also display a remarkable association with Rab11 a GTPases of the Rab family, known to be regulators of membrane traffic between organelles. Rab11 protein has been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN) [38, 39]. Thus, the major intracellular pool of Langerin is the Rab11_ BGs, which correspond to subdomains of the endosomal recycling compartment (ERC) of LCs [32]. Langerin induces the formation of BGs in areas where it accumulates (Valladeau et al., 2000; McDermott et al., 2004), but the cellular distribution and traffic of Langerin are controlled by Rab11A [33-40]. The formation of BGs depends on Langerin being addressed to an appropriate Rab11Adependent membrane environment. In this environment, the coordinated action of Rab11A and Rab11AвЂ“effector complexes creates the membrane platform required for BG formation. In the absence of this platform, the traffic of Langerin is altered and favors its routing to lysosomal compartments where it is degraded. Because Langerin recycles actively between the endocytic system and the plasma membrane, the formation of a Rab11A driven membrane platform must be viewed as a highly dynamic process. Such a role, as illustrated here for Rab11A, may lead directly or indirectly to the biogenesis of a specialized compartment. BGs may therefore correspond to a specialized membrane domain of the ERC, devoted to the loading of CD1a with glycolipids internalized and routed to the BGs by Langerin [32]. Rab coupling protein (RCP) depletion also decreased Langerin levels and numbers of BGs, although to a less significant extent than in the case of TfR. Rab11AвЂ“RCP complexes couldintervene in the routing of Langerin from degradative to recycling pathways [41]. Since it was not still clear the function of Birbeck granules, two opposing theories have been proposed. The previous first suggests that BGs could function as alternative endocytotic structures, specific to LCs [42-46]. However, under physiological conditions, BGs appended to the cell surface were an exceptional finding [47,48]. Moreover, there are no evidence that BGs participate in the internalization of Langerin, one of their essential constituents. Unlike in the physiological state, вЂњopen endedвЂќ [49] BG-like structures appended to the cell mem38 New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role brane were rapidly induced by treating LCs with cytochalasin D or latrunculin A, irrespective of the presence of an anti-Langerin mAb. One possible explanation for these results would be that BGs represent a transient endocytic intermediate that can no longer be pinched off the cell membrane in the presence of these drugs. However, not only BG-like structures were seen under these conditions but also a large number of coated pits, which were labeled by gold conjugated anti-Langerin mAb, consistent with the recognized effect of these drugs on classical receptor-mediated endocytosis [50, 51]. In addition, many of the BG-like structures were clathrin coated, or had labeled coated pits at their cytoplasmic terminal end. These findings indicate first that Langerin endocytosis is mediated by a classical clathrin coated pathway, regardless of whether the plasma membrane is linear or folded, as in the characteristic zipped formation of BG-like structures and, second, that BGs are not endocytotic structures. The second proposed theory regarding BG function is that they could represent secretory structures formed in the Golgi area and involved in transport of cargo molecules to the cell membrane. The disappearance of BGs from the pericentriolar area and concomitant appearance of BG-like structures at the cell membrane after cytochalasin D or latrunculin A treatment could signify that BGs act as transport intermediates вЂњdrivingвЂќ Langerin, and perhaps other molecules, from internal membranes to the cell surface. On arrival at the cell surface such BGs would fuse with the plasma membrane, in a similar manner synaptic vesicles originating from the early endosomal pathway [52]. Unzipping would follow, allowing delivery of their molecular content to the cell surface. However, immunoelectron microscopy revealed BGs in continuity with tubular structures and also occasionally as part of early endosomal tubular networks in the pericentriolar area, networks which became manifest after treatment with BFA. In addition, after internalization at 19.5В°C and temperature shift to 37В°C, Langerin was found both in the recycling compartments and in pericentriolar BGs. Thus it is apparent that BGs may simply represent membrane domains of tubular recycling endosomes, the morphology of which has been modified by the formation of molecular bridges. In this context, the elongated tubular configuration of the recycling compartment [53] could enable two membrane leaflets to come into close apposition, facilitating the generation of BGs. In contrast, such membrane interaction might not occur in early sorting endosomes because of their vesicular shape. The predominance of BGs in the ERC could also be explained if the molecules responsible for their formation transiently accumulate in this compartment during their traffic. These last findings substantiate the hypothesis that BGs represent morphological modifications of preexisting membranes, rather than specific transport intermediates [32]. One can only speculate as to which antigen-presenting molecules Langerin might deliver bound antigen; however, on the basis of our results, CD1a is a potential target-presen39 Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno ting molecule. It is demonstrated that endocytosed Langerin and CD1a molecules share a common intracellular fate in LCs, traveling through identical intracellular compartments, including pericentriolar BGs. Moreover, endocytosis and recycling of both molecules is arrested by LC maturation [51]. It was show that in a leprosy model infection LCs have a specialized role in the presentation of non peptide antigens to T cells, and that this function is mediated by the LCspecific pattern-recognition receptor langerin as well as the antigen-presenting molecule CD1a, which is highly expressed by these cells [54]. Because BGs form where Langerin accumulates in the ERC and are depleted when LCs are activated, they could in this context, serve as a loading compartment and/or a membrane reservoir for antigens before LC maturation. Langerhans cells precursor cell subsets The process of LC differentiation and their migration into the epidermis is not clearly Understood It is assumed that LCs have no single origin and that local conditions within the epidermis direct theirs development. Studies of chimeric mice suggest that under normal non inflammatory conditions, LCs are replaced entirely from a skin-resident LC precursor pool, which could be derived from dermal CD141 cells [55]. These LC precursors co-express Langerin and CD14, and have the potential to migrate through the dermalвЂ“ epidermal barrier to the suprabasal layer of the epidermis in response to controlled keratinocyte-derived CXCL14 and CCL20, once in epidermis, in the presence of keratinocytederived TGFb1, differentiate into immature resident LC characterized by a weak T cell stimulatory activity [56-58]. So the final differentiation of LC precursors depends on the cytokine environment of the epidermis (7). In fact the cutaneous cytokines granulocyteвЂ“ macrophage colony-stimulating factor (GMCSF), interleukin (IL)-15, and TGF-b1 contribute to the establishment of immature LC in the epidermis [57] Other evidences suggest that LC are of myeloid origin and can differentiate from monocytes or CD34+ precursors [59-63]. Many studies suggested that bone marrow- derived myeloid cutaneous lymphocyte associated antigen (CLA)-expressing LC precursors travel through peripheral blood through the dermis into the epidermis [64]. In an in vitro study it was demonstrated that CD141CD341 PBMCs migrate across the endothelium and initiate development of immunostimulatory DCs [65]. Moreover, it was reported that the rare CD161monocyte subtype (5% to10%of peripheral blood monocytes), which is dramatically increased under inflammatory conditions, differentiates into LCs [66]. 40 New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role It is interesting to note that no LCs of recipient origin were observed in transplanted human hands over a period of 4.5 years [67]. These results strongly suggest that under steady-state conditions, the replacement of human epidermal LCs from bone marrowвЂ“ derived precursors occurs very slowly. In contrast, inflammation induces replacement of emigrated LCs, largely through a bone marrowвЂ“derived precursor. Monocytes, MHC class II2 CD11c1 cells, and CD341 cellвЂ“ derived CD1a1 cells might give rise to development of LCs [66]. It is therefore of interest to note that while the generation of DC from monocytes requires GM-CSF and IL-4 only, the additional presence of TGFb1 in the cytokine milieu appears to be essential for the development of LC [60, 61]. Accordingly, TGFb1-deficient mice display a severe defect in LC, but not in DC, development [68]. In vitro experiments have shown that IL-15, in cooperation with GM-CSF, induces the differentiation of monocytes into cells that express LC markers, such as Ecadherin, CCR6 and Langerin but lacking the expression of conventional Birbeck granules [69]. Another study reports that Activin A, a protein abundantly produced in the skin during normal and pathological wound healing [70, 71] and inflammatory/autoimmune diseases induces the differentiation of human CD14+monocytes in Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ cells. These langerin+ cells can be induced to differentiate by Activin A within the dermis in the absence of epidermis. In agreement with these results, cells emigrated from skin explants could also be induced to differentiate into Langerin+/CD1a+ cells by the presence of Activin A in vitro. These findings are compatible with the description of CD14+, Langerin+ LC precursors located in the superficial and deep dermis, predominantly in perivascular areas [72, 73]. Since Activin A is abundantly produced during certain inflammatory conditions, it was proposed that this cytokine represents a new pathway, alternative to TGFb, responsible for LC differentiation during inflammatory/autoimmune conditions [74]. Recognition of antigen LC are able to bridge innate and adaptive immunity. They are able to interact directly with microorganisms at the periphery to produce effector cytokines and initiate or restimulate activation of T and B lymphocytes through antigen presentation. LC express different pattern recognition receptors such as C-type lectin receptors (CLR) langerin and Toll-like receptors (TLR), to bind and capture pathogens. These interactions result in activation of intracellular signalling pathways leading to LC activation and cytokine induction. LC like other migratory DC are in an immature, highly endocytic state while sessile in the skin/mucosa. However after pathogen binding to TLR and uptake, LC become mature 41 Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno and migratory, down regulating their endocytic and antigen processing capacity. Mature LC migrate to the lymph nodes where they are directly or indirectly involved in presentation of pathogen derived antigens on MHC Class I land II or non polymorphic MHC like CD1a to T cells, resulting in activation of antigen-specific T cells [75, 76]. LC maturation and migration from the Epidermis LC exhibit a very slow turnover under steady-state conditions compared with other DC subsets (including dDC), which undergo renewal at a much faster rate. During skin inflammation, LC turnover is rapidly and markedly increased. The increased LC turnover allows the recruitment of new bone-marrow-derived LC precursors to the epidermis, a process that requires the expression of CCR2 and CCR6 on LC precursors [25, 77, 78]. Skin contact with antigens is known to stimulate various epidermal cytokines, e.g. IL6, TNF-a, GM-CSF, and CXCL2/CXCL3 (macrophage inflammatory protein-2, MIP-2) [79], among which IL-1b and TNF-a are essential in DC migration [80, 81]. LC leave the epidermis by a mechanism that depends on the expression of chemotactic receptors, adhesion molecules and proteases [82-84]. Activation of Langerhans cells Antigen recognition and subsequent activation of LC start a change in expression pattern of cytokines and surface receptors, functional to accomplish antigen presentation into the lymph node also cytokine signalling results in altered expression of adhesion molecules, which facilitates DC migration out of the epidermis Once activated, LC In order to leave the epidermis, need to cross the basement membrane at the dermo-epidermal junction [21, 28]. IL-1b and TNF-a play a central role in the process of LC migration across the basement membrane [85, 86]. Upon initiation of the maturation process, LC produce IL-1b, which induces TNF-a secretion from adjacent keratinocytes [85, 86]. In brief, it is proposed that LC-derived IL-1b performs two functions. First, LC-derived IL-1b activates the LC in an autocrine loop through the IL-1 receptor 1 (IL-1RI). Second, IL-1b also stimulates epidermal keratinocytes (KCs) to secrete TNF-a, which acts in a paracrine manner to facilitate DC migration through TNFRII (87).TNF-О± contributes to decreasing the attachment between LC and keratinocytes by downregulating E-cadherin and by inhibiting the expression of CCR6, which renders LC insensitive to CCL20 produced by keratinocytes [85, 86]. TNF-a also induces the expression of a6b1 integrin on LC, which is important for their interaction with extracellular matrix proteins such as laminin that is present in the basement membrane of the epidermis [86, 88, 89]. The integrin LFA-1 (leukocyte function-associated antigen-1) is also implicated in skin DC migration to LN and the LFA-1 ligand ICAM-1 is expressed by lymphatic endothelial cells [86]. 42 New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role Simultaneously, production of epidermal basement membrane degrading enzymes, such as matrix metalloproteinases (MMPs), is upregulated in activated LC [90]. The MMP2, MMP-3, and MMP-9 have been described to facilitate in the passage of LC across the basement membrane. Subsequently, MMPs are also essential for migration of LC and DDC through the dermal tissue matrix, by cleavage of type IV collagen [91]. Therefore, earlier CXCR4 expression confers upon LC the capacity to migrate faster to the dermis where the CXCR4 ligand CXCL12 is increased during inflammation. Once in the dermis, LC complete their maturation and upregulate CCR7, allowing their final migration to LN. CCR7 is induced only at later stages of LC maturation because its ligands CCL19 and CCL21 are expressed in lymphatic endothelial cells that are not immediately underneath the epidermal layer, making them not readily available for LC that are still in the epidermis The temporal dissociation of CXCR4 and CCR7 expression would also permit LC to transiently dwell in the dermis before continuing their transit to the LN, potentially allowing these cells to interact with and deliver antigens to resident dDC [92]. About LC stimulatory capacity, as any other DCs they are able to activated different subsets of lymphocytes. It was seen, in different experimental models of skin pathology an involvement of LC in priming of adaptative response in terms of CD4 or CD8 T cells activation or B activation. Many works report that Differentiated LC primed T cells much more efficiently than dermal DC (dDC) or monocyte-derived DC. These results led to the classical view that LC play a prominent role in skin immunity by capturing and processing antigens in the epidermis in order to activate T cells in the skin-draining LN [93вЂ“94]. Studies on response to viruses and gram-positive bacteria, shows that freshly purified human LC express a restricted set of functional TLR, namely TLR2, TLR6, and TLR3, and are directly activated by their respective ligands, while cooperation of keratinocytes and other cell types is probably essential for response to Gram-negative bacteria and viruses in vivo. In particular, lack of type I IFN production precludes a direct effector role of LC in viral infections and further shows that the full development of an immune response in the skin does not solely rely on these cells. Of interest, activation of LC by different TLR agonists leads to specific cytokine secretion profiles. Particularly stimulation of TLR2 or TLR6 with PGN- lead to a specific IL-10 secretion, although the exact physiological relevance of this specific pattern remains to be determined, LC may contribute to the tolerance of commensal Gram-positive bacteria that colonize the skin of healthy individuals without causing inflammation [95]. Is interesting note that in many case reported the role played by LC is supported by other cells subsets especially skin Keratinocites and without this cross-talk the LCs shows very poor efficiency as APC. 43 Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno Some studies on Mice of inbred strains C57BL_6, OT-I (39), and OT-II [96], and TAP-1deficient [97] shows that LCs isolated from the skin can cross-present exogenous soluble and cell-associated antigen to CD8_ T cells. Furthermore, migratory LCs can take up material from keratinocytes and cross-present it. The final outcome of cross-presentation by LCs is the induction of IFN-Оі production and cytotoxicity in antigen-specificCD8_ T cells [98]. other study focused on in vitro LC response to TLR ligands shown that lc from BALB/c mice pre-treated with 2,4,6-Trinitrochlorobenzene (TNCB) and 2,4,6-trinitrobenzene, are incapable of responding to TLR ligands, as the expression of MHC class II and co-stimulatory molecules on LCs are not augmented by the TLR-mediated signal [99]. But it was also found that CpG is capable of stimulating LCs in a certain condition, resulting in enhancement of their haptenpresenting function. This stimulatory effect was exerted only when LCs co-existed with keratinocytes, as purified LCs mounted the CpG-induced enhancement at a lower level than did LCвЂ“ECs. It was also found that other TLR ligands derived from Gram-positive or negative bacteria, such as peptidoglycan and LPS, enhance the hapten-presenting ability of LCs by augmenting of keratinocyte cytokine production [100]. Therefore, keratinocytes serve as an up-regulator of acquired immunity upon exposure to microbes. Even if LCs are low-responsive to skin commensals, they can be stimulated with microbial elements indirectly via keratinocytes. Tolerogenic role of Langerhans cells Many studies on murine experimental models, after antigens or pathogens exposure, shows the presence of LC into draining lymph node but their role often seems to be much more regulatory than activator and is accomplished trough the help of other cells subset. Studies based on constitutive or transient depletion of LC in mice gave some unexpected and even contradictory findings, with reports suggesting an important role of LC in skin immunity [100-103], whereas other studies found that LC were dispensable for inducing skin-associated immune responses [104]. These disparate observations may be explained, at least in part, by the variable degree of deletion of other DC subsets that also express langerin From work with the two LangerinвЂ“DTRвЂ“EGFP mouse strains in the setting of an acute depletion of LC, it appears that when antigen is targeted preferentially to LC such as with low-dose hapten, then LC appear to be stimulatory and their absence leads to diminished CHS responses. However, when antigen is simultaneously targeted to LC and other cell subsets like dDC such as in the setting of normal hapten doses, then LC appear largely redundant and CHS responses are mediated by dDC. This is consistent with a model in 44 New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role which all skin DC have similar function and the number that migrate to the CLN carrying antigen determines the ultimate outcome [105]. More recent studies [28, 104] in which LC were selectively depleted, while other langerin1 DC subsets (including dDC) were preserved, did not show an essential role for LC in inducing contact hypersensitivity responses to either haptens or peptide antigens [10, 15] or in a model of skin allograft rejection [106]. Nonetheless, although LC may not be strictly required for skin immune responses in some settings, they might still be sufficient to trigger effective protective or pathogenic skin immune responses. Consistent with this possibility, allogeneic LC are sufficient to trigger skin graft-versus host disease in the absence of host-derived dDC [78]. Many studies on murine experimental models, after antigens or pathogens exposure, shows the presence of LC into draining lymph node but their role often seems to be involved in maintenance of peripheral tolerance. Recent observations on the dynamics and functions of CD207+ cells fuel speculation in a number of different inflammatory situations that LCs have a regulatory, rather than stimulatory, role [107-110]. In many cases LCs rather than important in the priming of a CD4+ mediated immune response in the sdLN, seems to lead to the development of a protective immune response [111-113] Another work described an otherwise normal mouse that constitutively lacks only epidermal LCs and used this model to demonstrate that the lack of LCs leads to an enhanced rather than inhibited CHS response then show via cell-transfer experiments that LCs act at the priming rather than effector phase of the response [114]. Other study on LC chimeric mice reports that PAF is involved in LC migration. However because PAFRв€’/в€’ mice generate a CHS reaction no different from controls, these findings support the hypothesis that LC are not involved in activating CHS. morover shows that dDC, but notLC, are sorted from the lymph nodes of hapten sensitized mice, probably dDC can present antigen to T cells [115]. In a murine model of parasite immunisation whith RA schistosoma larvae, Comparison of mice vaccinated VS mice exposed to non-protective normal parasites shows that presence of CD207+ cells in the sdLN does not correlate with the enhanced CD4+ cell proliferation and Th1 cytokine production that is a feature of mice exposed to the RA schistosome vaccine [112, 113, 116]. It is possible that CD207+ cells, despite expressing slightly greater amounts of MHCII, do not have an immune priming role but regulate events in the sdLN, or crosspresentv antigen to other APCs [98]. 45 Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno Final remarks LC were discovered in 1868 and well characterized morphologically (i.e. Birbek granules) from1961, but still now, despite several immunological studies LC are not so well functionally characterized especially in their regulatory role. The understanding of the mechanisms employed by the skin to determine between the stimulation of immune responses vs maintenance of tolerance needs further investigation. It is still controversial whether the high plasticity, characteristic of myeloid DC, like LC, suffice to determine between immunity or tolerance induction. In addition, whether LC have redundant, complementary, or even opposing functions respect to other skin cells population like DDC has not yet been clarified. Last works support the hypothesis that, owing to their high plasticity, skin resident DC and especially LC censor for foreign Ag but are still able to maintain the homeostasis of the skin. Functionally, it is probable that most of the LC and DDC remain dormant during the steady state whereas a low number gets activated and transports self-Ag from the skin to sDLN to present Ag in a tolerogenic pathway. In the presence of inflammatory insult, LC and DDC may have the ability to respond to the injury and to mature into potent APC while still being able to maintain tolerance to self-Ag. Is interesting to note that earliest works represents LC as more modulating than activating immune response in this way is quite difficult to profile in a secure manner the LC activity. Surely is very important the micro-environment meaning the cross-talk with other cells subsets, especially skin Keratinocytes, in fact cytokine production from these cells, subsequently to antigen contact, sign the destiny of LC which can so became traditionally APC, while if there is no cross-talk with keratinocytes presentation of antigen by LC to Limphocytes is not completed by second signal and do not induce lymphocytes activation. So the recently works from one hand reduce the classical role of LC as APC in activation of immune response to potentials pathogens, but from the other hand attribute to LC a new and more amazing role of cells involved in modulation of immune response and maintenance of peripheral tolerance to skin commensally bacteria. Regardless the LC paradigm, probably this remain a valid key to explain the role of LC in skin immunity, but surely the last and new point became perhaps the most important to describe LC activities in the immune system. About therapeutic use of LC more of scientific production indicate LC as possible target for vaccine administrated subcutaneously, this due to their role of APC. LCs, like all DCs, are cells of the innate immune system that can influence the genera46 New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role tion of an adaptive T cell response through their ability to take up proteins and to process and present them via MHC class II molecules to T cells [117]. Particularly, targeting of non peptide antigens to LCs via CD1a is a vaccine strategy that could be used for the generation of immunity to cutaneous pathogens [118]. The location of LCs in the epidermis allows the innate immune system to respond rapidly to microbial invaders in the skin. But, for set up a efficient vaccine is possible controversial to target only LC because their role of APC need the involving of other skin cells populations (Keratinocytes) which became as important as LC in activation of immune system, nevertheless LC remain interesting as target for vaccine. More interesting is the possible use of LC as down regulating cells against autoimmune disease which involved skin. As antigen-presenting cells in the epidermis, LCs control T cellвЂ“mediated allergenspecific immune responses. Allergens can penetrate the epidermis, mucosal barriers, or both and thus induce eczema in patients with atopic disorders. The local cytokine milieu in the epidermis influences the LC-regulated T-cell response against the encountered allergen toward silent elimination, allergy, or tolerance. The recent progress made in understanding the mechanisms by which LCs populate the skin opens new therapeutic strategies for the modulation of immune responses in atopic diseases. Through manipulation of LC development and biology in vitro or in vivo, new strategies for the treatment of allergic diseases could evolve. 47 Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno Epidermic enviroment (historical himage by Von Keith R. Porter and Mary A. Bonneville, EinfГјhrung in die Feinstruktur von Zellen und Geweben, Springer-Verlag, Berlin 1965, t.9) New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role Activation of skin immune system and different role of Langerhans cells: 1) mainly regulatory with large amount of antigen; 2) mainly pro-inflammatory with a poor amount of antigen, in this case is pivotal the cross-talk between Langerhans cells and keratinocytes. 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Both Langerhans cells and interstitial DC cross-present melanoma antigens and efficiently activate antigen-specific CTL. Eur J Immunol 2007; 37: 2657-2667. [95] Flacher V, Bouschbacher M, Verronese E, Massacrier C, Sisirak V, Berthier-Vergnes O, de Saint-Vis B, et 54 New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role al. Human Langerhans Cells Express a Specific TLR Profile and Differentially Respond to Viruses and Gram-Positive Bacteria1 The Journal of Immunology 2006; 177: 7959-7967. [96] Barnden MJ, Allison J, Heath WR, Carbone FR. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 1998; 76: 34-40. [97] Van Kaer L, Ashton-Rickardt PG, Ploegh H, Tonegawa S. TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4-8+ T cells. Cell 1992; 71: 1205-1214. [98] Stoitzner P, Tripp CH, Eberhart A, Price KM, Jung JY, Bursch L, Ronchese F, Romani N. Langerhans cells cross-present antigen derived from skin. Proc Nat Aca Sc USA 2006; 103: 7783-7788. [99] Mitsui H, Watanabe T, Saeki H. et al. Differential expression and function of Toll-like receptors in Langerhans cells: comparison with splenic dendritic cells. J Invest Dermatol 2004; 122: 95-102. [100]Sugita K, Kabashima K, Atarashi K, Shimauchi T, Kobayashi M, Tokura Y. Innate immunity mediated by epidermal keratinocytes promotes acquired immunity involving Langerhans cells and T cells in the skin Clin Exper Immunol 2006; 147: 176-183. [101]Bennett CL, van Rijn E, Jung S, Inaba K, Steinman RM, Kapsenberg ML, Clausen BE. Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity. J Cell Biol 2005; 169: 569-576. [102]Bennett CL, Noordegraaf M, Martina CA, Clausen BE. 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[107]Douillard P, Stoitzner P, Tripp CH, Clair-Moninot V, Ait-Yahia S, McLellan AD, Eggert A, Romani N, Saeland S. Mouse lymphoid tissue contains distinct subsets of langerin/CD207 dendritic cells, only one of which represents epidermal-derived Langerhans cells. J Invest Dermatol 2005; 125:983-994. [108]Itano AA, McSorley SJ, Reinhardt RL, Ehst BD, Ingulli E, Rudensky AY, Jenkins MK. Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cellmediated immunity. Immunity 2003; 19:47-57. [109]Kissenpfennig A, Malissen B. Langerhans cells - revisiting the paradigm using genetically engineeredmice. Trends Immunol 2006; 27:132-139. [110]Villadangos JA, Heath WR. Life cycle, migration and antigen presenting functions of spleen and lymph node dendritic cells: limitations of the Langerhans cells paradigm. Semin Immunol 2005; 17:262-272. [111]Sato H, Kamiya H. Role of epidermal LangerhansвЂ™ cells in the induction of protective immunity to Schistosoma mansoni in guinea-pigs. Immunology 1995; 84:233-240. [112]Hogg KG, Kumkate S, Mountford AP. IL-10 regulates early IL-12-mediated immune responses inducedby the radiation-attenuated schistosome vaccine. Int Immunol 2003; 15:1451-1459. [113]Hewitson JP, Hamblin PA, Mountford AP. Immunity induced by the radiation-attenuated schistosomevaccine. Parasite Immunol 2005; 27:271-280. [114]Kaplan DH, Jenison MC, Saeland S, Shlomchik WD, Shlomchik MJ. Epidermal Langerhans Cell-Deficient 55 Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno Mice Develop Enhanced Contact Hypersensitivity. Immunity 2005; 23: 611-620. [115]Fukunaga A, Khaskhely NM, Sreevidya CS, Byrne SN, Ullrich SE. Dermal Dendritic Cells, and not Langerhans cells, play an essential role in inducing an immune response. J Immunol 2008; 180: 3057-3064. [116]Kumkate S, Jenkins GR, Paveley RA, Hogg KG, Mountford AP. 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Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 Espressione immunoistochimica della orexina A, della orfanina FQ e della leptina nellвЂ™apparato respiratorio del ratto [Orexin A, Orphanin FQ, Leptin immunohistochemical expression in rat respiratory system] Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo e Vincenzo Tessitore Di.Me.S. вЂ“ Sezione di Istologia ed Embriologia вЂ“ UniversitГ degli Studi di Palermo (IT) Parole chiave: Orexina; Orfanina FQ; Leptina; Immunoistochimica; Sistema respiratorio Key words: Orexin; Orphanin FQ; Leptin; Immunohistochemistry; Respiratory system Riassunto. Recenti evidenze sperimentali riferiscono sulla imprevista presenza in distretti organici periferici, delle Orexine A e B, della Orfanina FQ e della Leptina, nuovi neuropeptidi ritenuti in origine, esclusivamente espressi da neuroni centrali nel cui ambito svolgono un ruolo oressizzante e anoressizzante. Con riferimento anche a recenti rilievi microanalitici e farmacologici, abbiamo esteso la nostra indagine, in precedenza condotta in alcuni distretti periferici, sulla possibile espressione immunoistochimica di tali messaggeri nellвЂ™apparato respiratorio del ratto normale. I risultati da noi ottenuti fanno rilevare che i neuro peptidi presi in esame sono espressi, sia pure con diversa intensitГ per ciascuno di essi, da diverse strutture respiratorie (albero tracheo-bronchiale e ghiandole annesse, bronchioli prossimo distali, bronchioli respiratori e alveoli). In aggiunta, la immunoreattivitГ espressa da ciascun messaggero interessa in modo non uniforme i diversi citotipi dellвЂ™epitelio mucosale bronchiolare (cellule ciliate, cellule di Clara, cellule neuroendocrine, miociti peribronchiali). Nel distretto alveolare, soltanto i pneumociti di II tipo esprimono immunopositivitГ , di variabile intensitГ , in relazione ai diversi neuropeptidi esaminati. I risultati ottenuti forniscono la base morfoistochimica per ritenere che tali sostanze al di fuori della giГ nota funzione centrale oressizzante e anoressizzante, possano svolgere nuovi ruoli periferici esercitati localmente modulando diversi aspetti della funzione respiratoria con meccanismo endocrino-paracrino, neurocrino e autocrino. Abstract. Recent studies document that the novel neuropeptides Orexins A and B, Orphanin FQ and Leptin considered since its first identification exclusively expressed by central neurons, may be also expressed by different peripheral tissues (GEP system, reproductive and urinary system) like our previous immunohistochemical researches has demonstrated. Following this method, we have considered interesting to carry out an immunohistochemical study on this neuropeptides expression and distribution in normal rat respiratory system. Our results indicate that these substances are expressed in all respiratory structures examined, but with different intensity. In addition, each substance is immunohistochemically expressed with different intensity in the various cytotypes of bronchiolar epithelium (Clara cells, neuroendocrine cells, ciliated epithelial cells, peribronchiolar smooth muscle cells). In the alveoli the immunoreactivity appears in pneumocytes type 2 even though with different intensity for every tested substances. The results obtained provide a morphoistochemical basis to understand the new additional peripheral roles, apart from their already orexigenic and anorexigenic functions, played by these regulatory peptides directly in the respiratory system with a autocrine, neurocrine, paracrine-endocrine mechanism. 57 Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore Introduzione Numerose evidenze sperimentali accumulate negli ultimi anni hanno rivelato la presenza nel polmone di un considerevole numero di neuropeptidi, alcuni dei quali sono distribuiti in terminali assonici e agiscono pertanto da neurotrasmettitori, attivando una mediazione peptidergica che si affianca alla giГ nota mediazione adreno-colinergica e a quella, piГ№ recente, nitrossidergica nella regolazione neurocrina delle funzioni polmonari. Altri neuropeptidi sono stati identificati in cellule neuroendocrine intraepiteliali, ove si comportano da neurormoni che autorizzano a considerare il polmone un vero e proprio organo endocrino [1]. Alcuni dati suggeriscono inoltre che sia i peptidi neurotrasmettitoriali che quelli neuroendocrini svolgono i loro ruoli non soltanto nella fisiologia ma anche nella patologia polmonare flogistica e neoplastica [2,3]. Le orexine (OXA e OXB), la leptina e la orfanina FQ sono nuovi neuropeptidi ritenuti in origine esclusivamente prodotti da neuroni del SNC, ove sono integrati in complessi circuiti dellвЂ™area ipotalamica e mesolimbica preposti alla regolazione dellвЂ™appetito e del bilancio energetico. LвЂ™orexina e la orfanina svolgono effetti oressizzanti, la leptina (ormone della sazietГ ) svolge effetti anoressizzanti. Tuttavia, indipendentemente dai loro effetti centrali, grande interesse hanno suscitato, assai recentemente, alcuni effetti svolti da alcuni di essi in distretti periferici. In proposito nostre precedenti ricerche hanno fornito documentazioni immunoistochimiche sulla imprevista presenza di tali peptidi in alcuni distretti periferici correlati (GEP, stomaco e ghiandole salivari) e non correlati (apparato genitourinario maschile) con la loro azione oressizzante e anoressizzante [4,5,6,7,8,9,10]. In aggiunta, ci sembrano significativi i risultati di una recente ricerca sugli effetti stimolanti respiratori indotti da microiniezioni di leptina nella parte respiratoria del nucleo del tratto solitario che esprime tra lвЂ™altro alte concentrazioni di recettori leptinici [11]. Ed ancora, topi transgenici mancanti della orexina mostrano una attenuazione della risposta ipercapnica ventilatoria durante la veglia ma non durante il sonno; la somministrazione di un antagonista del recettore della orexina mima questa anormalitГ , rimossa al contrario da somministrazione di orexina [12]. Con riferimento a questi imprevisti riscontri morfofunzionali, abbiamo ritenuto interessante indagare sulla possibile espressione immunoistochimica di tali neuropeptidi nelle diverse componenti strutturali dellвЂ™apparato respiratorio nel ratto, al fine di fornire la base morfoistochimica per la interpretazione di nuovi ruoli esplicati da tali neuropeptidi. I risultati preliminari di tale ricerca sono stati comunicati al 32В° Congresso della SocietГ Italiana di Istochimica (Messina 2007) [13] e al 61В° Congresso Nazionale della SocietГ Italiana di Anatomia ed Istologia (Sassari 2007) [14]. 58 Espressione immunoistochimica della orexina A, della orfanina FQ e della leptina nellвЂ™apparato respiratorio del ratto Materiali e metodi Sono stati impiegati 15 ratti Wistar trattati in accordo con la Convenzione di Helsinki sullвЂ™utilizzo degli animali nella ricerca biomedica. Gli animali sono stati sacrificati previa anestesia (50 mg/kg di Nembutal somministrato per via endoperitoneale). Sono stati prelevati frammenti di trachea, di bronchioli prossimali e distali, di bronchioli respiratori e di alveoli. I campioni sono stati immediatamente fissati in liquido di Bouin, inclusi in paraffina, sezionati e infine processati per la tecnica immunoistochimica mediante il kit EnVision+System HRP (AEC) (Dako Cytomation), utilizzando anticorpi anti-Orexina A e anti-Orfanina FQ (Chemicon International), anti-Leptina (Santa Cruz Biotecnology). Controlli negativi sono stati realizzati su sezioni adiacenti omettendo il passaggio dellвЂ™antisiero primario. Tutti i campioni sono stati studiati mediante fotomicroscopio Leica DMLB. Risultati Orfanina LвЂ™epitelio pseudostratificato della mucosa tracheobronchiale appare discretamente immunopositivo: spesso la immunoreattivitГ ГЁ addensata in forma microgranulare nel citoplasma perinucleare (Fig.1). Gli acini sieromucosi delle ghiandole intracoriali e sottomucose mostrano discreta e parcellare immunoreattivitГ (Fig.2). In corrispondenza della biforcazione tracheale si scorgono tronchi nervosi orfaninergici (Fig.3). LвЂ™epitelio delle diramazioni bronchiolari mostra intensa immunoreattivitГ non uniformemente distribuita nei vari citotipi (c. di Clara marcatamente immunoreative rispetto agli altri epiteliociti). LвЂ™anello miocitario peribronchiolare mostra debole positivitГ (Fig.4). Nel distretto alveolare la rimarchevole immunopositivitГ rimane limitata ai pneumociti di II tipo (Fig.5). Leptina LвЂ™epitelio mucosale tracheobronchiale mostra immunoreattivitГ sovente piГ№ intensa e di aspetto microgranulare nellвЂ™endoplasma perinucleare e nel bordo apicale delle cellule. Le ghiandole sieromucose tracheobronchiali mostrano discreta immunoreattivitГ non uniformemente estesa a tutti gli adenomeri (Fig.6). NellвЂ™epitelio bronchiolare si rileva discreta immunoreattivitГ a carico soprattutto delle cellule neuroendocrine (Fig.7). La muscolatura perivascolare e peribronchiale ГЁ intensamente immunopositiva (Fig.9). Parimenti reattivi appaiono alcuni mastociti endobronchiali. NellвЂ™interstizio peribronchiale si rilevano alcuni piccoli neuroni gangliari fortemente immunoreattivi (Fig.8). Nel distretto alveolare alcuni pneumociti di II tipo esibiscono debolissima immunoreattivitГ (Fig.9). 59 Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore Orexina Modesta immunoreattivitГ ГЁ presente nellвЂ™epitelio tracheobronchiale. LвЂ™epitelio bronchiolare appare discretamente immunopositivo con immunoreattivitГ distribuita disomogeneamente nelle cellule di Clara e negli altri epiteliociti mucosali. La muscolatura peribronchiale appare debolmente immunoreattiva. NellвЂ™epitelio alveolare si rileva scarsa immunopositivitГ a carico dei pnemociti di II tipo (Fig.10). Conclusioni I risultati da noi ottenuti indicano che lвЂ™apparato respiratorio del ratto si rivela una rimarchevole sorgente di neuropeptidi, testimoniata dalla capacitГ orfaninergica, orexinergica e leptinergica dei segmenti tracheobronchiali, bronchiolari e alveolari che si manifesta peraltro con reattivitГ immunoistochimica di diversa intensitГ per ciascuno dei neuropeptidi presi in esame. In aggiunta, la immunoreattivitГ espressa da uno stesso neuropeptide interessa in modo non uniforme i diversi citotipi che costituiscono i vari distretti respiratori considerati. CosГ¬ nellвЂ™epitelio mucosale bronchiale e bronchiolare le cellule di Clara, le cellule ciliate, le cellule a muco e le cellule neuroendocrine appaiono per ciascun neuropeptide non uniformemente reattive. NellвЂ™epitelio alveolare soltanto i pneumociti di II tipo si mostrano apprezzabilmente immunoreattivi e con immunopositivitГ di intensitГ variabile in relazione al tipo di neuropeptide espresso. I nostri rilievi forniscono la base morfoistochimica per prospettare la possibilitГ che i neuropeptidi presi in esame, al di fuori della loro giГ nota funzione oressizzante e anoressizzante centrale o periferica, ritenuta finora esclusiva, possano esplicare nuovi ruoli agendo direttamente e localmente sulle diverse componenti immunoreattive dellвЂ™apparato respiratorio, anche se la precisa funzione svolta da tali neuropeptidi nel polmone al presente non puГІ essere esattamente chiarita. Tuttavia il loro riscontro immunoistochimico in diverse strutture dellвЂ™apparato respiratorio suggerisce che essi possano agire con diversi meccanismi in maniera discriminata, controllando negli epiteliociti mucosali, con meccanismo autocrino paracrino, verosimilmente il trofismo epiteliale in epoca pre e postnatale, la secrezione mucipara e quella del surfattante alveolare, modulando nei terminali nervosi, con meccanismo neurocrino, la broncomotilitГ e la vasomotilitГ e pertanto la perfusione polmonare ed infine influenzando a distanza con meccanismo neuroendocrino lвЂ™attivitГ di organi lontani come prospettato per altri peptidi polmonari (1). 60 Espressione immunoistochimica della orexina A, della orfanina FQ e della leptina nellвЂ™apparato respiratorio del ratto Fig.1: Ratto, orfanina: discreta immunopositivitГ Fig.2: Ratto, orfanina: parcellare immunoreattivitГ negli adenomeri delle ghiandole sieromucose tradellвЂ™epitelio mucosale tracheale. Oc. 10x; Ob. 40x. cheali. Oc. 10x; Ob. 20x Fig.3 Ratto, orfanina: tronco nervoso orfaninergico Fig.4: Ratto, orfanina: intensa e disomogenea immutracheale. Oc. 10x; Ob. 20x. noreattivitГ dellвЂ™epitelio bronchiolare. Miociti peribronchiolari debolmente immunopositivi. Oc. 10x; Ob. 40x. Fig.5: Ratto, orfanina: intensa immunopositivitГ nei Fig.6: Ratto, leptina: disomogenea immunoreattivitГ pneumociti di II tipo. Oc. 10x; Ob. 40x. negli adenomeri delle ghiandole sieromucose tracheali. Oc. 10x; Ob. 20x. Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore Fig.6: Ratto, leptina: disomogenea immunoreattivitГ Fig.8: Ratto, leptina: immunopositivitГ delle cellule negli adenomeri delle ghiandole sieromucose trache- neuroendocrine nellвЂ™epitelio mucosale bronchiolare. ali. Oc. 10x; Ob. 20x. Oc. 10x; Ob. 40x. Fig.9: Ratto, leptina: neuroni gangliari leptinergici Fig.10: Ratto, leptina: miociti perivascolari e perinellвЂ™interstizio peribronchiolare. Oc. 10x; Ob. 63x. bronchiolari intensamente immunoreattivi. Debole immunopositivitГ dei pneumociti di II tipo. Oc. 10x; Ob.10x. Espressione immunoistochimica della orexina A, della orfanina FQ e della leptina nellвЂ™apparato respiratorio del ratto Bibliografia [1] Cutz E. Neuro-endocrine (APUD-type cells of the lung), in: вЂњUltrastucture of endocrine cells and tissuesвЂќ Motta PM (Eds), Chapter 13, 1984. [2] Vernooy JH, Drummen NE, Suylen RJ, Cloots RH, MГ¶ller GM, Bracke KR, Zuyderduyn S, Dentener MA, Brusselle GG, Hiemstra PS, Wouters EF. Enhanced pulmonary leptin expression in patients with severe COPD and asymptomatic smokers. Thorax. 2009 Jan; 64(1):26-32. Epub 2008 Oct 3. [3] Havemann K. et al. вЂњPeptide hormones in lung cancerвЂќ. Springer-Verlag 1985. [4] Tessitore V, Uzzo ML, Bonaventura G, Spatola GF. Immunohistochemical expression of Orexin A and Orexin Type 2 receptors in the rat gastrointestinal apparatus and endocrine pancreas. Eur J Histochem 2005; vol. 49 Suppl.1, p. 19. [5] Bonaventura G, Tessitore V, Uzzo ML, Spatola GF. Immunohistochemical distribution of orphanin FQ in the rat gastrointestinal apparatus and pancreas. Ital J Anat Embryol 2005; vol. 110 Suppl.1, Fasc3, p. 262. [6] Tessitore V. et al. Immunohistochemical studies on leptin, ghrelin, orexin, orphanin FQ and endocannabinoids expression in the stomach of lean and obese (fa/fa) zucker rats. First world congress on therapies against obesity. Parigi 18-19 maggio 2006. [7] Uzzo ML, Spatola GF, Mandracchia R, Buscemi M, Farina Lipari E, Lipari D, Valentino B, Gerbino A. Orphanin FQ and motility in seminal vesicles. Ital J Anat Embryol 2006; Vol.111, Suppl. 2 [3] 267. [8] Uzzo ML, Martorana A, Cappello F, Farina Lipari E, Valentino B, Lipari D, Aragona F, Buscemi M, Gerbino A. Immunohistochemical expression of orphanin FQ, atrial natriuretic peptide, and oxytocin in normal human seminal vesicles. Eur J Histochem 2007; 51: Suppl. 2,30. 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Espressione immunoistochimica di alcuni neuropeptidi e del recettore CB1 nel polmone di ratto. nota I: bronchioli e alveoli. Eur J Histochem 2007; vol. 51 (2), p. 24. [14] Bonaventura G, Cucco D, Spatola G., Uzzo M.L, Tessitore V. Immunohistochemical expression of some neuropeptides and CB1 cannabinoid receptor in rat lung. Note II: Trachea and Bronchi. Ital J Anat 2007; Embryol. Vol.112, Suppl. 1 [2] 44. 63 Heart, circulation Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 ISOLATED Left ventricular NON-COMPACTION, STROKE AND trabecular meshwork FIBROSIS [La non compattazione ventricolare sinistra isolata, вЂњstrokeвЂќ e fibrosi trabecolare] Giovanni Fazio, Ivana Basile, Monica Lunetta, Luciana DвЂ™Angelo, Loredana Sutera, Emiliano Maresi**, Giuseppina Novo, Aldo Gerbino* and Salvatore Novo Department of Cardiology, *Department of Experimental Medicine, Section of Histology and Embryology; **Department of Legal Medicine; University of Palermo (IT) Key words: Non-compaction, Stroke, Fibrosis, Recesses Parole chiave: Non-compaction, Stroke, Fibrosi, Recessi Abstract. Left ventricular non-compaction is a myocardium disease characterized by an increased and excessively prominent ventricular trabecular meshwork, interrupted by deep wall recesses. Although the presence of intertrabecular recesses undoubtedly is a favourable condition for thrombus formation, the real prevalence of this event in non-compacted hearts is still unknown. We assessed prevalence of strokes and echocardiographic findings of thrombi inside the left ventricle in a continuous series of 229 patients, and we detected that only 5 of them (2%) had a history of ischemic stroke or thrombus. Also, in macroscopic heart specimen in cases of left ventricular myocardium non-compaction shows many prominent trabeculae separated by recesses: by observing the wall meshwork, it is possible to notice fibrosis striae on the external borders of every trabecula. Peritrabecular fibrosis is due to the stress induced by blood on the left ventricular wall, which is further enhanced by turbulent flow into the wall recesses. If blood stasis into the left ventricle wall recesses is not presents it is reasonable to infer that non-compaction in itself does not predispose to the endoventricular thrombosis. Riassunto. La вЂњnon compattazione ventricolare sinistraвЂќ ГЁ una malattia caratterizzata dalla presenza di unвЂ™ipertrabecolatura della parete del ventricolo sinistro. Sebbene la presenza di trabecole e recessi, indubbiamente ГЁ una condizione favorevole per la formazione di trombi, la reale prevalenza di eventi emboligeni cerebrali ГЁ ancora sconosciuta. Abbiamo valutato la prevalenza di ictus in una serie continua di 229 pazienti affetti da non compattazione, e abbiamo rilevato che solo 5 di loro (2%) avevano una storia di ictus ischemico. Inoltre, nelle sezioni anatomo-patologiche macroscopiche di miocardio ventricolare sinistro non compattato mostrano molti eminenti trabeculae separati da recessi ГЁ possibile notare la presenza di fibrosi nelle trabecole piГ№ esterne. Verosimilmente tale fibrosi ГЁ dovuta allo stress indotto dal sangue sulla parete ventricolare sinistra, stress determinato da un flusso turbolento del sangue allвЂ™interno delle trabecole. Г€ nostra opinione quindi che se ГЁ presente un flusso turbolento non sarГ presente stasi, pertanto non sarГ presente un rischio aumentato di trombosi in questa malattia. 67 Giovanni Fazio, Ivana Basile, Monica Lunetta, Luciana DвЂ™angelo, Loredana Sutera, Emiliano Maresi, Giuseppina Novo, Aldo Gerbino, Salvatore Novo Figura 1 Introduction Left ventricular non-compaction is a myocardium disease characterized by an increased and excessively prominent ventricular trabecular meshwork, interrupted by deep wall recesses. Although the presence of intertrabecular recesses undoubtedly is a favourable condition for thrombus formation, the real prevalence of this event in non-compacted hearts is still unknown [1-4]. With regard to this matter, Sahin S. et al [1] reported an unusual case of cerebral cardioembolism in a 33-year-old man due to ventricular non-compaction, diagnosed by magnetic resonance imaging [1]. As the same Authors write, only 1 adult case of stroke among patients affected by non-compaction has been documented so far. Overall, just 3 isolated cases of thrombus formation due to non-compaction of the left ventricular myocardium were described in the literature [2-3]. In 2004 Stollberger and Finesterer reported in a review a low prevalence of ventricular thrombus findings and embolic events in patient populations with non-compacted myocardium [4]. These data were confirmed by the same Authors in a retrospective analysis of 62 patients [5]. We assessed prevalence of strokes and echocardiographic findings of thrombi inside the left ventricle in a continuous series of 229 patients, and we detected that only 5 of them (2%) had a history of ischemic stroke or thrombus. On the basis of the opposing results 68 Isolated left venticular non-compaction, Stroke and Trabecular Meshwork fibrosis gathered so far, it is not still clear if the presence of intertrabecular recesses can lead to thrombi formation, with possible thromboembolic consequences [1-10]. Observations and Conclusions So, a further clarification is needed: the Figure 1 shows a macroscopic heart specimen in a case of left ventricular myocardium non-compaction, in which many prominent trabeculae separated by recesses of varying depth are visible. By observing the wall meshwork, it is possible to notice fibrosis striae on the external borders of every trabecula. Peritrabecular fibrosis is due to the stress induced by blood on the left ventricular wall, which is further enhanced by turbulent flow into the wall recesses. Therefore fibrosis ultimately proves the presence of a turbulence flow pattern within the trabecular meshwork. According to the theory correlating myocardium non-compaction with endoventricular thrombus formation, blood stasis into the left ventricle wall recesses is likely to be the pathophysiologic link between these events by favouring coagulation activation. But the presence of peritrabecular fibrosis refutes this theory and the assumption on which it is based. Therefore, it is reasonable to infer that non-compaction in itself does not predispose to the endoventricular thrombosis. 69 Giovanni Fazio, Ivana Basile, Monica Lunetta, Luciana DвЂ™angelo, Loredana Sutera, Emiliano Maresi, Giuseppina Novo, Aldo Gerbino, Salvatore Novo References [1] Sahin S, Sekban A, Ayalp S, Karsidag S. An Unusual Cause of Cardioembolic Stroke: Isolated Left Ventricular Noncompaction. Neurologist 2008; 14(2):125-127. [2] Ritter M, Oechslin E, Sutsch G, Attenhofer C, Schneider J, Jenni R. Isolated noncompaction of the myocardium in adults. Mayo Clin Proc 1997;72:26-31. [3] Hascelik S, Yalnizoglu D, Kafali G, Celiker A,Cila A, Topcu M, Gurgey A. Stroke owing to noncompaction of myocardium. J Child Neurol 2003;18:437-9. [4] Stollberger C, Finsterer J. Thrombi in left ventricular hypertrabeculation/noncompaction - Review of the literature. Acta Cardiol 2004;59:341-4. [5] Stollberger C, Finsterer J. Left ventricular hypertrabeculation/noncompaction and stroke or embolism. Cardiology 2005;103:68-72. [6] Fazio G, Pipitone S, Iacona MA, MarchГ¬ S, MongiovГ¬ M, Zito R, Sutera L, Novo G, Novo S. Evaluation of diastolic function by the Tissue doppler in children affected by non-compaction. Int J Cardiol 2007;116:e60-2. [7] Fazio G, Sutera L, Corrado G, Novo S. The chronic heart failure is not so frequent in non-compaction. Eur Heart J 2007; 28:1269. [8] Fazio G, Corrado G, Pizzuto C, Zachara E, Rapezzi C, Sulafa AK, Sutera L, Stollberger C, Sormani L, Finsterer J, Benatar A, Di Gesaro G, Novo G, Cavusoglu Y, Baumhakel M, Drago F, Carerj S, Pipitone S, Novo S. Supraventricular arrhythmias in non-compaction of left ventricle: Is this a frequent complication? Int J Cardiol 2007. [9] Corrado G, Zachara E, Rapezzi C, Sulafa AK, Sutera L, Pizzuto C, Stollberger C, Sormani L, Finsterer J, Benatar A, Di Gesaro G, Cascio C, Cangemi D, Cavusoglu Y, Baumhakel M, Drago F, Carerj S, Pipitone S, Novo S. Ventricular tachycardia in non-compaction of left ventricle: is this a frequent complication? Pacing Clin Electrophysiol 2007; 30:544-6. [10] Fazio G, Sutera L, Vernuccio F, Fazio M, Vernuccio D, Pizzuto C, Di Gesaro G, Cascio C, Novo S. Heart failure and cardiomyopathies: a case report. G Ital Cardiol 2007; 8:129-32. 70 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 The Chromogranin A-derived peptides: structural and functional features in heart biology [Peptidi derivati dalla cromogramina A: caratteristiche funzionali e strutturali relative alla biologia del cuore] Valentina Di Felice*, Antonella Montalbano, Angela De Luca, Filippo Macaluso, Antonella Marino Gammazza, Francesco Cappello and Giovanni Zummo Human Anatomy Section вЂњE. LunaвЂќ, Department of Experimental Medicine, University of Palermo (IT) Key words: Vasostatin I, Vasostatin II, Natural peptides, Neuroendocrine tissue, Heart physiology Parole chiave: Vasostatina I, Vasostatina II, Peptidi naturali, Tessuto neuroendocrino, Fisiologia del cuore . Abstract Chromogranin A (CGA) is a 48 kDa acidic protein co-stored in secretory granules of several endocrine and neuroendocrine cells with other compounds and it is co-released with them by exocytosis. Either inside the granules or in extracellular fluid or into circulation, CGA is cleaved by tissue-specific proteolytic enzymes into smaller peptides with different biological activities. High levels of plasma CGA have been correlated to endocrine and neuroendocrine tumours, renal failure and heart failure and it has been recently demonstrated that CGA is produced not only by the diffuse neuroendocrine cells but also by cells of different embryologic origins. Moreover, new effects of CGA-derived peptides come out every day. The main CGA-derived peptides correlated to heart biology are Pancreastatin (PST) and Vasostatins (VSs). PST is a 49-amino-acid peptide, corresponding to bovine CGA248-293 and human CGA240-288, able to stimulate secretion in neonatal rat atrial cells. Vasostatin I (VS-I) and Vasostatin II (VS-II) correspond to the N-terminal fragments CGA1-76 and CGA1-113 respectively, and seem to be the best cardioregulatory peptides in mammals, since they counteract the ОІ-adrenergic-mediated positive inotropism in vertebrate hearts. These findings let us suppose that CGA and CGA-derived peptides may play an autoregolatory role in heart secretion. This review would be a summary of the possible relationships among CGA and CGA-derived peptides and heart physiology, biology and pathology. . Riassunto La cromogranina A (CGA) ГЁ una proteina acida di 48 kDa conservata nei granuli di secrezione di diverse cellule endocrine e neuroendocrine insieme ad altre sostanze e viene rilasciata insieme ad esse per esocitosi. Sia allвЂ™interno dei granuli o nel fluido extracellulare o nella circolazione, la CGA viene tagliata da enzimi tessuto-specifici in peptidi piГ№ piccoli che possiedono svariate attivitГ biologiche. Alti livelli plasmatici di CGA sono stati correlati a tumori endocrini e neuroendocrini, ad alterazioni renali e cardiache ed ГЁ stato recentemente dimostrato cha la CGA viene prodotto non solo dalle cellule del sistema neuroendocrino diffuso ma anche da cellule di diversa derivazione embriologica. Inoltre, vengono scoperti ogni giorno nuovi effetti di peptidi derivati dalla CGA. I principali peptidi derivati dalla CGA, correlati con la biologia del cuore, sono la Pancreastatina (PST) e le Vasostatine (VSs). La PST ГЁ un peptide di 49 aa, corrispondente alla CGA248-293 bovina e alla CGA240-288 umana, capace di stimolare la secrezione nelle cellule striali di ratto neonato. La Vasostatina I (VS-I) e la Vasostatina II (VS-II) corrispondono ai frammenti N-terminali CGA1-76 e CGA1-113 rispettivamente, e sembrano essere i migliori peptidi cardioregolatori nei mammiferi, dal momento che sono in grado di contrastare 71 Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso, Antonella Marino Gammazza, Francesco Cappello, Giovanni Zummo lвЂ™inotropismo positivo mediato dai recettori b-adrenergici nei cuori dei vertebrati. Queste scoperte ci lasciano supporre che la CGA e i peptidi da essa derivati possano svolgere un ruolo regolatore nella secrezione cardiaca. Questa вЂњreviewвЂќ rappresenta un riassunto delle possibili relazioni esistenti tra la CGA ed i suoi peptidi, e la fisiologia, la biologia e la patologia del cuore. Introduction Chromogranin A (CGA) is an acidic secretory protein detected in several endocrine and neuroendocrine tissues, co-stored and co-secreted with catecholamines. CGA undergoes tissue-specific proteolytic events, and it gives rise to several derived peptides with a wide range of functions and tissue targets, acting in an autocrine, paracrine and endocrine manner. High levels of CGA have been detected, also, in pathological conditions regarding the neuroendocrine, the renal and the cardiac system, postulating its possible involvement in the patho-physiology of these diseases. The purpose of this mini-review is to correlate the old and new knowledge about CGA and its derived-peptides with heart biology and physio-pathology, suggesting a modulation and cardiac-protective role for these molecules. CGA and CGA derived peptides: structural and functional features CGA is a 48 kDa acidic water-soluble protein, identified for the first time by K. Helle in 1966 in the secretory granules of bovine adrenal medullary chromaffin cells, and successively detected also in other endocrine and nervous tissues. It represents the 40% of the soluble proteins of the granules, is co-stored with other granule secretory molecules such as catecholamines, enzymes, neurotransmitters, calcium, and is co-released by exocytosis upon splancnic nerve acetylcholine stimulation. Only after twenty years from its discovery, two distinct groups obtained for the first time the primary sequence of bovine CGA (see Figure1) [3, 11]. The analysis of the primary sequence shows many important features: a signal sequence, composed of the first 18 amino acid residues of the primary pro-peptide transcript, targeting it to rough endoplasmic reticulum membrane for maturation; numerous polyglutamic acid clusters in several regions of the molecule conferring a net negative charge, and an isoelectric point at about 5; two cysteine residues at positions 17 and 38 forming a disulphide bridge important for many CGA activities; an RGD sequences at the residues 43-45, probably implicated in adhesive processes between the cells and the extracellular matrix; several sites of post-translational modifications, such as O-glicosilation, phosphorilation and sulphation; twelve cleavage sites recognized by endopeptidase enzymes, five of them situated in the N-terminus domain and the others in the C-terminus. CGA seems to have a double function: intracellulary, it binds both calcium and cate72 The Chromogranin A-derived peptides: structural and functional features in heart biology cholamines and it regulates the granule biogenesis acting as an вЂњon/off switchвЂќ; extracellulary, it acts as pro-hormone. CGA is important as a whole molecule, but now they are becoming very important the several smaller peptides derived from this secretory protein, which showed to have different autocrine, paracrine or endocrine biological functions. In fact, CGA can be degraded by specific proteases (such as Prohormone convertases) either inside the granules, outsides in the extracellular environment or in the blood stream. This process and the fragments derived have a tissue- and specie-specific pattern. The peptides derived from the cleavage of bovine CGA and their functions are listed in Table 1. Vasostatins correlation with the heart: old and new findings CGA and its derived fragments have been detected also in the cardiac tissue. Recently, Glattard and colleagues [8] investigated the pattern of N-terminal CGA derived peptides in the cardiac tissue, revealing the presence of several VS-I- and VS-II-containing CGA fragments. Vasostatin-I (VS-I) and Vasostatin-II (VS-II) derive from the cleavage at the first two pair of basic amino acids of the N-terminal domain of CGA, respectively K77-K78 and K114-R115 [15], giving rise to two peptides corresponding to the fragments CGA1-76 and CGA1-113 [7]. The prediction of the secondary structure of VS-I, the most abundant natural N-terminal CGA fragment, is compatible with an О±-helical orientation and shows three anphipatic domains: two in the N-terminal region, including the fragment CGA1-16 and the fragment CGA17-38 containing the cysteine-bridged loop, and the third in the C-terminus comprehending the fragment CGA47-66. Moreover, the latest was well characterized and named chromofungin for its capability to cross the fungi membranes blocking their growing. These fragments were named вЂњVasostatins (VSs)вЂќ because of their first role discovered: an inhibition of the vasoconstriction, evoked by the potent vasoconstrictor ET-1, on isolated segments of human internal thoracic artery and saphenous vein [1]. Subsequent evidences suggested a regulatory role for VSs acting via autocrine, paracrine or endocrine mechanisms in function of the cell/tissue target and the local peptide concentration [10]. VSs, in fact, promote the adhesion of fibroblasts and smooth muscle cells on extracellular matrix, exert autocrine inhibition of parathyroid hormone secretion in parathyroid cells, have an antimicrobial activity and, finally, have an effect on heart [20]. It has been well established that VSs exert a general negative effect on the physiology of eel, frog and rat hearts [2,6]. VSs and several other smaller CGA-N-terminal fragments (i.e.CGA7-57 , CGA1-40 , CGA4-16 CGA47-66), in fact, have been tested on isolated and perfused working hearts both in basal and isoproterenol (ISO)-stimulated conditions mimicking a ОІ-adrenergic response. They acted modulating negatively the hearts inotropism on basal condition and counteracting the positive inotropism stimulated by the ОІ-adrenergic agonist 73 Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso, Antonella Marino Gammazza, Francesco Cappello, Giovanni Zummo ISO. This effect seems to be due to the fragment containing the disulfide-bridge loop, because the N- terminal fragments CGA4-16 and CGA47-66 are able to counteract the ISO-stimulated inotropy only at higher concentration than the peptide CGA1-40 with the intact loop [19]. Moreover, VS-II shows a lower inotropic potency than VS-I, probably because of a different steric conformation. Both in eel, frog and rat hearts VS cardio suppressive effect depends on extracellular calcium influx, as previously showed by Aardal and Helle [1] in saphenous vein, and potassium channels. Furthermore, only in eel heart the intact endothelium is fundamental for VS activities. In fact, while in frog heart a treatment with Triton X-100 has no effect on VS activities, in eel heart it abolishes the VS-mediated inotropic effect. Moreover, recent studies clarified the role of the cytoskeleton in VS signal, both in eel and frog hearts, being involved in several cellular functions, such as, for instance, the modulation of some ion channels (i.e. Calcium channels or Sodium channels) [14]. Using specific cytoskeleton inhibitors in concentrations which did not affect the functional integrity of the contractile cardiac machinery, it has been demonstrated a total block of the VS negative inotropism. Thus the cytoskeleton integrity plays a crucial role in the VS action. Nevertheless, VS interaction with the cells has not been discovered yet and the pathway or pathways affected by VSs have not been well characterised yet, neither in mammals nor in other vertebrates. The most recent studies, in fact, deal with the insight of the possible cell-interaction way and with the characterization of a precise cellular pathway triggered or affected by VSs. A classical and specific receptor for VSs has not been discovered yet, thus two possible different hypotheses of interaction with the cells could be taken into account [20]. The extracellular hypothesis proposes an interaction either with the membrane phospholipids or with some membrane proteins, for instance integrins, and a subsequent interference with the function of cellular effectors (I.E ion channels, receptors, enzymes). Blois and colleagues using a Langmuir film balance apparatus at 37В°C demonstrated, in vitro, an interaction between VS-I at low concentration with membrane phospholipids in physiological conditions. The electrostatic and hydrophobic interaction affected the film fluidity [4]. On the other hand, we studied the effects of VS-I on 3D-cultured primary cardiomyocytes establishing its influence in extracellular matrix-cell interactions affecting the localisation of intracellular proteins such as endothelial nitric oxide synthase (eNOS) and HSP 90 [20]. The intracellular hypothesis suggests an internalisation of VS-I and its action inside the cell, as it has been demonstrated using rhodamine-labelled CGA 47-70 on smooth muscle layer of rat posterior cerebral artery [13]. Nevertheless, further studies have to be performed to clarify which hypothesis is correct, or whether both could be considered as combined interaction ways. Another aim of the last studies was to detect the pathways involved in VS action. 74 The Chromogranin A-derived peptides: structural and functional features in heart biology Figure 1. Schematic rapresentation of the main features of bovine CGA structure. (S-S) Disulphide bridge; (P) Phosphorylation; (G) Glycosilation; (t) Cleavage site. Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso, Antonella Marino Gammazza, Francesco Cappello, Giovanni Zummo Figure 2. Hypothetical sites of action and pathways involving Vasostatin-I. (AC) Adenylyn Cyclate; (TnI) Troponin I; (TnC) Troponin C. The Chromogranin A-derived peptides: structural and functional features in heart biology Previously works carried out in eel and frog heart preparations, showed important differences in the pathways affected by VS-I, due to species-specific features. The implication of endocardial endothelium (EE) and the Nitric Oxide (NO)-cGMP signal transduction pathway seems to be essential in mediating VS effects only in eel heart [6, 18]. In fact, it has been demonstrated that the use of either NOS or soluble guanylate cyclase (GC) or cGMP-activated protein kinase (PKG) (an important NO target) inhibitors abolishes completely the effects of VS-I. The negative VS-inotropism can be explained considering that the NOS-dependent NO production increases the level of cGMP which activates PKG. PKG, on one hand, phosphorilates the troponin I and causes the decrease of the affinity of troponin C for calcium, regulating negatively the cardiac contraction; on the other hand, it phosphorilates the alpha-subunit of the Gi/0 protein, which negatively affects adenylyn cyclase, decreases the cAMP levels and finally inhibits the L-type Ca current. Lastly, both pathways elicit the negative regulation of the cardiac contraction. Recently, Cappello and colleagues [16] studied, for the first time, the pathways affected by VS-I in mammalian heart, using as a model the rat heart. Using specific inhibitors, they demonstrated the involvements of the same NOS-NO-cGMP-PKG system as in the eel heart. Gallo and co-workers [12] detected whether anti-adrenergic effects of VS-I occurs directly on cardiomyocytes or on other cell types of the cardiac tissue. They demonstrated that the synthesis of NO induced by VS-I takes places not in cardiomyocytes but in EE and it depends on Phosphoinositide-3 kinase (PI3K) activation. Role of CGA and CGA derived peptides in heart physio-pathology High serum levels of CGA have been correlated to several kind of neuroendocrine tumours for long time [9]. Recently high levels of CGA have been also reported in patients with heart failure and myocardial infarction [5]. Ceconi and co-workers correlated the increase of serum CGA with the chronic heart failure (CHF) [5], which is a syndrome characterized by neuroendocrine activation, in which the serum levels of catecholamines, natriuretic peptides and several peptides signalling system increase. These work demonstrated for the first time that serum CGA levels increased in patients with CHF and it was related to the clinical severity of the syndrome. The increase in CGA was found already in the early stages of the failure and it was a predictive factor of mortality. Moreover, it has been speculated that the high CGA levels may represent the response of the organism to counteract the effects of an excessive neuroendocrine activation. On the other hand, being CGA a component of the neuroendocrine system, the increase of its levels could be just the consequence of the over stimulation of the neuroendocrine system [5]. It has been well established that CGA either into the neuroendocrine vescicles or in the blood stream undergoes proteolytic events, giving rise to several CGA derived peptides 77 Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso, Antonella Marino Gammazza, Francesco Cappello, Giovanni Zummo showing autocrine and paracrine effects. Moreover, it has been demonstrated that the cardiac tissue, in both in normal and pathological condition, is able to produce CGA [17]. Thus, CGA and its derived peptides could play an auto-endocrine regulation on the heart, counteracting an excessive adrenergic stimulation in normal condition and especially in pathological condition correlated with their high plasma levels. In this view CGA shows a role in myocardial protection. Recently, it has been, in fact, proposed a preconditioninglike effect of VS-I, if given before ischemia/reperfusion and it has been demonstrated a reduction of the infarct size after the administration of VS-I before I/R. Furthermore, it has been established the involvement of, at least, two different pathways: the NO-cGMP-PKG pathway and the adenosine-pathway. Indeed, the inhibition of eNOS blocked totally this effect and on the other hand the inhibition of A1-receptors reduced it about 50% [16]. Little is still known about the role of CGA and its derived peptides in physio-pathology of the heart and further studies are necessary. However, these molecules, acting as neurormones, could play a central role in the cardiac physio-pathology and could represent the main target of the new pharmacological therapy. Acknowledgments We thank the University of Palermo, Italy, for a doctoral scholarship to Antonella Montalbano and the Ministero della UniversitГ e della Ricerca, for financial support (MURST ex 60% Prof. Giovanni Zummo). List of abbreviations CGA = Chromogranin A VS-I = Vasostatin I VS-II = Vasostatin II PST = Pancreastatin TNFО± = Tumor necrosis factor О± ET-1 = Endothelin-1 ANF = Atrial Natriuretic Factor PLC = Phospholipase C IP3 = Inositol 1,4,5 triphosphate DAG = Diacylglycerol PKC = Protein Kinase C ISO = Isoproterenol eNOS = Endothelial nitric oxide synthase EE = Endocardial endothelium NO = Nitric oxide GC = Guanylate cyclase 78 The Chromogranin A-derived peptides: structural and functional features in heart biology PKG PI3K CHF = = = cGMP-activated protein kinase Phosphoinositide-3 kinase Chronic Heart Failure Table I. Bovine CGA derived peptides and their main function Peptides Vasostatin I (CGA1-76) Vasostatin II (CGA1-113) (rat-ОІgranin (CGA1-128)) Chromofungin (CGA47-66) Chromostatin (CGA124-143) Chromacin (CGA173-194) Function Inhibition vasoconstriction Promotion of adhesion Inhibition parathyroid hormone secretion Antimicrobial effects Negative inotropism Antimicrobial effects Inhibition chromaffin cells secretion Antimicrobial effects Pancreastatin (CGA248-293) Inhibition of glucose-stimulated insulin secretion Take part in lipid and glucose metabolism WE-14 (CGA316-331) No Data Parastatin (CGA347-419) Inhibition of parathormone and CGA secretion from parathyroid cells Catestatin (CGA344-364) Inhibition of catecholamine secretion; promotion histamine release in mast cells 79 Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso, Antonella Marino Gammazza, Francesco Cappello, Giovanni Zummo References [1] Aardal S, et al. 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Chromogranin A N-terminal fragments vasostatin-1 and the synthetic CGA 7-57 peptide act as cardiostatins on the isolated working frog heart. Gen Comp Endocrinol 2004; 136(2): 217-224. [7] Corti A, et al. Production and structure characterisation of recombinant chromogranin A N-terminal fragments (vasostatins) - evidence of dimer-monomer equilibria. Eur J Biochem 1997; 248(3): 692-699. [8] Glattard E, et al. Characterization of natural vasostatin-containing peptides in rat heart. Febs J 2006; 273(14): 3311-3321. [9] Gratton J P, et al. Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro. Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1. J Biol Chem 2000; 275(29): 22268-22272. [10] Helle KB, Metz-Boutigue M-H, and Aunis D. Chromogranin A as a Calcium-Binding Precursor for a Multitude of Regulatory Peptides for the Immune, Endocrine and Metabolic Systems. Curr Med Chem Immun, Endoc & Metab Agents 2001; 1: 119-140. [11] Iacangelo A, et al. Bovine chromogranin A sequence and distribution of its messenger RNA in endocrine tissues. Nature 1986; 323(6083): 82-86. [12] Malan D, et al. Microtubules mobility affects the modulation of L-type I(Ca) by muscarinic and betaadrenergic agonists in guinea-pig cardiac myocytes. J Mol Cell Cardiol 2003; 35(2): p. 195-206. [13] Mandala M, et al. Chromogranin A-derived peptides: interaction with the rat posterior cerebral artery. Regul Pept 2005; 124(1-3): 73-80. [14] Mazza R, et al. Crucial role of cytoskeleton reorganization in the negative inotropic effect of chromogranin A-derived peptides in eel and frog hearts. Regul Pept 2007; 138(2-3): 145-151. [15] Metz-Boutigue M H, et al. Intracellular and extracellular processing of chromogranin A. Determination of cleavage sites. Eur J Biochem 1993; 217(1): 247-257. [16] Pagliaro P, et al., Protection against ischemia/reperfusion injures by vasostatin-1 in the isolated rat heart, in вЂњPhysiological Society MeetingвЂќ. 2005: University of Oxford, UK. [17] Pieroni M, et al. Myocardial production of chromogranin A in human heart: a new regulatory peptide of cardiac function. Eur Heart J 2007; 28(9): 1117-1127. [18] Tota B, et al. Vasostatins and Negative Inotropy in Vertebrate Hearts. Curr Med Chem вЂ“ Immun, Endoc & Metab Agents 2004; 4: 195-201. [19] Tota B, et al. Peptides from the N-terminal domain of chromogranin A (vasostatins) exert negative inotropic effects in the isolated frog heart. Regul Pept 2003; 114(2-3): 123-130. [20] Tota B, et al. New biological aspects of Chromogranin A-derived peptides: Focus on vasostatins. Comparative Biochemistry and Physiology (Part A) 2007; 147: 11-18. 80 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise [Ruolo delle MMPs di matrice nellвЂ™angiogenesi del cuore indotta dallвЂ™esercizio fisico] Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina Dipartimento di Medicina Sperimentale, sezione di Anatomia Umana вЂњE. LunaвЂќ, UniversitГ degli Studi di Palermo (IT) Key words: Angiogenesis, Exercise, Heart, MMPs Parole chiave: Angiogenesi, Esercizio fisico, Cuore, MMPs Abstract. Angiogenesis induced by exercise has been observed in both cardiac and skeletal muscle and plays a fundamental role in maintaining tissue function adequate to the increase in metabolic requests. Mechanical and haemodynamic forces are strong starters of angiogenic process via regulation of secondary mediators such as vascular endothelial growth factor (VEGF). A crucial step of vessel sprouting is the degradation of the basement membrane and remodelling of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs). It has long been accepted that MMPs are involved in the angiogenesis, but the exact mechanisms are not well characterized. Cryptic fragments and neo-epitopes released by proteolysis of basement membrane components by MMPs can promote or block capillary growth. MMPs are also involved in the release and activation of growth factors embedded into ECM such as VEGF. In turn, it has been showed in vitro that VEGF can stimulate endothelial and smooth muscle cells to produce MMPs suggesting a reciprocal relationship between VEGF and MMPs. The expression patterns of MMPs are temporally dissociated by those of VEGF in vivo: MMPs and VEGF seem to be independently regulated and involved respectively in sprout formation and capillary proliferation. Little is known about the regulation of MMPs by exercise and the most studies were carried out in the skeletal muscle. The increase in MMP activity is an early and critical feature of angiogenesis in skeletal muscle when initiated by mechanical stimuli which lead to breakage of the basement membrane but not by stimuli such as shear stress that act via the luminal surface of vessels. Although MMPs seem to play a important role in sprouting angiogenesis induced by exercise, in our knowledge their function in exercised hearts has not been still tested. It is known that elevated levels of tissue inhibitors of the MMPs (TIMPs) may contribute to the accumulation of collagen content in the infarcted heart leading to myocardial fibrosis. Therefore, a positive modulation of MMP system by exercise in heart failure-related myocardial remodelling could lead to a ECM with a less fibrous component and more angiogenic sprouting in order to preserve the cardiac function. Riassunto. LвЂ™angiogenesi ГЁ stata osservata nel miocardio e nel muscolo scheletrico in risposta allвЂ™esercizio fisico e riveste un ruolo fondamentale nel mantenere lвЂ™attivitГ del tessuto adeguata allвЂ™aumento delle richieste metaboliche. Le forze meccaniche ed emodinamiche sono potenti iniziatori del processo angiogenico mediante la regolazione di mediatori secondari, come il fattore di crescita vascolare endoteliale (VEGF). Una fase cruciale per la formazione di nuovi capillari ГЁ la degradazione della membrana basale e il rimodellamento della matrice extracellulare (ECM) da parte delle metalloproteasi di matrice (MMPs). Sebbene sia noto da tempo che le MMPs sono coinvolte nellвЂ™angiogenesi, gli esatti meccanismi non sono stati ancora ben caratterizzati. Frammenti criptici e neo-epitopi rilasciati mediante la proteolisi dei componenti della 81 Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina membrana basale da parte delle MMPs possono promuovere o inibire la crescita capillare. Le MMPs sono anche coinvolte nel rilascio e nellвЂ™attivazione di fattori di crescita intrappolati nella ECM cosГ¬ come il VEGF. A sua volta, ГЁ stato dimostrato in vitro che il VEGF puГІ stimolare le cellule endoteliali e le cellule muscolari lisce a produrre le MMPs suggerendo una relazione reciproca tra il VEGF e le MMPs. I profili di espressione delle MMPs sono temporalmente dissociati da quelli delle MMPs in vivo: le MMPs e il VEGF sembrano essere regolati indipendentemente e coinvolti rispettivamente nella gemmazione del nuovo vaso e nella proliferazione capillare. Poco ГЁ noto circa la regolazione delle MMPs da parte dellвЂ™esercizio fisico e la maggior parte degli studi sono stati condotti sul muscolo scheletrico. LвЂ™incremento dellвЂ™attivitГ delle MMPs ГЁ una caratteristica cruciale e precoce dellвЂ™angiogenesi nel muscolo scheletrico qualora innescata da stimoli meccanici che portano alla rottura della membrana basale; tuttavia non da stimoli quali le forze da taglio che agiscono attraverso la superficie del lume dei vasi. Sebbene le MMPs sembrino rivestire un ruolo importante nellвЂ™angiogenesi indotta dallвЂ™esercizio fisico, nellвЂ™ambito della nostra conoscenza la loro funzione nei cuori allenati non ГЁ stata ancora studiata. Г€ noto che livelli elevati degli inibitori tissutali delle MMPs (TIMPS) possano contribuire allвЂ™accumulo del contenuto di collagene nei cuori infartuati portando a fibrosi del miocardio. Pertanto, una modulazione positiva del sistema delle MMPs da parte dellвЂ™esercizio nel rimodellamento del miocardio con insufficienza cardiaca potrebbe condurre ad una ECM con una minore componente fibrosa e con una maggiore angiogenesi tale da garantire la funzionalitГ del miocardio. Introduction Angiogenesis is the formation of new blood vessels from existing ones. A fundamental step of this process is the degradation of the basement membrane and remodelling of the extracellular matrix (ECM) in order to allow endothelial cells to migrate into the extravascular space where they proliferate and form abluminal sprouts which become functional on connection to other segments of capillary network. Subsequently, the sprouts acquire newly formed basement membrane and become surrounded by pericytes. For the any components of matrix, there are a large number of enzymes controlling stroma turnover. One important group are the matrix metalloproteinases (MMPs) that regulate the degradation of components of the ECM in conjunction with specific inhibitors (TIMPs) whose net effects determine the structure of the ECM at any particular point in time [1]. This process of ECM degradation need to be highly controlled and coordinated because unguarded dissolution of the matrix would result in a loss of the integrity and function of the microvasculature. Angiogenesis process occurs in the healthy adult organism in specific situations including endocrine changes such as in the female reproductive organs, hemodynamic and mechanical stress and humoral stimuli in response to exercise [2]. Capillary growth induced by exercise has been observed in both cardiac and skeletal muscle and it results to be essential for maintaining tissue function adequate to increased metabolic demand [3]. The present review points to the regulation of MMPs by exercise in the cardiac angiogenesis and potential implications that regular and prolonged exercise could have on the heart failure-related ECM turnover. 82 New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise Description of ECM components in cardiac tissue The myocardial ECM is a dynamic structure whose component proteins are maintained by a finely controlled homeostatic balance between deposition and degradation. Within the myocardium, myocytes are linked by an intricate lattice of ECM proteins which provide structural integrity to the tissue and a means for optimal vectoral transmission of force. Although myocytes occupy two thirds of the tissue volume, 70% of cells within the myocardium are fibroblasts [4]. The primary role of cardiac fibroblasts (CFs) appears to be the synthesis of ECM components and regulation of their turnover by the secretion and activation of proteolytic enzymes such as the MMPs. The lattice surrounding the myocytes includes a complex network of structural proteins (collagen and elastic fibers) and adhesive proteins (fibronectin, laminin) within a hydrated proteoglycan and glycosaminoglycan-rich milieu [5]. Fibrillar collagens are the most abundant of the ECM proteins and at least 28 distinct collagen types are expressed in vertebrates. Within the myocardium, collagen types I, III, IV, V, VI and VIII have been identified [6]. The fibrillar collagen types I and III coexist to form the collagenous network of the myocardium comprising 85% and 11% of the collagen in healthy mammalian hearts respectively [7]. Type I collagen fibrils have an extremely high tensile strength and in combination with the giant sarcomeric muscle protein, titin, largely determine the stiffness of the myocardium [8]. Type III collagen is more distensible than type I by comparison and is argued to maintain the structural integrity of the network. Other less abundant collagen types of the myocardium are types IV, V, VI and VIII. Type IV is expressed by myocytes and endothelial cells and does not form fibrils but instead assembles into an open network within basement membranes [7]. Type V coexists with type IV collagen in the basement membrane whilst both types V and VI collagens co-localize with collagen I and III fibrils in the interstitium. Type V appears to play contrasting roles to other collagens, as it is thought to inhibit endothelial cell adhesion and proliferation [9]. Type VIII collagen expression is predominant during development, yet is detectable at very low levels in the adult myocardium and is likely to play a role in the morphogenesis of cardiac valves [10]. To date, a full understanding of the roles of these less abundant collagens in the heart remains to be realized. Collagenous matrices exhibit high levels of resistance to both tensile forces and proteolytic degradation. These features are enhanced by the process of cross-linking which provides a final step in the maturation of collagen fibrils. Robinson et al. (1988) have identified elastic fibers associated with the coiled perimysial fibres of the collagen fibers axis and suggested that the elastic fibers counteract stretch in the myocardium and are responsible for passive recoil [11]. Fibronectin, laminin and collagen type IV not only constitute the structural support of the basement membranes, but are also involved in signalling and coupling the myocyte to the collagenous ECM scaffold [12]. 83 Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina Involvement of MMPs in the capillary growth MMPs, also called matrixins, are a family of zinc-containing endopeptidases that are capable of degrading various components of the ECM. There are two main types of MMPs: those that are secreted into the extracellular space and those that are membrane bound. The secreted MMPs are synthesized as inactive pro-enzymes and are activated by cleavage of the regulatory peptide sequence. This cleavage determines a conformational change and exposure of the catalytic domain to ECM substrate. Activation of MMPs is achieved by serine proteases, such as plasmin, and other MMPs. The membrane-type MMPs, or MTMMPs, undergo intracellular activation and are proteolytically active once inserted into the cell membrane. MT-MMPs constitute an important pathway for activation of other MMPs within the ECM [13]. Another important control point of MMP activity is through the presence of tissue inhibitors of the MMPs, called TIMPs. Four different TIMP species have been identified and bind to activated MMPs in a 1:1 stoichiometric ratio [14]. By contrast, MTMMPs do not appear to bind and be regulated by TIMPs. It has long been accepted that MMPs play an important role in angiogenesis, but the exact mechanisms are not well characterized. Recently, cryptic fragments and neo-epitopes released by proteolysis of basement membrane components result to be involved in the promoting or blocking angiogenesis (Table 1). For instance, both MMP-2 and MMP-9 expose a cryptic epitope within collagen IV that promotes angiogenesis. Fragments generated from NC1 domain of collagen IV, VIII, XV and XVIII can be released by cleavage involving MMPs and act as anti-angiogenic factors. Other components of basement membrane, such as laminin-5 and fibronectin, also harbor cryptic sites that are release by MMP activity (MMP-2, -3, -12, -13 and -14) and fragments generated can induce cell migration [15]. However, the mechanisms responsible for the release of all these fragments have not been clearly elucidated. MMPs are also involved in the release and activation of growth factors and cytokines embedded into ECM [1]. Vascular endothelial growth factor (VEGF) and transforming growth factor-ОІ (TGF-ОІ) represent two examples of such factors that are stored within the ECM and can be released by MMP proteolysis increasing their bioavailability. VEGF is involved in the process of angiogenesis because it enhances vascular permeability and promotes new vessel growth. Two different tumor models implicate MMP-9 in the release of VEGF from the ECM. In the insulinoma model, release of VEGF by MMP-9 is associated with an angiogenic switch, which promotes tumor progression [16]. In an ovarian carcinoma model, activated MMP-9 and, to lesser extent, MMP-2 are involved in increased bioavailability of VEGF in culture [15]. Even though these are tumor models, it is highly likely that similar mechanisms for the release of VEGF, leading to increased angiogenesis, occur in non-pathological conditions. Furthermore, VEGF can stimulate endothelial and 84 New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise smooth muscle cells to produce MMPs in vitro to facilitate migration and tube formation. All this suggests that a reciprocal relationship between VEGF and MMPs in different cell types in vitro exists. In vivo, much less is known about interactions between MMP activities and VEGF expression during angiogenesis, where temporal patterns of expression may differ from in vitro. Rivilis et al. (2002) showed that the increase in MMP preceded VEGF in response to skeletal muscle stretch whereas with stimulation, it lagged behind VEGF [17]. Moreover, during treatment with prazosin vasodilator, VEGF was increased very early on when MMP expression was actually slightly decreased. MMPs are unlikely to be important for endothelial proliferation since this proceeded unaltered in stimulated muscles treated with a MMP inhibitor whereas sprout formation was suppressed [18]. VEGF, on the other hand, might play a more prominent role in capillary proliferation as increases in its expression were dissociated temporally from those of MMPs. Thus, these factors appears to be regulated independently in vivo. Cardiac angiogenesis in response to exercise: role of MMPs In the literature there are controversial data about heart angiogenesis in response to exercise. It has been shown that various variables such as the age of the animals, kind and intensity of exercise may affect the coronary capillary growth. It is clear, however, mechanical and haemodynamic forces are strong starters of angiogenic process. For istance, the increased myocardial stroke volume and contractility induced by dobutamine mechanically can vary the capillary stretch and spreading throughout the cardiac cycle. In the same way, the increase in the coronary blood flow, stroke volume and contractility induced by exercise training may increase the shear stress, wall tension, capillary stretch and compression forces on vessels [19]. It has been reported that these stimuli can initiate angiogenesis process via regulation of secondary mediators such as VEGF. Indeed, several authors found that VEGF is up-regulated by ventricle wall stretching and increased dyastolic pressure [20,21]. Angiogenesis induced by exercise training appears to occur by vessel sprouting rather than intussusception process. Angiogenic sprouting consists into branching out of activated endothelial cells from an existing capillary, extending through the degraded surrounding matrix to form a cordlike structure [2]. Little is known about the regulation of MMPs by exercise and most studies were carried out in the skeletal muscle. In electrically stimulated skeletal muscles, total and active MMP-2 protein increased after 7 days coinciding with the appearance of angiogenic sprouts [18]. Both stretched and stimulated, but not vasodilator treated, muscles also showed increased expression of mRNA and protein for MMP-2 and MT1-MMP [17,18]. Moreover, inhibition of MMP activity prevented the growth of new capillaries in stimulated 85 Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina muscles; instead it did not affect the capillary-associated cell proliferation. These findings point to increased MMP activity as an early and critical feature of angiogenesis in skeletal muscle when initiated by mechanical stimuli which lead to breakage of the basement membrane but not by stimuli such as shear stress that act via the luminal surface of vessels. In addition, capillary-associated cell proliferation seems to precede and occurs independently of endothelial cell sprout formation [18]. In stretched muscles, lengthening of capillaries by changes in sarcomere length might be the main stimulus whereas in stimulated muscles, the repetitive shortening and relaxation of myocytes, and the 2-fold increase in capillary pressure and hence wall tension could act in combination. Moreover, cyclic strain applied to rat microvascular endothelial cells led to time-dependent upregulation of MT1-MMP mRNA and protein indicating the importance of physical tensile forces in the regulation of extracellular matrix degrading factors [19]. Although MMPs seem to play a important role in sprouting angiogenesis induced by exercise, in our knowledge their function in exercised hearts has not been still tested. Verzola et. al. studied the effect of acute swimming training with anaerobic component on MMP activity and myosin heavy chain gene expression in the rat myocardium. They found an increase in expression and activity of MMP-2 72, 96 and 120 hours after the beginning of exercise training suggesting precocious heart remodeling. This modification was associated with myocardium hypertrophy induced by training protocol suggesting that increased MMP activity is necessary to allow tissue growth in order to support the enhanced metabolic requests during exercise. However, vascular remodelling that might have contributed to the significant increase in heart weight has not been examined in this study [22]. In our previous work, we found that mouse heart hypertrophy induced by endurance training was significantly due to an increase in the capillary growth rather than increased myocardiocyte mass [23]. Moreover, we showed that both expression of VEGF receptors and MMP-2 and MMP-9 activity were involved in the angiogenic process of exercised mouse hearts and were differently regulated by intensity and time of training [24]. Exercise training is an important complementary intervention for heart health The benefits of exercise in terms of reduced risk of coronary disease and mortality are widely recognized. There is no doubt that the direct effects of repeated bouts of exercise on the myocardium вЂ“ improved contractile function, increased end-diastolic dimensions, resting bradycardia, induction of protective heat shock proteins, altered energy substrate utilization and improved anti-oxidant capacity [25] вЂ“ contribute significantly to the enhanced ventricular performance of the trained heart. Moreover, it is known that physical training programs are beneficial to the heart because chronic exercise increases myocardial vascularity and protects the myocardium against ischemic insult [26]. This highlights 86 New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise the significance of coronary vascular growth and remodelling as intrinsic to the protective benefits of exercise. Brown et al. (1994) showed a increase in the capillary density in pigs hearts with a region of infarction about 15% of the left ventricular free wall during induced bradycardia. After 4 weeks, capillary density was significantly reduced in the border zone of the infarct compared with that in undamaged left ventricle of the same hearts or control sham-operated pig hearts. When infarcted pig hearts were electrically paced to bradycardia for 4 weeks, maintaining a basal heart rate of 60вЂ“80 beats min-1 compared with around 110 beats min-1 in unpaced infarcted or normal conscious pigs, capillary densities in both the border zone and the undamaged left ventricle were significantly greater. This enlargement of the capillary bed most likely enabled more homogeneous tissue perfusion, contributing to the improvement in cardiac function of paced infracted hearts during inotropic challenge. Proliferation of capillaries after pacing was confirmed in non-infarcted hearts by an increase in proportion of those labelled by a cell proliferation marker [27]. In agreement with what has been said the knowledge of the mechanisms of vascular growth in response to exercise may be used to target specific portions of the coronary vascular tree for therapeutic purposes. A positive modulation of MMP system by exercise in heart failure-related myocardial remodelling could be an important complementary intervention. It is generally accepted that ECM and myocardial maladaptive is a unifying feature in the progression to heart failure [28]. For this reason an exercise-related reduction of TIMPs expression may improve the balance between MMPs and TIMPs and enhance the proteolytic activity, thus decrease the collagen accumulation in post-infarcted myocardium [29]. Indeed, Webb et al. (2006) demonstrated that TIMP-1 levels were higher at day 1 post-infarction and remained substantially elevated through day 180 in patient with myocardial infarct. The elevated TIMP-1 level may contribute to the accumulation of collagen content in the infarcted heart leading to myocardial fibrosis [30]. Moreover, a ECM with a less fibrous component could improve sprouting angiogenesis in response to exercise, thereby preserve the cardiac function. Conclusions Little is known about the signals that promote vascular growth and remodelling in the heart in response to exercise. It is still necessary to study as hearts that are trained and/ or affected by angiogenic stimuli such as haemodynamic and mechanical stresses (typical of physical activity) may improve angiogenesis. Furthermore, although MMP system appears to be involved in one of essential step of angiogenesis process, there are no data about their function in cardiac angiogenesis induced by exercise training. For this reason, it could be interesting to study the expression and activity of cardiac MMPs and TIMPs and their regulation by exercise. Moreover, at the present it is not possible to prescribe with 87 Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina adequate precision the kind, duration and intensity of exercise that may promote vascular growth. Therefore, developing the knowledge about the regulation of MMP system by exercise might allow to draw up specific training protocols for improving performance of healthy and diseased hearts. Table 1. Cryptic basement membrane fragments involved in angiogenesis ECM protein Fragment Function Collagen IV Cryptic epitope Angiogenic Collagen IV NC1 a1, arresten Anti-angiogenic NC1 a 2, canstatin Anti-angiogenic NC1 a 3, tumstatin Anti-angiogenic NC1 a 6 Anti-angiogenic Collagen VIII NC1 a1, vastatin Anti-angiogenic Collagen XV NC1, restin Anti-angiogenic Collagen XVIII NC1, endostatin Anti-angiogenic Perlecan C-term, endorepellin Anti-angiogenic Modified from Mott JD and Werb Z 2004 [15] 88 New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise References [1] McCawley L, Matrisian L. Matrix metalloproteinases: theyвЂ™re not just for matrix anymore! Curr Opin Cell Biol 2001; 13:534-40. [2] Bloor CM. Angiogenesis during exercise and training. Angiogenesis 2005; 8: 263-271. [3] Hudlicka O, Brown MD, Egginton S.: Angiogenesis in skeletal and cardiac muscle. Physiol Rev 1992; 72: 369-417. 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Specific temporal profile of matrix metalloproteinase release occurs in patients after myocardial infarction: relation to left ventricular remodeling. Circulation 2006; 114(10):1020-7. 90 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 ORAL CAPILLAROSCOPY: OUR EXPERIENCE [Capillaroscopia orale: nostra esperienza] Francesco Carini1, Giuseppe Alessandro Scardina3, Gianfranco Cocorullo4, Pierluigi Varia1, Chiara Lo Piccolo1, Angelo Leone2, Pietro Messina3 and Vincenzo Valenza1 Department of Experimental Medicine, Section of Human Anatomy; 2Department of Experimental Medicine, Section of Histology and Embryology; 3Department of Dental Stomatological Sciences, Section of Dental Medicine; 4Department of General and Urgent Surgery, University of Palermo (IT) 1 Key words: Capillaroscopy, Vasculitis, Oral mucositis Parole chiave: Capillaroscopia, Vasculite, Mucosite orale Abstract. Capillaroscopy has now definitely become an important and indispensable exam in the area of diagnosing vasculopathies and in particular micro angiopathies. The type of damages connected to these anatomy pathologies regarding the actual micro vascular unit work in a functional pathogenic of an extreme importance, in conditioning the course and the eventual aggravation of micro angiopathies properly called vasculity and also of macroangiopathies. In all, this underlines the great importance achieved by using certain instrumental exams which complements the clinical research, exactly because of the valid elements which have been given for a complete and precise morphological valuation for the surrounding circulation conditions. Such techniques, like the laser Doppler or the pletismography do not let us express a prognostic verdict on eventual risks with tropic damages, which are instead valued directly on realistic times with the aid of oral capillaroscopy, of whose use has permitted new morphological and functional acquisitions. The unharmful, no-invocatory and easily repeatable test and its moderate cost, also allows us to monitor and up-date the future state of peripheral vasculopathies, as well as the efficiency of the medicines which have possibly been used. Riassunto. La capillaroscopia ГЁ entrata a pieno diritto tra gli esami diagnostici indispensabili nellвЂ™ambito delle vasculopatie e in particolare nelle microangiopatie. Le lesioni anatomo-patologiche a carico dellвЂ™unitГ microvascolare svolgono una funzione patogenetica estremamente importante, se non addirittura fondamentale, nel condizionare il decorso e lвЂ™eventuale aggravamento sia di microangiopatie propriamente dette, vasculiti, sia di macroangiopatie. Tutto ciГІ sottolinea il grande valore assunto da alcuni esami strumentali quale complemento allвЂ™indagine clinica, proprio perchГ© capaci di fornire elementi validi per una piГ№ completa e minuziosa valutazione morfologica e funzionale delle condizioni circolatorie distrettuali. Varie tecniche, come il laser doppler o la pletismografia non ci consentono di esprimere un giudizio prognostico sullвЂ™eventuale rischio di lesioni trofiche, che sono invece valutate direttamente in vivo e in tempo reale con lвЂ™ausilio della capillaroscopia orale, il cui uso ha permesso nuove acquisizioni morfologiche e funzionali. La non invasivitГ , lвЂ™innocuitГ , lвЂ™agevole ripetibilitГ dellвЂ™esame, unite ai costi relativamente contenuti consentono inoltre di monitorare nel tempo lвЂ™andamento delle vasculopatie periferiche, nonchГ© lвЂ™efficacia dei farmaci eventualmente impiegati. 91 Francesco Carini, Giuseppe Alessandro Scardina, Gianfranco Cocorullo, Pierluigi Varia, Chiara Lo Piccolo, Angelo Leone, Piero Messina and Vincenzo Valenza Morfological parametres The experiment research was effectuated with a preference on the bottom lip and the capillaries are visible like curves in the skin papilla. A normal person has parallel curvatures, formed in mainly upside U, a regular size, without a large dilatation; either way they occupy in a homogeneous density in their reserved space. The morphology of the curves, the number and size of the vases, the color, Morfological parameters in a capillaroscopic image the visualization of the vein system, of micro-haemorrhage and the together of intravascular permit a diagnosis and to have a major number of information on the condition of the microcirculation in functional arteriopathies such as diabetes and in a less specified way, in artery-sclerosis. The use of capillaroscopy has though a precise electivity in allowing a rapid diagnosis of dermato sclerosis in the presence of a pre existing functional arteriopathy. To also keep in mind is that an aneurismatic dystrophy in the extreme flexible part of the capillaries, mega capillaries, stasis, hemorrhages aggregated or prodigious with the mega capillaries, to the point of an ischemic skin disorder with a capillary deduction, there is a possibility of verifying a such picture of the various states in a patient with a dermato-sclerosis. The valuation of these morphologic parameters is complex and needs an attentive analysis with expert training. It is for this exact reason and we are obligated, that to value, according to certain objective parameters that we can distinguish between morphologies and functional . Between the morphologic parameters we can distinguish: visibility, morphology of the ansae, an orientation of the ansae, the density of the capillary, the length of the curves in the veins and their diameters. Functional parameters A fundamental innovative characteristic regarding capillaroscopy is the dynamic study in vivo of the circulation. An abnormal biochemical solicitation, turns into a functional breakdown, to a complete penalization of the contracting elements of the area pre-capillary, in an excessive or longer spasm of the post-capillary veins. The insufficient microcirculation that it becomes into is characterized by a progressive slowing down till stopping for a hyper aggregability of the elements in the blood. The conventional capillary permits one to distinguish either a continuous flow or the 92 Oral Capillaroscopy: our experience ribbon-shaped flow, a granulose flow being present a micro aggregation of erythrocytes, an intermittent characterized by a visualization of plasmatic spaces visibly empty. The Video Capillary using fluorescent techniques represent a superior realistic of the morphologic phase of the study of capillaroscopy; in fact, in a conventional stereo-microscope, even though being able to receive quite important aspects of the capillary flow, we cannot quantify and neither value the real grade of the capillaryвЂ™s permeability. This last comment could also be expressed, in suggestive terms, by the grade of an interstice edema. However, it can be distinguished that an ordinary or conventional capillaroscopy, useful in the valuation of the morphological parameters, and that of a dynamic one, aimed towards a functional research, which either can or not use fluorescent tracers. These permit an even more precise valuation in the way that the microcirculation unit replies to pharmacological or physical stimulations, being in a good state of health or in a bad one. A capillaroscopic exam and a bioptic exam In spite of the vast quantity of contributions in the worldвЂ™s literature offered by conventional and dynamic, the conceptual scheme regarding the skinвЂ™s and mucosaвЂ™s micro circulation have not changed all that much: clinging to a par cellular and limited vision of the capillaries morph structure of certain skin and mucosa areas. The characteristic вЂњhairpinвЂќ disposition started off a large study on morphological data, in an attempt to correlate structural modifications of the ascending and descending ansae or of the apex of the ansae, or of their number and of an optimum visualization, to pathologic situations of the micro circulation of the body in general. Much of this data has contributed effectively to a much better comprehension and a codification of certain queries diagnostic differentials, with a particular attention to certain syndromes them being organic or functional, also for micro angiopathies, such as diabetes, rheumatism, sclera dermas and hypertension. The very rapid evolution of the microcirculation acknowledgment request a conceptual correction during its course, by going back to morphological and structural acquisitions, but with an even more dynamic impression than usual. With this to have a better knowledge of the most important aspects on in vivo circulation and not just because of the bi-optical findings. To answer the question if or not more capillary-copy-cal images are really un-replaceable for a correct diagnosis, the answer is negative; we will attenuate, in fact, even them being more aggressive, that will give us a certified diagnosis regarding the parameters and relation, as in an histological; the figure of an anatomy pathologist would not be less in terms of diagnosis. Diagnosis damages to the microcirculation, with the use of capillaroscopy orally, in a practical way, can be made apart from the fact of a bi-optical exam; and itвЂ™s for this reason that capillaroscopy is generally classified as in vivo and not in vitro in contradiction with the the researches on the bioptical statistics, as a 93 Francesco Carini, Giuseppe Alessandro Scardina, Gianfranco Cocorullo, Pierluigi Varia, Chiara Lo Piccolo, Angelo Leone, Piero Messina and Vincenzo Valenza complementary and supportive research with the clinical diagnosis. To also have a proper characteristic which gives us secure information and immediately, on the dynamic of the blood flow in the artery system regarding the whole of the organism and gives us data on the organic and functions of the vase illness at the moment, because of presence or absence of aggregated erythrocytes, takes us to a significant and of a great merit diagnosis regarding the capillaroscopy, to assume one integrant and un-replaceable fact which is prognostic. The experiences reached with this technique outlines, not needing help, but an operative proposal based on objective data ,difficult to refute, that the re-activity of the micro-vascular tissue system has towards the endogens, especially with the pharmacologic, which is represented as the most reliable for the hoped for solution, of the prognostic query of the micro vascular. The term re-activity its underlined that the whole complex of circumstances them being the function-anatomy that invests as a whole these so called micro vascular-tissues, whose energetic motor is represented by the modality functions of the small arteries, from the arteries pre-capillary and from the muscular components of the dispositive of the end arteries and of the blocking end arterial volume regulators and the speed of the endo capillary. The capillaries function structured passively, in the sense that accessibility flow, the velocity of transfer of the elements represented in the blood, the actual emetic mass, essentially depend on the systolic potency of the small arteries and arterials . Our experience 30 patients under examination ; medium age В±DS = 55,61В±2,55; range = 45-65). A capillaroscopy examination was effectuated on the mucosa of the lower lip near the attaching part of the tongue to the pavement of the mouth; this area was chosen because of the notable transparent mucosa and for a simpler application of the technique, the feeler, even if small, would have had difficulty entering in different areas. The patients were not chosen by chance; in fact these subjects had previously been through a bi-optical operation, in the same lip area, for pathologies in that area: to study properly analogies and differences of the microcircuit on two methods of diagnostic research, classic and un replaceable, the histology exam and an innovative and complementary, the video capillaroscopy with an optical tracker and with a polarized light. The methodic of video imaging biomicroscopy was carried out thanks to the utilization of the capillaroscopy with acknowledgements: in fact during the capillaroscopy the temperature of the room was kept between 21В° and 24В°, and the patient in resting conditions without smoking and or haven drunk alcohol for at least an hour before the test. The choice of the objective was easily taken, because in the examination of the micro circulation of the mucosa it is advisable to use contact objectives. The usage of these objectives could be compromised by the scarce consistency of 94 Oral Capillaroscopy: our experience the examination surface, for which focusing is very precarious; for an altogether vision, after having set the illumination intensity, that then was calibrated for all the other exams, a 50x was then applied. With this enlargement the capillaries are distinguished thanks the red colour of blood. To examine adequately the morphological aspects of a district micro circulation, which permits the identification of the regional microvascular, a 200x was then used which let us explore point to point all the morph structural characteristic of the capillaroscopy field. Having a diameter of 7,5 they appear to the observer as small points with a variable velocity in the single capillaries; appreciated though is the phenomenon of aggregations erythrocytes and the capillaries or of the smaller veins in whose flow is slowed down o momentarily still. The video capillaroscopy is constituted: a body central which contains a source of light of cold halogen from a lamp of 100w which comes from a device that automatically controls the luminous intensity; a tracker with optical fibers, which consists of a flexible cable measuring 2 meters which presents an optical-video terminal, which also contains a micro colored television camera and with a support for the dwelling of different objectives. Furthermore for the digitizing of the histological images we used LEICA IM10000 attached directly to the microscope, which gave us the possibility, once calibrated, the exact measurements, in fact, setting both the resolution and the enlargement, the histological image presented itself inside a crowded network scales and borders, within which it was permitted the correct recognition of the dimension of any cell, including the micro circulation. Valued capillaroscopic parameters: density of the capillaries (d-c); width of capillaries (LC); diameter of ansae efferent; diameter of ansae afferent (DAA). Valued histological parameter: capillaries density (d-c); width of capillaries (LC); presence hyper keratosis (IP). All of this at the end is to correlate affinity between the two different methods of research which altogether are different from each other, this demonstrates an major attend ability of the capillaroscopy data, by an accurate means of images digitalized so differently. Experimentation iconographic confrontation Capillary examination Using the media of 4 analyzed parameters we have :d-c 0 14 x mm2; LC media 0 11 В± 0.8 Вµ DAA Media 0 5 В± 2,90 Вµ; DAE media =6 В± 2,10 Вµ Histological examination The results of our observation is evidence that there are perfect analogies by the observation of the micro circulation in vivo and in vitro, with a small range error which turns out 95 Francesco Carini, Giuseppe Alessandro Scardina, Gianfranco Cocorullo, Pierluigi Varia, Chiara Lo Piccolo, Angelo Leone, Piero Messina and Vincenzo Valenza to be around a В±0.3 Вµ and also regarding the capillary density we can see a variation of В± 2 capillaries per mm2. There have also been found general values observed in the capillaroscopy studies of these years; or where the cap. density (d-c), determines the number of ansae present per surface unit shines between 12 and 16 capillary per mm2, and also the diameter of the afferent curvature is always smaller than the efferent one for a small micron: or rather is the first one really 4-8 Вµ, the second around 7-10 Вµ it has also been confirmed that the width of the capillary is around 9-14 Вµ. Conclusions All the capillaroscopy techniques and in particular the Video Capillaroscopy using an Optic Feeler and with Polarized Light offer scientific and practical starting points on a clinical plan for a global valuation of the damages to the microcirculation with a widespread ubiquity; itвЂ™s principal virtue consists in the possibility of valuing the anatomy-topographic differences to the entity of the microcirculationвЂ�s damage and it continues today and will so in the future to value this damage as it is that what represents the most original innovation: the possibility if monitoring the damage in each accessible body part, following, in subsequent phases, the evolution being positive or negative and associating with capillaroscopic a clinical and symptomatologic frame. 96 Oral Capillaroscopy: our experience References [1] Hettema ME, Zhang D, Stienstra Y, Oomen PN, Smit AJ, Kallenberg CG, Bootsma H. 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Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 GLI ANEURISMI DELLвЂ™AORTA ADDOMINALE [Abdominal aortic aneurysm] Guido Bajardi1, Felice Pecoraro2, Annamaria Mauro3, Umberto Marcello Bracale1 Cattedra di Chirurgia vascolare - 2Scuola di specializzazione in Chirurgia vascolare - 3Di.Me.S. Sezione di Istologia ed Embriologia, UniversitГ degli Studi di Palermo (IT) 1 Parole chiave: Aneurisma dellвЂ™Aorta Addominale, AAA, ACE, IL, MMPs, PAF, TIMP-2, Aterosclerosi, EVAR Key words: Abdominal Aortic Aneurysm, ACE, IL, MMPs, PAF, TIMP-2, Atherosclerosis, EVAR Riassunto. Nelle ultime due decadi si ГЁ registrato un incremento dellвЂ™incidenza dellвЂ™aneurisma dellвЂ™aorta addominale ( AAA) a causa di una maggiore aspettativa di vita nella popolazione, il fumo di sigaretta ed ai programmi di screening. Nei Paesi sviluppati lвЂ™AAA ГЁ responsabile dellвЂ™1.3% di tutte le morti negli uomini di etГ compresa tra 65 ed 85 anni. Sebbene lвЂ™AAA venga descritto come una massa pulsante nella regione dellвЂ™epicondrio od epigastrio, di solito resta misconosciuta fino alla rottura. LвЂ™AAA ГЁ comunemente definito quale incremento di 1В·5 volte del diametro rispetto al normale. LвЂ™AAA ГЁ stato considerato come manifestazione tardiva del processo aterosclerotico. Studi recenti mostrano che, almeno parzialmente, il processo dilatativo differisce da quello ostruttivo: lвЂ™AAA ГЁ caratterizzato da un processo degenerativo trans murale localizzato in un segmento arterioso specifico. I principali fattori di rischio per lвЂ™AAA sono il fumo, il sesso maschile, lвЂ™ipertensione, la broncopatia cronica ostruttiva, lвЂ™iperlipemia e la lostoria familiare di malattia. Anche lвЂ™infiammazione ГЁ stata coinvolta come uno dei principali protagonisti nel processo aneurismatico. LвЂ™aorta normale ГЁ un condotto fibro-elastico compliante, resistente e duraturo. Le principali caratteristiche di unвЂ™aorta normale sono legate allвЂ™elastina presente nella media ed al collagene presente nellвЂ™avventizia. Nello sviluppo ed evoluzione dellвЂ™AAA sia le fibre di elastina che di collagene vanno incontro ad un rimaneggiamento indirizzato verso la proteolisi. La riduzione delle cellule muscolari lisce ГЁ coinvolta nella formazione dellвЂ™AAA per quanto riguarda il rimodellamento vascolare. Anche la presenza costante del trombo endoluminale nellвЂ™AAA viene associato a condizioni di ipossia locale ed incremento dello stress parietale . soalmnete il 30-40% degli AA viene diagnosticato allвЂ™esame obiettivo. LвЂ™eco-color-doppler ГЁ la metodica diagnostica piГ№ semplice ed economica per la valutazione della presenza dellвЂ™AAA ed ГЁ usata convenzionalmente nei programmi di screening. La tomografia computerizzata ГЁ lвЂ™esame con la migliore accuratezza e viene utilizzata nel periodo preoperatorio od in soggetti sintomatici. I programmi di screening riducono significativamente la mortalitГ per AAA ( 43% negli uomini con etГ tra 65 e 74 anni). Il tasso di crescita per AAA di piccole dimensioni (в‰¤ 5.5 cm) ГЁ di 3.6-3.2 mm per anno ed incrementa al crescere dellвЂ™aneurisma. Solitamente gli AAA sono asintomatici fino alla rottura. Questa ГЁ caratterizzata da un improvviso dolore, shock, e presenza di massa pulsante. Il grado di shock dipende dalla localizzazione della rottura sulla parete aorica: una rottura antero-laterale ГЁ associata ad unвЂ™emorragia massiva in peritoneo. Differentemente una rottura posteriore puГІ essere tamponata nello spazio retro peritoneale e permettere una riparazione urgente dellвЂ™AAA. Una rottura tamponata ГЁ sempre seguita da una rottura maggiore nel giro di poche ore. La decisione di trattare in elezione un paziente dipende dal rischio di rottura rispetto al rischio operatorio nel contesto dellвЂ™aspettativa di vita del paziente. Il diametro massimo di 5.5 cm ГЁ il limite per lвЂ™indicazione al trattamento; questo ГЁ inferiore di 5 mm nelle donne. Altri parametri da considerare nella riparazione sono la crescita rapida dellвЂ™AAA (в‰Ґ1 cm/anno o в‰Ґ 0.5 cm/6 mesi), la presenza di sintomi e la conformazione ana- 99 Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale tomica (tortuositГ ed asimmetria). Il tasso di mortalitГ per la riparazione elettiva dellвЂ™AAA varia da 1.1% ad 8.0%. le ultime decadi hanno visto una riduzione della mortalitГ legata alla riparazione elettiva dellвЂ™AAA; tale tendenza perГІ non si ГЁ registrata in quelli rotti. Il tasso di mortalitГ per AAA rotti varia dal 30% al 70% per i pazienti che giungono vivi in ospedale. Il trattamento medico viene usualmente proposto ai pazienti in regime followup. Sono in corso studi per la valutazione dellвЂ™efficacia di doxicilina, roxithromicina e delle statine. Il trattamento chirurgico tradizionale consiste in una aneurismectomia ed innesto di una protesi in Dacron. LвЂ™intervento chirurgico puГІ essere complicato da problemi cardiaci, polmonari, renali ed emorragici. Il trattamento endovascolare (EVAR) ГЁ una metodica piГ№ recente che prevede il posizionamento di una endoprotesi dalle arterie feomrali evitando lвЂ™apertura dellвЂ™addome. Questo trattamento seppur meno invasivo puГІ essere complicato da endoleaks, endotension migrazione della protesi ed infezione delle ferite inguinali. Diversi trials randomizzati sono stati realizzati con lвЂ™intenzione di verificare quale sia il trattamento migliore. Attualmente questi studi hanno mostrato una mortalitГ perioperatoria piГ№ bassa dopo EVAR con risultati a lungo termina ancora da valutare. Alcuni aspetti come la prevenzione primaria con programmi di screening nella popolazione a rischio e lвЂ™identificazione di biomarkers predittivi di rottura dovrebbero essere investigati. Anche le indicazione al trattamento open ed EVAR dovrebbero essere valutate specialmente in quei pazienti con AAA rotto. Abstract. Last two decades have seen an increased incidence of abdominal aortic aneurysm (AAA) because of higher life-expectancy, smoking and screening program. In developing countries the AAA is responsible of 1.3% of all death in men between 65 and 85 years. Even if AAA has been defined as a pulsating mass in hypochondriac or epigastric region it usually remain asymptomatic until rupture. For AAA definition it has been proposed as a criterion that the infrarenal diameter should be 1В·5 times the expected normal diameter. AAA have been usually considered as a late manifestation of atherosclerotic disease. Recent studies show that aneurysm process differ, at least in part, from arterial obstructive disease: AAA is characterized by a transmural degenerative process usually localized in a specific arterial segment. Tobacco smoking, masculine sex, hypertension, chronic obstructive bronchopneumopathy, hyperlipemiaand family history are the most important AAA risk factor. Also inflammation have been involved in AAA process as one of the most important aspect. Normal aorta is a compliant, resistant and durable fibro-elastic conduit. The most important characteristics of normal aorta are linked to elastin in the tunica media and collagen in the tunica adventitia. In AAA development and evolution both elastin and collagen fibers turnover are pendent to proteolysis. The smooth muscle cells reduction play a relevant role in vessel remodeling and consequent AAA formation. Also the constant presence of thrombus into the AAA has been linked to local hypoxia and increased wall stress. Only 30-40% of all AAA is diagnosed on physical examination. Echo-color-doppler is the simplest and cheapest diagnostic method to evaluate the presence of AAA and is conventionally used in screening programs. Computed tomography is the exam with the best accuracy and it is used in the preoperative period or in symptomatic patients. Screening programs significantly reduce AAA related death (43% in men between 65 and 74 years). Growth rate for small AAA (в‰¤ 5.5 cm) is 3.6-3.2 mm/years and increase with AAA dimension. Usually AAA are asymptomatic until rupture. AAA rupture is characterized by sudden pain, shock and pulsating mass. Shock degree is dependent on the aortic wall rupture portion: an antero-lateral rupture is associated to a massive hemorrhage in peritoneal space. Differently a posterior rupture could be tamponade by the retroperitoneal space and give the opportunity for an urgent treatment of AAA. A tamponade rupture is always followed by a major rupture in hours. The decision to electively operate a patient depend on the risk rupture versus the risk related to operation in the context of overall life expectancy. Maximum diameter of 5.5 cm remains the gold standard as cut-off 100 Gli aneurismi dellвЂ™aorta addominale in the operation decision; this limit is 5 mm inferior in women. Other parameters involved in the decision to operate are rapid growth (в‰Ґ1 cm/year or в‰Ґ 0.5 cm/6 months) , symptoms presence and anatomic details (tortuosity and asymmetry). Mortality rate for elective AAA repair ranges between 1.1% and 8.0%. AAA management in the last decade have decreased elective aneurysm-related deaths differently this trend has not been registered in ruptured AAA. Mortality rate reported for ruptured AAA ranges between 30% and 70% for those who reach hospital alive. Medical treatment is usually administered for those patients who have AAA in the surveillance range. Further studies are required to evaluate the real action and activity of doxycyline, roxithromycin and statin therapy. Surgical treatment consists of an open surgery approach or an endovascular aneurysm repair (EVAR). The open surgical treatment contemplates an aneuryectomy and Dacron interposition graft. The main complications of the open surgery are cardiac, pulmonary, renal and hemorrhagic. A totally laparoscopic surgical repair is a feasible option. EVAR treatment is a relatively more recent option that consists in the endovascular position of the prosthesis from femoral arteries avoiding abdominal incision and general anesthesia. Most frequent complications after EVAR are endoleaks, endotension, device migration and wound infection. To evaluate the superiority of the two surgical treatment many randomized control trials have been proposed. Actually these studies show no survival improvement for AAA repair less than 5.5 cm; lower perioperative death after EVAR with unknown long-term results; and no better results in patient considered unfit for open surgery. Some aspects like primary prevention with screening programs in population at risk and the identification of biomarkers predictive of rupture should be investigated. Also better indication to open or EVAR treatment should evaluated especially in those patients with ruptured AAA. Introduzione LвЂ™Aneurisma dellвЂ™Aorta Addominale (AAA) rappresenta una malattia cronica degenerativa con severe implicazioni per la vita. Si ritiene che gli AAA possano essere causati da una forma di danno della parete aortica a cui si sovrappone la presenza di fattori degenerativi legati allвЂ™etГ , lвЂ™emodinamica, fattori di rischio sistemici e genetici anche se la natura o la sequenza precisa di questi eventi non ГЁ nota. Nei Paesi sviluppati lвЂ™AAA ГЁ responsabile dellвЂ™1.3% della mortalitГ tra gli uomini di etГ compresa tra 65 ed 85 anni. Nelle ultime due decadi si ГЁ assistito ad un incremento dellвЂ™incidenza dellвЂ™AAA causato dalla maggiore aspettativa di vita nella popolazione, lвЂ™incremento del numero dei soggetti fumatori ed anche per lвЂ™introduzione di programmi di screening ed il miglioramento delle apparecchiature diagnostiche1. LвЂ™AAA rappresenta la tredicesima causa di morte negli Stati Uniti2 ed ГЁ piГ№ frequente nei soggetti con storia di fumo [3]. Sebbene in alcuni casi lвЂ™AAA possa presentarsi con una sintomatologia vaga,caratterizzata da dolore dorsale od addominale, la maggior parte degli AAA restano asintomatici fino alla rottura3 ed ГЁ generalmente riferito come una massa pulsante localizzata alвЂ™ipocondrio sinistro od allвЂ™epigastrio [4]. Nel 1991, la Society for Vascular Surgery e lвЂ™International Society for Cardiovascular Surgery Ad Hoc Committee on Standards in Reporting hanno proposto come criterio di 101 Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale definizione dellвЂ™AAA unвЂ™aorta sottorenale di almeno 1.5 volte rispetto al diametro normale5. LвЂ™AAA puГІ presentarsi in una variante infiammatoria caratterizzata dalla presenza di una spessa parete, una marcata fibrosi perianeurismatica e retroperitoneale e dense adesioni che coinvolgono le strutture adiacenti [6]. Strategia di ricerca e criteri di selezione. La ricerca ГЁ stata condotta su MEDLINE per la letteratura recente con le parole chiave вЂ�AbdominalвЂ™, вЂ�AorticвЂ™, вЂ�AneurysmвЂ™, вЂ�TrialвЂ™, вЂ�RuptureвЂ™, вЂ�EVARвЂ™ and вЂ�open repairвЂ™. Queste sono state utilizzate singolarmente ed in associazione. Sono state selezionate principalmente pubblicazioni degli ultimi 5 anni e quelle maggiormente referenziate, anche se antecedenti Epidemiologia Ogni anno circa 300.000 persone negli Stati Uniti improvvisamente muoiono senza una diagnosi certa, di queste il 4-5% per una rottura misconosciuta di un AAA [7]. Nel Regno Unito la rottura dellвЂ™AAA causa circa 8.000 morti ogni anno [8]. Il tasso di mortalitГ globale per i pazienti con AAA rotto ГЁ compresa tra il 65% e lвЂ™ 85% [3,8], inoltre circa la metГ delle morti attribuite alla rottura di AAA avviene prima che il paziente raggiunga la sala operatoria [9,10]. Per prevenire la rottura negli Stati Uniti ogni anno vengono eseguiti circa 40.000 trattamenti in elezione degli AAA asintomatici. Comunque queste procedure causano circa 1500 morti [11]. LвЂ™AAA risulta essere piГ№ frequente negli uomini che nelle donne con un rapporto maschi/femmine che varia da 3/1 ad 8/1 [12-16]. La settima decade di vita ГЁ la piГ№ colpita presentandosi raramente prima dei 50 anni [15-19]. Ciononostante, visto che il fumo rappresenta uno dei principali fattori di rischio per lвЂ™AAA e visto lвЂ™incremento sia delle donne fumatrici che dei giovani fumatori, le caratteristiche epidemiologiche legate allвЂ™etГ ed al sesso dellвЂ™AAA probabilmente cambieranno in futuro [14,15]. Etiologia e fattori di rischio Anche se la reale causa della formazione dellвЂ™AAA rimane sconosciuta, questo ГЁ stato comunemente considerato una manifestazione terminale della patologia aterosclerotica [20]. Recenti studi clinici e di laboratorio indicano che il meccanismo patogenetico alla base degli AAA differisce, almeno in parte, da quello responsabile della patologia ostruttiva [21,22]. Infatti lвЂ™anatomia patologica degli AAA ГЁ caratterizzata da un processo degenerativo transmurale generalmente localizzato in uno specifico segmento arterioso, mentre la patologia ostruttiva ГЁ una malattia diffusa dellвЂ™intima che coinvolge i vasi lungo tutto lвЂ™albero arterioso. Defawe et al. sono giunti alle stesse considerazioni analizzando i livelli di due inibitori fisiologici delle proteasi (il Tissue inhibitor of metalloproteinase [TIMP-2] ed 102 Gli aneurismi dellвЂ™aorta addominale il Plasminogen Activator Inhibitor-1[PAI-1]) nel contesto della parete vasale responsabili di una diversa evoluzione verso una patologia dilatativa od ostruttiva [23]. Solo una limitata percentuale di AAA risulta essere in relazione a cause specifiche come traumi, infezioni (brucellosi, salmonellosi, tubercolosi), malattie infiammatorie (BehГ§et eTakayasu) [24,25] e malattie del tessuto connettivo (Sindrome di Marfan, Ehlers-Danlos tipo IV) [26]. Dal momento che non tutti i pazienti con aterosclerosi sviluppano un AAA, ulteriori fattori sono coinvolti nello sviluppo dellвЂ™aneurisma. Tra questi il fumo ГЁ responsabile dello sviluppo e della progressione aneurismatica [16,27-29] determinando nei soggetti fumatori una prevalenza di circa 4 volte superiore 3. Altri fattori di rischio coinvolti sono il sesso maschile, lвЂ™etГ , lвЂ™ipertensione, la bronco pneumopatia cronica ostruttiva, lвЂ™iperlipemia e la storia familiare della malattia [14-16]. La componente genetica , secondo Shibamura H. et al, potrebbe contribuire al 70% o piГ№ nella genesi dellвЂ™AAA [31]: i loci coinvolti nello sviluppo sono stati individuati sui cromosomi 19q13 e 4q31 [32,33], che includono i geni della risposta immune e dellвЂ™apoptosi [32]. Anche la tendenza familiare dellвЂ™AAA non ГЁ dovuta esclusivamente ad unвЂ™unica causa: il background genetico gioca un ruolo principale in associazione con fattori esterni [34]. LвЂ™associazione tra antigeni dellвЂ™ HLA di I [35] e di II [36] Classe ГЁ stata rilevata sia negli AAA di tipo infiammatorio che non infiammatorio [37], dando supporto allвЂ™ipotesi di una patogenesi comune immuno-mediata modulata da fattori HLA -indipendenti. Oltre al PAI-1 ed al TIMP-2 sono numerosi i geni e le molecole chiamate in causa nel processo di formazione dellвЂ™AAA: lвЂ™Angiotensin Converting Enzyme (ACE); lвЂ™Interleukina(IL) 10; le metallo proteinasi (MMPs 2,7,9,12); la methylenetetrahydrofolate reductase (MTHFR) ed il platelet activating factor (PAF) [38-45]. LвЂ™aneurisma dellвЂ™aorta addominale (AAA) ГЁ legato, tra gli altri fattori identificati, ad una eccessiva degradazione della matrice extracellulare (ECM) che rende la parete del vaso meno resistente alla dilatazione. Si ritiene che questa de-regolazione della degradazione della ECM sia dovuta allвЂ™aumento dellвЂ™espressione e dellвЂ™attivitГ delle metalloproteinasi di matrice (MMPs), un gruppo di enzimi collettivamente capaci di degradare praticamente qualsiasi componente della ECM [133]. Vari studi legano lo sviluppo dellвЂ™aneurisma dellвЂ™aorta addominale allвЂ™overespressione di diverse metalloproteinasi di matrice tra cui ricordiamo le MMP-1, -2, -3, -7, -8, -9, -12 e -13 [128,141,142]. Nella patogenesi della AAA si pensa sia coinvolta soprattutto la degradazione del collagene e delle fibre elastiche, per questo motivo, tra le varie MMPs, i primi candidati ad essere indagati come possibili responsabili della patologia sono state la MMP-9 e la MMP103 Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale 2, enzimi degradanti collagene ed elastina. GiГ da molti anni negli aneurismi aortici addominali ГЁ nota la presenza di livelli aumentati di MMP-9 [138] ed MMP-2 [129]. Altri studi riportano che la MMP-2 ГЁ la principale MMP espressa nei piccoli aneurismi mentre la MMP-9 ГЁ la principale espressa negli aneurismi di grandi dimensioni [131]. A sostegno di questi dati, esperimenti di delezione genica hanno mostrato che topi knock-out, privati del gene della MMP-9 o della MMP-2, non sviluppavano aneurismi dellвЂ™aorta addominale neanche dopo induzione con cloruro di calcio, al contrario di quanto avveniva in topi normali [135]. Inoltre la doxyciclina, un inibitore delle MMPs ad ampio spettro ha dimostrato in molti studi su modelli animali di ridurre lвЂ™incidenza di AAA [136,127]. LвЂ™overespressione della MMP-9 in particolare ГЁ stata spesso associata con una maggiore probabilitГ di rottura dellвЂ™aneurisma e con lo sviluppo di aneurismi di maggiori dimensioni [140,144]. Г€ stata anche studiata la possibile associazione tra polimorfismi dei geni per le diverse MMPs e sviluppo dellвЂ™AAA, ed in alcuni casi si ГЁ visto che determinate varianti geniche aumentavano la predisposizione allo sviluppo della patologia [132,143]. Per questi motivi le MMPs possono essere dei buoni target per lo sviluppo di terapie per la prevenzione e la cura dellвЂ™AAA e alcuni tentativi e trial clinici sono giГ in corso [134]. Gli inibitori tissutali delle metalloproteinasi di matrice (TIMPs) regolano lвЂ™attivitГ delle MMPs e possono influenzare lвЂ™equilibrio tra deposizione e degradazione della matrice extracellulare, ne consegue che anche lвЂ™espressione di queste molecole ГЁ stata ampiamente investigata come potenziale target di terapie contro lвЂ™aneurisma aortico addominale [139,130,137]. Anche il processo infiammatorio ГЁ oggetto di studio nella genesi dellвЂ™AAA per la presenza di cellule infiammatorie mononucleate nel tessuto aneurismatico che potrebbero intervenire nellвЂ™apoptosi delle cellule muscolari lisce (SMC) [46-50] e nella produzione di citochine pro infiammatorie (tumour necrosis factor-a [TNF-a], interferon-g [IFN-g], IL- 5 ed IL-6) [39]. Recenti studi avanzano lвЂ™ipotesi che la formazione dellвЂ™AAA sia la conseguenza di una alterata e persistente rispostaauto- immune [38,51] allвЂ™interno della parete aortica [50,52 53,54]. CiГІ ГЁ supportato dal riscontro istologico di follicoli simil-linfoidi organizzati nel contesto della parete aortica [39] la cui presenza fa ipotizzare anche unвЂ™etiologia infettiva [55] oltre che autoimmune (verso peptidi derivanti dalla degradazione dellвЂ™elastina o lipoproteine ossidate a bassa densitГ ) [56]. Fisiopatologia LвЂ™aorta normale ГЁ un condotto fibroelastico i cui requisiti funzionali nei confronti della deformazione al continuo stress emodinamico sono la compliance (elasticitГ ), la resistenza 104 Gli aneurismi dellвЂ™aorta addominale e la durata. Queste particolari necessitГ vengono soddisfatte dalle proprietГ biomeccaniche dellвЂ™elastina e del collagene, proteine fibrillari di matrice che si trovano nella tonaca media ed avventizia dellвЂ™aorta. Uno dei principali e precoci riscontri istologici del tessuto aneurismatico ГЁ la frammentazione delle fibre elastiche in associazione con la ridotta concentrazione dellвЂ™elastina durante la crescita aneurismatica fino al momento della rottura [57,58]. Il tessuto avventiziale, in cui il collagene ГЁ predominante, ГЁ responsabile della resistenza aortica in assenza dellвЂ™elastina contenuta nella media:la degradazione del collagene potrebbe rappresentare la causa finale di rottura [58]. NellвЂ™AAA ГЁ stato descritto un incremento del turnover del collagene [59]: uno sbilanciamento tra la degradazione del collagene e la sua sintesi potrebbe creare la condizione catabolica che porta alla rottura [60]. Le cellule muscolari lisce della media ed i fibroblasti avventiziali producono le proteasi responsabili dellвЂ™alterazione dellвЂ™elastina e del collagene [61]. La parete aortica aneurismatica ГЁ caratterizzata da uno spesso strato di infiltrato di monociti, linfociti B, Plasmacellule e Linfociti T, indice di un processo infiammatorio cronico concomitante [62-65]. Pertanto lвЂ™immunitГ potrebbe giocare un ruolo anche nello sviluppo dellвЂ™AAA [66,67], con la produzione di enzimi proteolitici dellвЂ™elastina e del collagene [68-71] e conseguente sbilanciamento verso la proteolisi [23,72]. Un evento chiave nello sviluppo dellвЂ™AAA, ГЁ la riduzione delle densitГ delle SMC della media elastica [73] che partecipano nel rimodellamento della parete vascolare [74]. Lo sviluppo dellвЂ™AAA ГЁ anche associato alla presenza del trombo murale nella maggioranza dei pazienti. Diversamente dalla patologia ostruttiva, il flusso ematico ГЁ mantenuto per il continuo rimodellamento delle componenti del trombo.. Sebbene il trombo possa ridurre significativamente lo stress della parete aneurismatica, il suo incremento continuo di spessore potrebbe condurre ad una condizione di ipossia locale nello strato interno della media e conseguente induzione della neovascolarizzazione ed infiammazione mediale [75,76]. Anche la relazione che il trombo murale possa avere con la quota figurata del sangue e con i fattori della coagulazione dovrebbe essere investigato. Diagnosi Solamente il 30-40% degli AAA viene riconosciuta allвЂ™esame obiettivo a seconda della dimensione [77] e di eventuale obesitГ addominale [78]. La sensibilitГ della palpazione bimanuale dellвЂ™area sopraombelicale incrementa con il diametro della lesione: 61% per AAA di diametro tra 3.0 e 3.9 cm, 69% per quelli tra 4.0 e 4.9, e del 76-82% per quelli superiori a 5 cm [78]. La radiografia standard addominale puГІ incidentalmente essere diagnostica se sono presenti delle calcificazioni nella parete aortica che permettono la visualizzazione della dilatazione. 105 Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale LвЂ™EcoColorDoppler (ECD) ГЁ la metodica diagnostica piГ№ semplice ed economica che puГІ misurare lвЂ™aorta in tutte le sue dimensioni con unвЂ™accuratezza di 3 mm [79]. LвЂ™ECD ГЁ ampiamente utilizzato non solo per la diagnosi iniziale ed il follow-up, ma anche per studi o programmi di screening. Quando il diametro dellвЂ™aneurisma ГЁ tale da richiederelвЂ™intervento chirurgico, la Tomografia Computerizzata (TC) rappresenta lГ¬ indagine piГ№ idonea per determinare la tipologia di trattamento ed eventuali anomalie che possono essere di notevole impedimento durante lвЂ™accesso chirurgico al вЂњcollettoвЂќ (vena cava sinistra, vena renale sinistra posteriore) [80]. LвЂ™esame TC puГІ anche visualizzare la presenza di sangue allвЂ™interno del trombo come segno predittivo di rottura imminente [81]. In caso di AAA di tipo infiammatorio, la TC evidenzia un importante spessore della parete e la presenza di fibrosi para-aortica [6]. Lo stravaso del mezzo di contrasto ГЁ diagnostico per rottura dellвЂ™AAA. Attraverso la diagnostica tridimensionale, la TC spirale e lвЂ™angio-TC possono fornire ulteriori dettagli anatomici, fondamentali in caso di programmazioni di procedure endovascolari [82]. La Risonanza Magnetica (RM), combinata con lвЂ™angio-RM, ГЁ unвЂ™ulteriore metodica da considerare soprattutto per lвЂ™utilizzo di mezzo di contrasto non nefrotossico, a differenza dellвЂ™angiografia tradizionale, che puГІ portare ad insufficienza renale ed embolizzazione distale. LвЂ™utilizzo dellвЂ™angiografia ГЁ attualmente indicato in caso di posizionamento di dispositivi endovasclolari Screening Lo screening su uomini con etГ tra 65 e 74 anni riduce significativamente la mortalitГ correlata allвЂ™AAA [2]. Tradizionalmente, il diametro ГЁ stato utilizzato quale principale segno nella progressione dellвЂ™AAA. LвЂ™esame ECD addominale ГЁ stato dimostrato quale metodica diagnostica accurata e riproducibile per studi di screening per patologia aneurismatica [83]. La prevalenza di AAA asintomatici negli uomini e donne con etГ superiore a 60 anni ГЁ compresa tra il 4% e lвЂ™8% e tra lo 0.5% e lвЂ™1.5%, rispettivamente [84]. LвЂ™US Preventive Services Task Force ha concluso che lвЂ™ECD puГІ ridurre la mortalitГ del 43% negli uomini con etГ compresa tra 65 e 75 anni [2]. Nel piГ№ grande studio di screening su popolazione statunitense il 90% degli AAA identificati era di dimensione inferiore a 5.5 cm di diametro [85]. La crescita media per piccoli AAA (в‰¤5.5 cm di diametro) ГЁ di circa 2.6-3.2mm/anno, e tale tendenza incrementa al crescere del diametro [86,87]. LвЂ™espansione dellвЂ™AAA in 1743 pazienti con follow-up a 7 anni ГЁ stata significativamente associata al diametro di partenza. Differentemente non ГЁ stata riscontrata associazione per etГ o sesso. Il fumo di sigaretta ГЁ stato associato con un incremento addizionale di 0.4 mm per anno. 106 Gli aneurismi dellвЂ™aorta addominale Presentazione Clinica Gli aneurismi non rotti sono generalmente asintomatici nella maggioranza dei pazienti. Sono essenzialmente diagnosticati incidentalmente durante un attento esame clinico, specialmente in pazienti visitati per patologia coronarica, periferica o cerebrovascolare nonchГ© durante screening su popolazione [10]. La rottura dellвЂ™AAA ГЁ fortemente correlata alla triade dolore improvviso (al centro dellвЂ™addome o al fianco con possibilitГ di irradiazione allo scroto), shock e presenza di massa addominale pulsante. LвЂ™appendicite e la colecistite acuta possono determinare una sintomatologia simile alla rottura dellвЂ™AAA [88]. Il grado di shock varia a seconda della localizzazione, della dimensione e del tempo intercorso dalla rottura. La rottura della parete anterolaterale nella cavitГ peritoneale ГЁ usualmente un evento drammatico associato a morte repentina. La maggioranza dei pazienti che giungono in ospedale vivi presentano, invece, una rottura della parete posterolaterale nello spazio retro peritoneale; una rottura tamponata determina una perdita ematica limitata. Tale evento ГЁ sistematicamente seguito da una rottura maggiore entro poche ore. LвЂ™evoluzione bifasica della rottura dellвЂ™AAA enfatizza lвЂ™importanza del periodo che intercorre tra le due rotture, che dovrebbe essere utilizzato per il trasferimento e lвЂ™intervento urgente . Solo raramente lвЂ™AAA si rompe spontaneamente nel duodeno [89]: la fistola aorto-duodenale si realizza di solito dopo un precedente intervento di riparazione per AAA [88,90]. Anche la rottura dellвЂ™AAA in vena cava ГЁ un evento che puГІ realizzarsi con quadro di edema apparente agli arti inferiori [91]. Indicazioni per il trattamento LвЂ™indicazione al trattamento viene posta in considerazione delle dimensioni dellвЂ™AAA, del rischio peri-operatorio e dellвЂ™aspettativa di vita del paziente. [92,93] In una review di 32 articoli di Norman et al. ha rilevato che la sopravvivenza a 5 anni dei pazienti sottoposti ad intervento per AAA rispetto alla popolazione generale ГЁ del 70% [94]. La chiave ГЁ naturalmente come incorporare le comorbiditГ del paziente rispetto allвЂ™aspettativa di vita [15,87]. Il diametro massimo dellвЂ™AAA resta la miglior metodica per la stima del rischio di rottura: lвЂ™United Kingdom Small Aneurysm Trial (UK SAT) e lвЂ™Aneurysm Detection and Management study (ADAM) suggeriscono quale limite per lвЂ™intervento di riparazione aneurismatica un diametro di 5.5 cm negli uomini [87,96] e 5 mm meno nelle donne [97]. Il rischio di rottura aneurismatica ГЁ significativo quando il diametro trasverso ГЁ superiore a 5.5 cm ed incrementa esponenzialmente al crescere della dimensione [98,99]. Anche se il diametro aneurismatico ГЁ il principale indice per la rottura, in taluni pazienti puГІ fallire: alcuni AAA vanno incontro a rottura anche a dimensioni inferiori [92]. Infatti ad un anno il rischio ГЁ del 9.4% per AAA tra 5.5 e 5.9 cm, 10.2% per AAA tra 6.0 e 6.9 cm. e 107 Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale del 32.5% per AAA superiori a 7.0 cm ed un tasso del 25.7% a 6 mesi per diametri superiori ad 8 cm [98]. La metodica вЂ�finite element analysis of AAA wall stressвЂ™ che usa ricostruzioni tridimensionali TC si ГЁ rilevata migliore sia nel differenziare gli AAA sintomatici prossimi alla rottura che nel predirne il rischio di rottura [93]. La crescita rapida di AAA (в‰Ґ 1 cm/annuo o в‰Ґ 0.5 cm in 6 mesi) e lвЂ™insorgenza di sintomi sono fattori indipendenti per lвЂ™indicazione allвЂ™intervento. Alcuni criteri anatomici possono incrementare il rischio di rottura come lвЂ™asimmetria e la tortuositГ vasale [97,100]. Aneurismi eccentrici, sacciformi o con blisters parietali hanno un rischio di rottura maggiore [101]. La familiaritГ rappresenta un fattore di rischio per lвЂ™AAA con un tasso di rottura precoce 4 volte superiore rispetto a casi sporadici [101]. Vi ГЁ stata ed ГЁ presente una costante ricerca per lвЂ™identificazione, di altri fattori che possano predire il rischio di rottura. Tre questi la MMP-9, che ГЁ stata direttamente correlata nella degradazione proteolitica della matrice extracellulare della parete aortica [102], potrebbe essere un utile marker misurandone i livelli plasmatici [103]. Dati preliminari ottenuti alla tomografia ad emissione di positroni per lвЂ™AAA hanno mostrato un maggiore captazione focale di 18-fluorodeoxyglucose (18F-FDG) nel contesto della parete aortica in pazienti con AAA di grandi dimensioni, ad espansione rapida o sintomatici. La captazione di18F-FDG rappresenta lвЂ™immagine funzionale dellвЂ™infiltrato infiammatorio e quindi potenziale tecnica non invasiva per lвЂ™identificazione di AAA instabili in fase di rottura [104]. La mortalitГ correlata alla riparazione dellвЂ™AAA varia in relazione allвЂ™ospedale ed alla equipe operatoria [88-105]. La mortalitГ media a 30 giorni risulta essere compresa tra lвЂ™1.1% e lвЂ™8.0% nel trattamento in elezione [88,106-109] e dovrebbe essere contenuta al di sotto del 2% nella maggioranza degli ospedali [108,109]. Nelle ultime decadi il miglioramento dei risultati nel trattamento elettivo non ГЁ stato seguito da risultati sovrapponibili nel trattamento per rottura, con una mortalitГ tra il 30% ed il 70% [3,10]. E, se considerata la mortalitГ combinata alla rottura, durante il trasferimento, allвЂ™ammissione ospedaliera ed allвЂ™intervento, solamente il 18% dei pazienti con AAA rotto sopravvive. Prance et al hanno suggerito cinque fattori di rischio preoperatori per la stima della mortalitГ legata allвЂ™AAA: (1) etГ superiore a 76 anni; (2) livelli di creatininemia superiori a 190 Вµmol/L; (3) emoglobina inferiore a 8g/dl; (4) perdita di coscienza; (5) ECG con evidenza di ischemia. Nel loro studio il tasso di mortalitГ fu del 100% quando il paziente presentava tre o piГ№ fattori di rischio decrescendo al 48%, 28% e 18% se i fattori di rischio presenti erano rispettivamente due, uno o nessuno [110]. Attualmente particolare importanza viene conferita alla вЂ�emostasi ipotensivaвЂ™, che consiste nellвЂ™infusione di liquidi limitatamente al raggiungimento di 108 Gli aneurismi dellвЂ™aorta addominale valori pressori sistolici di 90 mmHg, ed al riconoscimento della sindrome compartimentale addominale specialmente in relazione al trattamento endovascolare [88,111]. Trattamento medico La diagnosi di AAA ГЁ solitamente occasionale in corso di indagini per altre patologie o durante programmi di screening. Alla diagnosi il 90% di questi AAA risulta essere allвЂ™interno della zona di sorveglianza [84]. In tale periodo viene usualmente somministrata una terapia medica adiuvante. In particolare la terapia con statina ha dimostrato un effetto biologico pleiotropico inclusa la riduzione dei livelli di proteina C-reattiva circolante,che potrebbe essere rilevante nella patogenesi dellвЂ™AAA [112]. Ad oggi, comunque, non vi sono evidenze sufficienti per raccomandare la terapia con statina alla diagnosi di AAA. La somministrazione del propanololo ГЁ di beneficio controverso nellвЂ™AAA. Evidenze di Livello A dallвЂ™American College of Cardiology/ American Heart Association clinical practice guidelines indicano che il propanololo non inibisce la crescita aneurismatica [113]. Dati provenienti da studi su animali dimostrano che lвЂ™utilizzo degli ACE-inibitori o del losartan riducono il tasso di espansione aneurismatica [84]. LвЂ™utilizzo di roxitromicina, in caso di riscontro sierico di infezione da Chlamydia pneumoniae, e delle tetracicline ГЁ stato associato ad una riduzione del tasso di espansione aneurismatica [114,115]. UnвЂ™interessante target terapeutico nella farmacoterapia degli aneurismi potrebbe essere la risposta infiammatoria interferendo con la via delle MMPs. Anti-infiammatori non steroidei, come lвЂ™indometacina, sono stati in grado di inibire la crescita aneurismatica in modelli animali [116]. Risultati da studi clinici hanno suggerito che la doxiciclina previene la crescita aneurismatica negli uomini [117]. Ciononostante sono necessari ulteriori studi per chiarire il ruolo potenziale della doxicilina, della roxitromicina e della terapia con statine nella progressione della patologia aneurismatica [88]. Trattamento chirurgico Per la riparazione chirurgica dellвЂ™AAA sono disponibili due forme di trattamento. La riparazione chirurgica вЂ�openвЂ™ viene utilizzata ampiamente da piГ№ di 50 anni. PiГ№ recentemente, la riparazione endovascolare aneurismatica (EVAR) ГЁ stata sviluppata per offrire unвЂ™alternativa meno invasiva e sta superando in frequenza di utilizzo la metodica вЂ�openвЂ™ [11]. Il primo trattamento chirurgico dellвЂ™AAA fu realizzato da Charles Dubost il 29 Marzo 1951 mediante unвЂ™aneurismectomia ed interposizione di homograft [118]. Il trattamento odierno deriva da Creech, del gruppo di DeBakey, che ha pubbliacto nel 1966 una semplificazione dellвЂ™intervento mediante unвЂ™endoaneurismectomia ed interposizione protesica divenendo quello ad oggi piГ№ utilizzato [119]. Durante il trattamento chirurgico, lвЂ™addome ГЁ aggredito, di norma, mediante una lunga 109 Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale incisione mediana. Un approccio retro peritoneale viene raccomandato per pazienti con patologia polmonare cronica ostruttiva. Gli svantaggi di tale approccio includono sia lвЂ™impossibilitГ allвЂ™esplorazione del contenuto intraperitoneale che la difficoltГ dвЂ™accesso allвЂ™arteria iliaca destra. Una volta aperta la cavitГ addominale deve essere identificato il colletto aneurismatico per permettere il controllo dello stesso. In caso di colletto soprarenale od infrarenale potrebbe essere necessario posizionare una clamp al di sopra delle arterie renali. Le arterie iliache vengono controllate in maniera analoga. In taluni casi si rende necessario il repertare lвЂ™arteria mesenterica inferiore per il suo successivo reimpianto nella protesi aortica mediante patch di Carrell al fine di garantire un flusso soddisfacente al sigma ed al retto. Comunemente vengono utilizzate protesi in Dacron a вЂ�zero porositГ вЂ™ rese impermeabili dallвЂ™impregnazione con collagene, albumina o gelatina [88]. LвЂ™anastomosi prossimale ГЁ del tipo termino-terminale e la distale puГІ essere realizzata alla biforcazione aortica, alla biforcazione iliaca o sullвЂ™arteria femorale comune a seconda dellвЂ™estensione aneurismatica ed alla pervietГ delle arterie iliache esterne. Viene posta molta cura nel mantenere almeno unвЂ™arteria iliaca interna pervia ed nel diagnosticare preoperatoriamente una potenziale ischemia colica sinistra o del retto. Talvolta puГІ rendersi necessario lвЂ™impianto di un corto вЂ�side arm graftвЂ™ per riperfondere unвЂ™arteria iliaca interna. Negli uomini sessualmente attivi, la raccomandazione ГЁ di non dissecare la parete aortica sinistra e lвЂ™arteria iliaca comune sinistra. Le principali complicanze dopo trattamento chirurgico вЂ�openвЂ™ dellвЂ™AAA sono quelle cardiache, polmonari, renali ed emorragiche [120]. La paraplegia postoperatoria, una complicanza rilevante nella chirurgia per aneurismi toraco-addominali, ГЁ infrequente negli AAA. LвЂ™incidenza della paraplegia dopo trattamento EVAR o chirurgico viene riportata tra lo 0.21%121 e lo 0.25-0.9% rispettivamente [122]. Coggia et al hanno dimostrato, in uno studio preliminare, la fattibilitГ della riparazione chirurgia per via totalmente laparoscopica. Anche se tale tecnica risulta meno invasiva e riduce il trauma chirurgica, sono richieste piГ№ esperienza e miglioramento delle conoscenze per assicurare un beneficio reale di questa tecnica rispetto alla riparazione tradizionale [123]. Il trattamento EVAR, introdotto per la prima volta da Parodi nel 1991 [124], prevede il posizionamento di unвЂ™endoprotesi (stentgraft) allвЂ™interno dellвЂ™AAA e il suo fissaggio sulla parete sana dellвЂ™aorta e delle iliache. Non ГЁ necessaria una laparotomia, bensГ¬ la protesi endovascolare viene impiantata attraverso piccole incisioni femorali. DallвЂ™epoca del primo impiego di unвЂ™endoprotesi, lвЂ™uso delle tecniche endovascolari per gli AAA ha avuto larga diffusione e ad oggi sono stati impiantati migliaia di dispositivi in tutto il mondo. La tecnica ГЁ stata ampiamente studiata in diversi trial randomizzati, multicentrici, in Europa (DREAM, EVAR) e negli USA (OVER) che ne hanno dimostrato la fattibilitГ e lвЂ™efficacia [125]. Non tutti i pazienti portatori di AAA possono essere sottoposti ad un intervento endovascolare, 110 Gli aneurismi dellвЂ™aorta addominale ma sono necessarie caratteristiche anatomiche specifiche. La TC spirale e lвЂ™angioTC sono metodiche indispensabili per la scelta del piГ№ idoneo stentgraft. Lo stengraft ГЁ composto, quindi, da tessuto e stents di metallo giГ premontati su un sistema di rilascio. Sotto guida fluoroscopia, il sistema ГЁ inserito attraverso le arterie femorali su guide angiografiche fino al corretto posizionamento dello stentgraft al di sopra ed al di sotto del segmento aneurismatico. Il rilascio dello stentgraft in corretta posizione esclude il flusso ematico dalla sacca aneurismatica con conseguenteeliminazione della pressione sulla parete. La principale complicanza dopo EVAR ГЁ lвЂ™endoleak ossia lвЂ™incompleta esclusione dellвЂ™aneurisma dalla circolazione. Vi ГЁ la possibilitГ che lвЂ™endoleak possa risolversi spontaneamente, ma una parte di essi persistono e sono associati ad un rischio tardivo di rottura. Altre complicanze sono rappresentate dallвЂ™endotension, dalla migrazione del device e dalle infezioni delle ferite inguinali [88]. Le controindicazioni allвЂ™EVAR consistono in aspetti anatomo-morfologici attentamente valutati alla TC preoperatoria.. Sono stati pubblicati diversi trials controllati randomizzati (RCTs) per la valutazione del migliore trattamento dellвЂ™AAA. Lederle et al. hanno recentemente rivisto la letteratura analizzando solo RCTs per riparazione versus sorveglianza di AAA di piccole dimensioni, trattamento chirurgico вЂ�openвЂ™ versus EVAR e trattamento EVAR versus controllo per pazienti non candidabili a riparazione вЂ�openвЂ™. Nel gruppo riparazione versus sorveglianza di AAA di piccole dimensioni nГ© lвЂ™ADAM nГ© lвЂ™UKSAT trial hanno raggiunto una differenza statisticamente significativa per mortalitГ legata a tutte le cause (rischio relativo [RR] 1.1 [ 95% da 0.771.32]) o per mortalitГ legata allвЂ™AAA (RR 0.78 [95% CI; 0.56-1.10]). In entrambi gli RCTs le differenze della qualitГ di vita rilevate furono modeste tendendo a favorire, ad un anno, la riparazione chirurgica, sebbene la maggioranza degli AAA monitorati erano asintomatici. Nel gruppo trattamento chirurgico вЂ�openвЂ™ versus EVAR il Dutch Randomized Endovascular Aneurysm Management trial (DREAM), lвЂ™Eindhoven Stent Prothesis for Aneurysm Study (ESPAS), lвЂ™Endovascular Aneurysm Repair trial (EVAR)-1 ed il Montreal trial hanno rilevato che il trattamento EVAR riduce la mortalitГ legata a tutte le cause a 30 giorni rispetto al trattamento chirurgico вЂ�openвЂ™ (RR 1.6% vs. 4.8%; 0.33 [95%CI, 0.17-0.64]). La differenza nella mortalitГ precoce perГІ scompare entro 2 anni nel DERAM e nellвЂ™EVAR-1trial, risultando in nessuna differenza statisticamente significativa nella mortalitГ a medio termine. GLI RCTs pubblicati hanno mostrato una ridotta mortalitГ operatoria e un miglioramento nella qualitГ di vita precoce dopo trattamento EVAR ma non hanno rilevato alcuna differenza statisticamente significativa in termini di qualitГ di vita o mortalitГ relata a tutte le cause nel medio termine. Nel gruppo trattamento EVAR versus controllo per pazienti non candidabili a riparazione tradizionale solamente lвЂ™EVAR-2 trial ГЁ stato considerato. Lo studio non ha riportato alcuna differenza statisticamente significativa per quanto riguarda la mortalitГ per tutte le cause. Il trend fu rilevato favorevole per i soggetti sottoposti ad osservazione 111 Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale Fig 1: Foto intraoperatoria: AAA infiammatorio. Fig 2. TC di AAA infiammatorio con caratteristica вЂ�cuffвЂ™ perianeurismatica. Fig 3. Foto intraoperatoria: voluminoso trombo endo- Fig 4. AAA di grandi dimensioni che deforma il profilo aneurismatico. della parete addominale. Fig 5. TC addominale di AAA di grandi dimensioni che Fig 6. AAA rotto: voluminoso ematoma retro ed intradeforma il profilo della parete addominale. peritoneale. Gli aneurismi dellвЂ™aorta addominale Fig 7. Foto intraoperatoria: AAA di grandi dimensio- Fig 8. Protesi in Dacron retta 20mm aorto-aortica. niisolato con posizionamento di clamps ed apertura della parete. Fig 9. Protesi in Dacron biforcata 18-9mm aortobisiliaco. Fig 10. Rx addome: controllo EVAR. Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale (hazard ratio [HR] 1.21 [95% CI, 0.87-1.69]). Non ГЁ stata rilevata inoltre, alcuna differenza statisticamente significativa nella mortalitГ per AAA (HR 1.01 [95% CI; 0.55-1.84]). Le conclusioni degli Autori furono che la sopravvivenza non migliora operando AAA inferiori a 5.5 cm; il trattamento EVAR ГЁ associato ad una minore mortalitГ operatoria ma con risultati a lungo termine sconosciuti rispetto al trattamento chirurgico вЂ�openвЂ™ e lвЂ™EVAR non ha mostrato miglioramenti in pazienti che hanno controindicazioni mediche al trattamento chirurgico вЂ�openвЂ™ [126]. Conclusioni La sopravvivenza non ha mostrato miglioramenti per riparazioni di AAA inferiori a 5.5 cm; lвЂ™EVAR ГЁ associata a minore mortalitГ operatoria, re interventi piГ№ frequenti e risultati a lungo termine sconosciuti rispetto al trattamento chirurgico вЂ�openвЂ™. LвЂ™EVAR non ha mostrato miglioramenti in termini di sopravvivenza in pazienti che presentano controindicazioni mediche al trattamento chirurgico вЂ�openвЂ™. I risultati a lungo termine degli RCTs che valutano il trattamento chirurgico вЂ�openвЂ™ rispetto allвЂ™EVAR sono necessari e si attendono. Per gli uomini un valore appropriato di AAA per considerare lвЂ™intervento ГЁ un diametro di 5,5 cm e tale valore deve essere ridotto a 5 cm per le donne. Gli AAA inferiori a tale dimensioni dovrebbero essere sorvegliati mediante esame ECD con un intervallo di 6 mesi e corretti chirurgicamente nel caso in cui dovessero raggiungere le dimensioni appropriate oppure se la crescita durante lвЂ™intervallo tra due esami sia superiore a 0.5 cm o insorgano dei sintomi legati allвЂ™AAA. Le ultime 5 decadi sono state caratterizzate da un progressivo miglioramento nella diagnosi, terapia e prognosi nel trattamento dellвЂ™AAA. Sicuramente alcuni aspetti dovranno essere approfonditi da future ricerche riferendoci in particolare alla prevenzione primaria mediante programmi di screening, al riconoscimento della popolazione a rischio ed allвЂ™identificazione di eventuali biomarcatori predittivi di rottura. 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University of Palermo (IT) Key words: Heat shock proteins, Rheumatoid arthritis, Experimental arthritis, Molecular chaperones, Pathogenesis, Risk factors Parole chiave: Proteine da shock termico, Artrite reumatoide, Artrite sperimentale, Chaperonine, Patogenesi, Fattori di rischio Abstract. The pathogenesis of rheumatoid arthritis (RA) is very complex, the cell types involved have been characterized: monocyte/macrophages, T and B cells, synoviocytes and endothelial cells. Chronicity and destructive potential are characteristic features of the inflammatory response in the synovial membrane, it has proposed that an exogenous antigen, likely an infectious organism, targets the synovia and elicits a chronic immune response. A major effort has been made to determine which cytokines and inflammatory mediators are produced at the site of disease. Tissue residing and infiltrating cells secrete proinflammatory cytokines in situ, which likely have a role in ampliflyng and maintaining the inflammation. Heat shock proteins (HSPs) are a group of proteins that are present in all cells, they are induced when a cell undergoes various types of environmental stresses like heat, cold and oxygen deprivation but HSPs are also present in cells under perfectly conditions. Heat shock proteins trigger immune response through activities that occur both inside the cell and outside the cell, they play an important role in protein folding and assembly in the cells, defective HSPs can contribute to the pathogenesis of a number of disease, known as chaperonopathies. Bacterial HSPs are recognized by the immune system and thus may provide a missing link between an immune response to infection and autoimmunity, infact, the overwhelming presence of bacterial HSPs on the mucosal surfaces of the gastrointestinal tract, the expression of self-HSP at sites of inflammation and the cross-reactivity between bacterial and self-HSP, self-HSP reactive T cells are normally controlled by the immune system. The generation of self-HSP specific T cells may even be essential for the maintenance of self-tolerance in a healthy individual. Riassunto. La patogenesi dellвЂ™ artrite reumatoide (AR) ГЁ molto complessa, le cellule coinvolte nella patogenesi sono: monociti/macrofagi, linfociti T e B, sinoviociti e cellule endoteliali. Le caratteristiche principali dellвЂ™AR sono la cronicitГ e la totale distruzione della principale sede verso la quale si rivolge la risposta infiammatoria ossia: la membrana sinoviale; si presuppone che un antigene esogeno, verosimilmente un agente di natura infettiva, sвЂ™insedi in corrispondenza della membrana sinoviale ed eliciti lвЂ™attivazione della risposta infiammatoria verso la stessa. A seguito della risposta infiammatoria vengono prodotte in situ numerose citochine proinfiammatorie in grado di richiamare dal torrente ematico altre cellule pro-infiammatorie amplificando in tal modo e perpetuando la risposta infiammatoria quindi il danno a carico della membrana sinoviale. Le proteine da shock termico (HSPs) sono un gruppo di proteine presenti in tutti i tipi cellulari, la loro espressione viene indotta a seguito dellвЂ™esposizione a stress di varia natura come lвЂ™elevate o basse temperature e la privazione 125 Luana Lipari di O2 sebbene possono esser presenti anche in condizioni fisiologiche. Le HSPs attivano la risposta immune e partecipano ai processi di assemblaggio e ripiegamento di proteine allвЂ™interno della cellula stessa, pertanto possono contribuire alla patogenesi di numerose patologie note come chaperonopatie. Le HSPs batteriche sono riconosciute dal sistema immune ed attivando la risposta immune fungono da legame tra risposta immune attivata nei confronti di un agente di natura infettiva e lвЂ™autoimmunitГ , infatti lвЂ™overespressione di HSPs batteriche sulla mucosa del tratto gastrointestinale, lвЂ™espressione di HSP-self nel sito dвЂ™infiammazione determina una cross-reattivitГ tra HSPs batteriche e linfociti T diretti contro HSPs self innescando ma soprattutto amplificando la risposta immune nei confronti di strutture self dellвЂ™organismo stesso. Rheumatoid Arthritis: whatвЂ™s known on pathogenesis Rheumatoid arthritis (RA) is a systemic, chronic inflammatory disease that affects 0.51% of the population and causes progressive joint destruction that leads to the restriction of activities of daily living and deterioration of quality of life. The inflammatory process is characterized by infiltration of inflammatory cells into the joints, leading to the proliferation of fibroblast-like synoviocytes and the destruction of cartilage and bone. In synovial tissue of patients with RA, the infiltrating cells consist of macrophages, T cells, B cells, plasma cells, neutrophilis, mast cells, dendritic cells and natural killer cells. Migration of leukocytes into the synovium is a regulated multistep process involving interactions between leukocytes and endothelial cells and cellular adhesion molecules, as well as between leukocytes, chemokines and chemokine receptors. Chemokines are small, chemoattractant cytokines that play key roles in the accumulation of inflammatory cells at the site of inflammation [8]. Among risk factors: genetic and environmental agents Although the pathogenesis of RA has not yet been fully elucidated, it is considered to be a complex disease that is influenced by both genetic and environmental factors. Genetic influences that contribute to RA susceptibility have been demonstrated in both studies of twins and families, as well as in genome-wide linkage scans. These studies estimated that genetic factors are responsible for 50-60% of the risk of developing RA and that environmental factors may explain the remaining risk. This quantification was made by a classical approach to separating the quantitative influence of genetic factors in RA with nationwide studies of twins with RA [7]. Several infectious agents have been reported to be risk factors for RA, including human parvovirus B19 (B19), Epstein-Barr virus, retroviruses, alphaviruses, hepatitis B virus, Mycobacterium tuberculosis, Escherichia coli, Proteus mirabilis and Mycoplasma. Moreover, a large number of epidemiological studies have demonstrated an association between cigarette smoking and the development of RA. In particular, smoking is known to be associated with rheumatoid factor positive and anti peptide C citrullinate positive, and to interact 126 Heat shock proteins: key players in pathogenesis and therapy of rheumatoid arthritis with the HLA-DRB1 shared epitope alleles. In addition, some studies have suggested that smoking also influences RA disease severity, although this remains controversial. While the mechanisms responsible for the influence of smoking in RA are not clear, some studies have shown an association between RA and the toxic compounds found in cigarette smoke, such as nicotine, 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD) and reactive oxygen species. It was found that the mRNA and protein levels of aryl hydrocarbone receptor (AhR) were higher in RA synovial tissue than in osteoarthritis tissue, and that TCDD upregulated the expression of IL-1ОІ, IL-6 and IL-8 through binding to AhR, with this effect transmitted via the nuclear factor-kB and extracellular signal-regulated kinase signalling cascades. In addition, AhR expression in synovial cells was upregulated by TNFО± [30]. The increased risk of RA in women has lead to evaluation of the role of sex hormones in disease susceptibility. It is well known that the levels of male sex hormones, particularly testosterone, are lower in men who have RA. By contrast, the levels of female sex hormones do not differ significantly between RA patients and control individuals. It has been reported that bone erosion occurs more frequently in men than in women, and that arthritis tends to occur earlier in men. Furthermore, male gender is correlated with a higher risk of bone erosions and an accelerated disease course, while female gender is correlated with structural consequences of joint destruction. Joint surgery is performed more frequently in female RA patients than in male patients. In addition, gender influences the risk, as well as the pattern, of organ involvement in RA. Nodules, and rheumatoid lung disease are typical manifestations in men, whereas women typically develop sicca syndrome. Exogenous hormones have also been reported to influence RA disease risk [5]. It is widely accepted that RA frequently remits during pregnancy. Although the mechanism for this is unclear, it is reported that the amelioration of RA during pregnancy is associated with a disparity in HLA class II antigens between mother and fetus. These studies suggested that the maternal immune response to paternal HLA antigens may play a role in pregnancy-induced RA remission. By contrast, several studies have suggested that nulliparous women have an increased during the postpartum period, particularly after the first pregnancy. Subsequent investigations demonstrated that much of this increased risk is associated with breastfeeding, and that women who breastfeed after their first pregnancy are at the greatest risk of developing RA. It is also suggested that the association among breastfeeding, pregnancy and RA may be related to either increased prolactin levels or an abnormal, pro-inflammatory response to prolactin [4]. Recently, it has been showed that the amelioration of disease activity in the third trimester corresponded to the increased number of regulatory T cells that induced a pronounced anti-inflammatory cytokine milieu. These data suggests that pregnancy leads to a beneficial effect of regulatory T cells on RA disease activity [12]. 127 Luana Lipari Few reports have addressed the influence of diet on RA development and progression. The addition of omega-3 fatty acids to the diets of RA patients has been associated with improvement in RA. Furthermore, diets high in eicosapentaenoic acid have a favourable effect on the clinical outcome of RA. Although the association between diet and RA onset is unclear, it is accepted that such fatty acids compete with arachidonic acids, the latter of which are involved in inflammation. Some reports have demonstrated the influence of vitamins on RA, for example greater intake of vitamin D, primarily from fish and fish products, has been associated with a lower risk of RA. In addition, the vitamin K, which is primarily derived from vegetables and legumes could inhibit the proliferation of fibroblast-like synoviocytes and the development of collagen-induced arthritis [29]. Experimental arthritis as a research model Animal models of arthritis play an important role in unravelling mechanisms of chronic inflammation in rheumatoid synovial tissue. They are used extensively to study disease pathogenesis and to apply new treatment strategies for rheumatoid arthritis and juvenile idiopathic arthritis (JIA). However, regardless of their limitations and drawbacks, experimental arthritis models can provide useful information on both pathogenesis and novel venues for therapy. A good example is the ongoing study of the most commonly used models of experimental arthritis. Its clinical and pathological features mimic several human chronic arthritic diseases, including RA, reactive arthritis and JIA. It can be used as a model to study other aspects of chronic arthritis such as pain, neuro-endocrine dysbalance, cartilage degeneration and even physical therapy. Nevertheless, the model is probably best known for its role in the study of the immune pathogenesis of chronic synovial best known for its role in the study of the immune pathogenesis of chronic synovial inflammation and exploring novel immune interventions in arthritis. Over the years, studies on the role of T cell recognition in autoimmune diseases have been performed [1]. As a consequence of these studies, novel venues for possible immune intervention with Heat Shock Proteins (HSPs) in human chronic arthritis are currently being explored, as we are going to discuss later. Adjuvant arthritis: description of histopathological steps Adjuvant arthritis (AA) is one of the most widely used experimental arthritis models. The first classical model of AA has described in 1956 by a single intracutaneous injection with heat-killed mycobacteria incomplete FreundвЂ™s adjuvant (CFA). The presence of mycobacteria in CFA suggested an infectious cause of the polyarthritis; however, anti-inflammatory drugs, but not antibiotics, are successful in attenuating disease severity [23]. AA is induced by intradermal or footpad injection of heat-killed mycobacterial species, preferably in a fine suspension in a mineral or vegetable oil. The disease is restricted to susceptible rodents, 128 Heat shock proteins: key players in pathogenesis and therapy of rheumatoid arthritis mostly certain rat strains, such as Lewis, Buffalo, Sprague-Dawly and Wistar rats. Interestingly, arthritis can also be induced in humans through immunization with mycobacterial antigens. The pathogenesis of this BCG-induced reactive arthritis in man is still largely unknown, but the disease appears T cell driven, presumably with an important pathogenetic role for both CD4+ and CD8+ lymphocytes. Some data suggest that, as in AA, HSPs may play a triggering role [22]. Following AA induction with CFA, rats not only develop arthritis but also tract and a loss in body weight that starts 24-48 hours before the clinical onset of arthritis. AA is a symmetric polyarthritis, affecting primarily the peripheral joints and quite frequently the spine. The affected joints are red, swollen and painful. The onset of overt clinical arthritis is seen 10-14 days following the induction of AA with CFA. The first histopathological signs of arthritis, an accumulation of mononuclear cells in connective tissues adjacent to periostal surfaces, are already manifest 6 days after disease induction. Approximately 10 days after disease induction, the first radiological signs of inflammation become visible: localized osteoporosis, with erosive lesions and periostal reaction. The synovial infiltrate leads to pannus formation, resulting in cartilage deformation and severe destruction of the joint [21]. In the early stages of overt disease, the histopathological abnormalities of AA are characterized by the infiltration of mononuclear cells into the synovial tissue. This cell infiltrate consists primarily of lymphocytes, but also plasma cells and at later stages neutrophils are present. Lymphocytes play a central role in triggering AA. It was shown that adoptive transfer of the disease is possible with the injection of primed lymph node cells or thoracic duct lymphocytes from diseased animals into recipients rats without co-immunization with mycobacteria or any triggering antigen. During the past, it was shown that the induction of AA is T cell driven, it was generated a T cell line, A2, directed at M. tubercolosis, that is capable of transferring AA to irradiated recipient rats. The same T cell line is capable of vaccinating non-irradiated rats against subsequent AA induction with CFA immunization. Thus a microbial agent may pull the trigger and initiate a self-perpetuating autoimmune process in a susceptible individual [3]. Heat shock proteins: players of rheumatoid arthritis pathogenesis HSPs are highly conserved cellular proteins. They play an important role in protein folding and assembly in the cells, as so called molecular chaperones. Based on size, four main groups of HSPs can be distinguished; Hsp90, Hsp70, Hsp60 and the small-HSPs. HSPs can be considered immunologic important proteins [14,15,17,26]. Defective HSPs can contribute to the pathogenesis of a number of diseases, now known as chaperonopathies [13,16,18]. First of all, HSPs are called stress proteins because their production increases when a 129 Luana Lipari cell is confronted with an environmental stressor, such as a rise in temperature, hypoxia, irradiation, viral infection or exposure to toxic chemicals. Because of this, self-HSP expression is up-regulated at sites of cell stress, such as in damaged or inflamed tissues, irrespective of the cause of damage. In inflamed synovial tissue from both rats with AA and from patients with either RA or JIA, an increased expression of self-Hsp60 can be found in synovial lining cells [25]. Secondly, HSPs are found in cells of all eukaryotes and prokaryotes and are remarkably conserved during evolution. Human Hsp60 has 40-50% identical amino acid residues and an additional 20% conservative replacements in comparison to mycobacterial Hsp65, GroEL [6]. Thirdly, despite the high degree of homology among HSPs of different species, they have been found to be strong immunogens. Bacterial HSPs are easily recognized by the immune system, which in turn can lead to cross recognition of self-HSP, and thus may provide a missing link between an immune response to infection and autoimmunity [11]. However, in the healthy immune system this is not the case. Despite, or maybe as a consequence of, the overwhelming presence of bacterial HSPs on the mucosal surfaces of the gastrointestinal tract, the expression of self-HSP at sites of inflammation, e.g. during an infection, and the cross-reactivity between bacterial and self-HSP, self-HSP reactive T cells are normally controlled by the immune system. Over the years, evidence has mounted that the generation of self-HSP specific T cells may even be essential for the maintenance of self-tolerance in a healthy individual. From that point of view, self-HSP-specific T cells may effectively play a key regulatory role in the control of potential danger situations, such as inflammation [8]. A new approach for therapy of rheumatoid arthritis: the chaperonotherapy Following the initial discovery that the T cell epitope of the arthritogenic T cell clone A2b in AA is a Hsp60-derived sequence, several attempts have been made to induce arthritis with Hsp60, or peptides derived from Hsp60. To date, all these attempts have been unsuccessful, even when the arthritogenic epitope itself or self-Hsp60 was used. Intraperitoneal immunization with the 180-188 Mhsp65 epitope of A2b induces a specific immune response towards both the 180-188 peptide and whole M. tuberculosis [27]. However, such immunization fails to induce arthritis, but instead can prevent the subsequent induction of AA. Conversely, as immunization with MHsp65 protects in AA and in practically all forms of experimental arthritis. Since that initial discovery, many approaches using Hsp60 or Hsp60 derived peptides have been successfully applied to induce protection in AA. These approaches included, among others, oral and nasal tolerance induction and DNA vaccination [9, 10]. Bacterial HSPs epitopes are present to the mucosal surface of the gastrointestinal 130 Heat shock proteins: key players in pathogenesis and therapy of rheumatoid arthritis tract. This can lead to the expansion of tolerized T cells, specific for these bacterial epitopes, but capable of cross-reacting with conserved self-hsp epitopes. Such a mechanism can be functionally fully comparable with the induction of regulatory T cells in oral tolerance. Subsequently, when cells are confronted with stress, e.g., during joint inflammation, selfHSP-reactive T cells [24]. These tolerized self-HSP-reactive cells, induced by the bacterial flora of the intestinal tract, may be part of the regulatory network of the healthy immune system controlling inflammation [20]. There is evidence from experimental arthritis models that supports this concept. Conclusions Heat shock proteins have received broad interest over recent years. Initially, they were found to be dominantly immunogenic microbial antigens, then the raised presence of immunity to HSPs in virtually all conditions of inflammation including autoimmune diseases, transplantation rejection and atherosclerosis has emphasized the critical significance of immunity to HSP inflammatory. The observations that HSPs can be released and that they can directly or indirectly elicit potent immunoregulatory activities give a new perspective on the roles of HSP and antiHSP reactivity in autoimmunity, transplantation, vascular disease and other conditions. Indeed, HSPs have unique arthritis-protective properties that may play a role in determining the disease outcome, not only in experimental arthritis, but also in patients. [2, 28]. In conclusion, the interaction of Hsps with the immune system, and the potential use of chaperones as therapeutic agents, i.e. the chaperonotherapy [19], is a putative new field of research for next years. 131 Luana Lipari References [1] Berner B, Akca D, Jung T, Muller GA, Reuss-Borst MA. Analysis of Th1 and Th2 cytokines expressing CD4+ and CD8+ T cells in rheumatoid arthritis by flow cytometry. J Rheumatolol 2000; 27:1128-35. [2] Chen G, Hao J, Xi Y, Wang W, Wang Z, Li N, Li W. The therapeutic Effect of Vasoactive Intestinal Peptide on Experimental Arthritis is associated with CD4 CD25 T Regulatory Cells. Scand J Immunol 2008; 68:572-8. [3] Cope AP. T cells in rheumatoid arthritis. Arthritis Res Ther 2008;10(Suppl 1):S1. Epub 2008 Oct 15. [4] Cutolo M. Sex hormone adjuvant therapy in rheumatoid arthritis. Rheum Dis Clin North Am 2000; 26:881-95. [5] Gerosa M, De Angelis V, Riboldi P, Meroni PL. Rheumatoid arthritis: a female challenge. Womens Health (Lond Engl) 2008; 4:195-201. [6] Hromadnikova I, Nguyen TT, Zlacka D, Sedlackova L, Popelka S, Veigl D, Pech J, Vavrincova P, Sosna A Expression of heat shock protein receptors on fibroblast-like synovial cells derived from rheumatoid arthritis-affected joints. Rheumatol Int 2008; 28:837-44 Epub 2008 Jan 30. [7] Imboden JB. The Immunopathogenesis of Rheumatoid Arthritis. Annu Rev Pathol. Epub 2008 Oct 27. [8] Iwamoto T, Okamoto H, Toyama Y and Momohara S. Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients. FEBS Journal Epub 2008. [9] Kamphuis S, Kuis W, de Jager W, Teklenburg G, Massa M, Gordon G, Boerhof M, Rijkers GT, Uiterwaal CS, Otten HG, Sette A, Albani S, Prakken BJ. Tolerogenic immune responses to novel T-cell epitopes from heat-shock protein 60 in juvenile idiopathic arthritis. Lancet 2005; 366:50-6. [10] Kaufmann SH, Ladel CH, Flesch IE. T cells and cytokines in intracellular bacterial infections: experience with Mycobacterium bovis BCG. Ciba Found Symp 1995; 195:132-32; discussion: 132-6. [11] Kim HR, Kim EY, Cerny J, Mougdil KD. Antibody responses to mycobacterial and self heat shock protein 65 in autoimmune arthritis: epitope specificity and implication in pathogenesis. J Immunol 2006; 177:6634-41. [12] Kobayashi S, Momohara S, Kamatani N, Okamoto H. Molecular aspects of rheumatoid arthritis: role of environmental factors. FEBS Journal Epub 2008. [13] Macario AJL. Heat-shock proteins and molecular chaperones: Implications for pathogenesis, diagnostics, and therapeutics. Intl J Clin Lab Res 1995; 25:59-70. [14] Macario AJL, Lange M, Ahring BK, Conway de Macario E. Stress genes and proteins in the Archaea. Microbiol Mol Biol Reviews 1999; 63:923-967. [15] Macario AJL, Conway de Macario E. The molecular chaperone system and other anti-stress mechanisms in archaea. Front Biosci 2001; 6:d262-283. [16] Macario AJL, Conway de Macario E. Sick chaperones and ageing: A perspective. Ageing Res Rev 2002; 1:295-311. [17] Macario AJL, Conway de Macario E. The pathology of anti-stress mechanisms: A new frontier. Stress 2004; 7:243-249. [18] Macario AJL, Conway de Macario E. Sick chaperones, cellular stress, and disease. N Engl J Med 2005; 353:1489-1501. [19] Macario AJL, Conway de Macario E. Chaperonopathies and chaperonotherapy. FEBS Letters 2007; 581:3681-3688. [20] Massa M, Passlia M, Manzoni SM, Campanelli R, Ciardelli L, Yung GP, Kamphuis S, Pistorio A, Meli V, Sette A, Prakken B, Martini A, Albani S. Differential recognition of heat-shock protein dnaJ-derived epitopes by effector and Treg cells leads to modulation of inflammation in juvenile idiopathic arthritis. 132 Heat shock proteins: key players in pathogenesis and therapy of rheumatoid arthritis Arthritis Rheum 2007; 56:1648-57. [21] Mirshfiey A, Mohsenzadegan M. The role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis. Iran J Allergy Asthma Immunol 2008; 7:195-202. [22] Moudgil KD. Diversification of response to hsp65 during the course of autoimmune arthritis is regulatory rather than pathogenic. Immunol Rev 1998; 164:175-84. [23] Prakken B, Kuis W, van Eden W, Albani S. Heat shock proteins in juvenile idiopathic arthritis: keys for understanding remitting arthritis and candidate antigens for immune therapy. Curr Rheumatol Rep 2002; 4:466-73. [24] Prakken BJ, Roord S, Ronaghy A, Wauben M, Albani S, van Eden W. Heat shock protein 60 and adjuvant arthritis: a model for T cell regulation in human arthritis. Springer Semin Immunopathol 2003; 25:47-63. [25] Puga Yung GL, Le TD, Roord S, Prakken B, Albani S. Heat shock proteins (HSP) for immunotherapy of rheumatoid arthritis (RA). Inflamm Res 2003; 52:443-51. [26] Sedlackova L, Nguyen TT, Zlacka D, Sosna A, Hromadnikova D. Cell surface and relative mRNA expression of heat shock protein 70 in human synovial cells. Autoimmunity Epub. 2008 Jun 12. [27] Sheikhi A, Nazarian M, Khadem-Al-Melleh A, Nasab NM, Esmaeilzadeh A, Yahaghi N, Sheikhi R. In vitro effects of Mycobacterial bovis BCG-lysate and its derived heat shock proteins on cytokines secretion by blood mononuclear cells of rheumatoid arthritis patients in comparison with healthy controls. Int Immunopharmacol 2008; 8:887-92. Epub 2008 Mar 7. [28] Sherer Y, Gerli R, Bocci EB, Gilburd B, VaBistoni O, Shoenfeld Y. Heat-shock protein 65 autoantibodies are differently associated with early atherosclerosis in rheumatoid arthritis and in healthy subjects. Ann N Y Acad Sci 2007; 1108:408-13. [29] Strietholt S, Maurer B, Peters MA, Pap T, Gay S. Epigenetic modifications in rheumatoid arthritis. Arthritis Res Ther 2008; 10:219 Epub 2008 Oct 10. [30] Weyand CM, Goronzy JJ. Pathogenesis of rheumatoid arthritis. Med Clin North Am 1997; 81:29-55. 133 Brain, behavior Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 EFFECTS OF APOMORPHINE ON ORIENTING REACTION IN THE RAT [Effetti dellвЂ™Apomorfina nella reazione di orientamento nel ratto] Maurizio Casarrubea, Filippina Sorbera and Giuseppe Crescimanno Department of Experimental Medicine, Human Physiology Section, University of Palermo (IT) Key words: Dopamine, A9 and A10 neurons, Nigrostriatal Dopaminergic Neurons, Mesolimbic-Mesocortical Dopaminergic Neurons, Head turning, Sensorimotor integration. Parole chiave: Dopamina, Neuroni A9 e A10, Neuroni Dopaminergici Nigrostriatale, Neuroni Dopaminergici Mesolimbico-Mesocorticali, Rotazione del capo, Integrazione sensitivomotoria. Abstract. Apomorphine, a mixed D1 and D2 dopaminergic agonist, provokes rapid modifications of A9 and A10 neuronal firing. Aim of the research was to study the possible relationship between rapid apomorphine-induced modifications of midbrain dopaminergic neuron activity and rapid modifications of a motor response triggered by sensory stimuli (sensorimotor integration). With the aid of a videoanalysis system, effects of apomorphine (0.1, 0.5, 1.0, 2.0 mg/kg IP) on latency and duration of head orienting movement, induced by acoustic stimulation, were examined. During trials lasting 20 min, in a control group of animals, receiving only acoustic stimulation, latency and duration of head turning movement were respectively 184.21 + 48.30 ms and 257.82 + 49.19 ms. On vehicle + acoustic administered animals, no significant modifications of head turning parameters occurred. Following apomorphine administration, ANOVA test revealed significant decrease of head turning latency and duration. Post-hoc comparisons, between drug and corresponding control conditions, showed that a) the two larger doses provoked significant effects only on head turning latency; b) the slight increase, observed with doses of 0.1 and 0.5 mg/ kg, was no significant. An apomorphine activity on pre- or postsynaptic receptors of nigrostriatal and mesolimbicmesocortical dopaminergic neurons could explain the effects; hence, a change on neurotransmitter release, leading to an inhibition or facilitation of dopamine-mediated responses to environmental stimuli, may be hypothesized. Riassunto. LвЂ™apomorfina, un agonista dopaminergico sia D1 che D2, induce rapide modificazioni dellвЂ™attivitГ dei neuroni A9 e A10. Scopo della ricerca ГЁ stato di studiare la possibile relazione tra le rapide modificazioni dellвЂ™attivitГ neuronale indotte dallвЂ™apomorfina e le rapide modificazioni di una risposta motoria innescata da una stimolazione sensoriale (integrazione sensitivo-motoria). Per mezzo di un sistema di videoanalisi, sono stati esaminati gli effetti della somministrazione di apomorfina (0.1, 0.5, 1.0, 2.0 mg/kg IP) sulla latenza e durata del movimento di orientamento della testa (HT), indotto da stimolazione acustica. Durante sedute della durata di 20 minuti, in un gruppo di animali controllo che riceveva solo stimoli acustici, la latenza e la durata dellвЂ™HT erano rispettivamente 184.21 + 48.30 ms e 257.82 + 49.19 ms. Animali cui era somministrata soltanto la soluzione veicolo e ai quali erano erogati gli stimoli acustici, non esibivano significative variazioni dei parametri dellвЂ™HT. In seguito alla somministrazione di apomorfina, il test di significativitГ ANOVA mostrava significativi decrementi sia della latenza che della durata dellвЂ™HT. La post-hoc analisi dimostrava che: a) le due dosi maggiori di apomorfina provocavano effetti significativi solo sulla latenza dellвЂ™HT; b) solo un piccolo e non significativo effetto si osservava con le dosi di 0.1 e 0.5 mg/kg. Gli effetti osservati possono essere spiegati con unвЂ™azione dellвЂ™apomorfina sui recettori presinaptici dei neuroni dopaminergici nigrostriatali e mesolimbico-mesocorticali. In conseguenza di ciГІ, ГЁ possibile ipotizzare che una modificazione dellвЂ™entitГ del rilascio di neurotrasmettitore, possa condurre ad una alterazione delle risposte agli stimoli ambientali mediate dalla dopamina. 137 Maurizio Casarrubea, Filippina Sorbera, Giuseppe Crescimanno Introduction Presentation of novel auditory stimuli elicits a motor response which has been referred to as the orienting reaction. Modifications of this response have been considered as sensitive and reliable indexes of changes in attentive condition and sensory information processing speed [1-3]. Different neuronal systems, in which a dopaminergic (DA) activity has been demonstrated, seem to play a role in orientational capacities [4-8]. Among them, A9 neurons in pars compacta of the substantia nigra and A10 group neurons of ventral tegmental area (VTA) show an increased neuronal firing in response to sensory stimuli [9-15]. Moreover, lesions of these systems result in characteristic sensorimotor impairments and attention disorders with affected animals unable to perform a correct orientation toward incoming sensory stimuli [8,16-18]. It has been previously demonstrated that the rapid increase of neuronal firing, observed on A9 and A10 DA neurons, following sulpiride administration, is correlated to rapid modifications of specific behavioral activities needing sensorimotor integration [19]. Moreover, in freely moving rats, acute apomorphine (APO), a mixed D1 and D2 DA agonist directly binding to dopamine receptors [20], has been proved to provoke dose-dependent rapid changes of A9 and A10 neuronal firing [21]. Possible behavioral correlates of this last finding have not been fully examined. Aim of the present research, was to investigate possible relationships between rapid APO-induced modifications of midbrain DA neuron activity and rapid modifications of a motor response triggered by sensory stimuli (sensorimotor integration). To this purpose, after acute administration of different APO doses, the acoustically-evoked head turning (HT) movement was studied in rats by means of a videoanalysis system allowing a subtle frame-by frame scanning. Since HT is a component of the orienting reaction toward environmental stimuli, i.e. a response needing sensorimotor integration, HT latency and duration can be considered as useful parameters to evaluate possible rapidly-induced changes on sensorimotor processing speed [1,22,3]. Materials and methods Animals The study was performed on male wistar rats (250 - 300 g), housed in separate cages (room temperature 21В°C), with free access to food and water. Experimental sessions were carried out during light phase (from 0700 to 1900 h) of day-night cycle. 138 Effects of Apomorphine on Orienting Reaction in the Rat Apparatus experiments were carried out in a soundproof room, maintained at constant illumination and temperature. Illuminated and ventilated perspex boxes (30 x 30 x 30 cm) were used as observation cages. Two loud-speakers placed within the box walls (one on the left wall and the other on the right one), exactly in the centre and 5 cm from the bottom, allowed to induce an orienting reaction whenever the acoustic stimulus occurred. Loudspeakers were controlled by an acoustic stimulator (Coulbourn), whose output switched on a LED, placed in the front of the cage, indicating, on the monitor, stimulus start and duration. Stimuli (300 Hz, 2 s) were randomly delivered as for spatial localization and intertrial interval (from 1 to 5 min; a mean of six stimuli in each trial) to avoid habituation; their intensity was checked not to induce avoidance responses. Behavioral analysis Six groups of animals, each composed of 10 rats, were tested during one session only. One group received acoustic stimulation, one was stimulated and received vehicle solution IP, and four groups were stimulated and administered different doses of APO (0.1, 0.5, 1.0, and 2.0 mg/kg IP). AnimalвЂ™s habituation to novel environment was performed twenty-four hrs before trials and consisted of ten min exploration of the observation box, without delivering any acoustic stimuli. Experiments, lasting 20 min, were recorded on videotapes by a videocamera apparatus and analyzed via a videocassette recorder. Motor sequence was measured on playback in slow motion and frame-by-frame with a temporal resolution of 40 ms. HT latency and duration were calculated analyzing initial and final frame. HT initial time was considered to occur the frame preceding the start of head-turning movement following acoustic stimulation, and HT final time the frame preceding the end of head movement. Interval between onset of acoustic stimulus and beginning of head movement indicated HT latency, and interval between HT initial and final time indicated HT duration. Experiments were conducted in accordance with the European Communities Council Directive (86/609/EEC) regarding care and use of animals for experimental procedures. Drugs Apomorphine hydrochloride (Sigma Chemical Co., St. Louis, MO) was dissolved in saline and injected intraperitoneally at concentration of 1.0 mg/ml at a dose of 0.1, 0.5, 1.0 and 2.0 mg/kg. Control group received same volume of vehicle calculated at the 1.0 mg/kg dose. 139 Maurizio Casarrubea, Filippina Sorbera, Giuseppe Crescimanno Statistical analysis All values are expressed as means + S.D. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by the Newman-Keuls (N-K) post-hoc test for multiple comparisons. Values of p < 0.05 were considered significant. Unpaired StudentвЂ™s t-test (two-tailed) was used when comparison was made between vehicle and only acoustically treated group. Results Acoustic stimulation provoked in the rat a head orienting movement toward stimulus source. Animal position, relatively to the active loudspeaker, varied throughout trials along a 360В° degrees spectrum, depending on a) animal standing area in the box when the randomized acoustic stimulus occurred, and b) interstimulus animal activity. Frame-by-frame analysis of responses revealed a sequence of fixed motor acts: auricle erection, coordinated eye deviation, neck and head torsion, with drug beginning to take effect (i.e. when animals showed first behavioral change) 1-2 min post injection. Likely because of rapid metabolism of the compound [23], the specific behavioral effects, we investigated, lasted no longer than 20 min. Two specific parameters of motor sequence were analyzed: HT latency and duration. As to latency, the mean + S.D. was 184.21 + 48.30 ms and to duration 257.82 + 49.19 ms. Results, observed in animals only delivered acoustic stimulation, were not significantly modified by vehicle administration (mean + S.D. 181.15 + 50.66 ms for latency, p<0.892 unpaired StudentвЂ™s t-test, and 261.30 + 46.48 ms for duration, p<0.873 unpaired StudentвЂ™s t-test). Moreover, to study whether rat response may change depending on different frequencies (100 to 500 Hz, steps of 100 Hz) and durations (1, 2, 3 sec) of acoustic stimulus, control tests were carried out for each variation of parameters and for each dose of APO (two animals for each control test). Only slight and non significant modifications of HT latency and duration were observed. However, following acoustic stimuli at frequency of 500 Hz, coupled to all train durations, animals did not orient their heads, but exhibited a jerk interfering with the orienting response. When the drug began to be effective, low doses induced whisker movements, occasionally-occurred sequences of head oscillations and the typical snout-to-surface (of the box walls) contact. Larger doses also provoked licking, gnawing, sniffing, and rare climbing episodes. Neither retching, nor other signs of nausea or general discomfort were observed. Acoustically-evoked HT movements, occurring during sequences of oscillatory head movements, were not taken into consideration. On the other hand, a careful interstimulus analysis allowed to discriminate behavioral modifications if depending or not on acoustic activation. 140 Effects of Apomorphine on Orienting Reaction in the Rat ANOVA test showed that APO IP administration evoked a significant decrease of HT latency when compared to vehicle injected animals. As for HT latencies, means + S.D. for each dosage were 188.61 + 43.82 ms, 191.36 + 45.67 ms, 125.40 + 33.78 ms, 115.52 + 37.12 ms respectively, F(4,49)=7.46 p<0.0001 (fig. 1). As for HT durations, means + S.D. for each dosage were 282.56 + 38.17 ms, 274.45 + 46.86 ms, 243.29 + 43.90 ms, 231.44 + 41.63 ms respectively, F(4,49)=2.38, p<0.066 (fig. 1). Newman-Keuls post hoc comparisons between drug and corresponding control conditions (vehicle injected animals) revealed that the two larger doses of APO (1.0 and 2.0 mg /kg) produced significant decrease of HT latency (p<0.05), whereas they failed to induce significant modifications of HT duration. As for APO at 0.1 and 0.5 mg/kg, post-hoc analysis showed that both doses failed to induce significant changes. Discussion Results of present research clearly indicate that APO provokes significant modifications of the orienting movement induced by an acoustic stimulation. Specifically, a decrease of HT latency was observed. Moreover, post-hoc analysis revealed one main, significant effect, i.e. a decrease of HT latency with the two larger doses which, on the other hand, failed to induce significant modifications on HT durations. A slight, non significant increases of both HT parameters resulted from post-hoc analysis of APO effects with the lower doses. These findings give rise to some interesting questions: 1) which central mechanisms are influenced by APO and, 2) which APO properties could explain the effects. 1) Nigrostriatal and mesocorticolimbic DA systems have been demonstrated to be closely involved in orienting behavior and in regulation of attentive condition [4-6,11,13,24,25]. Accordingly, changes in A9 and A10 neuronal activity, e.g. trains of burst firing, were observed in response to different sensory stimuli and during orientation [9-15]. Lower doses of APO inhibited A9 and A10 DA neuron firing, on the contrary larger doses increased it [21]. On the other hand, a slowed reaction capacity was observed in rats treated with the DA antagonist chlorpromazine [26] or О±-flupenthyxol [27]. Our results suggest a possible correlation between rapid APO-induced modifications of A9 and A10 neuronal firing and rapid modifications of specific parameters of a sensory-triggered motor response (sensorimotor integration). Since HT, which follows an acoustic stimulation, can be considered as a component of the orienting reaction toward environmental stimuli, changes of HT latency might depend on APO influence on DA systems regulating sensorimotor integration and attention shift [28]. These functions have been suggested to be under the control of both mesolimbicmesocortical system, where DA neurons of VTA change their activity after presentation of different kinds of sensory stimuli [11,29,15,25] and of neostriatum, which shares these 141 Maurizio Casarrubea, Filippina Sorbera, Giuseppe Crescimanno properties [9,7,12,14], and also receives input from brain areas associated with sensory as well as motor processing [30]. Moreover, dopamine receptor agonists, like bromocriptine and APO, induced an acute recovery of function from severe neglect produced by unilateral destruction of posterior parietal cortex in rats. This effect seems to be due to an activity on a DA system underlying directed attention and involving medial agranular cortex and its connection to the neo-striatum [31]. In addition, nigrostriatal dopamine system has long been associated with control of motor and postural mechanisms [12]; hence, APOinduced modifications of HT duration may arise from a specific influence on neurons of this system. 2) As to specific drug properties, implicated in the observed effects, it has been previously demonstrated that APO administration resulted in a significant restoration of orientation to all modalities of stimuli [17]. Further interesting remarks are suggested by the slight increase of HT latency and duration, when doses of 0.1 and 0.5 mg/kg were administered. If on the one hand, this effect resembles inhibition of operant motor performance obtained in rats trained to press force-sensitive beams [24], on the other hand, these delayed or weaker motor responses may be explained taking into account that dopamine autoreceptors are more sensitive to the direct-acting dopamine agonist APO than postsynaptic receptors are [32]. In parallel studies, different effects were obtained. In fact, APO caused dose-related modifications of lever-press responding for reward in an instrumental motor task, i.e., a general increase was observed both when stimuli were paired to conditioned reinforcers, and when they were not [33]. Moreover, single i.v. APO injection to freely moving rats has been reported to decrease A9 DA neuron activity [21,9]. Thus, effects of lower APO doses on DA neuronal activity and on orienting parameters may be due to selective activation of autoreceptors with a decrease of dopamine release and synthesis [34-36]. On the contrary, since larger doses have been proved to influence postsynaptic receptors, inducing specific behavioral effects [37,38], an increased APO-induced DA activity might provoke a facilitation of dopamine-mediated response to environmental stimuli, thus resulting a decrease of response latency and a facilitation of orienting shift. In addition, APO stimulates both D1 and D2 receptors and this activity has been mimicked in experiments where intracerebral or systemic injections of D1 and D2 agonists were carried out provoking enhancement of the rewarding effect of brain stimulation [39]. These evidences suggest that modifications of HT parameters could not be exclusively dependent on pre-or-postsynaptic drug activity, but also on a specific D1 and/ or D2 mediation [40]. In conclusion, drugs affecting DA activity can be effectively used to identify nature, timing and duration of different stages of sensorimotor integration, a function often damaged in diseases arising from an altered DA transmission. 142 Effects of Apomorphine on Orienting Reaction in the Rat Effects of different doses of apomorphine (0.1, 0.5, 1.0, 2.0 mg/kg IP, hatched columns) and vehicle solution (blank columns) on HT latency and duration. 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Differential roles of dopamine D1 and D2 receptors in the nucleus accumbens in attentional performance on the five-choice serial reaction time task. Neuropsychopharmacology 2007; 32: 273-83. 145 Organogenesis Philogenesis Clinical Embryology Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 The balance of Hsps expression during pregnancy: the role of placenta [Il bilancio dellвЂ™espressione delle HSPs nella gravidanza: il ruolo della placenta] Simona Corrao1#, Giampiero La Rocca1#, Rita Anzalone1, Francesca Magno1, Tiziana Loria1, Melania Lo Iacono1, Everly Conway de Macario2, Alberto JL Macario2, Francesco Cappello1* and Felicia Farina1 #The two authors contributed equally to this work. 1Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana, UniversitГ degli Studi di Palermo (IT); 2University of Maryland Biotechnology Institute, Center of Marine Biotechnology, Baltimore MD (USA). *Corresponding author Key words: Placenta, Placental Pathologies, Pregnancy, Heat Shock Proteins, Chaperones, Immune Regulation, Immune Tolerance Parole chiave: Placenta, Patologie Placentari, Gravidanza, Proteine da Shock Termico, Chaperoni, Regolazione Immunitaria, Tolleranza Immunitaria Abstract. The outcome of pregnancy depends on a series of events that start from embryo implantation and maintenance and end, under normal conditions, with a full-term delivery, passing through those events that underlie correct embryo development. Placenta has a key role in all of these processes. Placenta constitutes a highly differentiated organ of extra-embryonic origin with a structure characterized by a successive series of membranes: amnion, chorion, trophoblast of chorionic villi, and decidua, through which mother/foetus metabolic exchanges occur. Chorionic villi and respective capillaries are chiefly responsible for metabolic exchange. Any deviation in placental structures causes foetus damage, resulting in miscarriage, or other adverse effects. In pregnancy maintenance, inhibition of the immune response is essential to prevent a reaction against the semi-allogeneic embryo. Here we present the placentaвЂ™s components of the chaperoning system, including heat-shock proteins (Hsps) and chaperones. Hsps, many of which are chaperones, are involved in a pivotal manner during placentation, embryogenesis, and immune response regulation to allow foetal growth. Hence, a better understanding of the various aspects of these proteins (synthesis, modifications, structure, gene regulation, and function), and of those cellular processes associated with Hsps in the placenta is of primary importance for the recognition and treatment of placental pathologies. Riassunto. La riuscita di una gravidanza ГЁ legata ad una serie di eventi che iniziano dallвЂ™impianto e mantenimento dellвЂ™embrione e che si concludono, in condizioni normali, con il parto a termine, passando attraverso quei meccanismi che sono alla base del corretto sviluppo embrionale. In tutti questi processi, la placenta svolge un ruolo fondamentale. La placenta ГЁ un organo altamente differenziato di origine extra-embrionale; la sua struttura ГЁ caratterizzata da una serie di membrane: lвЂ™amnios, il corion, il trofoblasto dei villi coriali e la decidua, attraverso cui avvengono gli scambi metabolici madre/feto. I villi coriali ed i relativi capillari sono i principali responsabili degli scambi metabolici. Ogni modificazione della struttura placentare si puГІ riflettere in un danno al feto, con effetti avversi che possono culminare nellвЂ™aborto. Nel mantenimento dello stato di gravidanza ГЁ essenziale lвЂ™inibizione della risposta immunitaria nei confronti del embrione semi-allogeneico. In questo articolo, noi presentiamo le componenti placentari del sistema chaperonico, comprendente pro- 149 Simona Corrao, Giampiero La Rocca, Rita Anzalone, Francesca Magno, Tiziana Loria, Melania Lo Iacono, Everly Conway de Macario, Alberto JL Macario, Francesco Cappello, Felicia Farina teine da shock termico (Hsps) e chaperoni. Le Hsps, molte delle quali sono chaperoni, sono coinvolte in modo cruciale durante la placentazione, lвЂ™embriogenesi e la regolazione della risposta immune, per permettere la crescita fetale. Quindi, una migliore comprensione delle caratteristiche biomolecolari di tali proteine (sintesi, modificazioni, struttura, regolazione genica e funzione) e dei meccanismi cellulari ad esse associati nella placenta potrebbero assumere una importanza fondamentale nel riconoscimento e nel trattamento delle patologie placentari. Morphology of placenta and physiological implications Placenta is a component of extra-embryonic annexes. It is a highly differentiated organ in which reside the mechanisms that create the conditions for embryo implantation and correct growth of the foetus, i.e. respiration, nourishment, and metabolic exchanges. Placenta also has a controlled and complex endocrine activity. Placental structure varies throughout pregnancy. The trophoblast is the first layer that is formed during transition from morula to blastocyst stage. The next layer is the hypoblast, which originates inside the trophoblast and beneath the inner cell mass of the blastocyst. Finally, the chorion originates from the trophoblast that is backed by mesoderm [1, and refs. therein]. The serial organization of human placenta membranes at term from the side of the foetus to the side of the uterus consists of amnion, chorion, trophoblast of chorionic villi, and decidua, (Fig.1) [2]. During placentation, vascularization is the most important event that induces the formation of continuous capillaries, allowing metabolic exchanges at the mother/ foetus interface. Maintenance of the structure of the capillary endothelium is necessary for correct vascular permeability [3]. Foetal blood is separated from the maternal blood by three tissue layers that constitute the placental barrier: foetal capillary endothelium, stromal connective tissue of the villus, and chorionic epithelium of the villus (or trophoblast). The trophoblast is constituted of two layers, the cytotrophoblast (inner proliferative zone) and the syncytiotrophoblast (outer terminally differentiated epithelium) (Fig.1). All of the placental structures, especially the villous tree, are involved in the finely regulated functional interaction between mother and foetus. Full-term pregnancy depends on a globally good condition of placental components. Pre-term birth is a common cause of perinatal morbidity and death, often related to infections or utero-placental vascular disease [4]. The thickness of the villous epithelium depends on correct proliferation of cytotrophoblast cells and their recruitment into syncytiotrophoblast. Since the villous epithelium is involved in metabolic exchanges, any deviation of its thickness beyond the physiological range will affect diffusive conductance. Alterations in placental barrier permeability due, for example, to chronic inflammation and/or immune complex formation, will contribute to the development of pregnancy-related disorders such as pre-eclampsia [5,6] and intrauterine growth restriction (IUGR) [6,7,8]. 150 The balance of Hsp expression during pregnancy: the role of placenta Fig.1: Organization of human placenta at term. A, amnion; B, chorion; C, villous tree; D, foetal capillary; E, syncytiotrophoblast; F, cytotrophoblast; G, decidua. Other structures such as umbilical vein (H) and arteries (I), and maternal arteries (J) and veins (K) are also shown. Oxygen and nutrients exchanges between maternal oxygenated blood and foetal deoxygenated blood occur in the intervillous space (L). Placenta as a secretory organ: hormones and Hsps and their involvement in placental development and pathogenesis In addition to its fundamental role in connecting anatomically and physiologically mother and foetus, the placenta has an important endocrine activity during pregnancy that allows embryo implantation. In the early stages of pregnancy, the placenta produces si151 Simona Corrao, Giampiero La Rocca, Rita Anzalone, Francesca Magno, Tiziana Loria, Melania Lo Iacono, Everly Conway de Macario, Alberto JL Macario, Francesco Cappello, Felicia Farina gnificant amounts of progesterone and estrogens, substituting and influencing the ovaryвЂ™s activity. The placental endocrine activity includes the release of Chorionic Gonadotrophin (hCG); Placental Lactogen (hPL); growth factors such as Epidermal Growth Factor (EGF), Insulin-like Growth Factor (IGF)-I and -II, and Platelet-derived Growth Factor (PDGF); and chemokines and cytokines [9]. Placenta also releases Vascular Endothelial Growth Factor (VEGF) [10]. These hormones act together modifying the uterine structure and creating the correct environment to ensure embryo survival and growth. In addition to being a part of the endocrine system, placenta also contributes to the chaperoning system. For instance, the decidua has also a role in the establishment of pregnancy possibly with the participation of Hsps as suggested by the fact that during the first trimester of pregnancy, both the decidua and placenta produce a series of Hsps [11,12]. Hsps are highly conserved cellular proteins, many of which are the products of stressinducible genes present in all organisms (from bacteria to man) and many of which are chaperones. These proteins along with other molecules are part of the chaperoning system whose main function is the maintenance of protein homeostasis [see article by Macario and Conway de Macario in this volume]. Hsps and chaperones are major components of the anti-stress mechanisms that are activated in all cell types that undergo endogenous or exogenous stress, and prevent and correct molecular damage [13]. Participation of Hsps in various steps of reproduction has been reported, including spermatogenesis and oogenesis [14], probably because of their roles in the maintenance of a high cell-proliferation rate and in the maturation of newly synthesised proteins as they are chaperones. The trophoblast cells of blastocyst proliferate rapidly during implantation, differentiating into inner cytotrophoblast and outer syncytiotrophoblast, all of which are accompanied by a series of invasion mechanisms on endometrial connective tissue [rev. in 9]. Possible roles of Hsps in protein synthesis and cell-damage prevention at these stages are likely, so these proteins would be key players in foetal development and in the outcome of pregnancy. Hsp27 is actively transcribed during trophoblast differentiation, during which acts as a regulator of actin filaments structure and dynamics. Moreover, Hsp27 expression levels are related to expression of molecules involved in trophoblast invasion and motility such as MMP2 and iNOS [15]. Hsp27 interacts with isoform beta (ERb) of the estrogen receptor, acting as a co-repressor. Since ERb is over-expressed in vascular injury, Hsp27 could also be involved in vascular disease [16, and refs. therein]. An open question is whether Hsp27 has a role in vascular disease-related pre-term birth. Other Hsps, such as Hsp90 and Hsp60, were investigated in order to determine their involvement in stress-related pre-term labour. The levels of these two Hsps were unchanged during the first trimester of pregnancy and also did not change following the onset of labour [17]. An immunohistochemical investigation was carried out on the levels of 152 The balance of Hsp expression during pregnancy: the role of placenta Hsp70 and Hsp90 in chorionic villi of women who experienced first trimester miscarriage compared to the levels in the placentas of women who had full-term, normal delivery [18]. Both, Hsp70 and Hsp90 (Hsp90A and Hsp90B isoforms) were found increased in the cases with miscarriage compared to full-term placentas. These findings suggest participation of the Hsps investigated in the miscarriage mechanisms and/or reflect the stressful situation inherent to pregnancy and miscarriage. An association between over-expression of Hsp70 and endothelial activation and immune response in placental vascular disease has been reported [19]. Hsp70 might interact with proteins involved in cell cycle modulation in embryo development and thus play a role in the whole process of pregnancy at the placental, foetal, and maternal levels [rev. in 20]. Since a successful pregnancy depends on first cleavage steps of the zygote and, therefore, on embryo development and cell differentiation, and because of ethical problems in carrying out studies on human embryos, most of the information about involvement of Hsps in embryogenesis comes from studies in animal models. For instance, in an in vitro mouse model, using antibodies specific for Hsp60, Hsp70, and Hsp90, an impairment of embryo development was shown after 3, 5, and 7 days respectively [21]. Hsp70 has also been found extracellularly and thereby participates, along with other Hsps, in the regulation of the immune system [22,23]. Microbial Hsp60 and Hsp70, which can originate from bacteria in genital tract infections and penetrate into circulation before or during pregnancy, stimulate the immune system to produce antibodies. These antibodies can cross-react with the human Hsps, inducing autoimmunity, pro-inflammatory responses and, probably, pre-term delivery [24] or other adverse pregnancy outcomes [25]. In women with unexplained recurrent spontaneous abortions, elevated levels of the Th1 cytokines (IFNОі and TNFО±) were observed, compared to women with a normal pregnancy; the latter showed, instead, higher Th2 cytokines production than the other women [26]. All these observations reveal that a very interesting aspect of placental physiopathology is its involvement in the regulation of the immune system. The foetus possesses a half paternal chromosomal inheritance and, therefore, represents a homologue graft against which the maternal immune system might react. Pregnancy establishment seems to be tightly related to Hsps release. An important role is probably performed by Hsp10 during the first stages of pregnancy. In fact, Hsp10 was previously known as Early Pregnancy Factor (EPF) because of its release during the first stages of development, within 24h after oocyte fertilization and continuing until, at least, up to the first half of gestation [27]. EPF was first purified and characterized from various sources, including human pregnancy sera [28], and human platelets, showing a molecular weight of 10,843 Da and an amino-acid sequence approximately 70% identical to that of the rat mitochondrial chaperonin 10 (Cpn10), the homologue of human Hsp10 [29]. Further 153 Simona Corrao, Giampiero La Rocca, Rita Anzalone, Francesca Magno, Tiziana Loria, Melania Lo Iacono, Everly Conway de Macario, Alberto JL Macario, Francesco Cappello, Felicia Farina analyses demonstrated that the EPF gene coding sequence has high similarity with the sequence of the human Hsp10 gene [30]. EPF is thought to be the extracellular form of Hsp10 [31,32]. Current knowledge about EPF and its participation in the suppression of the immune system and, thus, in facilitating the establishment of pregnancy, lead us to hypothesize that Hsp10 plays a crucial role in various aspects of the embryosвЂ™ life and beyond. In fact, the downregulation effect of Hsp10 on immune cells was also seen in ovarian cancer [33]. Marked inhibition was observed of the expression of the signal-transducing zeta chain associated with the T cell receptor (TCR), known as CD3-zeta [33]. Also, it was observed that during pregnancy a T-cell signaling modulation by pregnancy-associated exosomes (which have been isolated from the blood of pregnant women) [34], which suggests that the exosomes might be involved in the mechanism of Hsp10/EPF release. Likely, EPF is also involved in embryonic development, as seen in in vivo and in vitro mouse models [35]. These observations regarding EPF suggest that Hsp10 could play key roles in cell differentiation during placentation and, consequently, any modifications in its expression levels could have an impact on the progression of pregnancy. Conclusions and perspectives The complex mechanisms that take part in all the events starting from oocyte fertilization and leading to a full-term gestation are finely regulated by an intricate network of hormones, proteins, and immune response-related molecules. The balance between endogenous and exogenous stresses can influence placental functions, leading to damage that could have and impact on embryo fate. The role of the chaperoning system, Hsps in particular, in normal and pathological placenta is not yet fully understood. Further studies about the functions of Hsps and chaperones should be carried out in order to give a definitive response to as yet unanswered questions such as, how do Hsps participate in the interaction between mother and foetus through the placenta? And, what kind of pathways are induced or blocked by Hsps in pregnancy-related pathological conditions? These and other similar questions represent a stimulating challenge to basic and applied scientists alike who are interested in the chaperoning system and its role in health and disease. 154 The balance of Hsp expression during pregnancy: the role of placenta References [1] Enders AC, Blankenship TN. Comparative placental structure. Adv Drug Deliv Rev 1999; 38:3-15. [2] Benirschke K. Remarkable placenta. Clin Anat 1998; 11:194-205. [3] Firth JA and Leach L. Structure and permeability in human placental capillaries- a review. Trophobl Res 1997; 10:205-213. [4] Baker AM, Braun JM, Salafia CM, Herring AH, Daniels J, Rankins N, Thorp JM. 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Modulation of estrogen signaling by the novel interaction of Heat shock Protein 27, a biomarker for atherosclerosis, and Estrogen Receptor ОІ. Mechanistic insight into the vascular effects of estrogen. Artherioscler Thromb Vasc Biol 2005; 25:e10-e14. [17] Divers MJ, Bulmer JN, Miller D, Lilford NJ. Placental heat shock proteins: no immunohistochemical evidence for a differential stress response in pre-term labour. Gynecol Obstet Invest 1995; 40:236-243. [18] Sotiriou S, Liatsos K, Ladopoulos I, Arvantis DL. A comparison of heat shock proteins (HSP) 70 and 90 on chorionic villi of human placenta in normal pregnancies and missed miscarriages. Clin Exp Obstet Gynecol 2004; 31:185-190. [19] Liu Y, Li N, You L, Liu X, Li H, Wang X. HSP70 is associated with endothelial activation in placental vascular diseases. Mol Med 2008; 14:561-566. [20] Luft JC and Dix DJ. Hsp70 expression and function during embryogenesis. Cell Stress Chaperones 1999; 4:162-170. 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[26] Raghupathy R, Makhseed M, Azizis F, Hassan N, Al-Azemi M, Al-Shamali E. Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions. Cell Immunol 1999; 196:122-130. [27] Morton H, Rolfe BE, Cavanagh AC. Ovum factor and early pregnancy factor. Curr Top Dev Biol 1987; 23:73-92. [28] Mehta AR, Eessalu TE, Aggarwal BB. Purification and characterization of Early Pregnancy Factor from human pregnancy sera. J Biol Chem 1989; 264:2266-2271. [29] Cavanagh AC, Morton H. The purification of early-pregnancy factor to homogeneity from human platelets and identification as chaperonin 10. Eur J Biochem 1994; 222:551-560. [30] Summers KM, Murphy RM, Webb GC, Peters GB, Morton H, Cassady AI, Cavanagh AC. The human early pregnancy factor/chaperonin 10 gene family. Biochem Mol Med 1996; 58:52-58. [31] Cavanagh AC. Identification of early pregnancy factor as chaperonin 10: implications for understanding its role. 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Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 Expression of cytokeratins in human umbilical cord at term [Espressione delle citocheratine nel cordone ombelicale umano a termine] Annamaria Mauro, Maria Buscemi and Aldo Gerbino Departiment of Experimental Medicine, Section of Histology and Embryology, School of Medicine, University of Palermo (IT). Key words: Human umbilical cord, Cytokeratin, Immunohistochemistry, RT-PCR Parole chiave: Cordone ombelicale umano, Citocheratine, Immunoistochimica, RT-PCR Abstract. Background. Cytokeratins are the proteins that costitute intermediate filaments. In the cytoplasm, keratin filaments extend from the nuclear membrane to the inner side of the plasma membrane. This link is important for the structural organization of the cytoplasm and for many cellular processes such as mitosis, cell movement, cell differentiation and adhesion. The pattern of expression of cytokeratins is frequently organ or tissue specific and depends mainly on the type of epithelium, the level of differentiation and the stage of development. The expression of cytokeratins in umbilical cord has been investigated by many groups, but these previous studies gave only fragmentary informations: each single study focused only on few types of cytokeratins and usually concerned only their expression at protein level. Aim. With this paper we tryed to make light on the pattern of expression of a wide range of cytokeratins in human umbilical cord both at protein and at RNA level. Results. Through immunohistochemistry we detected in the amniotic epithelium of human umbilical cord at term immunoreactivity for cytokeratin clone AE1/AE3 (Pankeratins) antibody, clone MNF116 (antibody against cytokeratins 1, 5, 10 and 14) and clone 34ОІE12 (antibody against cytokeratins 5, 6, 8, 17 and 19). RT-PCR analysis revealed gene expression for cytokeratin 1, 5, 6, 8, 10, 14 and 17. Riassunto. Premessa. Le citocheratine sono le proteine costituenti dei filamenti intermedi. Nel citoplasma i filamenti di cheratine si estendono dal nucleolemma fino alla faccia interna della membrana plasmatica; questa connessione ГЁ importante per lвЂ™organizzazione strutturale del citoplasma e per molti altri processi cellulari come la mitosi, il differenziamento cellulare e lвЂ™adesione. Il pattern di espressione delle citocheratine ГЁ spesso organo-specifico o tessuto-specifico e dipende principalmente dal tipo di epitelio, dallo stadio di differenziamento e dallo stadio di sviluppo. LвЂ™espressione delle citocheratine nel cordone ombelicale ГЁ stata studiata da diversi gruppi, ma tali precedenti studi hanno dato soltanto informazioni frammentarie; ogni singolo studio si concentrava soltanto su pochi tipi di citocheratine ed in genere riguardava solo la loro espressione a livello proteico. Obiettivo. Con questo lavoro abbiamo cercato di far luce sul pattern di espressione di un vasto gruppo di citocheratine nel cordone ombelicale umano sia a livello proteico che di RNA. Risultati. Per immunohistochimica abbiamo rilevato nellвЂ™epitelio amniotico del cordone ombelicale umano a termine immunoreattivitГ per AE1/AE3 (anticorpo contro pancheratine), per il clone MNF116 (anticorpo contro le citocheratine 1, 5, 10 e 14) e per il clone 34ОІE12 (anticorpo contro le citocheratine 5, 6, 8, 17 e 19). LвЂ™analisi per RT-PCR ha rivelato espressione genica per le citocheratine 1, 5, 6, 8, 10, 14 e 17. 157 Annamaria Mauro, Maria Buscemi, Aldo Gerbino Introduction Cytokeratins (CKs) are the proteins that build intermediate filaments. These filaments, along with actin microfilaments and microtubules, constitute the cytoskeleton of epithelial cells and their primary function is to provide mechanical strength to cells. The classification of cytokeratins (CK1-CK20) is based on the catalogue of Moll et al. [1]. There are two types of cytokeratins: the low weight, acidic type I cytokeratins and the high weight, basic or neutral, type II cytokeratins. The high molecular weight cytokeratins comprise subtypes CK1 to CK8. The low molecular weight cytokeratins comprise subtypes CK9 to CK20. Cytokeratins are made of a central О±-helix rich domain and non-О±-helical N- and C- terminal domains. The О±-helical domain comprises four segments in which a seven-residue pattern is repeated. Into this repeated pattern, the first and fourth residues are hydrophobic and the charged residues show alternate positive and negative polarity, resulting in the polar residues being located on one side of the helix. This central domain of the chain provides the molecular alignment in the keratin structure and makes the chains form coiled dimers in solution. The end-domain sequences of cytokeratin chains contain in both sides of the rod domain the subdomains V1 and V2. The subdomains V1 and V2 contain residues enriched by glycines (Gly) and/or serines (Ser), the former providing the cytokeratin chain a strong insoluble character and facilitating the interaction with other molecules. Cytokeratins are usually found in tetramers comprising two type I cytokeratins and two type II cytokeratins. Two dimers of cytokeratin associate into a keratin tetramer by antiparallel binding (Fig.1). This cytokeratin tetramer is considered to be the main building block of the cytokeratin chain. By head-to-tail linking of the cytokeratin tetramers, the protofilaments are originated, which in turn intertwine in pairs to form protofibrils. Four protofibrils give place to one cytokeratin filament. In the cytoplasm, the keratin filaments form a complex network which extends from the surface of the nucleus to the cell membrane. This association between the plasma membrane and the nuclear surface provides important implications for the organization of the cytoplasm and cellular communication mechanisms. Apart from the relatively static function provided in terms of supporting the nucleus and providing tensile strength to the cell, the Fig.1: Graphic representation of a cytocheratins cytokeratin networks undergo rapid depolyme- tetramer. 158 Expression of cytokeratins in human umbilical cord at term rization which is important for many cellular processes such as mitosis, cell movement and differentiation. Cytokeratins also interact with desmosomes and hemidesmosomes, thus collaborating to cell-cell adhesion and basal cell-underlying connective tissue connection. The expression of cytokeratins is frequently organ or tissue specific. The subsets of cytokeratins which an epithelial cell expresses depends mainly on the type of epithelium, the moment in the course of terminal differentiation and the stage of development. Thus this specific cytokeratin fingerprint allows the classification of all epithelia upon their cytokeratin expression profile. Furthermore this applies also to the malignant counterparts of the epithelia (carcinomas). The main clinical implication is that the study of the cytokeratin profile by immunohistochemistry techniques is a tool of immense value widely used for tumor diagnosis and characterization in surgical pathology. Cytokeratin 1 (CK1) is specifically expressed in the spinous and granular layers of the epidermis, usually associated with cytokeratin 10. Mutations in these genes have been associated with bullous congenital ichthyosiform erythroderma. Cytokeratin 5 (CK5) is specifically expressed in the basal layer of the epidermis and is usually found as a heterotetramer with two keratin 14 molecules. Mutations in these genes have been associated with a complex of diseases termed epidermolysis bullosa simplex. Cytokeratin 6 (CK6) is expressed with family members CK16 and/or CK17 in the filiform papillae of the tongue, the stratified epithelial lining of oral mucosa and esophagus, the outer root sheath of hair follicles, and the glandular epithelia. Mutations in these genes have been associated with pachyonychia congenita. Cytokeratin 8 (CK8), together with cytokeratin 18, was first identified in liver and as marker of mouse embryonal carcinoma (EC) and embryonic stem (ES) cell differentiation. Investigation on early mouse embryos confirmed that the differentiation of the inner cell mass of mouse blastocysts to trophoblast derivatives and extra-embryonic endoderm parallels the induction and accumulation of CK8 and CK18 [2]. Cytokeratin 10 (CK10) is an acidic (type I) cytokeratin. It is synthesized in the suprabasal cell layers of the human epidermis as well as in several epithelial tumours, squamous metaplasias and transformed keratinocytes [3]. Cytokeratins 10 is closely related to CK11 and they are mostly coexpressed with the basic cytokeratin 1. Cytokeratin 14 (CK14) is an acidic (type I) cytokeratin, it is usually coexpressed with cytokeratin 5. Cytokeratin 17 (CK17) is involved in the formation and maintenance of various skin appendages, specifically in determining shape and orientation of hair. CK17 is also considered a marker of basal cell differentiation in complex epithelia and recognizes cervical stem cells [4]. 159 Annamaria Mauro, Maria Buscemi, Aldo Gerbino The expression of CK17 protein has been found increased in cancerous tissues compared with normal tissues in cervical squamous cell carcinoma, laryngeal squamous cell carcinoma, oesophageal squamous cell carcinoma and lung cancers [5]. The different epithelia of the human body express cytokeratins which are not only characteristic of the type of epithelium, but also related to the degree of differentiation. The following pattern of expression of cytokeratins is usually found in the different types of epithelia. One-layered epithelium, generally referred to as вЂњsimple epitheliumвЂќ primarily expresses cytokeratins 8 and 18 and often cytokeratins 7, 19 and 20. Stratified epithelia are subdivided into the keratinizing, the non-keratinizing and transitional type. The keratinizing and non-keratinizing epithelia have a basal cell layer which invariably expresses cytokeratins 5 and 14. The suprabasal cell layers express cytokeratins 4 and 13 in noncornifying epithelia and cytokeratins 1 and 10 in cornifying epithelia. The transitional epithelium of the urinary bladder expresses cytokeratins 4 and 13 together with the cytokeratins of the вЂњsimpleвЂќ epithelia (CK7, 8, 18, 19 and 20). In combined epithelia, morphologically characterized by a basal cell layer and a layer of columnar cells both in contact with the basement membrane, the basal cells express cytokeratins 5, 14 and 17, while the luminal cells can express different combinations of cytokeratins 7, 8, 18 and 19. Squamous epithelia with a high cell turnover express cytokeratins 6 and 16. CKs can be expressed also in non-epithelial cells; in a few mesenchymal cells such as certain smooth muscle cells (particularly vascular wall and myometrium), myofibroblasts, arachnoidal cells, fibroblastic reticulum cells of lymph nodes and rarely endothelial cells, as well as cells of the umbilical cord and foetal myocardium, CK8 and CK18 have been identifyed in addition to the original mesenchymal intermediate filaments (vimentin, desmin). CKs can be expressed in non-neoplastic epithelial lesions. Upon various kinds of damage, epithelial cells may change their pattern of CK expression. The changes may indicate an immature CK pattern of regenerative cells or a metaplastic process which sometimes is not morphologically evident [1]. Carcinomas generally express CK patterns, which at least in part represent the pattern of the putative cells of origin. The consequent profile, which reflects both epithelial type and differentiation status, may therefore be useful in tumour diagnosis. Certain carcinomas may lose the ability to express one or more CKs found in the putative tissue of origin while other types of carcinoma may switch on certain CKs. Non-epithelial tumours are usually CK negative. However, in a number of mesenchymal tumours CK positivity, usually faint or focal and limited to CK8, CK18 and CK19, has been demonstrated [1]. Recent publications have classified breast cancers on the basis of expression of cytok160 Expression of cytokeratins in human umbilical cord at term eratin 5 and 17 at the RNA and protein levels, and demonstrated the importance of these markers in defining sporadic tumours with bad prognosis; expression of basal cytokeratins (CK5, 6, 14, 17) is correlated with adverse clinical outcome for breast cancer [6,7,8]. In the field of diagnostic medicine the antibodies against cytokeratins are usefull in the distinction of epithelial malignancies from sarcomas and lymphomas of various types. Cytokeratin subtype expression patterns are however also being used in the distinction of different types of epithelial malignancies. Umbilical cord is the structure that connects the fetus to the placenta. Umbilical cord at term is made of three vessels (two arteries arranged in coils around a vein) surrounded by the WhartonвЂ™s jelly, a mucous connective tissue rich of water, described as a threedimensional spongy network of interlacing collagen fibers, bundles of glycoprotein microfibrils and an interfibrillar soluble phase [9] which are responsible for its elasticity and its mechanical properties [10]. The epithelium of the human umbilical cord at term is composed of between one and five layers of flattened cells, nuclei and recognizable organelles are absent from some of the superficial cells and their cytoplasm contains a loose meshwork of filamentous material. The plasma membrane is thickened. WhartonвЂ™s jelly cells resemble mesenchymal fibroblasts. Ultrastructural studies indicate that their intrinsic properties are also similar to smooth muscle cells [11] and they are therefore considered as myofibroblasts. The endothelium of the umbilical vessels is made of a single layer of endothelial cells containing cisternae of endoplasmic reticulum, mitochondria and Golgi apparatus. Large accumulations of glycogen and Weibel & Palade bodies are present [12]. The expression of cytokeratins in umbilical cord has been investigated by many groups; Bader et al. [13] and Bea et al. [14] showed the presence of low levels of cytokeratin 8, 18 and 19 in smooth muscle cells of the blood vessel walls of the human umbilical cord, while Mizoguchi et al. [15] investigated through immunohistochemistry the presence of several cytokeratins in the umbilical epithelium. Mahdi et al. [16] found CK1 expression in human umbilical vein endothelial cells (HUVEC). In literature, previous studies gave only fragmentary informations regarding cytokeratins expression in human umbilical cord; the single studies focused only on few types of Cytokeratins and usually concerned only their expression at protein level (immunohistochemical or immunoblotting investigations). With this paper we want to make light on the pattern of expression of a wide range of cytokeratins in human umbilical cord both at the protein level through immunohistochemistry and at RNA level through RT-PCR analysis. 161 Annamaria Mauro, Maria Buscemi, Aldo Gerbino Materials and Methods Samples The analysis was performed on human umbilical cords at term obtained from caesarian delivery. The umbilical cords were provided by the Gynecologic and Obstetric clinic of the University вЂњPaolo GiacconeвЂќ in Palermo. Immunohistochemistry Human umbilical cords were collected in physiological solution. One section (1 cm thick) was dissected from each umbilical cord and fixed in formalin solution. After fixation the tissue was dehydrated in a graded series of alcohols, cleared in xylene and paraffin embedded. Sections of 7 Ојm were cut on a Leica microtome RM2145, dryed overnight at 37В°C and then stored at R.T. until use. On the day of the experiment slides were dewaxed in xylene and rehydrated in a graded series of alcohols. Slides were then transferred into distilled water for 5 min. Samples were subjected to enzymatic pre-treatment with pepsin (Dako, code S3002) for 10 min at 37В°C. The immunohistochemistry was performed using the вЂњDakoCytomation EnVision + System-HRP (AEC)вЂќ kit from Dako, following the manufacturerвЂ™s instructions. Briefly: Sections were covered with the вЂњPeroxidase blockвЂќ reagent and incubated 5 min at room temperature. The samples were rinsed once in PBS buffer pH 7.2. The sections were covered with the antibody solution and incubated at 4В°C O/N. Mouse anti human cytokeratin clone AE1/ AE3 antibody (Dako; 1:50 dilution), mouse anti human cytokeratin clone 34ОІE12 antibody (Dako; 1:50 dilution) and mouse anti human cytokeratin clone MNF116 antibody (Dako; 1: 50 dilution) were used. The antibodies were diluted in a 0.1% BSA solution. Samples were rinsed twice in PBS pH 7.2 and then incubated with the вЂњPeroxidase Labelled PolymerвЂќ reagent for 30 min. Samples were rinsed twice in PBS pH 7.2, then incubated with the вЂњSubstrate-ChromogenвЂќ reagent and immediately observed under a light microscope; the reaction was carryed on until the stainig appeared (2-10 min). Reaction was stopped rinsing the slides in distilled water. Negative control sample was treated in the same way but omitting primary antibody. Slides were coverslipped using the вЂњDakoCytomation Faramount Aqueous Mounting MediumвЂќ. The specimens were observed under a Leica DM1000 light microscope. Total RNA extraction Samples of human umbilical cord were frozen in liquid nytrogen immediately after delivery and stored at -80В°C until use. Total RNA extraction was accomplished using the вЂњillustra RNAspin Mini KitвЂќ (Amersham Biosciences, Milan) following the manufacturerвЂ™s in162 Expression of cytokeratins in human umbilical cord at term structions. RNA yield was evaluated spectrophotometrically (A260/A280) and RNA aliquots were stored at -80В°C until use. Reverse-Transcription (RT) Polymerase Chain Reaction (PCR) For RT reaction 2 Ојg RNA was used. To avoid DNA contamination in the RNA samples DNAse digestion was performed using вЂњAMPD1 kitвЂќ (SIGMA). RT reaction was performed using the вЂњEnhanced avian HS RT-PCR kitвЂќ (SIGMA) following the manufacturerвЂ™s instructions. Briefly: 1Ојl random nonamers and 1 Ојl anchored oligo (dT)23 were added to the DNAse digestion product and incubated at 70В°C for 10 min to denature the sample. Then 2 Ојl 10x Buffer, 1 Ојl deoxynucleotide mix, 1 Ојl RNAse inhibitor, 1 Ојl Enhanced AMV Reverse Transcriptase enzyme and DEPC water were added to the sample. The RT reaction was performed in 20 Ојl total volume at 42В°C for 50 min, followed by 95В°C 5 min to inactivate the enzyme. The PCR was performed using the вЂњPCR enzyme Selection Kit- High specificityвЂќ (Invitrogen) following the manufacturerвЂ™s instructions. Briefly: 2 Ојl of template DNA, 0.5 Ојl of the Primers mix (200 nM final concentration) and 22.5 Ојl of the вЂњPlatinum Super MixвЂќ were mixed together. The reaction was cycled for 94В°C 3 min, then 40 cycles of 94В°C 60 sec, 56В°C 60 sec, 72В°C 60 sec, with a final extension at 72В°C 10 minutes. GAPDH gene was used as internal positive control. The PCR products were visualized on 2% agarose gels stained with ethidium bromide. Primers sequences are showed in table 1. Table 1: Names, sequences and amplification product size of the PCR primers used in this work. Primer name GAPDH human forward GAPDH human reverse Cytokeratin-1 forward Cytokeratin-1 reverse Cytokeratin-5 forward Cytokeratin-5 reverse Cytokeratin-6 forward Cytokeratin-6 reverse Cytokeratin-8 forward Cytokeratin-8 reverse Cytokeratin-10 forward Cytokeratin-10 reverse Cytokeratin-14 forward Cytokeratin-14 reverse Cytokeratin-17 forward Cytokeratin-17 reverse Primer nucleotide sequence GAG TCA ACG GAT TTG GTC GT TTG ATT TTG GAG GGA TCT CG agg agg tgg acg tgg tag tg gag ggc aga cag gac cat aa caa gcg tac cac tgc tga ga tca tac tgg gcc ttg acc tc ggg ttt cag tgc caa ctc ag ata gct gcc tcc ggc tct aca tcg aga tcg cca cct ac aga cac cag ctt ccc atc ac tcg cta ctg tgt gca gct ct cgg aac ttc cct ctc ctt ct gac cat tga gga cct gag ga ggc tct caa tct gca tct cc cag ttc acc tcc tcc agc tc tca cct cca gct cag tgt tg Product size 238 bp 220 bp 252 bp 226 bp 272 bp 185 bp 224 bp 325 bp 163 Annamaria Mauro, Maria Buscemi, Aldo Gerbino Results AE1/AE3 antibody reacts with the basic, high molecular weight cytokeratins 1, 2, 3, 4, 5, 6, 7 and 8, and with the acidic low molecular weight cytokeratins 10, 13, 14, 15, 16 and 19. The immunoreactivity for this antibody is localized in the amniotic epithelium of human umbilical cord (Fig.2,3). The fibroblasts of WhartonвЂ™s jelly didnвЂ™t react with AE1/AE3 antibody so that we belive they donвЂ™t express any type of cytokeratin. Endothelial cells and vessels walls do not express cytokeratins. Cytokeratin antibody, clone 34ОІE12, recognizes cytokeratin 1, 5, 10 and 14. Immunohistochemistry showed the expression of these types of cytokeratins (or at least one of them) in the cells of the umbilical epithelium (Fig.4,5), while all the other regions of the umbilical cord didnвЂ™t react with the antibody. MNF116 antibody recognizes cytokeratin 5, 6, 8, 17 and 19. The immunoreactivity for this antibody is localized only in the cells of the umbilical epithelium (Fig.6, 7) while all the other cells of the umbilical cord seems not to express these types of cytokeratins. The transcriptional expression of the genes encoding for these proteins was investigated through Reverse Transcription-Polymerase Chain ReCK1 CK5 CK6 CK8 CK10 CK14 CK17 action (RT-PCR). The analysis revealed the expression of cytokeratins 1, 5, 6, Fig. 8: Representative picture of RT-PCR analysis performed on human umbilical cord. Lane 1: 100bp DNA ladder; lane 2: 8, 10, 14 and 17 (Fig.8). Cytokeratin 8 and 14 were detec- cytokeratin-1 amplification product (220 bp); lane 3: cytokeratin-5 amplification product (252 bp); lane 4: cytokeratin-6 amted in the totality (100%) of umbilical plification product (226 bp); lane 5: cytokeratin-8 amplification cords tested, CK 5 in the 83% of the product (272 bp); lane 6: cytokeratin-10 amplification product samples and CKs 1, 6, 10 and 17 in (185 bp); lane 7: cytokeratin-14 amplification product (224 bp); lane 8: cytokeratin-17 amplification product (325 bp). the 66% of the samples. Conclusions The immunohistochemical expression of all the three cytokeratinвЂ™s antibodies used in this work is limited to the amniotic epithelium of human umbilical cord. The immunoreactivity for all these three antibodies is intense and clear and appears to be specific with the positive cells stained with dark brown granules and no background reactivity. The RT-PCR analysis confirmed the immunohistochemical data showing the transcriptional expression of all the cytokeratins testeded CK-1, 5, 6, 8, 10, 14 and 17. 164 Expression of cytokeratins in human umbilical cord at term Fig.2: AE1/AE3 immunoreaction in the amniotic epi- Fig.3: AE1/AE3 immunoreaction in amniotic epithethelium of human umbilical cord (40X). lium (100X). Fig.4: Cytokeratin antibody, clone 34ОІE12, immuno- Fig.5: Cytokeratin antibody, clone 34ОІE12, immunoreactivity in amniotic epithelium (40X). reactivity in amniotic epithelium (100X). Fig.6: Cytokeratin antibody, clone MNF116, immuno- Fig.7: Cytokeratin antibody, clone MNF116, immunoreactivity in amniotic epithelium (40X). reactivity in amniotic epithelium (100X). Annamaria Mauro, Maria Buscemi, Aldo Gerbino References [1] Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins: pattern of expression in normal epithelia, tumors and cultured cells. Cell 1982 Nov; 31(1):11-34. [2] Maurer J, Nelson B, CeceГ±a G, Bajpai R, Mercola M, Terskikh A, Oshima RG. Contrasting expression of keratins in mouse and human embryonic stem cells. PLoS ONE 2008; 3(10):e3451. [3] Meinhold-Heerlein I, Brandstetter T, Kommoss F, Bettendorf H, Hagedorn M, Bauknecht T. Localization of cytokeratin 10 mRNA in human epidermis using nonradioactive in situ hybridization as a routine method. Arch Dermatol Res 1998 May; 290(5):286-8. [4] Martens JE, Arends J, Van der Linden PJ, De Boer BA, Helmerhorst TJ. Cytokeratin 17 and p63 are markers of the HPV target cell, the cervical stem cell. Anticancer Res 2004; 24: 771вЂ“775. [5] Wei K-J, Zhang L, Yang X, Zhong L-P, Zhou X-J, Pan H-Y, Li J, Chen W-T, Zhang Z-Y. Overexpression of cytokeratin 17 protein in oral squamous cell carcinoma in vitro and in vivo. Oral Diseases 2009; (15): 111вЂ“117. [6] Gusterson BA, Ross DT, Heath VJ, Stein T. Basal cytokeratins and their relationship to the cellular origin and functional classification of breast cancer. Breast Cancer Res 2005; 7(4): 143вЂ“148. [7] Livasy CA, Karaca G, Nanda R, Tretiakova MS, Olopade OI, Moore DT, Perou CM. Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol 2006 Feb; 19(2):264-71. [8] Chen MH, Yip GW, Tse GM, Moriya T, Lui PC, Zin ML, Bay BH, Tan PH. Expression of basal keratins and vimentin in breast cancers of young women correlates with adverse pathologic parameters. Mod Pathol 2008 Oct; 21(10):1183-91. Epub 2008 Jun 6. [9] Meyer FA WhartonвЂ™s Jelly of the Umbilical cord; in: Extracellular Matrix. Tissue Function, W.D. Comper, ed Harwood Academic Publishers, Amsterdam 1996. Vol. 1, 443-456. [10] Sobolewski K, Bankokski E, Chyczewski L, Jaworski S. Collagen and glycosaminoglycans of WhartonвЂ™s jelly. Biol Neonate 1997; 71(1):11-21. [11] Can A, Karahuseyinoglu S. Concise review: Human umbilical cord stroma with regard to the source of fetus-derived stem cells. Stem cells 2007 Nov; 25(11):2886-95. [12] Parry EW, Abramovich DR. The ultrastructure of human umbilical vessel endothelium from early pregnancy to full term. J Anat 1972 Jan; 111(Pt 1):29-42. [13] Bader BL, Jahn L, Franke WW. Low level expression of cytokeratins 8, 18 and 19 in vascular smooth muscle cells of human umbilical cord and in cultured cells derived therefrom, with an analysis of the chromosomal locus containing the cytokeratin 19 gene. Eur J Cell Biol 1988 Dec; 47(2):300-19. [14] Bea F, BГ¤r H, Watson L, Blessing E, KГјbler W, Kreuzer J, Jahn L. Cardiac alpha-actin in smooth muscle cells: detection in umbilical cord vessels and in atherosclerotic lesions. Basic Res Cardiol 2000 Apr; 95(2):106-13. [15] Mizoguchi M, Ikeda S, Suga Y, Ogawa H. Expression of cytokeratins and cornified cell envelope-associated proteins in umbilical cord epithelium: a comparative study of the umbilical cord, amniotic epithelia and foetal skin. J Invest Dermatol 2000 Jul; 115(1):133-4. [16] Mahdi F, Shariat-Madar Z, Todd RF 3rd, Figueroa CD, Schmaier AH. Expression and colocalization of cytokeratin 1 and urokinase plasminogen activator receptor on endothelial cells. Blood 2001 Apr; 15;97(8):2342-50. 166 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 The evolutionary course of Vertebrate foot, from Fish to Man [Il corso evolutivo del piede dei Vertebrati, dal pesce allвЂ™uomo] Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello and Elvira Farina Lipari Department of Experimental Medicine, University of Palermo (IT) Key words: Humans, Anthropoids, Primates, Reptiles, Amphibians Parole chiave: Uomo, Antropoidi, Primati, Rettili, Anfibi Abstract. The evolutionary course of Vertebrates foot starts from the posterior (or pelvic) fins of aquatic Vertebrates (Fishes) and from these it follows a long way through Amphibians, Reptilians, archaic Mammals, Primates and modern Man. So, a functional analogy can be seen between the Fish fins and the Vertebrate limbs. During the evolution, the foot becomes вЂњa mechanic complexвЂќ with longitudinal and transverse arches united to constitute a вЂњstructural unityвЂќ that plays a role of supporting the body weight and discharging it to the ground while allowing a walking movement. In this review we do an excursus of the evolution of vertebrate foot, from fish to Man. Riassunto. Il corso evolutivo del piede dei Vertebrati inizia dalle pinne posteriori (o pelviche) di vertebrati acquatici (Pesci) e da questo esso segue un lungo percorso attraverso gli Anfibi, Rettili, Mammiferi arcaici, Primati e Uomo moderno. Pertanto, una analogia funzionale puГІ essere osservata tra le pinne dei pesci e gli arti dei Vertebrati. Durant lвЂ™evoluzione, il piede diviene вЂњun complesso meccanicoвЂќ con archi longitudinali e trasversali uniti a costituire una вЂњunitГ strutturaleвЂќ che svolge un ruolo di supporto al corpo per scaricare il peso sulla terra durante il cammino. In questa review noi facciamo un excursus della evoluzione del piede dei Vertebrati, dai pesci allвЂ™uomo. 1. The fish fins Fishes appear in the middle Paleozoic era, in a period included between 450 and 330 million years ago. Among the most advanced pluricellular primordial organisms, they are endowed with lengthen body, head, tail, nervous system, an impair skin fold and two pair ones. From the impair skin fold derive the impair fins: the dorsal, tail and anal fins; from the pair folds derive the pectoral and pelvic fins (Fig.1a). These fins were originally unarmed, successively armed with contractile and skeletal elements; consequently they take on the role of body appendix able to grant the wavering tail movement, making it stable and orienting its direction [4,9]. In particular, Fish fins framework, initially made of cartilaginous tissue formed by little and flexible laminae and sequentially by bone tissue, allows fish mo- 167 Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello, Elvira Farina Lipari vements into the aquatic habitat without having, however, any supporting role for the body weight. Besides we must signal that, while we can certainly recognize a functional correspondence or analogy between pair Fish fins and terrestrial Vertebrate limbs, it is not demonstrable the homology or morphological correspondence [7]. 2. The amphibian foot Amphibian foot of late Paleozoic era, included between about 330 and 240 million years ago, is considered the progenitor of Vertebrate foot (Fig.1b). Already in this era we can recognize a general organization of pentadactyl base (fivetoed) and the three constitutive parts are outlined yet: a proximal (tarsus), a middle (metatarsus) and a distal one (phalanges). In the tarsus of Amphibian foot there are eleven bones (Fig.1c) ordered in two transverse rows, a proximal one and a distal one. The proximal row is made by three (variable) bones, the tibial (ti), the intermedium (i) and the fibular (fi) one which is in relationship with tibia (T) and fibula (F); the distal row has five tarsal bones (T1-T5) each of them in relationship with the correspondent metatarsal bone. Between the proximal row and the distal one there are mainly three central bones, a proximal central (cp) and two distal central (cd) ones. Besides, there are two inconstant adding members: one along the medial margin, the prehallux 168 Fig.1: a) Fins in a Fish belonging to the Palaeozoic era (between 450 and 330 million years ago). b) Pentadactyl basic organization of Primitive Amphibian foot of the late Palaeozoic era (between 330 and 240 million years ago). Its anatomical parts were already defined (tarsus, metatarsus and phalanges). c) Tarsal bones of Amphibian foot with two additional inconstant members, the prehallux ray (Ph) and the postminimus ray (Pm). (T: tibia, F: fibula, Ti: tibial, i: intermedium, fi: fibular, cp: central proximal, cd: central distals, T1-T5: metatarsals); from Morton 1948, modified. d) Metatarsal bones (M1-M5) and phalanges* of Amphibian foot toes from the first to the fifth; digital formula: 2,3,4,5,4. The evolutionary course of Vertebrate foot, from Fish to Man ray (Ph) and the other one along the lateral margin, the postminimum ray (Pm). Concerning metatarsal bones and phalanges (Fig.1d), there are five metatarsal bones and in the five toes there are totally eighteen phalanges with a digital formula, 2,3,4,5,4, characteristic in Amphibian foot, where the first toe is constituted of two phalanges, the second of three, the third of four, the fourth of five and the fifth of four. The primitive Amphibian pentadactyl foot is wholly constituted of thirty-four skeletal segments. In Amphibians, bones composing foot show an incomplete ossification, and its longitudinal axis seems to be located on the same plan of the leg one. So Amphibian foot, being partially able to support the body weight against the terrestrial force of gravity, allows these partially terrestrial Vertebrates a certain raising of the animal centre of gravity from the ground, permitting the crawling movement of the body on the ground. In this precarious static condition, the load imbalance on the foot lateral margin determines the increasing number of lateral toes phalanges, and consequently the digital formula 2,3,4,5,4 [7]. 3. The reptilian foot In the late Paleozoic era and in Mesozoic era, during the Reptiles age, which is included between 240 and 85 million years ago, by reaching correct connection between proportion and sturdiness, foot, being able to raise the animal centre of gravity, takes the role of organ suitable for translating it on the ground: bones that constitute it, lined in an anteriorposterior axis direction, as for a longitudinal axis that form an angle with the leg axis, allow foot movements of dorsal-plantar flexion in the talocrural articulation. Making a comparison between the Amphibian skeletal foot components and the Reptilian one, in the latter while we find a complete mineralization of the bones which constitute it for bearing charges, we register a numerical reduction in the tarsus bones from eleven to seven, because of the fusion or reabsorbing of some of them [17]. In particular (Fig.2a): The intermedium (i) melts with central proximal (cp) in order to compose the talus; The fibular (fi) increases remarkably in its dimensions and volume, constituting the heel, disposing under the talus and entering in contact with the ground; The tibial (ti) progressively reabsorbs; The two central distal (cd and cd) melt together in order to compose the navicular; About the five tarsal bones, T1, T2 and T3 form respectively the medial cuneiform, the intermedium and the lateral while T4 and T5 melt together in order to compose the cuboid, which is the bone with the main volume in this row; The components of prehallux and postminimun ray, if present, progressively reabsorb; The five metatarsal bones remain as they were; The toes bones, phalanges, ordered in a linear succession, modify (Fig.2b) the digital 169 Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello, Elvira Farina Lipari formula of Amphibian foot (2,3,4,5,4) into a 2,3,3,3,3 formula, where the first toe is composed by two phalanges, proximal and distal, and the other four toes by thre phalanges, proximal, middle and distal. This digital formula is successively found again in Mammal and Primate foot. Totally, Reptile foot, compared to Amphibian one, presents a great reduction, (about 25%), of skeletal segments; from thirty-four bones in Amphibian foot to twenty-six in Reptilian one; besides these ones, we must consider even the sesamoid bones, which are present along the tendons path of the long flexor muscles of foot toes. 4. The mammal foot The archaic Mammals settled on earth during the end of Cretaceous period of Mesozoic era and during the Eocene and Oligocene periods of Cenozoic era, between about 85 and 35 million years ago. These Mammals, whose ancestry were actually represented by Therapsid group of вЂњmammals-likeвЂќ reptiles, during the Triassic period of Mesozoic era, were present on earth after the Jurassic mammals extinction [5]. These Mammal foot registers: the complete and definitive disappearance of marginal toes; the acquisition by the talus of a pulley, the trochlea, which inserts into wedge-shaped articular socket of the ankle joint and of a head for 170 Fig.2: a) Reptilian foot tarsal bones in the Mesozoic era (between 240 e 85 million years ago). We observe: fusion between the intermedium (i) and central proximal (cp) bones, which form the talus; increment of the fibular (fi) dimension which forms the calcaneus; fusion between two central distals (cd+cd) which forms the navicular; tarsal bones T1,T2,T3 which form the cuneiform medial, the intermedium and the lateral; fusion of the tarsal bones T4 and T5 which form the cuboid; progressive tibial (ti) reabsorption and prehallux ray (ph) (from Stark 1955, modified). b) Metatarsal bones (M1-M5) and phalanges* of Reptilian foot toes from first to the fifth; digital formula: 2,3,3,3,3. The evolutionary course of Vertebrate foot, from Fish to Man the talocalcaneonavicular joint; the definitive positioning of the heel under the talus with the plantar flexor muscles which insert on the heel; the steady union of tarsal bones with each other; the sturdiness and divergence of first metatarsal; the prehensile characteristic of hallux, which is developed and abducted; the long toes with the formula 2,3,3,3,3; the foot longitudinal axis which involves the talus, the heel, the lateral cuneiform, the three metatarsal and the third toe, that is the centre of the lever action of dynamic foot (Fig.3a). With the complete acquisition of all these morphostructural elements, foot is already conformed for his primitive role of gripping organ which allows the arboreal phase in these Mammals. Archaic Mammal foot, briefly, presents all the components of Reptilian foot that for this reason can be placed in the centre of the Vertebrate foot evolutionary course. Once that Mammals defined the essential and fundamental skeletal structures, a great variety of modifications took place in thoracic and pelvic limbs in toto. In Modern Mammals, appeared less than 35 million years ago, during the Miocene, Pliocene and Pleistocene period of Cenozoic era, proper evolutionary lines defined in conFig.3: a) Foot skeleton of archaic Mammals, settled on earth during the end of Cretaceous period, between 85 and 35 million years ago. We observe: divergence of the marginal toes; presence of trochlea and talus head; divergence of the first metatarsal and hallux; long toes, with formula 2,3,3,3,3; foot longitudinal axis passing through the third ray. b) During the evolutionary course from the Monkeys to Man, we observe the formation of the longitudinal medial arch; the heel gains the sustentaculum tali and the functional axis of the third ray in Monkey moves medially in Ape, between the first and the second ray; instead in modern Man it involves the second ray. c) The gradual changes of metatarsal bones torsion, symbolized by the oblique lines and the arrows, is clear on foot of Monkey, Ape and Man (from Morton 1948, modified). d) The arboreal Monkeys foot: angulated leverage axis. The semiterrestrial Apes foot: partial correction of the leverage axis. Lengthening of the first metatarsal bone causes a reduction in the angle BEF; adduction of the bone to DвЂ™ further reduces the angle to BEFвЂ™. The terrestrial Human foot: the leverage axis forms a straight line AB. A-B: The functional axis, tarsal and metatarsal portion; A-F: The angulated leverage axis, tarsal and metatarsal portion; BEF and BEFвЂ™: The angle; C-D and C-DвЂ™: Line between the heads of metatarsal I and II which act as the fulcrum; E-F: Line of metatarsal portion of leverage axis perpendicular to C-D; G-H: The direction of body movement. (From Morton 1948, modified). 171 Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello, Elvira Farina Lipari nection with the animal way of life, the role they are intended to have and the habitat. Limbs can be transformed, for example, in flight organ (Bats), in running organ (Equidae), paddles for swimming (Cetaceans), in limbs with gripping function thanks to the opposition of thumb and hallux with the other fingers or toes, as we can see in Primates and Man. A particular interest is in the evolutionary course variations of the extreme distal of thoracic and pelvic limbs relative to the number of toes, which are pair (in artiodactyls) or impair (in perissodactyls). In the extreme distal of Horse limb we can find that only the third toe supports the body weight and lean on the ground with the distal phalange endowed with a hoof, in Ruminants there are the third and the fourth toes, in Swine there are the third and the fourth toes but also the second and fifth ones, that however do not lean on the ground, in Hippopotamus there are four toes while in Marsupials there are five toes. 5. The primate foot Mammal order is represented by Primates which include Monkeys and Anthropoids Apes (Chimpanzee, Gibbon, Orang, Gorilla). Anthropoids apes are considered the most advanced existent Primates and they use thoracic and pelvic limbs for moving among the trees [15], the so called вЂњbrachiatingвЂќ; these Primates during the Miocene period of Cenozoic era, were preceded by a primitive stock of quadrupedal Primates and, in a very long space of time, they underwent a fundamental modification in their posture that changed from horizontal to vertical for a bigger development of superior limbs, for the accentuate shoulder mobility and for the pelvic limbs moving in a direct continuity with the trunk [8,10]. We usually believe that between pronogrades Anthropoids and ortogrades Hominids and between these ones and Modern Man there are still some connections, unknown by our knowledge and interpretation, which could explain and clarify particular aspects about form and actual structure [1]. During the Cenozoic era, probably before the beginning of Miocene period, about 40 million years ago, Man inherited form unknown arboreal bipeds progenitors, a foot endowed with a short and mobile hallux [6]. So, Primate and Man foot derives from a unique ancestor, the Australopithecus, whose structures had not reach the definitive and peculiar organization [16]. This ancestor reaches the terrestrial bipedism with a gradual transfer of locomotory functions on pelvic limbs, with the heel leaning on the ground and with the reduction of foot grasping function on the ground and his assuming lever functions for lifting and pushing body against the force of gravity. For defining and achieving the evolutionary course towards human foot, we need the intervention of further modifications regarding: the constitution of the medial longitudinal arch; the moving of foot functional axis between the first and the second ray; the toes 172 The evolutionary course of Vertebrate foot, from Fish to Man reduction in length; the torsion of lateral metatarsal bones; the first metatarsal bone divergence. The medial longitudinal arch takes form during the evolutionary course between Monkeys and Anthropoids apes, the heel medial surface, besides, is endowed with a very important bracket shaped relief, the sustentaculum tali, and foot functional axis moves medially from the third ray, involving in Man the second ray (Fig.3b). The reduced fingers length is necessary because they have not a role as grasping for climbing anymore. The medial torsion of lateral metatarsal bones and their flexion, that converge towards the first metatarsal, can be easy recognized (Fig.3c) in Chimpanzee foot, it is indicated by the oblique lines and arrows, and it characterizes the foot role as an arboreal organ; in terrestrial Gorilla we can notice that the metatarsal bones torsion is remarkable reduced, instead, in Man, the torsion is practically disappeared and metatarsal bones plantar turned. The first metatarsal divergence is evident looking at the Chimpanzee, Gorilla and Man feet (Fig.3d). In the Cimpanzee arboreal foot, it is clear the first metatarsal divergence and shortness: between the metatarsal anterior part of lever axis and the foot functional axis there is an angle included; the direction of body on ward translation movement is realized on the metatarsal part of lever axis, thanks to a movement of foot external rotation, from which it derives the characteristic walking of these Monkeys; in the terrestrial Gorilla foot, the lengthening and the partial adduction towards the lever axis of first metatarsal determines a reduction of the abduction angle, in Man foot, the complete adduction of first metatarsal determines the disappearing of the abduction angle, and the lever axis coincides with the functional axis which goes the second ray [7]. The finger length, the lateral metatarsal bones torsion, the first metatarsal bone divergence then represents the morphostructural conditions right for an arboreal bipedism, but not for the terrestrial bipedism of Human foot. 6. The human foot The skeletal elements of Human foot (Fig.4) are twenty-eight and are represented by seven bones at tarsal level (talus, heel, navicular, medial, intermedium and lateral cuneiform, cuboid); five bones at metatarsal level; fourteen phalanges at toe level (two phalanges, a proximal and a distal one, for the first toe, three phalanges, a proximal, a middle and a distal one, for the second, third, four and five toe); there are also two other canonical bones: the sesamoid hallux bones [14]. In the embryonic and foetal life, during the human foot ontogenesis, bones that composed it, at the beginning, present a linear series disposition on a sole plane, as is in the hand; successively, following a series of genetic messages, it occurs the so called вЂњpodalic windingвЂќ where the medial longitudinal arch rises, wrapping around the lateral longitudinal 173 Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello, Elvira Farina Lipari arch, making an helicoidally structure known as plantar vault; in the wake of this вЂњpodalic windingвЂќ, the skeletal elements of hindfoot overlaps, while in the forefoot and midfoot they dispose on the same plane [2]. Human foot is therefore made up by a dome-shaped bone system (fig.4) with the dorsum looking upward, posteriorly convex and anteriorly flattened; the sole is concave and comparable to a tripod resting on three points: posteriorly, the calcanear tuberosity, anteriorly and medially the head of the first metatarsal, anteriorly and laterally the head of the five metatarsal. Actually, load share that burdens on the forefoot uses for leaning against the ground six points so represented: two points on the two sesamoids of first metatarsal head, four points, one for each ones, on the second, third, fourth, fifth metatarsal heads [12]. Each foot, from an architectonic point of view, is actually represented by the curvilinear section of a half-vault, which basis is represented by the lateral border of the foot and the summit by the medial border; a complete dome is formed only when the two medial borders of the foot are approached together. Therefore Human foot can be defined as a complex mechanical construction with a Fig.4: Human foot bones seen from the dorsal surface (a) and from the medial margin (b,c). We can observe a 90В° angle between the leg and the foot at talocrural region (c); it is also evident the almost complete disappearing of grasping functions. 174 The evolutionary course of Vertebrate foot, from Fish to Man curvilinear axis with the medial longitudinal arch, more important than the lateral one, is higher and with a strong resilience; a lateral longitudinal arch, on the contrary, is much flatter and rigid in contact with the ground. The transverse arches of the foot run from side to side; they are numerous and they link the two longitudinal arches. Mainly we describe two main transverse arches: a posterior one lying the midfoot and the forefoot, at a Lisfranc line, and an anterior one in the forefoot, at a metatarsophalangeal line [12]. The longitudinal and transversal arches, complete and fixed together by strong ligaments, especially plantar, constitute a real вЂњstructural unityвЂќ that has two important function: to support the body weight and to serve as a lever to propel the body forward in walking and running. 7. Exegeses and views Greater anatomic differences distinguish the terminal segment of the lower limbs of Homo sapiens from the corresponding segment of the upper limb than in another Primates or Mammals. The anatomy of the foot is more characteristic of the human species than the anatomy of the hand; the erect bipedal habit account for these differences. The evolutionary course of Vertebrate foot, from Fishes to Man, reach its finish line through a series of successive improving valuable thanks to the following objective data (Fig. 4): foot and leg rotation of 180В° with the first ray is medially arranged, on the same plane of the other toes; horizontal direction and 90В° position compared to leg in order to amplify the supporting surface; completely vault-like morphological order with a plantar niche turning down in a way to consent an adequate protection for its contents against the mechanical stresses; foot bones volume and dimension, particularly at hindfoot level, where talus and heel must support the body weight and convey it to the ground; adequate and perfect inserting of astragalic trochlea into the wedge-shaped articular socket of the ankle joint for constituting together a powerful lever able to support and lift the body weight against the gravity, also allowing step pushing, riding, jumping [13]. During Human foot evolutionary period, at the forefoot level, we assist to the almost complete disappearing of grasping function (Fig.4) which shows through: atrophy of toe phalanges, which are shorter, more little and less mobile than the other Primates ones; hallux, that usually is the longest, only supports the foot static functions and the dynamical ones transmitted during the locomotion; first ray reduced mobility and loss of the other toes opposing function; hallux position on the plane of other toes with a flexor surface plantar-like turned, as for the other lateral toes, to constitute a unique complex suitable for its propulsive function; sturdiness of metatarsal bones, which are more mobile at head level and endowed with a larger possibility of executing movements of dorsal-plantar bending and limited move175 Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello, Elvira Farina Lipari ment of abduction and adduction compared to the axis toe represented in human foot by the second toe, partially fixed. 8. Conclusions Did the evolutionary process of Vertebrate foot effectively complete and exhaust its course? Would it not be possible to have more transformations in diversified or modified aspects under an organizing point of view, during the time? It is well-known that, through chemical-physical factors, it is possible to introduce changes inside the genetic molecule and that genetic mutation can realize, without any logic concatenation, even naturally and not only experimentally. The actual Human modus vivendi is under everyoneвЂ™s attention; with the correct exceptions, we donвЂ™t know or only partially know the pleasure in using the inferior limbs; yet terrestrial bipedism was the great conquest that diversify us from the other Primates! It becomes more and more strong an invitation to walk, even because this activity is good for health, it is useful to take weight under control, reduces risk in cardiovascular disease (-30%). Even if we share this problematic, we express our perplexity and our worry because, in a very long time, according to the vertebrate evolutionary course rules, it can intervene modifications in the organizing order, in the lower limb and, in particular, in the foot not in a positive, but in a negative sense, or even in a regressive one, in the wake of a gradual process of atrophy ex non usu. In this perspective, only posterity in the future and after several decennials, could verify the rightness of our not really optimistic forecasts. 176 The evolutionary course of Vertebrate foot, from Fish to Man References [1] Bruno G. Origine e perfezionamento del piede umano. Riv Biol 1958; 50:297-310. [2] Gravante G, Pomara F, Russo G, Amato G, Cappello F, Ridola C. 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Cour Forsch-Inst Senckenberg 2004; 243:35-45. [10] Richmond BG, Jungers Wl. Orrorin tugenensis femoral morphology and the evolution of human bipedalism. Science 2008; 319:1599-1601. [11] Ridola C, Cappello F, Palma A. Anatomy of the foot. Med Sport 2002; 55:229-240. [12] Ridola C, Palma A. Functional anatomy and imaging of the foot. It. J Anat Embryol 2005; 106:85-98. [13] Ridola CG, Cappello F, Lipari D, MarcianГІ V, Palma A. Clinical anatomy of joints of the foot. Med Sport 2006; 59:497-508. [14] Ridola CG, Cappello F, MarcianГІ V, Farina-Lipari E, Palma A. The synovial joints of the human foot. It J Anat Embryol 2007; 112:61-80. [15] Sellers WI, Cain GM, Wang W, Crompton RH. Stride lenght, speed and energy costs in walking of Australopithecus Afarensis: using evolutionary robotics to predict locomotionof early human ancestors. J R Soc Interface 2005; 2:431-441. [16] Senut B, Pickford M, Gommery D, Mein P, Cheboi K, Coppens Y. First hominid from the Miocene (Lukeino Formation, Kenya). C R Acad Sci 2001; Paris 332:137-144. [17] Stark D. вЂќEmbryologyвЂќ. Stuttgart, Thieme, 1955. [18] вЂњTerminologia AnatomicaвЂќ, International Anatomical Terminology. FCAT, Stuttgart, Thieme, 1998. 177 Gastrointestinal tract Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 Orexina e Orfanina FQ nuovi neuropeptidi regolatori delle funzioni del Sistema Gastroenteropancreatico. Uno studio immunoistochimico [Orexin and orphanin FQ in GEP system. An immunohistochemical study] Giuseppe Bonaventura, Giovanni Francesco Spatola, Maria Laura Uzzo e Vincenzo Tessitore Di.Me.S. - Sezione di Istologia ed Embriologia - UniversitГ degli Studi di Palermo (IT) Parole chiave: Orexina, Orfanina FQ, Sistema gastroenteropancreatico, Immunoistochimica Key Words: Orexin, Orphanin FQ, GEP system, Immunohistochemistry Riassunto. La orexina A (OXA) e B (OXB) chiamate pure ipocretina 1 e 2 e la orfanina FQ sono membri di una famiglia di neuropeptidi ritenuti in origine esclusivamente prodotti da neuroni dellвЂ™area ipotalamica posterolaterale ove regolano lвЂ™assunzione alimentare e il peso corporeo attivando specifici recettori e svolgendo un ruolo oressizzante. In aggiunta la orfanina FQ che presenta somiglianze strutturali con i neuropeptidi oppioidi (enkefaline-endorfine) ГЁ stata implicata nella nocicezione. PiГ№ di recente, evidenze sperimentali fanno ritenere che tali neuropeptidi svolgano i loro effetti al di fuori del S.N.C. in distretti periferici correlati o non correlati con lвЂ™equilibrio nutrizionale. In riferimento a tali acquisizioni registrate al di fuori del SNC abbiamo svolto indagini immunoistochimiche sulla espressione dei neuropeptidi summenzionati nel Sistema Gastroenteropancreatico del ratto normale. I nostri risultati forniscono documentazioni immunoistochimiche che autorizzano ad ammettere che sia la Orexina A con il suo recettore tipo 2 che la orfanina FQ possono essere espresse perifericamente da molti citotipi costitutivi del Sistema GEP. In aggiunta, i nostri reperti forniscono la base morfoistochimica per lвЂ™interpretazione del ruolo funzionale svolto dallвЂ™orexina e dallвЂ™orfanina in tali distretti suggerendo la possibilitГ che esse possano esercitare unвЂ™azione diretta di tipo periferico sulle strutture summenzionate esplicando nuovi ruoli con meccanismi neurocrini, endocrini e autocrino-paracrini. Abstract. The orexin A (OXA) and B (OXB) ipocretin 1 and 2 also called and the orphanin FQ are members of a family of neuropeptides originally considered to be exclusively produced by hypothalamic neurons in the posterolateral region, where regulate food intake and body weight by activating specific receptors, and playing a orexigenic role. In addition, the orphanin FQ presenting structural similarities with the opioid neuropeptides (enkefalin-endorphins) has been implicated in nociception. More recently experimental evidences suggest that these neuropeptides play their effects outside the CNS in peripheral districts related or not related to the nutritional balance. In reference to these acquisitions verified outside the CNS, we investigated the immunohistochemical expression of these neuropeptides in the Gastroenteropancreatic System of normal rats. Our immunohistochemical results provide the documentation that authorizes to admit that the orexin A with its receptor type 2 and the orphanin FQ can be expressed by several peripheral citotypes of the GEP system. In addition, our findings provide the morphoistochemical basis for the functional interpretation of the role undertaken by orexin and orphanin in these districts and suggest the possibility that they may bring a direct peripheral action on these structures playing new roles probably with neurocrine, endocrine and autocrineparacrine mechanism. 181 Giuseppe Bonaventura, Giovanni Franco Spatola, Maria Laura Uzzo, Vincenzo Tessitore Introduzione LвЂ™orexina A (OXA) e B (OXB), chiamate pure ipocretina 1 e 2 e la orfanina FQ sono membri di una famiglia di neuropeptidi ritenuti in origine esclusivamente prodotti da neuroni centrali dellвЂ™area ipotalamica posterolaterale ove regolano lвЂ™assunzione alimentare e il peso corporeo attivando specifici recettori e svolgendo un ruolo oressizzante. In aggiunta lвЂ™orfanina FQ che presenta somiglianze strutturali con i neuropeptidi oppioidi (enkefaline-endorfine) ГЁ stata implicata nella nocicezione, donde la denominazione alternativa di nocicettina ad essa attribuita. Successive precisazioni hanno chiarito che la orfanina ГЁ estesamente distribuita nel S.N.C. [1] per cui ГЁ stato ritenuto che essa sia implicata non soltanto nei complessi e discussi fenomeni nocicettivi ma anche nei fenomeni di apprendimento e di memoria, nellвЂ™ansia e nella epilessia. PiГ№ di recente aldilГ di tali neuroeffetti centrali, evidenze sperimentali fanno ritenere che tali neuropeptidi svolgano i loro effetti al di fuori del S.N.C. in distretti periferici correlati o non correlati con lвЂ™equilibrio nutrizionale [2-12]. Tutto ciГІ ovviamente dischiude dal punto di vista clinico-pratico nuove prospettive farmacoterapeutiche. In riferimento a tali acquisizioni registrate al di fuori del SNC abbiamo svolto indagini immunoistochimiche sulla espressione dei neuropeptidi summenzionati nel Sistema Gastroenteropancreatico del ratto normale. I risultati preliminari di tale ricerca sono stati comunicati al 31В° Congresso della SocietГ Italiana di Istochimica (Pisa 2005) e al 59В° Congresso della SocietГ Italiana di Anatomia e Istologia (Sorrento, 2005) [13,14]. Materiali e metodi Sono stati impiegati 10 ratti Wistar trattati in accordo con la Convenzione di Helsinki sullвЂ™utilizzo degli animali nella ricerca biomedica. Gli animali sono stati sacrificati previa anestesia (50 mg/kg di Nembutal somministrato per via endoperitoneale). Sono stati prelevati lo stomaco, lвЂ™intestino tenue e il pancreas. I campioni sono stati immediatamente fissati in liquido di Bouin ed inclusi in paraffina. I campioni sono stati sezionati, processati per la tecnica immunoistochimica e svelati mediante kit di rivelazione Envision + System HRP (AEC) (DAKO Citomation), utilizzando anticorpi policlonali anti Orexina A (Chemicon AB3098), anti Orexina-2 Receptor (Chemicon AB3094) data lвЂ™indisponibilitГ commerciale di un anticorpo per il recettore tipo 1 ed un anticorpo policlonale anti Orfanina FQ (Chemicon AB5515). Per la identificazione delle cellule principali gastriche sono stati impiegati il blue di toluidina e la ematossilina ferrica. Contemporaneamente i controlli negativi sono stati realizzati su sezioni adiacenti processate in base allo stesso protocollo omettendo il passaggio dellвЂ™antisiero primario. Tutti i campioni sono stati studiati mediante fotomicroscopio Leica DMLB. 182 Orexina e Orfanina FQ nuovi neuropeptidi regolatori delle funzioni del Sistema Gastroenteropancreatico. Uno studio immunoistochimico Risultati Nello stomaco Orexina A: intensa immunopositivitГ si evidenzia in cellule endocrine incastonate nel contesto degli adenomeri tubulari delle ghiandole della regione del fondo gastrico, le cui cellule parietali e zimogeniche mostrano una attivitГ orexinergica immunoistochimicamente assai debole (Fig.1-2). Nessuna immunopositivitГ ГЁ rilevabile a carico delle cellule principali. Nella regione antropilorica la immunopositivitГ per la orexina A ГЁ piГ№ evidente ed estesa essendo piГ№ abbondanti le cellule endocrine presenti soprattutto nella porzione mediobasale delle ghiandole tubulari ramificate. Sia nella regione del fondo gastrico che nella regione antropilorica il rilievo immunoistochimico del recettore OX2R ГЁ assai scarso e limitato ad alcuni citotipi endocrini presenti soprattutto nella regione mediobasale delle ghiandole. Discreta immunopositivitГ al recettore tipo 2 mostrano alcune cellule del corion mucosale che attorniano i fondi ghiandolari al limite con la sottomucosa. Fig.1: Stomaco di ratto вЂ“ regione antrale: intensa immunoreattivitГ OXA delle cellule endocrine (frecce nere); debole immunoreattivitГ a carico delle cellule parietali (frecce azzurre) - Ob. 63x Fig.2: Stomaco di ratto вЂ“ regione del corpo: intensa immunoreattivitГ orexinergica di cellule endocrine (frecce); scarsa immunoreattivitГ OXA in numerose cellule parietali - Ob. 63x Fig.3: ImmunoreattivitГ N/OFQ delle cellule endocrine Fig.4: ImmunoreattivitГ N/OFQ delle cellule principali (freccie) e delle cellule principali delle ghiandole della delle ghiandole della regione fundica (freccie verdi) e immunonegativitГ delle cellule parietali (freccie regione fundica - 40x nere) - 40x 183 Giuseppe Bonaventura, Giovanni Franco Spatola, Maria Laura Uzzo, Vincenzo Tessitore Orfanina FQ: nella regione antropilorica intensa immunopositivitГ si rileva in isolate cellule endocrine presenti negli adenomeri tubulari (Fig.3). Sia nella regione antropilorica che nella regione fundica discreta immunoreattivitГ ГЁ evidenziabile suggestivamente a carico delle cellule principali (Fig.4) identificate in sezioni adiacenti a quelle trattate immunoistochimicamente con il blu di toluidina e con la ematossilina ferrica. Le cellule parietali sono immunoistochimicamente negative (Fig.4). In alcune sezioni si scorgono pure fini fibre orfaninergiche tra gli adenomeri o tra i citotipi secernenti. Fig.5: Intestino tenue di ratto: immunopositivitГ ore- Fig.6: Intestino tenue di ratto: intensa immunoespressione dellвЂ™OX2R xinergica degli enterociti mucosali. Nel corion villare degli enterociti mucosali dei villi. Assai marcata immunoespressionumerosi citotipi immunopositivi - Ob. 40x ne dellвЂ™OX2R in grosse cellule dello stroma villoso - Ob. 20x Fig.7: Stomaco di ratto вЂ“ regione antrale: immunoe- Fig.8: ImmunoreattivitГ N/OFQ negli enterociti villaspressione OXA in piccoli neuroni submucosali ente- ri (freccie nere); cellule caliciformi immunonegative rici e negli epiteliociti degli adenomeri вЂ“ Ob. 40x (freccie verdi) - Ob. 20x Fig.9: Pancreas endocrino di ratto: marcata reattivitГ Fig.10: Intensa immunoreattivitГ N/OFQ pancreatica orexinergica dei citotipi endocrini - Ob. 40x nei citotipi insulari e negli acini - Ob. 20x 184 Giuseppe Bonaventura, Giovanni Franco Spatola, Maria Laura Uzzo, Vincenzo Tessitore NellвЂ™ intestino tenue (duodeno, digiuno-ileo) Orexina A: rilevante immunopositivitГ orexinergica ГЁ estesa uniformemente al citoplasma degli enterociti mucosali (Fig.5). Consistente immunoreattivitГ ГЁ rilevabile anche a carico delle cellule secernenti delle cripte di Galeazzi-LieberkГјhn concentrata soprattutto nel loro settore apicale. NellвЂ™asse stromale dei villi e nel corion perighiandolare si rinvengono numerosi citotipi, verosimilmente appartenenti al Sistema Immune Secretorio (linfociti-macrofagi) che esibiscono assai intensa immunopositivitГ (Fig.5). Cellule endocrine scaglionate tra gli enterociti mucosali tra le cellule esocrine delle cripte di galeazzi esibiscono intensa immunoreattivitГ . La espressione immunoistochimica del recettore OX2R ГЁ vistosa da parte degli enterociti e delle cellule secernenti esocrine dei segmenti intestinali prese in esame. NellвЂ™asse stromale dei villi si rinvengono alcuni grossi citotipi che esprimono intensamente il recettore tipo 2 (Fig.6). Sia nellвЂ™ambito gastrico che enterico una numerosa subpopolazione di neuroni del plesso mioenterico di Auerbach e del plesso sottomucoso di Meissner esibiscono discreta immunoreattivitГ (Fig.7). Orfanina: intensa positivitГ per la orfanina si rileva uniformememnte estesa nel citoplasma medioapicale degli enterociti mucosali assorbenti (Fig.8 freccia nera). Le cellule caliciformi e lвЂ™asse dei villi risultano immunoistochimicamente negativi (Fig.8 freccia verde). Isolate cellule endocrine esprimono intensa immunoreattivitГ . Nel pancreas: Orexina A: intensa immunopositivitГ ГЁ estesa uniformemente a tutti i citotipi dellвЂ™isola langheransiana (Fig.9). Il recettore OX2R ГЁ espresso in maniera meno rilevante in tutti i citotipi endocrini. Orfanina: tutti i citotipi insulari esprimono assai intensamente immunoreattivitГ orfaninergica. Nel pancreas esocrino le cellule acinose esprimono discreta immunoreattivitГ orfaninergica di aspetto fioccoso soprattutto concentrata nella porzione citoplasmatica medioapicale (Fig.10). Nel connettivo stromale si rileva rimarchevole immunopositivitГ a carico di tronchicini nervosi. Conclusioni I nostri risultati forniscono la documentazione immunoistochimica che autorizza ad ammettere che sia lвЂ™Orexina A con il suo recettore tipo 2 che la Orfanina FQ neuropeptidi ritenuti in origine prodotti esclusivamente nei neuroni centrali possono essere espressi perifericamente anche da alcuni citotipi costitutivi del Sistema GEP. Questa doppia sede di nascita cerebrale e gastroenteropancreatica rafforza il concetto che per questi messag185 Orexina e Orfanina FQ nuovi neuropeptidi regolatori delle funzioni del Sistema Gastroenteropancreatico. Uno studio immunoistochimico geri, cosГ¬ come accade per altri neuropeptidi, possa esistere un asse correlativo cervellointestino (brain-gut axis). In aggiunta, i nostri reperti forniscono la base morfoistochimica per lвЂ™interpretazione del ruolo funzionale esplicato dallвЂ™orexina e dallвЂ™orfanina in tali distretti suggerendo la possibilitГ che esse possano esercitare unвЂ™azione diretta di tipo periferico sulle strutture summenzionate eplicando nuovi ruoli verosimilmente svolti con diversi meccanismi: neurocrino, suggerito dalla immunopositivitГ dei neuroni orexinergici e orfaninergici del sistema enterico, con il quale esse potrebbero controllare lвЂ™attivitГ motoria gastroenterica e la vasomotilitГ ; endocrino suggerito dalla loro espressione immunoistochimica in cellule endocrine gastroenteropancreatiche con un possibile conseguente controllo sulla secrezione acida gastrica intestinale e pancreatica modulando il senso dellвЂ™appetito e lвЂ™omeostasi glicemica; autocrino-paracrino suggerito dalla loro espressione immunoistochimica nelle cellule adelomorfe gastriche, negli enterociti, nelle cellule degli adenomeri intestinali e nei citotipi esocrini pancreatici, tramite il quale questi neuropeptidi potrebbero essere implicati nel controllo della funzione secretoria gastrica ed intestinale, dellвЂ™assorbimento dei nutrienti, del trasporto ionico ed infine della funzione secernente esocrina del pancreas. 186 Giuseppe Bonaventura, Giovanni Franco Spatola, Maria Laura Uzzo, Vincenzo Tessitore Bibliografia [1] Neal CR Jr et al.: Localization of orphanin FQ (nociceptin) peptide and messenger RNA in Central Nervous System of the rat. J Comp Neurol 1999; 406:503. [2] Bonaventura G. Rilievi immunoistochimici della orfanina nellвЂ™apparato genitourinario del ratto. Exper Med Rev (eds A Gerbino, G Zummo, G Crescimanno) вЂњPlumeliaвЂќ Bagheria, Palermo 2008; Vol.1, pp.271277. [3] Takahashi N. et al.: Stimulation of gastric acid secretion by centrally administrered Orexin-A in conscious rats. Biochem Biophys Res Comm 1999; 254:623-627. [4] Kirchgessner A, Liu M. Orexin synthesis and response in the gut. Neuron 1999, 24:941-951. [5] Krzystof W et al. Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies. Life Sciences 2000; 66 (5):449-454. [6] Naslund E. et al.: Localizzation and effects of orexin on fasting motility in the rat duodenum. Am J Physiol Gastointest Liver Physiol 2002; 282:G470-G479. [7] Yazdani A, et al. Functional significance of a newly discovered neuropeptide, orphanin FQ, in rat gastrointestinal motility. Gastroenterology 1999; 116(1):108-17. [8] Osinski MA, Brown DR. Orphanin FQ/nociceptin: a novel neuromodulator of gastrointestinal function? Peptides 2000; 21:999. [9] Giuliani S. et al. Effect of nociceptin on heart rate and blood pressure in anaesthetized rats. Eur J Pharmacol 1997; 333:177-179. [10] Fischer A, et al. Nociceptin-induced inhibition of tachykinergic neurotransmission in guinea pig bronchus. J Pharmacol Exp Ther 1998; 285:902-907. [11] Shah S, et al. Nociceptin inhibits non-adrenergic non-cholinergic contraction in guinea-pig airway. Br J Pharmacol 1998; 125:510-516. [12] Giuliani S, et al. The inhibitory effect of nociceptin on the micturition reflex in anaesthetized rats. Br J Pharmacol 1998; 124:156-672. [13] Bonaventura G, et al. Immunohistochemical distribution of orphanin FQ in the rat gastrointestinal apparatus and pancreas. It J Anat Embryol 2005; 110/Suppl.1 (3):262. [14] Tessitore V, et al. Immunohistochemical expression of Orexin A and Orexin Type 2 receptors in the rat gastrointestinal apparatus and endocrine pancreas. EJH 2005;49 (Suppl.1):19. 187 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 EFFETTI DEL TRATTAMENTO CRONICO CON UN ANTAGONISTA DEL RECETTORE CB1 (RIMONABANT) SULlвЂ™APOPTOsi pancreatica DI RATTI ZUCKER OBESI E DIABETICI. UNO STUDIO IMMUNOISTOCHIMICO [Effects of cronical treatment with a CB1 receptor angatonist (Rimonabant) on pancreatic apoptosis of obese and diabetic Zucker rats. An immunohistochemical study] Vincenzo Tessitore, Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola e Maria Laura Uzzo Di.Me.S. - Sezione di Istologia ed Embriologia - UniversitГ degli Studi di Palermo (IT) Parole chiave: CB1, Rimonabant, Apoptosi, Pancreas, Ratti Zucker Key words: CB1, Rimonabant, Apoptosi, Pancreas, Zucker rats Riassunto. Recenti ricerche indicano che vi ГЁ una iperattivazzione del sistema endocannabinoide (EC) in ratti e umani obesi. In una precedente pubblicazione abbiamo dimostrato che nei ratti Zucker obesi e diabetici vi ГЁ un danno apoptotico nelle cellule B degli isolotti pancreatici. Inoltre, il recettore CB1 ГЁ iperespresso da parte di tutti i citotipi endocrini in confronto con gli analoghi citotipi dei ratti normali in cui lвЂ™immunoreattivitГ del CB1 ГЁ limitata alle cellule A. Inoltre, altri AA. hanno dimostrato che lвЂ™attivazione del recettore CB1 per i cannabinoidi induce una intolleranza al glucosio nei ratti. Gli studi clinici in umani obesi trattati con un antagonista del recettore CB1 dei cannabinoidi (Rimonabant) hanno mostrato una riduzione del peso corporeo e il miglioramento della resistenza insulinica. Con riferimento a quanto dimostrato abbiamo studiato gli effetti del trattamento cronico con Rimonabant sulla apoptosi delle cellule B nel pancreas endocrino di ratti Zucker obesi e diabetici (ZDF) utilizzando come indicatore immunoistochimico di apoptosi la Caspase-9 e valutando la presenza di nNOS / iNOS implicati nel danno cellulare. I nostri risultati dimostrano che il trattamento con Rimonabant riduce significativamente lвЂ™incidenza di apoptosi nelle isole di ratti ZDF rispetto a quelle dei ratti normali non-diabetici. Questi risultati forniscono una base morfoistochimica a supporto dellвЂ™uso di questo farmaco nella sindrome metabolica associata con lвЂ™obesitГ . Abstract. Recent reports suggest that there is an overactivation of the endocannabinoid system (EC) in obese humans and rats. In a previous publication we have shown that in obese and diabetic Zucker rats the pancreatic islets show B-cells apoptotic damage. In addition the CB1 receptor is overexpressed by all endocrine cells in comparison with normal rat islets in which CB1 immunoreactivity is restricted to A-cells. Also, other AA. have demonstrated that activation of cannabinoid CB1 receptor induce glucose intolerance in rats. Clinical trials in obese men with a cannabinoid CB1 receptor antagonist (Rimonabant) have shown a reduction of body weight and improvement of insulin resistance. With references to findings we have studied the effects of chronic treatment with Rimonabant on B-cells apoptosis in endocrine pancreas of the Zucker diabetic fatty rats (ZDF) underlining immunohistochemically Caspase-9 as an indicator of apoptosis and nNOS/iNOS activities implicated in cellular damage. Our results show that Rimonabant treatment significantly decreases the apoptotic incidence in the islets of ZDF rats compared with those of non-diabetic normal rats. These findings provide a morphohistochemical basis to support the use of this drug in metabolic syndrome associated with obesity. 189 Vincenzo Tessitore, Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo Introduzione Recenti evidenze sperimentali suggeriscono che gli endocannabinoidi (EC) regolano lвЂ™appetito e il peso corporeo rappresentando uno dei piГ№ importanti sistemi oressizzanti del nostro organismo. Tale effetto si esplica sia con un meccanismo di azione ritenuto in un primo tempo esclusivamente centrale sia anche con un meccanismo periferico piГ№ di recente acquisito, entrambi svolti per attivazione del recettore CB1 espresso sia da neuroni dellвЂ™ipotalamo e del sistema limbico (sistema endocannabinoide centrale) sia da cellule di organi periferici (muscolo, fegato, organo adiposo, sistema gastroenteropancreatico, ghiandole salivari) (sistema endocannabinoide periferico) [1]. Nuove evidenze sperimentali indicano che gli EC modulano le funzioni del pancreas esocrino ed endocrino, nuovo target periferico nellвЂ™azione degli EC nel controllo dellвЂ™equilibrio nutrizionale [2,3] tramite interazione con i recettori pancreatici CB1 e CB2. La identificazione recettoriale pancreatica fa supporre anzitutto che gli endocannabinoidi esercitano un controllo diretto sul pancreas con meccanismo autocrino o paracrino-endocrino e dischiude implicitamente nuove prospettive terapeutiche nellвЂ™ambito della patofisiologia pancreatica esocrina ed endocrina con lвЂ™impiego di farmaci che interferiscono in modo vario con i suddetti recettori. Nel pancreas esocrino, molte strutture (cellule acinari, cellule duttali, nervi) esprimono immunoistochimicamente nel ratto e nei soggetti normali sia il recettore CB1 che il recettore CB2 [2], tuttavia i risultati degli studi sullвЂ™esatto ruolo svolto in situazioni patofisiologiche dagli EC in questo distretto, sono ancora frammentari e per alcuni aspetti controversi. Ad esempio la attivazione nellвЂ™uomo dei recettori CB1 e CB2 con bassi dosaggi di agonisti dei cannabinoidi attenua la risposta infiammatoria nella pancreatite acuta, mentre alte dosi la incrementano [4]. Per contro lвЂ™antagonista AM251 del recettore CB1 prolunga la sopravvivenza del ratto affetto da severa pancreatite acuta [5]. Per ciГІ che riguarda i rapporti tra EC e pancreas endocrino la recente letteratura fornisce rilievi non univoci per quanto attiene la identificazione dei citotipi endocrini capaci di esprimere i recettori CB1 e CB2 tali dispareri sono verosimilmente asccrivibili sia allвЂ™impiego di modelli sperimentali diversi di volta in volta, sia allвЂ™adozione di tecniche non similari. In nostre precedenti ricerche abbiamo per la prima volta fornito documentazioni immunoistochimiche in vivo e nel ratto normale relative allвЂ™espressione del CB1 nelle cellule A (Fig.1); utilizzando ratti Zucker obesi ed iperfagici la immunoreattivitГ per il CB1 si estende a tutti i citotipi insulari interessando non soltanto i citotipi A, come avviene nel ratto normale (Fig.2) [3]. Jian PicГІ e coll hanno identificato in vitro nel topo la presenza del recettore CB1 nei citotipi A e del recettore CB2 nei citotipi A e B [6]. BermГ№dez-Silva e coll hanno documentato nellвЂ™uomo immunofluorescenza per il CB1 nelle cellule A [7]. William G. Tharp e coll hanno rilevato nei ratti Zucker lвЂ™espressione del CB1 colocalizzato con la somatostatina nelle cellule D [8]. Matias e coll hanno dimostrato in vitro la presenza di endocannabinoidi nelle cellule 190 Effetti del trattamento cronico con un antagonista del recettore cb1 (rimonabant) sul pancreas apoptotico di ratti zucker obesi e diabetici. Uno studio immunoistochimico B del ratto (rat insulinoma RIN-m5F ОІ-cells) e dei topi DIO [9]. Starowicz e coll nei topi DIO hanno documentato lвЂ™espressione immunoistochimica del CB1 in cellule A insieme agli enzimi di sintesi dei cannabinoidi e di enzimi degradanti cannabinoidi nelle cellule B [10]. Г€ stato infine dimostrato che in animali sottoposti a diete iperlipidiche e nellвЂ™obesitГ umana il sistema endocannabinoide ГЁ iperattivato e che nei vari targets centrali e periferici il CB1 ГЁ iperespresso [3,9-12]. Questi risultati fanno ritenere che gli EC esplichino un ruolo cruciale nellвЂ™obesitГ , per cui per il suo trattamento ГЁ stato recentemente proposto lвЂ™impiego di un antagonista del CB1. Г€ stato pure documentato che lвЂ™attivazione del recettore CB1 induce intolleranza al glucosio e diminuzione della secrezione insulinica da parte delle beta cellule il che suggerisce un ruolo di controllo degli EC sulla omeostasi glicemica [13,14]. LвЂ™insieme di questi dati ha stimolato a proporre nellвЂ™obesitГ e nelle comorbilitГ associate lвЂ™impiego di un antagonista del CB1 il Rimonabant, che in numerosi trials clinici ha prodotto in pazienti obesi e diabetici non soltanto una significativa riduzione del peso corporeo ma anche un aumento della insulinoresistenza e del profilo glicemico [15-18]. Con riferimento a quanto sopra, abbiamo studiato lвЂ™effetto di un trattamento cronico con Rimonabant sulla apoptosi Fig.1: Pancreas endocrino ratto normale: CB1 immuno- Fig.2: Pancreas endocrino ratto Zucker: CB1 immureattivitГ sostanzialmente limitata alle cellule A - 40x noreattivitГ omninsulare - 40x Fig.3: Pancreas esocrino ratto Zucker non trattato: Fig.4: Pancreas esocrino ratto Zucker trattato: nointensa immunoreattivitГ caspasica nei nuclei delle tevole riduzione dellвЂ™attivitГ caspasica nucleare del cellule acinose - 20x comparto acinoso - 20x 191 Vincenzo Tessitore, Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo delle beta cellule nel pancreas endocrino di ratti Zucker obesi e diabetici (ZDF) valutando immunoistochimicamente la Caspasi 9 come indicatore dellвЂ™apoptosi e le attivitГ enzimatiche NOS implicate spesso nel danno cellulare attraverso una iperproduzione di NO. Materiali e metodi Sono stati utilizzati ratti Zucker obesi e diabetici (ZDF) della Harlan Italy S.r.l. sottoposti a dieta tipo Harlan Tekland, Rodent diet # 8640. Gli animali sono stati divisi in due gruppi. Al primo gruppo ГЁ stato somministrato tramite sondino gastrico Rimonabant (10mg pro Kg/pro die per 28 giorni). Il secondo gruppo riceveva dosi orali di Carbossimetilcellulosa come veicolo (5ml proKG/pro die per 28 giorni). Di ciascun animale ГЁ stato controllato il peso corporeo e su ciascuno ГЁ stato effettuato un prelievo ematico per il monitoraggio dei valori glicemici sia il 1В° giorno di trattamento che il 28В° giorno. Tutti gli animali sono stati sacrificati allo scadere dei 28 giorni. I campioni di pancreas prelevati sono stati fissati in liquido di Bouin e inclusi in paraffina. Alcune sezioni seriate ottenute, dello spessore di 8 Fig.5: Pancreas endocrino ratto Zucker non trattato: Fig.6: Pancreas endocrino ratto Zucker trattato: signifiisola apoptotica; immunoreattivitГ caspasica dei ci- cativa riduzione dei fenomeni apoptotici con immunototipi residui - 20x reattivitГ caspasica limitata a qualche citotipo - 40x Fig.7: Pancreas endocrino ratto Zucker non trattato: Fig.8: Pancreas endocrino ratto Zucker trattato: immunoisola apoptotica; intensa immunoreattivitГ nNOS nei reattivitГ nNOS estesa come di norma a molti citotipi del parenchima insulare sostanzialmente non apoptotico - 40x citotipi residui - 40x 192 Effetti del trattamento cronico con un antagonista del recettore cb1 (rimonabant) sul pancreas apoptotico di ratti zucker obesi e diabetici. Uno studio immunoistochimico Ој, sono state colorate con ematossilina eosina per lo studio morfologico e altre sono state processate con un anticorpo anti Caspase-9 (Sigma) and anti nNOS/iNOS (Transduction Laboratories) by Envision + System HRP (AEC) (DAKO Citomation). Tutte le sezioni sono state osservate mediante microscopio Leica DM1000. Risultati e conclusioni Pancreas Esocrino: Nel comparto acinoso dei ratti non trattati le cellule acinari e duttali rivelano intensa attivitГ caspasica nucleare in molti citotipi (Fig. 3). Negli animali trattati i citotipi esocrini non esibiscono immuno reattivitГ caspasica nucleare e rivelano solo scarsa reattivitГ citoplasmatica (Fig.4). In entrambi i gruppi la immunoreattivitГ iNOS e nNOS ГЁ assai debole. Pancreas Endocrino: Nei ratti non trattati si evidenzia una destrutturazione dellвЂ™architettura insulare allвЂ™esame morfologico con EE. Nei citotipi residui la immunoreattivitГ caspasica nucleare ГЁ assai intensa denunciando un elevato grado di apoptosi (Fig.5). La immunoreattivitГ nNOS ГЁ intensa e limitata a quei citotipi endocrini che sfuggono ai fenomeni apoptotici (Fig.7), la iNOS ГЁ assai poco evidente. Nei ratti trattati le isole non presentano sostanzialmente, allo studio morfologico, fenomeni apoptotici come dimostra la immunoreattivitГ caspasica limitata a pochi citotipi endocrini (Fig.6). La immunoreattivitГ nNOS ГЁ discretamente estesa come di norma ai citotipi dellвЂ™isola (Fig.8), la iNOS ГЁ sostanzialmente assente. I risultati da noi ottenuti dimostrano che la somministrazione di un antagonista del CB1 (Rimonabant) puГІ modificare la funzione esocrina pancreatica per cui gli EC modulandola direttamente possono essere implicati, quindi, nel meccanismo fisiopatologico delle malattie esocrine del pancreas [2]. Per ciГІ che attiene il pancreas endocrino i nostri reperti fanno ritenere che il Rimonabant esercita un freno sulla comparsa della destrutturazione apoptotica a carico delle isole di Langherans migliorando lвЂ™insulite data la deflessione nei ratti trattati della immunoreattivitГ caspasica insulare, da noi considerata un indice proapoptotico. La somministrazione di un antagonista del CB1 blocca secondo la nostra interpretazione una sequenza di eventi che nel ratto Zucker non trattato esitano nella alterazione insulare e nel conseguente dissesto glicemico; infatti la documentata iperattivazione del CB1 svincolata dal freno che nel ratto Zucker la leptina non puГІ esercitare come di norma causa una iperfagia incontrollata e protratta con conseguente lipotossicitГ insulare e distruzione delle beta cellule. Il Rimonabant con la sua azione antagonista sul CB1 insulare garantisce la integritГ morfofunzionale delle beta cellule e la normale insulinopoiesi. Il Rimonabant ГЁ capace quindi di svolgere sul piano clinico non soltanto un effetto anoressizzante ma anche metabolico sulle comorbiditГ associate allвЂ™obesitГ (diabete) Data la sovrapponibilitГ dei reperti immunoistochimici attinenti alla NOS nei due gruppi di animali non sembra che essa sia coinvolta nel determinismo della destrutturazione apoptotica. 193 Vincenzo Tessitore, Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo Bibliografia [1] Cota D, Woods SC. The role of the endocannabinoid system in the regulation of energy homeostasis. Curr Opin Endocr Diab 2005; 12: 338-351. [2] Izzo AA, Camilleri M. Emerging role of cannabinoids in gastrointestinal and liver diseases: basic and clinical aspects. Gut 2008; 57(8):1140-55. Epub 2008 Apr 8. [3] Tessitore V, et al. Immunohistochemical study and densitometric comparision on CB1 receptor expression in the gastrointestinal tract and pancreas of obese (fa/fa) and lean Zucker rats. It J Anat Embryol 2006; 111 (Suppl 12):256. [4] Michalski CW, et al. Cannabinoids ameliorate pain and reduce disease pathology in cerulein-induced acute pancreatitis. Gastroenterology 2007; 132(5):1968-78. Epub 2007 Feb 21. [5] Matsuda K, et al. The cannabinoid 1 receptor antagonist, AM251, prolongs the survival of rats with severe acute pancreatitis. Tohoku J Exp Med 2005; 207(2):99-107. [6] Juan-PicГІ P, et al. Cannabinoid receptors regulate Ca 2+ signals and insulin secretion in pancreatic ОІ-cell. Cell Calcium 2006; 39:155-162. [7] BermГ№dez-Silva FJ, et al. Presence of functional cannabinoid receptors in human endocrine pancreas. Diabetologia 2008; 51:476-487. [8] Tharp WG, et al. The cannabinoid CB1 receptor is expressed in pancreatic Оґ-cells. Biochem Biophys Res Comm 2008; 372:595-600. [9] Matias I, et al. Regulation, function, and dysregulation of endocannabinoids in models of adipose and ОІ-pancreatic cells and in obesity and hyperglycemia. J Clin Endocrinol Metab 2006; 91(8): 31713180. [10] Starowicz KM, et al. Endocannabinoid dysregulation in the pancreas and adipose tissue of mice fed with a high-fat diet. Obesity 2008; 16:553-565. [11] Di Marzo V, et al. The role of endocannabinoids in the regulation of gastric emptying: alterations in mice fed a high-fat diet. Br Journ of Pharm 2008; 153:1272-1280. [12] Engeli S, et al. Activation of the peripheral endocannabinoid system in human obesity. Diabetes 2005; 54:2838-2843. [13] BermГ№dez-Silva FJ, et al. Activation of cannabinoid CB1 receptors induces glucose intolerance in rats Eur J of Pharm 2006; 531:282-284. [14] Masanori N, Toshihiko Y. Cannabinoids inhibit insulin secretion and cytosolic Ca 2+ oscillation in islet ОІ-cells via CB1 receptors. Regulatory Peptides 2008; 145:49-53. [15] Ravinet Trillou C, et al. CB1 cannabinoid receptor knockout in mice leads to leanness, resistance to dietinduced obesity and enhanced leptin sensitivity. Int J Obes Relat Metab Disord 2004; 28(4):640-8. [16] Ravinet Trillou C, et al. Anti-obesity effect of SR141716, a CB1 receptor antagonist, in diet-induced obese mice. Am J Physiol Regul Integr Comp Physiol 2003; 284(2):R345-53. Epub 2002 Oct 24. [17] Vickers SP, et al. Preferential effects of the cannabinoid CB1 receptor antagonist, SR 141716, on food intake and body weight gain of obese (fa/fa) compared to lean Zucker rats. Psychopharmacology (Berl). 2003; 167(1):103-11. Epub 2003 Mar 11. [18] Hildebrandt AL, et al. Antiobesity effects of chronic cannabinoid CB1 receptor antagonist treatment in diet-induced obese mice. Eur J Pharmacol 2003; 462(1-3):125-32. 194 Tumor process Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici [Heredofamilial and pediatric GISTs: biomolecular and clinical aspects] Antonio Russo1, Sergio Rizzo1, Daniele Fanale1, Valentina CalГІ1, Nello Grassi2, Gianni Pantuso2, Luigi Sandonato2, Michele Frazzetta3, Giuseppe Cicero1, Massimo Cajozzo4, Aldo Gerbino5, Giuseppe Badalamenti1, Nicola Gebbia1 e Viviana Bazan1 Sezione di Oncologia Medica, 2Sezione di Oncologia Chirurgica e 3Sezione di Chirurgia Generale, Dipartimento di Discipline Chirurgiche ed Oncologiche; 4UnitГ di Chirurgia Toracica; 5Sezione di Istologia ed Embriologia, Di.Me.S., UniversitГ degli Studi di Palermo (IT) 1 Parole chiave: Tumori stromali gastrointestinali; Mutazioni attivanti; KIT; PDGFRA; Carney-Stratakis; Neurofibromatosi Key words: Gastrointestinal stromal tumors; Germline mutations; KIT; PDGFRA; Carney-Stratakis; Neurofibromatosis Riassunto. I tumori stromali gastrointestinali (GISTs) sono rare neoplasie sporadiche che insorgono prevalentemente in etГ avanzata; tuttavia sono stati riportati casi di aggregazione familiare e pediatrici. Tali casi hanno caratteristiche clinicopatologiche e molecolari distinte da quelli sporadici. Infatti nei GISTs pediatrici mancano le mutazioni attivanti in KIT e PDGFRA comunemente riscontrabili negli adulti. Le analisi dei profili di espressione genica dei GISTs pediatrici mostrano caratteristiche peculiari e suggeriscono unвЂ™origine comune rispetto ai GISTs degli adulti. La review tratta inoltre i GISTs familiari con mutazioni germinali nei geni KIT e PDGFRA e le forme che non presentano queste mutazioni come la triade di Carney, la sindrome di Carney-Stratakis, e la neurofibromatosi di tipo 1. Abstract. Gastrointestinal stromal tumors (GISTs) are rare sporadic tumors that typically occur late in life; however, there also are reports of familial and pediatric cases. These rare subsets of GISTs have clinicopathologic and molecular features distinct from their sporadic counterparts. Most pediatric GISTs lack the gain-of-function mutation in KIT or PDGFRA commonly found in adult cases. Gene expression profiling studies of pediatric GISTs show distinct molecular signatures, suggesting a unique origin as compared with adult GISTs. The review discusses also familial GISTs with germline KIT or PDGFRA genes mutations and forms that donвЂ™t harbour mutations in these genes such as Carney triad, Carney-Stratakis syndrome, and neurofibromatosis type 1. Introduzione I tumori stromali gastrointestinali (GISTs) sono tumori rari la cui lвЂ™incidenza ГЁ stata chiarita di recente, dopo la ridefinizione dei criteri diagnostici in seguito al riconoscimento del caratteristico profilo immunoistochimico di queste neoplasie. Questi tumori inizialmente erano infatti considerati eccezionali, con una incidenza in 197 Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan passato stimata a circa 1,5 casi per milione di abitanti allвЂ™anno, ma oggi non sono piГ№ tali dato che la reale incidenza di queste neoplasie ГЁ 10 volte superiore a quanto precedentemente ipotizzato e pari a circa 1000 casi allвЂ™anno in Italia e a circa 4500-5000 casi negli USA. I GIST rappresentano meno dellвЂ™1% di tutte le neoplasie maligne, ma sono i piГ№ comuni tumori mesenchimali del tratto gastroenterico (0,1-3 % in questa sede). Si presentano in piГ№ del 99% dei casi in forma sporadica ma esiste una minima percentuale di GIST eredo-familiari caratterizzati da mutazioni germinali in diversi geni di suscettibilitГ . In particolare si distinguono forme familiari quando nella stessa famiglia piГ№ soggetti sono affetti dallo stesso tipo di tumore e forme ereditarie quando si riesce a dimostrare che la predisposizione allo sviluppo della neoplasia ГЁ trasmessa geneticamente dai genitori ai figli. Aspetti biomolecolari dei GIST: c-KIT e PDGFRA. La genetica molecolare non solo ha svelato il meccanismo coinvolto nella genesi dei GIST, ma, anche, ci ha consegnato un marcatore immunofenotipico il c-KIT. La maggior parte dei GIST (90-95%) mostrano mutazioni attivanti di c-KIT con immunopositivitГ KIT/CD117 pari circa al 95%. Tale marcatore consente quindi nel 90-95% dei casi di produrre una diagnosi molto affidabile. c-KIT ГЁ un oncogene che mappa sul cromosoma 4q12 ed ГЁ costituito da 21 esoni codificanti. Nel gene sono presenti 4 hot spot di mutazione che cadono in ordine di frequenza negli esoni 11, 9, 13 e 17. KIT ГЁ una glicoproteina transmembrana di 145KD con attivitГ tirosin-chinasica che ГЁ unвЂ™attivitГ enzimatica indispensabile per lo sviluppo (differenziamento) e la crescita delle cellule (proliferazione). In condizioni non patologiche il recettore c-KIT ГЁ una molecola che ha attivitГ TK cioГЁ ГЁ un вЂњinterruttore cellulareвЂќ che se attivato in seguito allвЂ™interazione del proprio ligando (SCF) con la porzione extracellulare del recettore attraverso processi di dimerizzazione innesca un pathway intracellulare cioГЁ avvia una cascata di segnali il cui fine ГЁ lвЂ™aumento della proliferazione, il differenziamento e lвЂ™inibizione dellвЂ™apoptosi. Entrando piГ№ in dettaglio, il legame di una molecola di SCF con ciascuno dei monomeri c-KIT dГ luogo alla formazione di un c-KIT dimero. LвЂ™interazione di due recettori adiacenti (omodimero) ГЁ mediata proprio da due molecole di SCF. La omodimerizzazione comporta cambiamenti strutturali dei recettori con attivazione della tirosinchinasi intracellulare che catalizza anzitutto la auto-fosforilazione a livello di specifici residui tirosinici, che antagonizza lвЂ™auto-inibizione ed ГЁ necessaria per la trasduzione a valle del segnale conseguente allвЂ™interazione del ligando col recettore. Tra i pathways attivati ci sono: PI3-Kinasi/AKT, RAS/RAF/MAPK e JAK/STAT. Nella situazione patologica la maggior parte delle mutazioni di questo gene innescano 198 GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici una omodimerizzazione spontanea che, in definitiva, provoca unвЂ™autoattivazione con un vantaggio proliferativo e un effetto anti-apoptotico. Sappiamo che le mutazioni del gene c-KITvengono riconosciute come lвЂ™evento patogenetico principale (90% dei casi), ma non rappresentano lвЂ™unico evento patogenetico. Infatti esistono GIST che non presentano mutazioni in c-KIT, ma, con rapporto mutualmente esclusivo, nel gene PDGFR-alfa (fattore di crescita di derivazione piastrinica) codificante per una molecola con attivitГ tirosin-kinasica con funzione sostanzialmente analoga a c-KIT. Strutturalmente il recettore ГЁ costituito da tre porzioni principali: 1. Una porzione extracellulare contenente i siti di legame con il ligando, costituita da 5 domini Ig-simili. In questa regione si riconoscono: -- il dominio di legame col ligando, vale a dire quella parte che lega specificatamente il ligando o fattore di crescita costituita da 4 domini Ig-simili; -- il dominio di dimerizzazione che ГЁ alla base dellвЂ™attivazione del recettore. 2. Una porzione transmembranaria 3. Una porzione intracellulare contenente lвЂ™attivitГ tirosin-chinasica, suddivisa in due domini: -- il dominio iuxtamembrana o loop auto-inibitorio (codificato dallвЂ™esone 11 e capace di auto-inibire lвЂ™attivitГ chinasica in assenza dello specifico ligando) sito al di sotto della membrana cellulare -- il dominio chinasico (catalitico) che ГЁ la parte attiva del recettore cioГЁ la parte che ha la proprietГ biochimica di trasferire un gruppo fosfato dallвЂ™ATP ad un certo substrato. In particolare il dominio TK1contiene il sito di legame per lвЂ™ATP mentre il dominio TK2 contiene lвЂ™attivitГ fosfotransferasica. Hot spot di mutazioni di c-KIT e PDGFRA. PiГ№ dellвЂ™90% dei GIST presentano mutazioni attivanti a livello del gene c-KIT (in circa il 90-95% dei casi) o meno frequentemente del gene PDGFRA (circa il 5-10% dei casi). In KIT sono stati identificati quattro hot-spots a livello degli esoni 9, 11, 13 e 17. Esiste una gerarchia nelle mutazioni dei GIST. LвЂ™esone maggiormente interessato ГЁ lвЂ™esone 11, in circa il 70-80% dei casi di GIST, che codifica per il вЂњloop auto-inibitorioвЂќ. Questo dominio esercita lвЂ™azione auto-inibitoria nei confronti della tirosin-chinasi del recettore. LвЂ™esone 9, risulta mutato nel 10-20% dei GIST. LвЂ™esone 9 codifica per il quinto dominio simil-immunoglobulinico della regione extracellulare del recettore. Meno frequenti (1-5%) sono le mutazioni puntiformi a carico degli esoni 13 e 17 che codificano per i domini tirosin-chinasici della porzione intracitoplasmatica del recettore. 199 Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan Il gene PDGFRA codifica per il recettore del fattore di crescita di derivazione piastrinica e condivide con c-KIT circa il 35% della sequenza aminoacidica. Anche questa ГЁ una molecola con attivitГ tirosin-chinasica. Il pathway che segue allвЂ™attivazione del recettore ГЁ sostanzialmente analogo a quello di c-KIT. Modello di progressione tumorale dei GIST Le mutazioni attivanti in c-KIT o in PDGFRA si osservano pressochГЁ nellвЂ™intera popolazione cellulare dei GIST, mentre le alterazioni cromosomiche si osservano solo in una percentuale di cellule neoplastiche. CiГІ suggerisce che le mutazioni di c-KIT rappresentano unвЂ™evento precoce, presumibilmente, aventi un ruolo di iniziazione della trasformazione neoplastica, mentre le successive alterazioni citogenetiche sono coinvolte nella progressione neoplastica. I GIST a basso gradohanno spesso un cariotipo normale o presentano la perdita del braccio corto del cromosoma 1p o la perdita del cromosoma 14 o 22 (50% dei casi). I GIST ad alto grado sono spesso accompagnati da perdita da almeno 3 delezioni cromosomiche (1p, 9p,11p o 22p). I GIST metastatici sono caratterizzati da perdita nel cromosoma 9p associata a guadagno in 5q, 8q, 17q e 20q. Inoltre presentano LOH di c-KIT e diminuzione dellвЂ™espressione di TP53 e MSI. Bisogna, tuttavia, sottolineare che questa sequenza di eventi rappresenta una semplificazione e che non tutti i GIST acquisiscono alterazioni genetiche nellвЂ™ordine riportato. Gist pediatrici Sono definiti pediatrici i GIST diagnosticati entro il diciottesimo anno dвЂ™etГ . I GIST pediatrici finora descritti rappresentano tra lвЂ™1 e il 2% di tutti i casi di GIST, e quindi in etГ giovanile sono tumori raramente diagnosticati. La ricerca di base sulla biologia dei GIST ГЁ stata ostacolata dalla raritГ della malattia e dalla mancanza di modelli sperimentali. Nonostante tali limitazioni, le conoscenze acquisite dallo studio dei GIST negli adulti e dei tipi tumorali ad essi correlati (per es. paragangliomi), oltre alla nascita di nuove metodiche (per es. tecniche di microarrays) hanno permesso significativi passi in avanti nella comprensione delle rare forme pediatriche. Questi tumori possono avere una differente patogenesi rispetto ai GIST degli adulti, poichГ© possono mancare le mutazioni nei geni KIT o PDGFRA (sono cioГЁ GIST wild-type). Questo dato potrebbe suggerire che siano attivi allвЂ™interno delle cellule altri meccanismi di attivazione di KIT o pathways oncogeniche non correlati ad esso. Nella maggior parte dei GIST pediatrici esaminati, non ГЁ stata riscontrata alcuna anomalia citogenetica o alterazioni degli esoni 9, 11 o 13 di KIT. Tra i pochi GIST pediatrici sottoposti ad analisi mutazionale e riportati in letteratu200 GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici ra, solo nellвЂ™11% dei casi ГЁ stata evidenziata mutazioni nei geni KIT o PDGFRA, equamente distribuite. Questo dato ГЁ differente da quello osservato nei GIST adulti sporadici in cui le mutazioni in KIT sono 10 volte piГ№ comuni delle mutazioni in PDGFRA. Nelle forme pediatriche tali mutazioni sono identificate prevalentemente nei maschi. Infatti, ГЁ stato riportato un solo caso di paziente di sesso femminile con una mutazione in PDGFRA. Probabilmente queste mutazioni rappresentano degli eventi molecolari casuali. Si potrebbe ipotizzare la presenza di mutazioni localizzate in altri esoni di KIT e PDGFRA, differenti da quelle riscontrate negli adulti, ma quando ГЁ stato effettuato il sequenziamento diretto del gene non sono state identificate nuove mutazioni in KIT. Nonostante non ci siano generalmente mutazioni a carico di KIT nelle forme pediatriche di GIST, tale gene risulta spesso espresso ed il suo pathway attivato. In relazione allвЂ™esiguo numero di casi ad oggi identificati, le alterazioni genetiche alla base dellвЂ™insorgenza di un GIST pediatrico sono comunque poco note e forse sono implicate anche alterazioni epigenetiche o mutazioni ipomorfiche (in porzioni genomiche che regolano il gene coinvolto). Infatti, il grado di attivazione misurato dalla fosforilazione della corrispondente proteina in tali pazienti risulta essere simile a quello riscontrato nei GIST adulti KIT-mutati. Inoltre, risultano attivati anche i geni a valle di KIT: MAPK, Akt, S6 e mTOR . Questi dati avvalorano lвЂ™ipotesi che KIT ed i geni a valle di esso possano essere importanti target terapeutici anche nei pazienti pediatrici. La maggior parte dei GIST pediatrici che non presenta mutazioni in KIT o PDGFRA ГЁ caratterizzata da pochi riarrangiamenti cromosomici su larga scala. Effettuando esperimenti di microarrays mediante SNP analysis, ГЁ stato osservato che la quasi totalitГ dei GIST pediatrici wild type mostrano preservazione dellвЂ™eterozigositГ e copy number diploide lungo lвЂ™intero genoma. Questo dato concorda con precedenti valutazioni di GIST in etГ pediatrica mediante citogenetica classica, che hanno evidenziato cariotipi diploidi. Mediante successiva analisi CGH (ibridazione genomica comparativa), il 60% dei 13 GIST wild type pediatrici non mostrava alterazioni cromosomiche. Nel restante 40% di tumori con variazioni cromosomiche, ГЁ stato osservato che lвЂ™anomalia piГ№ comunemente identificata era la delezione 1q, riscontrata in 3 casi. I profili di espressione genica dei GIST pediatrici sono differenti da quelli dei GIST adulti wild type e sono caratterizzati dallвЂ™incrementata espressione dei seguenti geni: BAALC, IGF1R, FGF4, CRLF1, PLAG1 e NELL1. Inoltre, in uno studio del 2005, Prakash S. et al. hanno analizzato lвЂ™espressione genica di campioni tumorali di GIST provenienti da bambini, giovani adulti (etГ inferiore ai 30 anni) e adulti, trovando unвЂ™alta espressione dei geni PHKA1, FZD2, NLGN4, IGF1R e ANK3 nei primi due gruppi di pazienti. I GIST che si sviluppano in giovani adulti, con caratteristiche simili a quelle osservate nelle forme pediatriche, mostrano profili di espressione genica piГ№ simili a quelli dei GIST pediatrici wild type. Tuttavia, gli esperimenti di gene expression descritti sono stati effettuati usando un numero esiguo di 201 Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan campioni tumorali e i risultati non sono stati confermati in seguito a causa della difficoltГ di ottenere campioni di tessuto disponibili per lвЂ™analisi dellвЂ™RNA. LвЂ™espressione di IGF1R (вЂњinsulin-like growth factor 1 receptorвЂќ) ГЁ 5 volte piГ№ alta nei GIST wild type pediatrici che in quelli wild type degli adulti. IGF1 e il suo recettore (IGF1R) giocano un ruolo chiave nella crescita e proliferazione cellulare e nello sviluppo. LвЂ™analisi dellвЂ™espressione proteica di IGF1R nei GIST, mediante western blotting e immunoistochimica, mostra che tale proteina ГЁ espressa in tutti i GIST, ma che i suoi livelli di espressione sono molto piГ№ alti nel GIST wild type . IGF1R ГЁ attivato in molti GIST, ma i suoi livelli di attivazione non sono correlati alla sua espressione. Г€ stato osservato, inoltre, che, durante la stimolazione con IGF1, lвЂ™inibizione di IGF1R da parte di piccole molecole riduce la proliferazione di una linea cellulare di GIST KIT-mutata. Questi dati supportano il ruolo di IGF1R come potenziale target terapeutico nei GIST pediatrici. Come nei GIST degli adulti, le manifestazioni cliniche piГ№ frequenti al momento della diagnosi comprendono il sanguinamento gastro-intestinale e lвЂ™anemia ad esso correlata e talvolta ГЁ presente una massa addominale palpabile, voluminosa e tale da determinare compressione intestinale. Ai GIST insorti in etГ pediatrica possono associarsi alcuni tumori, e cioГЁ lвЂ™osteosarcoma e il neuroblastoma. Circa il 10% dei GIST pediatrici si presentano nel contesto della triade o della diade di Carney (vedi piГ№ oltre), di conseguenza altre caratteristiche suggestive di questo complesso, in particolare lвЂ™iperpigmentazione e la sindrome da inappropriata secrezione di catecolamine associata ai paragangliomi, devono essere attentamente ricercate al momento della valutazione iniziale di un paziente pediatrico affetto da GIST. LвЂ™istotipo piГ№ comune ГЁ a cellule epitelioidi o a morfologia mista fusata/epitelioide. Il decorso clinico, in genere indolente, ГЁ tuttavia meno prevedibile rispetto allвЂ™adulto, anche prendendo in considerazione i fattori prognostici comunemente considerati (dimensioni, indice mitotico e sede del tumore). Altra caratteristica comune ГЁ la multifocalitГ nel contesto della regione anatomica sede di insorgenza che condiziona lвЂ™insorgenza di recidive locali dopo diversi anni dallвЂ™intervento chirurgico. Prima dellвЂ™introduzione dei farmaci a bersaglio molecolare (in primo luogo Imatinib), nella terapia dei GIST la chirurgia rappresentava lвЂ™unico trattamento possibile e costituisce a tuttвЂ™oggi lo standard quando il tumore ГЁ radicalmente resecabile. Nonostante la dissezione linfonodale non sia raccomandata negli adulti, nei GIST pediatrici, per lвЂ™incidenza di metastasi linfonodali relativamente elevata ГЁ prevista lвЂ™asportazione della stazioni linfonodali sospette al momento dellвЂ™intervento chirurgico. LвЂ™intervallo tra i controlli successivi allвЂ™intervento chirurgico dovrebbe prevedere, oltre allвЂ™esame obiettivo, il ricorso periodico ad esami di imaging quali ecografie, TC, RM e PET, che dovrebbero essere effettuati per i primi 2 anni ogni 3 mesi e ogni 6 mesi nei due anni successivi, infine annualmente. come del resto negli adulti. 202 GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici Prima dellвЂ™introduzione dellвЂ™Imatinib mesilato, lвЂ™intervento chirurgico con intento radicale veniva proposto anche a pazienti con malattia localmente avanzata ovvero non resecabile in maniera completa oppure metastatica. Tuttavia i dati disponibili evidenziano che la resezione completa di lesioni secondarie non si accompagna a guarigione e lвЂ™ulteriore ripresa di malattia si verifica nella quasi totalitГ dei casi (90-100%). Nei GIST pediatrici il quadro ГЁ sovrapponibile . La chirurgia estesa a scopo di debulking, cui si ricorreva in passato, in mancanza di altre terapie mediche efficaci, non appare piГ№ una opzione praticabile in epoca di Imatinib. I GIST, analogamente ai tumori mesenchimali, sono neoplasie scarsamente responsive alla chemioterapia. Studi randomizzati con antracicline, taxani, ifosfamide ed altri agenti variamente combinati hanno registrato tassi di risposta inferiori al 10%. LвЂ™Imatinib mesilato ha permesso di ottenere risultati decisamente migliori rispetto ad ogni tipo di chemioterapia o altro trattamento. La dose ottimale di Imatinib nei pazienti pediatrici, come terapia iniziale, varia da 230 a 400 mg/die e puГІ essere aumentata a 600800mg/die allorchГЁ la malattia ГЁ in progressione; in alternativa possono essere utilizzati Sunitinib o Nilotinib. Gist eredo-familiari I tumori stromali gastrointestinali (GIST) eredo-familiari rappresentano complessivamente meno dellвЂ™1% di tutti i GIST e sono caratterizzati da mutazioni ereditate in diversi geni di suscettibilitГ . Li possiamo distinguere in GIST con mutazioni che interessano KIT e PDGFRA, che ГЁ lвЂ™evento patogenetico principale in oltre il 90% dei GIST sporadici, e GIST con mutazioni su altri geni. GIST eredo-familiari con mutazioni di KIT e PDGFRA Le eventuali mutazioni germinali attivanti i geni KIT e PDGFRA vengono trasmesse con modalitГ autosomica dominante e sono state riportate in un numero esiguo di famiglie. Le manifestazioni cliniche negli individui portatori di mutazioni germinali del gene KIT variano in relazione allвЂ™esone coinvolto. La, che codifica per il dominio iuxtamembranario del recettore di membrana KIT, rappresenta un evento fondamentale e molto precoce nella oncogenesi dei GIST. Quando ereditata per via germinale comporta iperplasia delle cellule interstiziali di Cajal (ICC) che evolve precocemente verso la formazione di GIST multipli; comporta inoltre mastocitosi ed iperpigmentazione della cute, specialmente a livello perineale, ascellare, delle mani e della faccia (urticaria pigmentosa). La presenza di disordini della pigmentazione e la mastocitosi sono giustificate dal fatto che KIT ГЁ coinvolto anche nella melanogenesi e nello sviluppo delle mastcellule. 203 Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan LвЂ™associazione di GIST ed iperplasia generalizzata delle ICC supporta lвЂ™origine di questi tumori da cellule staminali mesenchimali CD34+ lungo la via di differenziazione delle ICC (Fig.1). LвЂ™attivazione di KIT ГЁ sufficiente per determinare una iperplasia delle ICC (stimolo proliferativo alle cellule progenitrici dei GIST), ma sono necessari altri eventi oncogenici per generare un GIST. I domini tirosin-chinasici del recettore comportano invece GIST multipli dello stomaco e dellвЂ™intestino tenue in assenza di evidenti disordini cutanei. Anche le non comportanopigmentazione della pelle o mastocitosi ma i GIST possono essere associati ad anomalie nella peristalsi esofagea. LвЂ™etГ media di insorgenza di questo gruppo di GIST eredo-familiari ГЁ 46 anni, di poco inferiore allвЂ™etГ media di insorgenza dei GIST sporadici dellвЂ™adulto (circa 50 anni) e fuori dal range indicato per i GIST pediatrici. Le loro fasi iniziali appaiono biologicamente simili a quelle del GIST sporadico, con altrettanto simili meccanismi di progressione citogenetica e comparabili profili di espressione genica. Le mutazioni germinali in KIT e PDGFRA sono prevalentemente simili a quelle trovate nei GIST sporadici. Tuttavia, in due famiglie con 204 GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici GIST ereditario sono state riscontrate due mutazioni mai viste nel GIST sporadico, rispettivamente in KIT e PDGFRA (Asp419del e Tyr555Cys). Inoltre, un recente studio ha riportato il caso di un paziente che ha sviluppato lipomi e GIST e che presentava una mutazione germinale (Asp561Val) in PDGFRA. La Tabella 1 riassume quanto esposto evidenziando anche le principali correlazioni genotipo-fenotipo di questo gruppo eredo-familiare. Tabella 1- GIST eredo-familiari con mutazioni di KIT e/o PDGRFA: Principali correlazioni genotipo-fenotipo Esone 11 KIT (V559A, delV559, W557R) Urticaria pigmentosa; mastocitosi Esone 13 KIT (K642E) Nessuna pigmentazione della pelle o mastocitosi Esone 17 KIT (D820Y) Nessuna pigmentazione della pelle o mastocitosi; anomalie nella peristalsi esofagea GIST eredo-familiari con mutazioni su altri geni: Il complesso di Carney (da nome di un noto anatomo-patologo della Majo Clinic che ha dedicato la propria attivitГ allo studio di queste patologie) comprende la Triade di Carney, descritta per la prima volta nel 1977, e la Diade di Carney/Stratakis. La Triade di Carney ГЁ una sindrome ad insorgenza giovanile (etГ media circa 20 anni) caratterizzata da GIST gastrici multipli ad insorgenza multifocale che si presentano in associazione ad altri tumori: paragangliomi extra-surrenalici e condromi polmonari. Un attento follow-up di questi pazienti ha mostrato un aumentato rischio anche di adenomi cortico-surrenalici. NellвЂ™85% dei casi ГЁ colpito il sesso femminile con decorso in genere lento ed indolente (80% dei pazienti vivi a 20 anni); le recidive locali, cosГ¬ come le metastasi epatiche linfonodali e peritoneali sono poco frequenti. I geni KIT, PDGFRA e SDH risultano wild type. La Diade di Carney-Stratakis ГЁ caratterizzata da GIST gastrici e paragangliomi multipli. LвЂ™etГ media di comparsa ГЁ 19 anni ed alcuni pazienti possono presentare, insieme al GIST, anemia ed emorragia gastrointestinale in assenza di evidenza di paraganglioma. Nella maggior parte dei pazienti con sindrome di Carney-Stratakis non si riscontra la presenza di mutazioni in KIT e PDGFRA, bensГ¬ mutazioni germinali o delezioni nei geni SDH B, C o D che codificano per le subunitГ B, C e D della succinato deidrogenasi (SDH, complesso mitocondriale II). La predisposizione allo sviluppo di questi tumori ГЁ ereditata come carattere autosomico dominante a penetranza incompleta. La succinato deidrogenasi ГЁ un sistema enzimatico che ha un duplice ruolo nel ciclo di Krebs e nella catena di trasporto degli elettroni nei mitocondri. La SDH ГЁ costituita da 4 205 Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan subunitГ codificate dai geni SDHA, SDHB, SDHC e SDHD, formati rispettivamente da 15, 8, 6 e 4 esoni. I primi due geni, localizzati nel cromosoma 5p15 e nel cromosoma 1p35, codificano per le due subunitГ catalitiche, mentre gli ultimi due codificano per le due subunitГ idrofiliche transmembrana. Mutazioni germinali nel gene tumor-suppressor SDH erano state precedentemente associate al paraganglioma ereditario (PGL1) e al feocromocitoma e dovrebbero essere ricercate in pazienti con GIST familiare e paragangliomi. La Tabella 2 riassume le principali caratteristiche del Complesso di Carney Г€ stata riportata unвЂ™aumentata incidenza (circa 200 volte) di GIST in pazienti con Neurofibromatosi di tipo 1. La NF1 ГЁ una tra le piГ№ comuni malattie autosomiche dominanti, e colpisce nel mondo 1 su 2500вЂ“3000 nati, con una prevalenza di circa 1 su 4000вЂ“5000 individui nella popolazione generale. Il tratto viene trasmesso in maniera autosomica dominante, con una penetranza del 100% a espressivitГ variabile in quanto esistono anche forme segmentali o a mosaico. Il background genetico dei pazienti influenza invece la manifestazione dei fenotipi correlati. Il gene NF1 codifica per una proteina, detta neurofibromina, di circa 220-250 kDa, con funzione di proteina inducente la funzione GTPasica (GAP) delle proteine della famiglia Ras. La sua azione comporta la stimolazione dellвЂ™attivitГ GTPasica di Ras, con la conseguente 206 GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici conversione del complesso mitogenico Ras-GTP nella forma inattiva Ras-GDP. Pertanto la perdita di questa proteina determina la permanente attivazione di Ras, condizione tipica di molti tumori sporadici in cui mutazioni attivanti di Ras ne eliminano lвЂ™attivitГ catalitica. A supporto di questa funzione, ГЁ stato osservato che linee cellulari umane e murine prive del gene NF1 (NF1-null) che presentano un fenotipo iperproliferativo possono essere riportate alla normalitГ con lвЂ™inibizione dellвЂ™attivitГ di Ras. La proteina Ras ГЁ anche implicata nelle vie di trasduzione del segnale attivate da c-KIT, ed infatti i pazienti con NF1 spesso presentano GIST multifocali nellвЂ™intestino tenue, piГ№ raramente nello stomaco. LвЂ™incidenza di GIST in questo sottogruppo di pazienti ГЁ circa il 5-25%. Anche questi insorgono in assenza di mutazioni in KIT e PDGFRA, prevalentemente nel sesso femminile e hanno un decorso clinico indolente. Gist eredo-familiari e principi di counselling oncogenetico LвЂ™identificazione dei geni di suscettibilitГ , coinvolti nelle varie sindromi tumorali ereditarie ha fornito la base molecolare dei test genetici, che permettono di individuare tra gli individui a rischio quelli portatori della mutazione germline, considerati вЂњad alto rischioвЂќ di sviluppare uno specifico tumore. Questa nuova acquisizione della genetica molecolare ha evidenziato la necessitГ di impiegare il counselling oncogenetico per venire incontro alle esigenze di tutti quei soggetti, affetti da tumore o sani, che desiderano comprendere il rischio di ricorrenza di una malattia genetica allвЂ™interno della famiglia e programmare una adeguata gestione clinica. Il numero di laboratori di ricerca e di professionisti del settore che si dedicano al counselling oncogenetico sta crescendo enormemente, visto lвЂ™aumento della richiesta da parte di oncologi e altri medici a cui pervengono individui con forti fattori di rischio dвЂ™insorgenza di tumori eredo-familiari, che vengono indirizzati alla consulenza genetica. Il primo fattore di rischio fra tutti che deve far pensare ad un tumore ereditario ГЁ la precoce etГ dвЂ™insorgenza, anche in assenza di anamnesi familiare positiva. Nel caso dei GIST eredo-familiari, lвЂ™etГ a cui si fa riferimento ГЁ circa 45 anni. La presenza dello stesso tipo di tumore in diversi membri da un solo lato (paterno o materno) della famiglia o lвЂ™associazione di vari tumori dovuti a mutazioni di KIT, PDGFRA e SDH (per esempio paragangliomi e condromi) costituiscono fattori di rischio molto forti per identificare famiglie candidate al counselling genetico. Il counselling oncogenetico (CGO) ГЁ un atto medico, rivolto alla persona nella sua globalitГ . Si svolge presso centri specializzati e prevede un approccio multidisciplinare ed integrato tra diverse figure professionali, quali il medico oncologo, il genetista e lo psicologo; ciascuno di questi specialisti svolge un ruolo ben determinato ed interviene a livelli differenti delle varie fasi di cui il counselling si compone (fase pre-test, fase del test 207 Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan genetico, fase post-test, follow-up). La multidisciplinarietГ tiene conto dei diversi aspetti e dei diversi bisogni della persona a rischio di cancro ereditario. Gli individui a rischio di sviluppare una neoplasia vengono sottoposti allвЂ™analisi della storia personale e familiare attraverso la ricostruzione dellвЂ™albero genealogico nella fase pre-test. Le principali caratteristiche della storia personale che suggeriscono la natura ereditaria di un GIST comprendono la diagnosi di GIST in etГ precoce, GIST primitivi multipli, o diagnosi di GIST e un altro tumore (ad esempio paragangliomi, adenomi, condromi, neurofibromi). La ricostruzione della storia familiare del soggetto (probando), rappresenta un momento fondamentale del processo, in quanto la dettagliata descrizione dellвЂ™albero genealogico consente agli specialisti di effettuare una diagnosi piГ№ corretta, prevedere la prognosi piГ№ accuratamente e, quindi, consigliare il consultante sulle decisioni da intraprendere. Г€ necessario raccogliere per almeno tre generazioni le informazioni personali e cliniche dei parenti di primo (figli, fratelli, genitori), secondo (nonni, zii, nipoti) e terzo grado (cugini, prozii) del probando in modo da facilitare lвЂ™identificazione di pattern ereditari presenti eventualmente nella famiglia. Per la corretta costruzione di un вЂњpedigreeвЂќ ГЁ necessario raccogliere il maggior numero di informazioni possibile, considerando: a) sia il ramo paterno che materno; b) la paternitГ , la consanguineitГ , lвЂ™uso di tecnologie di fecondazione assistita. Г€ importante raccogliere informazioni sia sui familiari affetti da patologie tumorali che sui familiari non affetti. Per ogni familiare affetto da tumore ГЁ necessario valutare: a) il tipo e la sede del tumore primitivo; b) lвЂ™etГ dвЂ™insorgenza alla diagnosi del tumore primitivo; c) lвЂ™etГ attuale se lвЂ™individuo ГЁ ancora in vita o lвЂ™etГ del decesso e la causa della morte; d) lвЂ™esposizione a carcinogeni (per es., uso di tabacco, esposizione a radiazioni); d) altri problemi di salute significativi (per es., presenza di malattie genetiche note che possono predisporre allo sviluppo di tumori). Per ogni familiare non affetto da tumore ГЁ necessario esaminare: a) lвЂ™etГ attuale o lвЂ™etГ del decesso; b) se deceduto, la causa del decesso; c) gli interventi chirurgici che possono aver ridotto il rischio di insorgenza del cancro; d) se routinariamente sottoposti a screening per la diagnosi precoce di tumore; e) lвЂ™esposizione a carcinogeni; f) altre patologie. In particolare, le caratteristiche che possono suggerire la predisposizione genetica allвЂ™insorgenza di un GIST comprendono la presenza di: -- due o piГ№ parenti con familiaritГ di I grado con GIST -- un membro della famiglia che ha avuto diagnosi di GIST e un altro con diagnosi di un altro tumore primitivo raro -- un membro della famiglia con GIST che ha una storia personale o familiare delle tipiche manifestazioni cutanee, multifocalitГ o risulta portatore di mutazioni nel gene NF1. 208 GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici NellвЂ™immagine di seguito riportata (Fig.3) viene illustrato lвЂ™albero genealogico di una famiglia affetta da Diade di Carney-Stratakis, caratterizzato dalla verticalitГ della trasmissione ereditaria per 4 generazioni e dallвЂ™associazione dei casi di GIST con paragangliomi. Da notare infine la precoce etГ di insorgenza della malattia rispetto ai casi sporadici. Dopo la valutazione della storia personale e familiare segue la fase del test genetico. LвЂ™identificazione di una mutazione genetica puГІ consentire, grazie alle correlazioni genotipo-fenotipo associate alla sindrome, di inquadrare le manifestazioni cliniche nellвЂ™ambito delle sindromi precedentemente descritte. La fase post-test prevede la consegna del referto, e nei portatori di mutazione, il follow up allo scopo di adottare programmi di sorveglianza individualizzati. 209 Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan Bibliografia [1] Agaram NP, Laquaglia MP, Ustun B, et al. Molecular characterization of pediatric gastrointestinal stromal tumors. Clin Cancer Res 2008; 14:3204-15. [2] Bolland M, Benn D, Croxson M, et al. Gastrointestinal stromal tumour in succinate dehydrogenase subunit B mutation-associated familial phaeochromocytoma/paraganglioma. ANZ J Surg 2006; 76:763-4. [3] Braconi C, Bracci R, Bearzi I, et al. Insulin-like growth factor (IGF) 1 and 2 help to predict disease outcome in GIST patients. Ann Oncol 2008; 19:1293-8. [4] Carballo M, Roig I, Aguilar F, et al. Novel c-KIT germline mutation in a family with gastrointestinal stromal tumors and cutaneous hyperpigmentation. Am J Med Genet A 2005; 132:361-4. [5] Carney JA, Stratakis CA. Familial paraganglioma and gastric stromal sarcoma: a new syndrome distinct from the Carney triad. Am J Med Genet 2002; 108:132-9. [6] Chiarugi M, Galatioto C, Lippolis P, Zocco G, Seccia M. Gastrointestinal stromal tumour of the duodenum in childhood: a rare case report. BMC Cancer 2007; 7:79. [7] Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors. J Clin Oncol 2004; 22:3813-25. [8] Cypriano MS, Jenkins JJ, Pappo AS, Rao BN, Daw NC. Pediatric gastrointestinal stromal tumors and leiomyosarcoma. Cancer 2004; 101:39-50. [9] Hartmann K, Wardelmann E, Ma Y, et al. Novel germline mutation of KIT associated with familial gastrointestinal stromal tumors and mastocytosis. Gastroenterology 2005; 129:1042-6. [10] Isozaki K, Terris B, Belghiti J, Schiffmann S, Hirota S, Vanderwinden JM. Germline-activating mutation in the kinase domain of KIT gene in familial gastrointestinal stromal tumors. Am J Pathol 2000; 157:1581-5. [11] Janeway KA, Liegl B, Harlow A, et al. Pediatric KIT wild-type and platelet-derived growth factor receptor alpha-wild-type gastrointestinal stromal tumors share KIT activation but not mechanisms of genetic progression with adult gastrointestinal stromal tumors. Cancer Res 2007; 67:9084-8. [12] Kuroiwa M, Hiwatari M, Hirato J, et al. Advanced-stage gastrointestinal stromal tumor treated with imatinib in a 12-year-old girl with a unique mutation of PDGFRA. J Pediatr Surg 2005; 40:1798-801. [13] Li FP, Fletcher JA, Heinrich MC, et al. Familial gastrointestinal stromal tumor syndrome: phenotypic and molecular features in a kindred. J Clin Oncol 2005; 23:2735-43. [14] McWhinney SR, Pasini B, Stratakis CA. Familial gastrointestinal stromal tumors and germ-line mutations. N Engl J Med 2007; 357:1054-6. [15] Miettinen M, Lasota J. Gastrointestinal stromal tumors: review on morphology, molecular pathology, prognosis, and differential diagnosis. Arch Pathol Lab Med 2006; 130:1466-78. [16] Miettinen M, Lasota J, Sobin LH. Gastrointestinal stromal tumors of the stomach in children and young adults: a clinicopathologic, immunohistochemical, and molecular genetic study of 44 cases with longterm follow-up and review of the literature. Am J Surg Pathol 2005; 29:1373-81. [17] OвЂ™Riain C, Corless CL, Heinrich 17. Nishida T, Hirota S, Taniguchi M, et al. Familial gastrointestinal stromal tumours with germline mutation of the KIT gene. Nat Genet 1998; 19:323-4. [18] Pasini B, McWhinney SR, Bei T, et al. Clinical and molecular genetics of patients with the Carney-Stratakis syndrome and germline mutations of the genes coding for the succinate dehydrogenase subunits SDHB, SDHC, and SDHD. Eur J Hum Genet 2008; 16:79-88. [19] Prakash S, Sarran L, Socci N, et al. Gastrointestinal stromal tumors in children and young adults: a clinicopathologic, molecular, and genomic study of 15 cases and review of the literature. J Pediatr Hematol Oncol 2005; 27:179-87. 210 GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici [20] Price VE, Zielenska M, Chilton-MacNeill S, Smith CR, Pappo AS. Clinical and molecular characteristics of pediatric gastrointestinal stromal tumors (GIST). Pediatr Blood Cancer 2005; 45:20-4. [21] Rubin BP, Antonescu CR, Scott-Browne JP, et al. A knock-in mouse model of gastrointestinal stromal tumor harboring kit K641E. Cancer Res 2005; 65:6631-9. [22] Sommer G, Agosti V, Ehlers I, et al. Gastrointestinal stromal tumors in a mouse model by targeted mutation of the Kit receptor tyrosine kinase. Proc Natl Acad Sci U S A 2003; 100:6706-11. [23] Tarn C, Rink L, Merkel E, et al. Insulin-like growth factor 1 receptor is a potential therapeutic target for gastrointestinal stromal tumors. Proc Natl Acad Sci U S A 2008; 105:8387-92. 211 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 The impact on the patientвЂ™s adjustment to breast cancer of the caregiverвЂ™s burden and distress [LвЂ™impatto del carico emotivo e del distress del caregiver sullвЂ™adattamento alla malattia del paziente affetto da carcinoma della mammella] Gianluca Lo Coco1, Elena Foddai1,2, Salvatore Gullo1, Maria Viviana Cicero1, Giovanna Manna1, Francesca Paola Guadagna1,2, Viviana Bazan2, Maria Buscemi4, Massimo Cajozzo3, Giuseppe Bronte2, Sergio Rizzo2 and Antonio Russo2 Department of Psychology; 2Section of Medical Oncology, Department of Surgery and Oncology, 3Unit of Toracic Surgery, 4Histology and Embryology Section, Di.Me.S., UniversitГ di Palermo (IT) 1 Key words: Breast cancer, Caregiver, Distress, Adjustment Parole chiave: Carcinoma della mammella, Caregiver, Distress Abstract. Introduction: Breast cancer involves continually stressful situation caused by the threat of the illness for both patient and caregiver. Impairment of coping and support processes can result in this context. The aim of the present study was to obtain further knowledge regarding the relationship between the breast cancer patient and the caregiverвЂ™s psychological distress. Methods: Participants were recruited in an outpatient oncology clinic. Measures included Impact of Events Scale (IES), Family Strain Questionnaire (FSQ), Mental Adjustment to Cancer (MAC) and Profile of Mood States (POMS). Patients completed a measure of psychological adjustment to their cancer and caregivers underwent a measure of caregiver strain. Results: A sample of 56 patients with breast cancer and their respective caregivers was included in the study. Mean age of patients was 56 (range: 30-75). The IES-Avoidance scores of patients were significantly correlated with the IES-Intrusion of caregivers (p < .05), the POMS-Anxiety (p < .01), Depression (p < .01) and Hostility (p < .01) scores of their caregivers. The POMS-Confusion scores of caregivers were correlated with the IES-A scores of patients. Conclusions: Our findings indicated that the caregiver is as likely to experience psychological distress as the cancer patient, and lends weight to the assertion that both members of the dyad experience similar levels of distress. Moreover, we found that the hopelessness and the sense of the uncontrollability of cancer in the patients are predicted by the vigour and the emotional burden of the caregiver. Riassunto. Introduzione: Il carcinoma della mammella frequentemente genera situazioni stressanti legate alla gravitГ della malattia, nel paziente cosГ¬ come nel caregiver. In questo contesto possono verificarsi alterazioni nel coping e nel processo di supporto. Lo scopo del presente studio ГЁ quello di investigare la relazione tra il distress psicologico del paziente affetto da carcinoma della mammella e il suo caregiver. Metodi: I partecipanti sono stati reclutati in un DH di oncologia. Come questionari sono stati somministrati lвЂ™Impact of Events Scale (IES), il Family Strain Questionnaire (FSQ), il Mental Adjustment to Cancer (MAC) e il Profile of Mood States (POMS). I pazienti hanno completato un questionario sullвЂ™adattamento psicologico alla loro malattia mentre al caregiver ГЁ stato proposto un questionario relativo al proprio malessere. Risultati: Il campione comprendeva 56 pazienti con carcinoma della mammella e i loro rispettivi caregiver. LвЂ™etГ media dei pazienti ГЁ stata di 56 anni (range: 30-75). Il punteggio dellвЂ™IES-evitamento dei pazienti ГЁ risultato significativamente 213 Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna, Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo, Antonio Russo correlato con lвЂ™IES-pensieri intrusivi dei caregiver (p < .05), con i punteggi del POMS-Ansia (p < .01), Depressione (p < .01) e OstilitГ (p < .01) dei loro caregiver. I punteggi del POMS-Confusione dei caregiver ГЁ risultato correlato con i punteggi dellвЂ™IES-Evitamento dei pazienti. Conclusioni: I nostri risultati suggeriscono che il caregiver ha una probabilitГ di sperimentare un distress psicologico proporzionale a quella del paziente, e avvalora la tesi secondo la quale entrambi i membri della diade sperimentano livelli simili di distress. Inoltre, abbiamo riscontrato che il senso di disperazione e di incontrollabilitГ della malattia da parte del paziente affetto da carcinoma puГІ essere predetto dal vigore e dal carico emotivo del caregiver. Introduction The experience of cancer may heavily depend on patientsвЂ™ interpersonal context and, notably, their primary caregivers. Caregivers of cancer patients may take an active role in key decisions concerning treatment options and provide emotional and instrumental support to the patient [1]. There is a general consensus that breast cancer poses a major stressor for patients as well as caregivers and that patients and partners are involved in each otherвЂ™s coping and support processes [2]. Significantly, an appropriate adjustment to cancer depends on the totality of the cognitive, emotional and behavioural responses to the diagnosis on the part of both the patient and the significant other. Most studies concerning breast cancer has reported that this life-threatening illness produces high stress in both patients and families, and a number of studies have found a significant correlation between distress in patients and partners [3]. Moreover Hodges [4], in a meta-analytic investigation conducted on 21 independent samples regarding the relationship between the psychological distress of various types of cancer patients and their caregivers confirm the positive association between patientsвЂ™ and caregiversвЂ™ psychological distress. Most studies of couples in which the woman has cancer have focused on breast cancer patients. Several studies indicates that the psychological distress of breast cancer patients is higher than that of their spouses [5,6], while others found that spouses present more psychosocial problems than patients [7,8]. Northouse [9] described husbandsвЂ™ level of distress as similar to that of their wives and higher, up to 18 months after diagnosis, than that in non-patient populations. A recent meta-analysis has indicated that differences in distress within couples coping with cancer could clearly be ascribed to gender: women were found to report more distress than men, regardless of whether they were the individuals with cancer or the partners [10]. Several studies have documented that the primary caregiver of the cancer patient is in a continually stressful situation caused by the threat of the illness and, simultaneously, by the assumption of the role of main informal social support resource for the patient in the illness process. Previous research has consistently found that a good marital relationship predicts better emotional adaptation to breast cancer [11,12]. In contrast, social constraints and unresponsiveness of family members in talking about the illness 214 The impact on the patientвЂ™s adjustment to breast cancer of the caregiverвЂ™s burden and distress have been related to poor mental health among cancer patients [13]. Emotional distress and depression in the caregiver can have important consequences for the diagnosed patient and can negatively affect his/her adjustment and how the couple copes with challenges [14,15]. Overall, it appears that perceived reactions of the patientвЂ™s caregiver represent an important influence on the womanвЂ™s response to the diagnosis and treatment of this disease [16]. Caregivers are at the center of the support network for the patient, and it is therefore extremely important to reach a detailed appreciation of their needs and experiences. Given this, it seems important to know more about how caregiver reactions relate to the patientвЂ™s emotional well-being, whether the effect is an adverse one or a beneficial one. The aims of this study were to determine the correspondence between the psychological distress of cancer patients and their caregivers, and ascertain the nature of this relationship. The following specific questions were raised. Research Question 1: Is there a significant positive correlation between breast cancer patient and caregiver psychological distress? Research Question 2: Is the distress of the caregiver a predictor of the patientвЂ™s poor adjustment to breast cancer ? Patients and methods Participants For enrolment participants should be new and returning cancer patients seen at the medical oncology clinic at the University of Palermo (Italy), with their caregivers. As caregiver we considered a person indicated by the patient as the вЂњprimary informal caregiver.вЂќ Procedures Patients were approached in the medical oncology clinic before or after their treatment appointments and asked to participate in the study. If a patient was interested, the research assistant explained the informed consent form to the patient and asked for the patientвЂ™s permission to contact his/her caregiver to request his/her participation. Exclusion criteria for patients were: prior history of psychiatric hospitalization (3 participants were excluded), and major concurrent disease (6 participants were excluded). Approximately 85% of the patients contacted who met eligibility criteria agreed to participate. Patients and caregivers who agreed to participate were separately interviewed before leaving the clinic. 215 Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna, Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo, Antonio Russo Measures Impact of Events Scale (IES) [17]. Psychological distress was assessed using a shortened version of the Impact of Events Scale. This scale has been seen to offer a reliable assessment of intrusive thoughts related to stressful events and conscious avoidance of feelings and ideas about the events [18]. The shortened version of the scale includes eight items (four for each subscale). Patients and caregivers respond to statements about the frequency (0 = not at all, 1 = rarely, 3 = sometimes, 5 = often) in the prior 7 days of intrusive thoughts related to the patientвЂ™s cancer (e.g. вЂњI thought about the diagnosis when I didnвЂ™t mean toвЂќ) and to the frequency of avoidance behaviors (e.g. вЂ�I kept away from reminders of the cancerвЂ™). In the present study, CronbachвЂ™s alphas for patients for the Intrusion and the Avoidance scale were .72 and .89, respectively, and .73 and .82 for caregivers. Family Strain Questionnaire (FSQ) [19]. The FSQ consists of a semi-structured interview and questionnaire that can be administered in a total of about 20 min. The semi-structured interview was focused on the caregiverвЂ™s knowledge of/beliefs about the disease and the way in which it is managed. The questionnaire consists of 35 dichotomous items (yes/no) covering five factors: Emotional Burden (EB); Problem of Social Involvement (SI); The Need for Knowledge of the Disease (KD); Quality of Family Relationships (FR); Thoughts about Death (TD). Nine non-factorial dichotomous items concern the caregiverвЂ™s attribution of problems relating to social stigma. As the вЂ�yesвЂ™ answers are attributed a score of 1, the higher the score for each area, the greater the problems involved, with the exception of satisfaction of family relationships (in which a high score indicates good relationships). The FSQ has been validated in Italy and has good psychometric properties. In this study CronbachвЂ™s alpha ranged from .72 (TD) to .87 (EB). Mental Adjustment to Cancer (MAC) [20]. The MAC is a 40 items scale which identifies four behavioral styles of coping: fighting spirit, fatalism, helplessness/hopelessness, and anxious preoccupation. PatientsвЂ™ responses were rated on a 4-point scale. Fighting Spirit (FS): identifies the patientвЂ™s tendency to see cancer as a challenge and to take an active role in therapy and recovery, and to adopt an optimistic attitude. Helplessness (H): measures cognitive schema and corresponding behaviors characterized by a sense of the uncontrollability of cancer. Stoic Acceptance/Fatalism (F): which indicates resignation and passive acceptance of the illness and the therapy, without personal involvement. Anxious Preoccupation (AP): evaluates anxiety and diffuse worry about cancer and its possible recurrence, associated with excessive search for reassurance and information. In the present study, internal consistency (CronbachвЂ™s alpha) of the subscales of MAC were: FS=.76, H=.69, AP=.67, F=.62, respectively. 216 The impact on the patientвЂ™s adjustment to breast cancer of the caregiverвЂ™s burden and distress Profile of Mood States (POMS) [21]. Patients and caregivers were asked to rate the Profile of Mood States (POMS), a 65-items measure designed to assess subjective mood states, including positive and negative affects. Responses were on a four-point scale, ranging from вЂњNot at all like thisвЂќ to вЂњVery much like thisвЂќ. The POMS yields a score for total mood disturbance based on six subscales: Anxiety (A), Depression (D), Hostility (H), Confusion (C), Vigor (V), and Fatigue (F). In this study, all the alpha coefficients for the six POMS scales ranged from .84 (C) to .95 (D) (patients); the alpha coefficients ranged from .76 (F) to .89 (A) for caregivers. Statistical analyses Means and standard deviations were computed for demographic and medical information as well as for psychosocial variables. Paired-sample t tests were conducted to compare mean differences between patients and caregivers scores in IES and POMS. Pearson correlation coefficients were calculated to examine the bivariate associations among study variables. Six separate stepwise regressions were constructed to examine the role of family strain (FSQ) and mood states (POMS) of caregivers in predicting the patient adjustment to cancer (IES-I, -A; MAC-FS, -H/H, -AP, F). The equations had 2 steps: in step 1, we entered the age of the patient and caregiver as control variables, in step 2 we entered the independent variables with a stepwise procedure. The stepwise regression procedure describes how much more each independent or predictor variable has contributed to the prediction from the preceding predictor variables, and it is generally used in exploratory procedures. In this study, we adopted a more restrictive level of .01. Results Fifty-six patient-caregiver couples participated. Demographic and medical characteristics of the sample are presented in Table 1. Patients had a mean age of 56 (range: 30-75). Eighty percent were married, and sixty-six percent had at least a high school diploma, and in terms of occupation, sixty-seven percent were unemployed. Eighty-five percent underwent surgical treatment or surgery and chemotherapy. Caregivers had a mean age of 48.8 (range: from 23 to 75), and 55.5% were female. Fifty-five percent were the patientsвЂ™ spouses, twenty-seven percent were the children, and eighteen percent had other relationships (parents, sisters). Only 44% were employed caregivers. 217 Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna, Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo, Antonio Russo Table 1. Demographic and medical characteristics Demographic and medical characteristics Mean age Sex male female Role partner son other Status single (or separated/widowed) married Education less than high school high school or more Employment status Employed Unemployed Type of cancer breast colon and other Stage I II and III Range time since diagnosis Therapy surgery or surgery + chemio palliative Patients (N = 56) Caregivers (N = 56) 56,02 (11,36) 48,85 (13,92) 12,5% 87,5% 44,6% 55,4% - 55,4% 26,8% 17,9% 19.6% 80.4 23.2% 76,8% 66,1% 32,1% 58,9% 41,1% 32.2% 67.8% 44,6% 55,4% 73.2% 26.8% 72.2% 27.8% 1 - 10 months 85.7% 14.3% A first step in the analysis was the comparison between patients and caregivers in terms of psychological distress (IES & POMS). Only the IES-Avoidant subscale was higher in cancer patients than caregivers (t = 2.02 p < .05). Table 2 shows zero-order correlations for the observed variables. The IES-Avoidance scores of patients were significantly correlated with the IES-Intrusion of caregivers (p < .05), the POMS-Anxiety (p < .01), De218 The impact on the patientвЂ™s adjustment to breast cancer of the caregiverвЂ™s burden and distress pression (p < .01), and Hostility (p < .01), scores of their caregivers. The IES-Intrusion of patients were correlated with the FSQ-Emotional Burden and KD scores of their caregivers (p < .05; p < .01, respectively). The FSQ-Emotional Burden of caregivers was correlated with the MAC-Helplessness (p < .05). The POMS-Confusion scores of caregivers were correlated with the IES-A scores of patients. Table 2. Zero-order correlations (PearsonвЂ™s r) between patients and caregivers Patients IES Intr POMS A POMS D POMS H POMS V POMS F POMS C IES Intr IES Av MAC FS MAC H/H MAC AP MAC F ,034 ,231 ,025 ,214 -,064 ,188 -,080 -,129 ,131 ,180 -,013 ,148 ,128 ,174 ,335* ,105 -,048 -,073 ,160 ,218 -,161 ,170 ,087 ,254 IES Av POMS A ,095 ,089 ,052 ,111 ,124 ,098 -,033 ,346** ,113 -,062 -,122 -,067 POMS D ,138 ,176 ,091 ,057 ,174 ,088 ,038 ,433** ,028 ,042 -,072 ,087 POMS H Caregiver POMS POMS V F ,180 -,225 ,156 -,232 ,226 -,050 ,102 ,407(**) ,178 -,288(*) ,113 -,062 ,083 -,095 ,362** -,185 ,076 ,264* -,105 -,398** -,129 -,152 -,167 -,110 ,322(*) ,212 ,246 ,104 ,334(*) ,192 -,027 ,216 ,136 -,068 ,044 -,170 POMS C ,277* ,338* ,296* -,095 ,276* ,211 ,104 ,374** ,052 ,062 ,059 ,106 FSQ EB FSQ SI FSQ KD FSQ RF FSQ KD ,065 ,189 ,056 -,114 ,108 ,054 ,150 -,006 -,060 ,100 ,062 ,147 ,039 -,094 ,048 -,182 -,196 -,212 -,155 -,076 -,013 ,195 -,060 -,084 ,057 -,081 ,063 -,078 -,063 ,015 ,294* ,179 ,408** -,003 ,208 ,112 ,184 ,123 -,095 ,232 -,180 ,022 -,164 -,058 -,082 ,296* ,230 ,232 ,189 ,243 ,057 ,070 ,077 -,045 -,020 ,016 ,058 -,058 ,083 ,046 * Correlation is significant at the 0.05 level. With regard to the regression analyses (see the statistical analyses section), the first block of equations (see Table 3) was conducted on the IES Intrusion and Avoidance scores of patients. The first final model was significant (F = 3.255, p < .01), and revealed that the FSQ-KD (ОІ = .40, p < .001) was the only predictor of IES-Intrusion of patients. The second equation showed that age of the patient had quite a significant effect (p < .05). A second block of multiple regression analyses was conducted on the MAC scores of patients as the dependent variables. In the final model both the POMS-Vigour and the age of patient were predictors of the MAC-Helplessness/Hopelessness (ОІ = -.36, p < .01; ОІ = .42, p < .01, respectively). The FSQ-EB showed a low effect on MAC Helplessness/Hopelessness (p < .05). 219 Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna, Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo, Antonio Russo Table 3. Hierarchical multiple-regression analyses predicting patientвЂ™s adjustment to cancer ОІ p I вЂ“ IES Intrusion FSQ вЂ“ KD -.40 .001 I вЂ“ IES Avoidance Age of patients POMS - C .29 -.52 .05 .001 II - MAC Helplessness/Hopelessness Age of patients POMS - V FSQ - EB .42 - .36 .24 .01 .01 .05 Adj R2 .14 F 3.26 .22 3.92 .01 .33 7.70 .00 p p of R2 change .01 Only significant variables and those approaching significance are included in the table. The Table displays the standardised regression coefficients (ОІ), the significance of R squared increment provided at each step (p of R2 change), and the overall Adjusted R squared (Adj R2). IES Intrusion and Avoidance = subscales of Impact Event Scale; MAC Helplessness/Hopelessness = subscale from Mental Adjustment to Cancer; FSQ KD, EB = knowledge of disease, emotional burden, subscales of Family Strain Questionnaire; POMS вЂ“ C = confusion, subscale of Profile of Mood States. Discussion In this study, the aim was to test if there exists a positive relationship between breast cancer patient and caregiver psychological distress, and if the caregiverвЂ™s distress may be a predictor of the patientвЂ™s poor adjustment to cancer. Our findings confirm a significant positive relationship between patient and caregiver psychological distress. Caregiver distress of (POMS-Confusion, Hostility, Depression, and Anxiety) is positively correlated with the patientвЂ™s avoidance of feelings and ideas about the cancer (IES-AV). Interestingly, the Vigor (POMS) of the caregiver is positively correlated with the Vigor and Fighting Spirit, and negatively with the Fatigue and the Helplessness of the patient. These findings seem to indicate that the caregiver is as likely to experience psychological distress as the cancer patient, and lends weight to the assertion that both members of the dyad experience similar levels of distress [3]. In our study, only the IES-Avoidant scale is higher in breast cancer patients than their caregivers. A cursory review of the literature regarding distress in breast cancer patients and their caregivers suggests that it is not cumulative and that it is beset by quite contradictory claims. Studies have reported that patients are as distressed as caregivers, that patients with cancer are more distressed than their partners, that caregivers are more distressed than patients. Our findings are consistent with previous research which has shown that the difference between patient and caregiver psychological distress appears to vary over time [4], and that 220 The impact on the patientвЂ™s adjustment to breast cancer of the caregiverвЂ™s burden and distress from the 3-month assessment period onwards, no significant difference in distress between patient and caregiver levels exist. One critical issue in the interpretation of findings regarding distress in patients and caregivers appears to be gender. Studies that find greater distress among partners may have confounded the patientвЂ“partner role with gender. Moreover, the findings of the multiple regression analyses show that different aspects of the caregiverвЂ™s psychological distress may predict the adjustment to breast cancer and distress of the cancer patient. The need of hear the disease (FSQ-KD) of the caregiver predicts the patientвЂ™s intrusive thoughts related to cancer (IES-Intrusion). The hopelessness and the sense of the uncontrollability of cancer in the patient are predicted by the vigour and the emotional burden of the caregiver. These findings seem to suggest the association between the psychological distress of caregiver and patient, and they might well be interpreted as support for the notion that patient-caregiver pairs react as an interdependent emotional system. These findings are also consistent with previous research on women with breast cancer, which shows that the family caregiverвЂ™s vulnerability is heightened because he or she finds himself/herself in a double and conflictive role. As the primary supporter, he/she must assume new roles in the household and provide tangible as well as emotional support; and at the same time must cope with the distress emanating from the significance of the patientвЂ™s diagnosis in terms of suffering and the threat to life [22]. Further research will be necessary to investigate how the distress of caregivers may influence the patientвЂ™s distress. Moreover, some important variables, such as gender and role, need to be taken into account, in order to study the relationship between the breast cancer patient and his/her caregiver. A recent meta-analysis [4] which covered 21 studies of distress in people with cancer and their caregivers outlined that while these caregivers were mainly partners, they also included siblings, children, and close friends. In our sample, only 55% of the primary caregivers indicated by patients are the spouses. The available literature suggests caution about assuming that partners of people with breast cancer have substantial caregiving responsibilities, in terms of providing assistance with medical management or the activities of daily life. Rather than being accepted uncritically as a synonym for partners of people with cancer, use of the term вЂњcaregiverвЂќ requires justification with explicit reference to instrumental caregiving tasks or demands of home management or functional limitations of the person with cancer. Finally, attention needs to be directed toward factors other than breast cancer as direct influences of distress in these couples and to mediators and moderators of the cancer experience. A limitation of this study was the modest size and the heterogeneous characteristics of both the samples of patient and caregiver. Results should therefore be considered tentative and exploratory until confirmed by further research studies. Despite these limitations on generalizability, our findings seem to offer useful information in the attempt to reach a clearer understanding concerning the experiences of breast cancer patients. 221 Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna, Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo, Antonio Russo References [1] Pistrang N, Barker C. The partner relationship in psychological response to breast cancer. Soc Sci Med 1995; 40: 789-797. [2] Manne S, Ostroff J, Sherman M, et al. CouplesвЂ™ support-related communication, psychological distress, and relationship satisfaction among women with early stage breast cancer. J Consult Clin Psychol 2004; 72: 660-670. [3] Manne SL. Cancer in the marital context: A review of the literature. Cancer Invest 1998; 16(3):188-202. [4] Hodges LJ, Humphries GM, MacFarlane G. A meta-analytic investigation of the relationship between the psychological distress of cancer patients and their caregivers. Soc Sci Med 2005; 60:1-12. [5] Ben-Zur H, Gilbar O, Lev S. Coping with breast cancer: patient, spouse and dyad models. Psychosom Med 2001; 63:32вЂ“39. [6] Hoskins CN. Adjustment to breast cancer in couples. Psychol Rep 1995; 77:435-454. [7] Goodyear-Smith F, Buetow S. Power issues in the doctor-patient relationship. Health Care Analysis 2001; 9: 449-462. [8] Yeo W, Kwan WA, Teo PML, et al. Psychosocial impact of breast cancer surgeries in Chinese patients and their spouses. Psychooncology 2004; 13:1 32-139. [9] Northouse L. Sharing the cancer experience: Husbands of women with initial and recurrent breast cancer. In Baider L, Cooper C L, & Kaplan De-Nour A. (ed), вЂњCancer and the familyвЂќ. Wiley, New York 1996, pp 304-317. 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Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 LA CONSULENZA GENETICA IN ONCOLOGIA: IMPLICAZIONI BIOMOLECOLARI E CLINICHE [Oncogenetic counselling: biomolecular and clinical implications] Antonio Russo1, Sergio Rizzo1, Valentina CalГІ1, Loredana Bruno1, Laura La Paglia1, Nello Grassi2, Gianni Pantuso2, Michele Frazzetta3, Massimo Cajozzo3, Domenico Guarneri4, Giuseppe Lo Dico4, Maria Buscemi5, Nicola Gebbia1 e Viviana Bazan1 Sezione di Oncologia Medica e Centro di Riferimento Regionale per la Caratterizzazione e lo Screening Genetico dei Tumori Ereditari, Dipartimento di Discipline Chirurgiche ed Oncologiche; 2Sezione di Oncologia Chirurgica, Dipartimento di Discipline Chirurgiche ed Oncologiche; 3Sezione di Chirurgia Generale, Dipartimento di Discipline Chirurgiche ed Oncologiche; 4Ginecologia ed Ostetricia, Dipartimento Materno Infantile; 5 Sezione di Istologia ed Embriologia, Di.Me.S., UniversitГ degli Studi di Palermo (IT) 1 Key words: Genetic counselling, Hereditary breast cancer, Ovarian cancer, Genetic testing Parole chiave: Consulenza genetica, Cancro ereditario della mammella, Cancro dellвЂ™ovaio, Test genetici Abstract. Oncogenetic counselling is a multistep process. The aim is to evaluate the risk that the client carries a heritable or germline mutation in a cancer susceptibility gene. The process begins by collecting information about the clientвЂ™s personal medical history and family history to assess heritable cancer risk. Then the multidisciplinary team, including a oncologist, a psychologist and a geneticist, should acquire informed consent for genetic testing when applicable, and propose options for medical management with regard to the risk category. Riassunto. Il вЂњcounselling oncogeneticoвЂќ ГЁ un processo di comunicazione multistep finalizzato alla valutazione del rischio di essere portatore di una mutazione germinale in un gene di suscettibilitГ per tumore ereditario. Tale processo ГЁ suddiviso in varie fasi partire dalla raccolta delle informazioni relative alla storia personale e familiare al fine di stabilire il rischio di neoplasia ereditaria. Il team multidisciplinare, composto da un oncologo, da uno psicologo e da un genetista, se il paziente ГЁ candidato allвЂ™esecuzione del test genetico deve acquisire il consenso informato allвЂ™analisi molecolare e proporre le appropriate opzione per la gestione clinica in funzione del rischio. Introduzione Il counselling oncogenetico (Consulenza Genetica Oncologica - CGO) ГЁ un processo di comunicazione multistep e multidisciplinare che consente di individuare: - soggetti вЂњad alto rischioвЂќ di essere portatori di mutazioni germinali nei geni di suscettibilitГ coinvolti nelle varie sindromi tumorali ereditarie, che sono quindi candidati al test genetico (membri di famiglie con nota predisposizione ai tumori ereditari o individui sani con una forte storia familiare per determinati tumori o con altre caratteristiche suggestive di sindromi ereditarie di predisposizione al cancro); 223 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan - soggetti вЂњa basso rischioвЂќ per i quali non sarГ necessario sottoporsi al test. Le nuove acquisizioni ottenute nel campo della genetica molecolare hanno consentito di identificare la predisposizione genetica allo sviluppo di specifiche neoplasie e hanno evidenziato la necessitГ di impiegare la CGO per venire incontro alle esigenze di tutti quei soggetti affetti da tumore o sani che desiderano comprendere il rischio di una malattia genetica allвЂ™interno della famiglia. Negli ultimi quindici anni, tali acquisizioni hanno permesso ai membri delle famiglie a rischio in base alla loro etГ o alla loro condizione patologica di intraprendere strategie di chemioprevenzione e trattamenti medici e chirurgici adeguati. Il numero dei Centri di ricerca e di professionisti del settore che si dedicano alla CGO sta crescendo enormemente, di pari passo allвЂ™aumentata richiesta di oncologi e clinici, a cui pervengono individui a rischio dвЂ™insorgenza di tumori eredo-familiari che devono essere indirizzati alla CGO. La principale terminologia utilizzata nella CGO ГЁ riportata in Tabella 1. Tabella 1. Principale terminologia utilizzata nella consulenza oncogenetica. Albero Genealogico: rappresentazione grafica, secondo le relazioni di parentela, di tutti gli individui che compongono piГ№ generazioni di una stessa famiglia. Esso fornisce indicazioni sullвЂ™eventuale modalitГ di trasmissione ereditaria della malattia. Allele: ГЁ una delle possibili forme o varianti di un gene. Per ogni gene (in un determinato locus sul cromosoma) esistono due alleli, ereditati dal padre e della madre. Quando i due alleli sono uguali, lвЂ™individuo ГЁ definito omozigote per quel specifico tratto; se gli alleli sono diversi, lвЂ™individuo ГЁ definito eterozigote. Cellula germinale (ga- spermatozoi e ovuli. mete): Cellula somatica: tutte le cellule del corpo eccetto quelle germinali. EreditarietГ autosomi- la presenza di una singola copia del gene mutato (eterozigosi) ГЁ sufficiente per il manifeca dominante: starsi della patologia. Sono colpiti entrambi i sessi ed ГЁ trasmessa da entrambi i sessi. Il figlio dellвЂ™unione di un affetto con un non-affetto ha il 50% di probabilitГ di essere affetto. EreditarietГ autosomi- ГЁ necessario che entrambe le copie del gene sui due cromosomi omologhi di derivazione ca recessiva: paterna e materna siano mutate per il manifestarsi della patologia. Gli individui affetti di solito sono figli di individui non affetti. I genitori degli individui affetti di solito sono portatori asintomatici. Sono colpiti entrambi i sessi. Ciascun figlio ha la probabilitГ di essere affetto del 25%. Eterozigosi: quando in un locus sono presenti due differenti alleli nei due cromosomi omologhi. Fenotipo: insieme dei caratteri che lвЂ™individuo manifesta, frutto dellвЂ™interazione tra genotipo e fattori ambientali. DNA microsatellite: sequenze di DNA molto semplici (2-5 paia di basi) ripetute (tandem) anche molte volte, disperse nel genoma, e situate il piГ№ delle volte al di fuori delle porzioni codificanti di un gene. La loro funzione non ГЁ ancora del tutto nota e la frequenza di ripetizione di queste sequenze puГІ essere utilizzata come markers di instabilitГ genomica. 224 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche Esone: segmento genico rappresentato nellвЂ™RNA messaggero dopo il processo di eliminazione dagli introni (maturazione, vedi Splicing). I singoli esoni possono contenere DNA codificante e/o DNA non codificante (sequenze non tradotte). FamiliaritГ : presenza, nellвЂ™albero genealogico di piГ№ familiari affetti da neoplasia. Fenotipo: insieme dei caratteri che lвЂ™individuo manifesta, frutto dellвЂ™interazione tra genotipo e fattori ambientali. Gene di suscettibilitГ : gene la cui alterazione patogena ГЁ alla base di una sindrome ereditaria di predisposizione ai tumori. Genotipo: ГЁ la costituzione genetica di un individuo, data dalla totalitГ dei geni presenti nel suo genoma. InstabilitГ dei DNA mi- fenomeno caratteristico di alcune cellule tumorali in cui, durante la replicazione del DNA, il crosatelliti (MSI): numero di copie ripetute dei DNA microsatelliti ГЁ soggetto a cambiamenti casuali. Locus: sito di una specifica sequenza di DNA (frequentemente un gene o un marcatore) sul cromosoma. Mutazione founder: specifica mutazione patogena presente solo o prevalentemente in alcune popolazioni o gruppi etnici. Omozigosi: quando in un locus ГЁ presente lo stesso allele nei due cromosomi omologhi. Penetranza: capacitГ di una variante genica di manifestarsi fenotipicamente quando presente. Polimorfismo: esistenza di almeno due varianti alleliche non patologiche in un locus di un gene, ciascuna delle quali presente in una popolazione con una frequenza maggiore dellвЂ™1%. Il polimorfismo dovuto alla variazione di un singolo nucleotide ГЁ denominato SNP (Single Nucleotide Polymorphism). Probando: ГЁ il primo membro affetto di una famiglia a rischio di tumore ereditario ad essere studiato. In molti casi ГЁ il membro piГ№ giovane affetto da neoplasia a cui viene proposto il test genetico per la ricerca dellвЂ™alterazione del gene di suscettibilitГ . In caso di esito positivo, a questвЂ™ultima puГІ far seguito lвЂ™indagine in altri membri della stessa famiglia, ammalati o sani. Viene anche indicato come вЂњcaso indiceвЂќ (index case) ovvero propositus/proposita (rispettivamente maschio/femmina). Riarrangiamenti: comprendono inserzioni, delezioni o duplicazioni di ampi frammenti genomici, non rilevabili con le metodiche comunemente usate per lвЂ™identificazione delle mutazioni puntiformi, e di significato in genere patogeno. Splicing: processo di maturazione del trascritto primario di RNA messaggero che prevede lвЂ™eliminazione degli introni. Lo splicing alternativo ГЁ il processo attraverso il quale, mediante un diverso arrangiamento degli esoni durante la maturazione dellвЂ™RNA messaggero, da uno stesso gene possono derivare diverse proteine. Tumore Ereditario: neoplasia caratterizzata dalla presenza di alterazioni note in geni di suscettibilitГ , che si trasmettono ai discendenti. Tumore Familiare: aggregazione di piГ№ casi di neoplasia nella stessa famiglia, spesso a precoce insorgenza che puГІ suggerire la presenza di alterazioni in geni di suscettibilitГ , e quindi un tumore ereditario, anche se le alterazioni stesse non vengono riconosciute. PuГІ anche dipendere dal fatto che i membri di una stessa famiglia sono spesso esposti agli stessi fattori di rischio ambientali o relativi agli stili di vita. 225 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan A. Fasi del Programma di Counselling oncogenetico La consulenza ГЁ un processo di accertamento e comunicazione offerto al probando e si articola in diverse fasi ben distinte tra loro. La CGO ГЁ un atto medico che si svolge presso centri competenti e prevede un approccio multidisciplinare ed integrato tra diverse figure professionali, quali il genetista, il medico oncologo e lo psico-oncologo; ciascuno di questi specialisti svolge un ruolo ben determinato ed interviene a livelli differenti delle varie fasi di cui il counselling si compone. La multidisciplinarietГ tiene conto dei diversi aspetti e dei diversi bisogni della persona a rischio di cancro ereditario. Gli individui a rischio di sviluppare una neoplasia di tipo ereditario vengono sottoposti prima allвЂ™analisi della storia personale e successivamente a quella della storia familiare attraverso la ricostruzione dellвЂ™albero genealogico e successivamente alla valutazione del rischio personale. Fondamentalmente il programma di consulenza prevede quattro fasi: fase pre-test, test genetico, fase post-test e follow-up. 1. Fase pre-test Questa prima fase ГЁ molto importante per fronteggiare i problemi clinici, psicologici ed etici che insorgono durante tutto il processo di consulenza. La fase pre-test solitamente prevede un primo momento in cui il probando e/o i suoi familiari vengono informati sui concetti di base (genetici, epidemiologici, clinici) dei tumori eredo-familiari e sui metodi diagnostici e preventivi esistenti; quindi si procede allвЂ™esame della storia personale e alla costruzione dellвЂ™albero genealogico, raccogliendo informazioni sulla storia personale e familiare del probando. Si procede quindi alla stima del rischio di insorgenza di un tumore attraverso lвЂ™anamnesi della famiglia e/o con programmi statistici di calcolo del rischio. La raccolta del consenso informato ГЁ lвЂ™altro elemento fondamentale di questa fase. a. Raccolta della storia personale e familiare Г€ un momento fondamentale, in cui vengono raccolte tutte le informazioni necessarie, che possono aiutare lo specialista a far luce sulla reale origine genetica della malattia. La raccolta dei dati clinici deve comprendere la storia personale del soggetto a rischio (probando) vale a dire lвЂ™etГ attuale e gli altri fattori di rischio. Il primo fattore di rischio fra tutti ГЁ la precoce etГ dвЂ™insorgenza di un tumore anche in assenza di storia familiare. Nel caso dei tumori della mammella, lвЂ™etГ a cui si fa riferimento ГЁ circa 45 anni, invece per quelli colorettali lвЂ™etГ considerata ГЁ circa 50 anni. NellвЂ™analisi della storia personale bisogna tenere conto che esistono varie patologie ereditarie che presentano piГ№ frequentemente particolari istotipi (fenotipo вЂњtriple negativeвЂќ del BC nei carrier di mutazioni del gene BRCA1, lвЂ™istotipo midollare del carcinoma della tiroide, lвЂ™istotipo diffuso del carcinoma gastrico). 226 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche Anche la presenza di adenomi multipli, amartomi o polipi del colon in etГ giovanile sono indicativi di sindromi ereditarie. Un attenzione particolare deve essere posta nella valutazione della storia personale quando viene diagnosticato un tumore raro, quale ad esempio il carcinoma alla mammella in un maschio, il carcinoma midollare della tiroide o il retinoblastoma, che hanno spesso unвЂ™origine ereditaria. Inoltre ГЁ necessaria la valutazione di caratteristiche suggestive di sindromi ereditarie di predisposizione al cancro. Gli esempi piГ№ classici sono i disordini della tiroide nella sindrome di Cowden, le cheratocisti odontogeniche nella sindrome di Gorlin, i tumori desmoidi, le anomalie dentarie della FAP, i nevi displastici nel melanoma familiare. Allo stesso modo ГЁ importante valutare quei soggetti affetti da piГ№ tumori primitivi. La ricostruzione della storia familiare del probando, rappresenta un momento fondamentale del processo, in quanto la dettagliata descrizione dellвЂ™albero genealogico consente agli specialisti di effettuare una diagnosi piГ№ corretta, prevedere una prognosi piГ№ accurata e, quindi, consigliare il probando sulle decisioni da intraprendere. Г€ necessario raccogliere per almeno tre generazioni le informazioni personali e cliniche dei parenti di primo (figli, fratelli, genitori), secondo (nonni, zii, nipoti) e terzo grado (cugini, prozii) del probando in modo da facilitare lвЂ™identificazione di pattern ereditari presenti eventualmente nella famiglia. La presenza dello stesso tipo di tumore in diversi membri da un solo lato della famiglia o lвЂ™associazione di tumori dovuti a mutazioni in un singolo gene, come quelli di mammella/ ovaio e prostata o quelli colon/utero, ovaio, stomaco e apparato urogenitale, costituiscono altri criteri utilizzati per selezionare le famiglie candidate per il counselling genetico. Per la corretta costruzione di un albero genealogico o di un вЂњpedigreeвЂќ ГЁ necessario, quindi, raccogliere il maggior numero di informazioni possibile, considerando: a) sia il ramo paterno che materno; b) la paternitГ , la consanguineitГ , lвЂ™uso di tecnologie di fecondazione assistita; c) il gruppo etnico di appartenenza. Un ruolo molto importante ГЁ determinato dalla conoscenza del gruppo etnico di appartenenza dellвЂ™individuo in esame. Infatti, la conoscenza del gruppo etnico puГІ influenzare le decisioni riguardanti il test genetico perchГ© ГЁ noto che mutazioni specifiche (le cosiddette вЂњfounder mutationsвЂќ) possono verificarsi con maggiore frequenza in popolazioni selezionate. Per esempio, come detto in precedenza, i soggetti ebrei Ashkenazi hanno un rischio alto di essere portatori di 3 mutazioni founder dei geni BRCA1/2 (185delAG, 5382insC del gene BRCA1 e 6174delT del gene BRCA2) e della mutazione I1307K del gene APC. Inoltre, ГЁ importante raccogliere informazioni sia sui familiari affetti da patologie tumorali che sui familiari non affetti. Per ogni familiare affetto da tumore ГЁ necessario valutare: a) il tipo e la sede del tumore primitivo; b) lвЂ™etГ dвЂ™insorgenza alla diagnosi del tumore primitivo; c) lвЂ™etГ attuale se lвЂ™individuo ГЁ ancora in vita o lвЂ™etГ del decesso e la causa della 227 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan morte; d) lвЂ™esposizione a carcinogeni (per es., uso di tabacco, esposizione a radiazioni, esposizione allвЂ™uso di terapie ormonali per lunghi periodi di tempo o comunque senza adeguate interruzioni); d) altri problemi di salute significativi (per es., presenza di malattie genetiche note che possono predisporre allo sviluppo di tumori). Per ogni familiare non affetto da tumore ГЁ necessario esaminare: a) lвЂ™etГ attuale o lвЂ™etГ del decesso; b) se deceduto, la causa del decesso; c) gli interventi chirurgici che riducono il rischio di insorgenza del cancro; d) se routinariamente sottoposti a screening per la diagnosi precoce di tumore; e) ogni lesione preneoplastica tipica della sindrome in questione; f) lвЂ™esposizione a carcinogeni; g) altri problemi di salute significativi. Gli aspetti piГ№ salienti di un вЂњpedigreeвЂќ che fanno supporre lвЂ™insorgenza di un tumore ereditario sono: la precoce etГ di insorgenza, la presenza dello stesso tipo di tumore in un gruppo ristretto di parenti; una inusuale etГ di insorgenza del tumore; due o piГ№ tumori primitivi in uno stesso familiare; evidenza di una ereditarietГ autosomica dominante; presenza di tumori bilaterali; presenza di tumori associati a note sindromi familiari, caratteristiche fisiche associate a note sindromi ereditarie e naturalmente la presenza di una mutazione di un gene di suscettibilitГ nella famiglia (Fig. 1). b. Verifica della storia familiare. Dato che le sindromi ereditarie di predisposizione al cancro vengono identificate fondamentalmente mediante lвЂ™attenta analisi della storia familiare, ГЁ necessario che le informazioni ottenute durante la costruzione del pedigree siano molto accurate. Purtroppo spesso ciГІ non si verifica perchГ© il probando non ricorda bene o ha informazioni incomplete o inesatte. Pertanto, ГЁ buona norma cercare sempre di verificare quanto riportato dal probando visionando i referti anatomo-patologici, i certificati di morte e/o i registri tumori. In figura 2 ГЁ mostrato lвЂ™esempio di un pedigree costruito considerando solamente quanto riportato verbalmente dal probando e quello reale modificato dopo aver consultato i referti anatomo-patologici. Come si puГІ notare, il proFigura 1: Albero genealogico di una famiglia probabilmente affetta da bando ricordava che nella sindrome tumorale ereditaria. 228 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche propria famiglia vi erano stati giГ dei casi di tumore, ma i suoi ricordi erano estremamente imprecisi. Solamente dopo aver consultato i referti anatomo-patologici lo specialista ha potuto ricostruire in modo esatto lвЂ™albero genealogico del probando, dal quale si evinceva la possibile presenza di una sindrome ereditaria mammella/ovaio. c. DinamicitГ della storia familiare. Un altro aspetto da considerare ГЁ la dinamicitГ della storia familiare, poichГ© questa non ГЁ statica, ma varia col trascorrere del tempo. Г€ quindi necessario aggiornare periodicamente lвЂ™albero genealogico inserendo ad esempio le eventuali nascite, i decessi, lвЂ™insorgenza di lesioni preneoplastiche, di nuovi tumori e le ricadute. Ogni volta che viene acquisita una nuova informazione al pedigree ГЁ opportuno riconsiderare la significativitГ della storia familiare, dal momento che queste nuove informazioni possono influire sulla valutazione del rischio, sui programmi di sorveglianza, e sulle raccomandazioni cliniche fornite al probando (Fig. 3). d. Identificazione e comunicazione del rischio LвЂ™obiettivo principale di questa fase ГЁ quello di fornire ai probandi informazioni accurate Figura 2: Verifica della storia familiare. Albero genealogico costruito tenendo conto di quanto riportato dal paziente (a) e dello stesso realizzato dopo aver consultato i referti anatomo-patologici (b) 229 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan sul proprio rischio di sviluppare un tumore. La CGO ГЁ offerta ad individui che sono ad alto e medio rischio di tumore ereditario e/o familiare; i soggetti a basso rischio sono tutti coloro che sviluppano un carcinoma in etГ avanzata e non hanno storia familiare di tumore. Nel caso dei tumori eredo-familiari di mammella/ovaio, gli individui a medio rischio (a basso rischio ГЁ la popolazione in genere) sono i soggetti con parenti di I grado che hanno avuto diagnosi di tumore alla mammella sotto i 50 anni di etГ oppure soggetti con parenti di I o II grado dallo stesso lato della famiglia che hanno avuto diagnosi di tumore alla mammella o allвЂ™ovaio sempre sotto i 50 anni di etГ . La definizione del rischio di essere portatori di mutazione si basa su differenti sistemi di valutazione. I sistemi empirici, basati essenzialmente sul numero di casi di tumore nella storia familiare o sullвЂ™etГ alla diagnosi, possono essere utilmente impiegati per selezionare le famiglie da sottoporre a CGO. Chiaramente i modelli matematici sono piГ№ affidabili dei sistemi empirici in quanto riescono effettivamente a fornire una stima del rischio di essere portatori di mutazione in un gene di suscettibilitГ . Se per esempio prendiamo in esame la sindrome HBOC, in questi casi vengono utilizzati due metodi analitici per il calcolo del rischio medio di sviluppare un carcinoma della mammella: a) il modello di Claus, che si basa sulle informazioni relative alla storia familiare di I grado della stessa linea parentale, ma prende in considerazione unicamente i carcinomi mammari presenti e b) il modello di Gail, che tiene conto sia dei fattori ormonali, a cui ГЁ stata soggetta la donna nel corso della vita, che i fattori di familiaritГ solo dei parenti di primo grado affetti con esclusione della storia familiare paterna. Ma il modello di Gail non risulta uno strumento adeguato alla valutazione del rischio di sindrome ereditaria, perchГ© tarato per stimare la probabilitГ dвЂ™insorgenza di BC nella popolazione generale. Gli individui ad alto rischio sono quei soggetti che fanno parte di famiglie, che presentano precoce etГ di insorgenza di carcinoma di mammella ed ovaio, o bilateralitГ dei tumori, associazione di tumore alla mammella e allвЂ™ovaio e presenza di carcinomi mammari Figura 3: DinamicitГ della storia familiare. Albero genealogico della maschili. Tali soggetti vengono stessa famiglia dopo follow up di due anni. 230 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche sottoposti durante la consulenza ad alcuni modelli matematici piГ№ specifici, quali BRCAPRO e Couch model che estendono le informazioni ottenute a livello familiare, tenendo conto anche dei casi di carcinoma ovarico presenti e danno un calcolo matematico di probabilitГ di essere portatore di mutazione nei geni di suscettibilitГ BRCA1 e BRCA2 (Tabella 2). Tabella 2. Modelli di calcolo del rischio di carcinoma della mammella per la popolazione generale e per i soggetti ad alto rischio Gail EtГ del menarca EtГ del primo parto Numero dei parenti di I grado affetti Numero di precedenti biopsie mammarie Presenza di iperplasie atipiche Claus EtГ alla diagnosi Numero dei parenti di I grado affetti da BC BRCAPRO EtГ alla diagnosi Parenti di I e II grado affetti da BC o CO EtГ del paziente EtГ di morte del parente non affetto Quando viene utilizzato il BRCA-PRO software il test genetico viene proposto dal genetista quando la probabilitГ a priori di essere portatori di mutazione ГЁ almeno del 10%. Prima di comunicare il rischio al probando ГЁ importante valutare il suo grado di comprensione e di percezione del rischio. Lo specialista deve sempre considerare che il probando di frequente non ha molta dimestichezza con i concetti di probabilitГ statistica, di rischio relativo e assoluto, di proporzione, per cui deve trovare il metodo migliore (mediante numeri, parole o grafici) per comunicare al probando la stima del rischio assicurandosi che questвЂ™ultimo gli abbia dato un giusto peso. Infatti, il probando spesso tende a sovrastimare la propria probabilitГ di ammalarsi soprattutto se in famiglia ci sono stati altri casi di tumore. Di conseguenza, la percezione personale del rischio non sempre corrisponde al valore oggettivo del rischio statistico calcolato. La scelta del metodo di comunicazione del rischio ГЁ fondamentale perchГ© una stessa informazione data usando metodi differenti puГІ essere recepita in maniera diversa. Anche in questo caso ГЁ importante il sostegno offerto dallo psicologo in sede di consulenza. Una volta effettuata la stima del rischio dovrГ essere ottenuto dal probando il consenso ad effettuare il test genetico. Questa decisione deve essere presa liberamente dal probando, informato circa i vantaggi ed i limiti di un test genetico. 231 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan e. Consenso informato Ottenere il consenso informato scritto prima della fase di testing ГЁ necessario per chiarire: a) i rischi, i benefici, i limiti e le alternative al test genetico; b) il significato di un risultato positivo, negativo, indeterminato o con varianti di sequenza di significato clinico sconosciuto (inconclusivo); c) le possibili ripercussioni di un simile risultato non soltanto a livello personale e familiare, ma anche sociale. Le informazioni che il consenso informato deve contenere riguardano le opzioni di stima del rischio di insorgenza di un tumore alternative al test genetico, la possibilitГ che il test sia indeterminato o inconclusivo. Spesso vi ГЁ la possibilitГ di disagio psicologico personale e familiare o la possibilitГ di discriminazioni nel settore lavorativo e assicurativo. Г€importante che si evinca la volontГ di comunicare il risultato del test ad altri membri della famiglia. Il probando dovrГ inoltre accettare il potenziale uso futuro (per es. per nuove tecniche) del materiale a disposizione e gli eventuali costi del test. B. Fase del test genetico I test molecolari disponibili sono molteplici, con tempi di indagine differenti e con diversi limiti in rapporto alla differente sensibilitГ e specificitГ . LвЂ™eterogeneitГ genetica delle sindromi rende la scelta del test molto difficile. Differenti sindromi possono essere accompagnate dallo sviluppo di un particolare tipo di tumore; cosГ¬, per esempio, i tumori ereditari del colon possono essere conseguenza di poliposi adenomatosa familiare (FAP), cancro colorettale ereditario non poliposico (HNPCC), sindrome di Peutz-Jegher (PJS), Juvenile polyposis syndrome (JPS), ognuno con una diversa base genetica; oppure diversi geni possono essere responsabili della stessa sindrome (HNPCC puГІ essere dovuta ad alterazioni in uno dei 6 geni del mismatch repair). *** Anche lвЂ™eterogeneitГ allelica puГІ condizionare il test genetico. Differenti mutazioni nello stesso gene possono conferire un diverso rischio o essere associate a un differente fenotipo. Se, per esempio, in linea generale le mutazioni di APC sono associate a centinaia/ migliaia di polipi colici e alla FAP classica, alcune mutazioni specifiche possono essere responsabili di forme intermedie con un minor numero di polipi e un piГ№ basso rischio di sviluppare il cancro al colon-retto, o predisporre ad alterazioni a carico della retina invece che allo sviluppo di tumori desmoidi. Pertanto, la selezione di un appropriato test genetico per un dato individuo richiede profonde conoscenze cliniche e molecolari ed il rapido aggiornamento delle tecniche di screening genetico che subiscono continuamente aggiornamenti e/o miglioramenti. 232 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche Figura 4: Struttura dei geni BRCA 1. Alterazioni genetiche nelle principali Sindromi di predisposizione ereditaria Carcinoma ereditario della mammella e/o dellвЂ™ovaio (HBOC). Le mutazioni dei geni BRCA non ricadono allвЂ™interno di вЂњhot spotsвЂќ, ma sono uniformemente distribuite lungo lвЂ™intero gene (Fig. 4). Le mutazioni identificate nei geni BRCA a partire dalla loro scoperta e le rispettive frequenze vengono catalogate in un registro internazionale denominato Breast Cancer Information Core Database (BIC; http//research.nhgri.nih.gov/bic). Nel database BIC sono state riportate oltre 600 mutazioni patologiche sia per BRCA1 che per BRCA2. Spesso si osserva una prevalenza di alcune di esse in rapporto allвЂ™area geografica di appartenenza del portatore (mutazioni вЂњfounderвЂќ o ricorrenti, Tabella 3). Tabella 3. Mutazioni вЂњfounderвЂќ dei geni BRCA in diverse popolazioni BRCA1 BRCA2 Europa 233 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan вЂў Finlandia IVS11+3A>G вЂў Francia вЂў Islanda вЂў Italia - Calabria - Sardegna - Sicilia 3600del11 вЂў Norvegia 1675delA 816delGT 3347delAG 1135insA вЂў USA/Israele (ebrei Ashkenazi) 185delAG 5382insC 9345 +1G>A 7708 C>T 8555 T>G 5579insA 6174delT and 995delG 5083del19 8765delAG 4843delC Il 70-80% delle alterazioni genetiche sono patogene e comportano la formazione di una proteina tronca di dimensione piГ№ piccola. Si tratta principalmente di mutazioni frameshift, nonsense, piccole inserzioni e delezioni e in minima parte missense. I riarrangiamenti genomici (grosse duplicazioni e delezioni) costituiscono il 3-5% delle alterazioni genetiche patogene e sono principalmente a carico di BRCA1. Oltre il 40% del gene BRCA1 ГЁ costituito da sequenze piccole sequenze ripetitive localizzate nelle regioni introni che denominate Alu, che giustificano lвЂ™elevata percentuale di riarrangiamenti genomici che si verificano in questo gene. Dal punto di vista funzionale, oltre alle alterazioni genetiche patologiche si distinguono varianti di sequenza di significato sconosciuto e mutazioni puntiformi sinonime (Tabella 4). Tabella 4. Mutazioni puntiformi -- Frame-shift (mutazione con slittamento del modulo di lettura): modifica della sequenza di un gene per inserzione/delezione di un singolo nucleotide. -- Sinonima (вЂњsilenteвЂќ): sostituzione di un singolo nucleotide che determina un codone diverso ma codificante per lo stesso amminoacido. -- вЂњNonsenseвЂќ: sostituzione non sinonima di un singolo nucleotide che produce un codone di stop e quindi la formazione di una proteina tronca. -- вЂњMissenseвЂќ (di senso errato): sostituzione non sinonima di un singolo nucleotide con cambiamento di un aminoacido. Le mutazioni вЂњmissenseвЂќ possono essere clas- 234 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche sificate in conservative (sostituzione di un amminoacido con un altro chimicamente simile) o non conservative (sostituzione di un amminoacido con un altro che ha una catena laterale completamente diversa). Esempi: Normale sequenza Sostituzione AВ®T Delezione di T Inserzione di T CAT GAG AAT CTT GAG AAT CAG AGA AT CTA TGA GAA N.B. Le mutazioni frame-shift sono sempre patogene, cosГ¬ come le nonsense. Le mutazioni missense possono non comportare la compromissione della funzione della proteina e quindi non essere patogene. Le sinonime sono ovviamente sempre prive di significato patologico. B. Carcinoma ereditario del colon retto Sindrome di Lynch (HNPCC) Il 70% dei soggetti portatori di mutazione nelle famiglie affette da HNPCC hanno mutazioni germinali nei geni hMSH2 e hMLH1. Altri geni quali hPMS1, hPMS2 e hMSH6 sono coinvolti in misura minore, ma anche le loro mutazioni manifestano alta penetranza. Tali geni sono oncosoppressori ed appartengono al gruppo dei geni вЂњcaretakersвЂќ coinvolti nei processi di riparazione del DNA ed in particolare degli errati accoppiamenti dei nucleotidi durante la fase di replicazione вЂњmismatchвЂќ, impedendo cosГ¬ la trasmissione di alterazioni geniche. La perdita della eterozigosi nella cellula somatica del tessuto bersaglio, che nel caso di hMSH2 ГЁ la cripta del colon dove si trovano le cellule epiteliali attivamente proliferanti, comporta la genesi del вЂњfenotipo ipermutabileвЂќ la cui espressione ГЁ lвЂ™instabilitГ delle sequenze dei microsatelliti nel DNA (MSI) e lвЂ™accumulo delle mutazioni necessarie alla trasformazione neoplastica in rapida sequenza. LвЂ™instabilitГ genomica a livello microsatellitare ГЁ un evento precoce nella rapida progressione adenoma-carcinoma dellвЂ™HNPCC mentre non si ritrova frequentemente nei carcinomi colorettali sporadici e non ГЁ necessariamente associata alla perdita di eterozigosi a livello dei geni coinvolti nel вЂњmismatch repairвЂќ (MMR). Questi sono localizzati in 3 diversi cromosomi e codificano per proteine che agiscono sottoforma di eterodimeri che lavorano in sequenza per riconoscere e riparare i вЂњmismatchвЂќ del DNA. Poliposi adenomatosa familiare (FAP) Il gene di suscettibilitГ della FAP ГЁ lвЂ™APC, localizzato sul cromosoma 5 (5q21). Tale gene codifica per una proteina citoplasmatica che riveste un ruolo chiave nella regolazione di 235 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan apoptosi, ciclo cellulare, interazione ed adesione intercellulare, processi di migrazione e metastatizzazione. La maggior parte delle mutazioni identificate nel gene APC portano alla formazione di proteine tronche. Sino al 30% dei pazienti presentano mutazioni вЂњde novoвЂќ. Le mutazioni del gene APC sono presenti a livello somatico in circa lвЂ™80% dei tumori colorettali sporadici, e, a livello germinale in piГ№ del 70% delle famiglie con FAP ed in circa il 25% delle famiglie con la cosiddetta вЂњattenuated FAPвЂќ (AFAP). In particolare nei soggetti con AFAP, le mutazioni si localizzano preferenzialmente nella porzione 3вЂ™-terminale del gene APC e negli esoni soggetti a splicing alternativo. In alcuni pazienti con FAP o AFAP ГЁ stata riscontrata evidenza di mosaicismo somatico, ovvero la presenza di mutazione solo in alcune sottopopolazioni cellulari. In altre forme di AFAP vengono ereditate in maniera autosomica recessiva mutazioni del gene MYH codificante per una glicosilasi coinvolta nel meccanismo di riparazione dei danni al DNA a singola elica, denominato base excision repair (BER). Le mutazioni bialleliche di questo gene sono presenti in circa il 25-30% dei soggetti AFAP ed in una minima frazione di soggetti con FAP. LвЂ™eventuale consanguineitГ tra i membri della famiglia che rende conto di alcune mutazioni bialleliche, potrebbe falsare il test genetico. Le correlazioni genotipo-fenotipo delle alterazioni del gene APC sono state descritte ampiamente e spiegano lвЂ™eterogeneitГ clinica dei pazienti. Ad esempio, la forma aggressiva di FAP con esordio precoce, caratterizzata da un numero molto alto di adenomi, ГЁ associata con mutazioni nei codoni compresi tra il 1250 e il 1464 (in particolare nel codone 1309), invece lвЂ™AFAP ГЁ associata con mutazioni allвЂ™estremitГ 5вЂ™ o 3вЂ™ del gene APC e negli esoni soggetti a splicing alternativo. 2. Test di screening a) Materiale biologico Il DNA genomico viene estratto o da sangue o da tessuto. Sangue. LвЂ™estrazione del DNA richiede la lisi dei linfociti e la digestione delle proteine della membrana plasmatica e degli istoni , attraverso tamponi contenenti sodio dodecilsolfato (SDS) e lвЂ™enzima Proteinasi K, con conseguente rilascio del DNA in essi contenuto. Segue una fase di purificazione e concentrazione del DNA mediante precipitazione in soluzione alcolica. Accanto a tale metodica ГЁ possibile disporre di kit commerciali basati sul principio della cromatografia per filtrazione che accorciano i tempi di estrazione e purificazione. Tessuto. Il DNA genomico puГІ essere estratto da prelievo bioptico o chirurgico di tessuto neoplastico fresco, congelato o fissato in formalina ed incluso in paraffina. LвЂ™utilizzo di tessuti paraffinati fornisce DNA di qualitГ inferiore rispetto a quella del tessuto fresco. 236 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche Il DNA estratto viene siglato con un codice nel rispetto della privacy e conservato a 4В°C, per poter essere utilizzato nel percorso diagnostico molecolare che prevede successivamente una metodica di amplificazione del DNA. Se opportunamente conservato il DNA puГІ essere utilizzato anche a distanza di tempo (diversi anni) per ulteriori indagini. b) Amplificazione del DNA La reazione di polimerizzazione a catena (PCR) ГЁ una tecnica estremamente sensibile che consente in vitro una amplificazione rapida, su base enzimatica (DNA polimerasi), di uno specifica sequenza di DNA. Per ogni reazione di PCR viene utilizzato un controllo negativo, in cui il DNA stampo viene sostituito con acqua. c) Metodiche per lo screaning mutazionale Una volta amplificato il DNA, si ha a disposizione materiale sufficiente per la ricerca e lвЂ™identificazione delle mutazioni genetiche, per le quali possono essere utilizzati metodi indiretti e diretti. I metodi indiretti, quali ad esempio la dHPLC (denaturing High Performance Liquid Chromatography o cromatografia liquida denaturante), rivelano la presenza di alterazioni del DNA e la loro localizzazione, ma non sono in grado di caratterizzarle. Viceversa i metodi diretti quali ad esempio il sequenziamento permettono di identificare e caratterizzare le mutazioni nei principali geni di suscettibilitГ . Principali metodi indiretti. dHPLC (denaturing High Performance Liquid Chromatography o cromatografia liquida denaturante). Г€ una cromatografia a scambio ionico in condizioni denaturanti che rappresenta una metodica di pre-screening genetico. Essa permette di analizzare in poco tempo un elevato numero di pazienti e di visualizzare la presenza di mutazioni geniche puntiformi. Tra i vantaggi si ha lвЂ™automatizzazione della tecnica, lвЂ™elevata sensibilitГ (> 98%), alta accuratezza, elevato rendimento, rapida velocitГ dвЂ™analisi ed i costi ridotti. Nella figura 5a sono riportati i cromatogrammi (profili di eluizione) che identificano rispettivamente un profilo wild type, un mutato ed un polimorfismo. Come si evince dal grafico il picco che identifica la mutazione ГЁ distinguibile da quello relativo al polimorfismo. Il limite di questa tecnica consiste nel fatto che, pur identificando la presenza di una mutazione del gene interessato, non consente di specificare di quale mutazione si tratti (per esempio, nelle mutazioni puntiformi non chiarisce quali basi azotate siano interessate). Nella Sindrome di Lynch, vengono eseguiti preventivamente dei test di screening quali lвЂ™analisi dellвЂ™MSI e lвЂ™immunoistochimica (IHC). Tali analisi necessitano del tessuto tumorale del membro piГ№ giovane allвЂ™interno della famiglia e consentono di discriminare quale dei 237 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan Figura 5: Mutazione nellвЂ™esone 7 del gene BRCA2 (V211I) geni MMR studiare evitando analisi mutazionali inutili in termini di costi e di tempo. LвЂ™IHC fornisce perГІ solo indicazioni sulle alterazioni geniche (mutazioni puntiformi o riarrangiamenti genomici) che determinano la formazione di proteine tronche non funzionali. La possibilitГ che alcune mutazioni missense non determinino la mancata espressione delle proteine, riduce lвЂ™utilitГ diagnostica di tale metodica. LвЂ™analisi della MSI prevede lвЂ™identificazione di specifiche sequenze dei microsatelliti nel DNA della linea somatica tumorale (markers microsatellitari) mediante lвЂ™utilizzo di primers marcati con fluorocromi che si appaiano alla sequenza bersaglio per dare inizio alla sintesi del filamento complementare e la loro amplificazione mediante PCR. I prodotti di amplificazione vengono separati, secondo le loro dimensioni (espresse in coppie di basi) mediante elettroforesi capillare in un sequenziatore automatico, e queste dimensioni paragonate a quelle dei frammenti contenuti nello standard. Tale metodo ha una sensibilitГ pari al 93% per i portatori di mutazione nei geni MMR, ma, paragonato allвЂ™IHC, non offre indicazioni su altri geni che potrebbero essere alterati. Si procede, quindi, allвЂ™analisi mutazionale dei geni MMR. Se viene rilevata una determinata mutazione in un membro affetto, si effettuerГ il test sugli altri membri non affetti. Se invece non ГЁ stata identificata alcuna mutazione nel membro affetto, non ГЁ indicato il test sugli altri membri della famiglia in quanto risultati falsi negativi possono essere dovuti alla presenza di altri geni MMR sconosciuti. Nelle famiglie con una storia familiare molto sospetta, inoltre, ГЁ consigliato comunque lo screening mutazionale qualunque sia il risultato dellвЂ™analisi microsatellitare. Principali metodi diretti. Sequenziamento automatico diretto (Direct Sequencing DS). A seguito dellвЂ™analisi dHPLC, nei campioni che mostrano un profilo di cromatogramma 238 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche alterato rispetto al profilo wild type viene effettuato il DS. In questi casi il DS permette di identificare e caratterizzare il tipo di mutazione puntiforme con una sensibilitГ di circa il 100%. Questa metodica prevede lвЂ™utilizzo di dideossinucleotidi marcati con un fluorocromo diverso per ciascun nucleotide, usati come terminatori, cioГЁ come ultimo nucleotide, durante la polimerizzazione della catena di DNA. I frammenti ottenuti a seguito della polimerizzazione, vengono separati mediante elettroforesi capillare e quindi colpiti da un raggio laser, che eccita i vari fluorocromi. La conseguente emissione di fluorescenza viene raccolta da una cella fotoelettrica e riportata in un grafico denominato elettroferogramma, composto da una sequenza di picchi colorati corrispondenti alla sequenza dei nucleotidi diversamente marcati. Nella figura 5b sono riportati un elettroferogramma tipico di un wild type ed uno in cui ГЁ possibile osservare il doppio picco che indica la presenza di una mutazione puntiforme del tipo missense (cambio del nucleotide 859 da G ad A) con effetto patogeno a carico del gene BRCA2. A causa di questa mutazione, infatti, lвЂ™aminoacido isoleucina sostituisce la valina con la formazione di una proteina non piГ№ funzionale. Le alterazioni genetiche alla base delle sindromi ereditarie di predisposizione possono comprendere riarrangiamenti genomici come nel caso del gene BRCA1 in cui questi si formano, per ricombinazione omologa, a causa dellвЂ™alta frequenza di sequenze ripetute come quelle Alu nelle regioni introniche. I riarrangiamenti genomici sono costituiti da grosse delezioni-duplicazioni e vengono ricercati nei casi risultati negativi con le tecniche comunemente usate per lo screening delle mutazioni puntiformi (dHPLC e DS). Tali alterazioni possono essere identificate con il saggio MLPA, acronimo di Multiple Ligation-dependent Probe Amplification. Dal momento che i riarrangiamenti genomici comportano variazioni del numero di copie degli esoni del gene di suscettibilitГ , lвЂ™MLPA consente di identificare queste variazioni quantitative che potranno essere meglio caratterizzate successivamente mediante sequenziamento. In particolare si utilizzano sonde oligonucleotidiche che si appaiono alle sequenze adiacenti gli esoni codificanti il gene. Quindi i prodotti di legame vengono amplificati e separati, mediante elettroforesi capillare, secondo la loro lunghezza. Nella figura 6 ГЁ riportato un elettroferogramma relativo al gene BRCA2 (a) tipico di un wild type (controllo) ed uno (b) in cui ГЁ possibile osservare in corrispondenza dellвЂ™esone 10 un picco marcatamente piГ№ basso che indica una delezione dellвЂ™esone 10. Negli ultimi anni sono state sviluppate metodiche di sequenziamento capaci di produrre in tempo reale notevoli quantitГ di sequenze di lunghezza inferiore rispetto al sequenziamento classico ma ad un costo minore e soprattutto ad una velocitГ maggiore. Il pirosequenziamento ГЁ una di queste tecniche: esso prevede la rilevazione del pirofosfato rilasciato dalla DNA polimerasi dopo lвЂ™incorporazione di uno o piГ№ nucleotidi. Il pirofo239 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan sfato inorganico viene trasformato in ATP (sulfurilasi) e queste molecole vengono utilizzate dalla luciferasi per ossidare la luciferina e produrre un segnale luminoso che viene rilevato da unвЂ™apposita camera fotosensibile (CCD). LвЂ™intensitГ di segnale ГЁ proporzionale al numero di nucleotidi incorporati e viene tradotta in un grafico (fluorogramma) in cui ad ogni base corrisponde un picco di colore specifico. Tale metodica ГЁ particolarmente indicata per la ricerca di mutazioni вЂњfounderвЂќ in specifiche popolazioni o per lвЂ™analisi dei DNA microsatelliti a partire da frammenti di DNA di lunghezza limitata (circa 100 nucleotidi). 3. Fase post-test Anche tale fase del counselling ГЁ di estrema importanza, poichГ© prevede la comunicazione del risultato del test e delle sue implicazioni. Prevede una discussione sulle misure di sorveglianza, di prevenzione e/o cure esistenti, sui possibili trattamenti chirurgici profilattici. Inoltre viene discussa la possibilitГ di offrire il test genetico anche ad altri membri della famiglia che pur essendo asintomatici potrebbero essere a rischio di sviluppare una neoplasia. Figura 6: Elettroferogramma relativo ad analisi con MLPA del gene BRCA2. La freccia indica lвЂ™esone 10. (a) In alto gli esoni del gene BRCA2 wild type (controllo). (b) In basso, in corrispondenza dellвЂ™esone 10 un picco marcatamente piГ№ basso che indica una delezione dellвЂ™esone 10. 240 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche 1. Comunicazione del risultato del test e delle sue implicazioni Il test genetico viene effettuato allo scopo di migliorare la stima del rischio tumorale. Tale rischio ГЁ dato dalla ricostruzione della storia personale e familiare del probando e dal risultato del test genetico stesso. La stima ГЁ importante per proporre ai soggetti вЂњa rischioвЂќ e alle loro famiglie un appropriato piano di sorveglianza diverso da quello proposto alla popolazione generale. Nonostante la scoperta di nuove tecniche e lo sviluppo delle piГ№ moderne tecnologie, non tutte le mutazioni possono essere identificabili; inoltre anche se il test dovesse risultare positivo esso indica la probabilitГ ma non la certezza del 100% che il portatore sviluppi la patologia tumorale. Г€ importante che il genetista comunichi al probando il risultato del test utilizzando un linguaggio semplice e molto diretto, che eviti di aumentare lo stato dвЂ™ansia del probando. Prima di procedere, ГЁ opportuno che lo specialista chieda un secondo consenso informato per accertarsi che il probando sia ancora disposto a conoscere lвЂ™esito del proprio test, avendo considerato a fondo il possibile impatto che il risultato potrebbe avere sulla propria famiglia, sulla vita professionale e sul suo stato emotivo. A volte, infatti, puГІ succedere che durante la fase di testing (la cui durata ГЁ variabile in base al tipo di test effettuato) il probando cambi idea e non voglia piГ№ conoscere lвЂ™esito del test. In questo caso, il probando potrГ comunque scegliere di sottoporsi periodicamente a specifici controlli clinici e, in futuro, conoscere il risultato del proprio test qualora lo desideri. Anche in questa fase, dal punto di vista psicologico vengono toccati elementi fondamentali nellвЂ™equilibrio individuale, per cui la presenza dello psico-oncologo puГІ supportare gli stati ansia del probando per i propri familiari e per il proprio futuro. 2. Risultato del test genetico Il risultato del test genetico puГІ essere positivo, negativo, indeterminato o inconclusivo (Fig. 7). a) LвЂ™esito ГЁ positivo quando si identifica una alterazione genetica patogena a cui si puГІ associare un rischio alto di sviluppare una neoplasia (nel caso in cui il probando sia sano) oppure di sviluppare un secondo tumore (nel caso in cui il probando sia un individuo affetto in precedenza dalla neoplasia, ma possibilmente guarito). In caso di risultato positivo dovrГ essere adottata una strategia multidisciplinare di sorveglianza intensiva o altre procedure di riduzione del rischio. b) LвЂ™esito di un test genetico effettuato a scopo predittivo in un individuo non affetto, cioГЁ quando una alterazione responsabile di predisposizione genetica ГЁ giГ stata identificata in un familiare di primo grado, ГЁ negativo se lвЂ™individuo non lвЂ™ha ereditata. CiГІ implica che il soggetto ГЁ sfuggito al rischio neoplastico su base genetica e presenta un rischio di 241 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan sviluppare la patologia pari a quello della popolazione generale. In questo caso le raccomandazioni per lo screening ricalcano quelle della popolazione generale. c) LвЂ™esito ГЁ indeterminato quando nel probando non viene identificata alcuna variante di sequenza o riarrangiamento genico. Tale risultato puГІ dipendere da diversi fattori: вЂў PuГІ trattarsi di un falso negativo: nella famiglia ГЁ presente una alterazione genica non riscontrabile con le tecniche comunemente impiegate (es. dHPLC, sequenziamento, MLPA). вЂў LвЂ™aggregazione familiare potrebbe non essere attribuibile ai geni di suscettibilitГ studiati, (es. BRCA1/BRCA2 in caso di carcinoma della mammella) ma essere dovuta a altri geni ad alta, media o bassa penetranza. вЂў Fenocopia cioГЁ un fenotipo causato da fattori ambientali che mimano nel risultato un carattere geneticamente determinato (Fig. 8). вЂў Il consenso al test potrebbe essere stato rifiutato per ragioni personali. In caso di risultato indeterminato il rischio di tumore si basa sulla storia personale e familiare e devono essere proposti programmi di sorveglianza personalizzati. d) Il test ГЁ definito inconclusivo se viene identificata una variante di sequenza di significato clinico ancora non conosciuto (variants of uncertain significance o VUS), escludendo i polimorfismi cioГЁ le varianti alleliche non patologiche in un locus di un gene. Nella maggior parte dei casi tale esito ГЁ dovuto ad una mutazione missense nella regione codificante del gene che comporta una sostituzione aminoacidica il cui effetto sulla funzione della proteina non ГЁ prevedibile, ma possono anche essere presenti inserzioni/ delezioni in frame nelle porzioni introniche o nelle regioni 5вЂ™ UTR o 3вЂ™ UTR. Le VUS possono essere вЂњnuoveвЂќ cioГЁ non precedentemente riportate in letteratura, ovvero giГ вЂњnoteвЂќ ma con significato ancora incerto. Anche in questo caso sulla base della storia personale e familiare viene quantificato il rischio individuale e pianificate le strategie di sorveglianza. 3. Implicazioni del risultato del test per i familiari Un test genetico non coinvolge solamente chi ad esso si sottopone, ma anche i membri della sua famiglia. Lo specialista ГЁ tenuto a rispettare il segreto professionale e in nessun caso puГІ contattare direttamente i familiari del probando e/o comunicare il risultato del test se il probando stesso non ha fornito il suo consenso. Alla fine verrГ consegnata al probando una relazione scritta contenente lвЂ™esito del test e gli eventuali suggerimenti clinici in rapporto al rischio individuale. Tra i rischi previsti per il test genetico vi ГЁ lo stress psicologico a cui viene sottoposto il probando e gli eventuali cambiamenti che possono avvenire nella dinamica familiare. CiГІ poichГ© lвЂ™idea di una patologia potenzialmente ereditaria non viene ovviamente ben 242 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche Figura 7. Fasi del Counselling Oncogenetico. a) Test genetico diagnostico. b) Test genetico predittivo. CI: Consenso Informato Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan Figura 13: Esempio di fenocopia. Nel probando con carcinoma della mammella a 35 anni non viene identificata la mutazione 633delC di BRCA1 responsabile della stessa neoplasia negli altri familiari. Pertanto il probando ГЁ una fenocopia. accettata dal probando e/o dai suoi familiari che talvolta possono vederla come un fattore discriminante nella societГ . Allo scopo di minimizzare questi rischi, ГЁ stata introdotta la figura dello psico-oncologo a coadiuvare il ruolo del genetista e del medico oncologo. Implicazioni psicologiche del test genetico La presenza dello psico-oncologo allвЂ™interno del CGO ГЁ un modo nuovo ed efficace per rispondere ai bisogni del probando aiutandolo a maturare con consapevolezza le scelte necessarie a mantenere la propria qualitГ di vita. Si tratta di scelte a forte valenza emozionale in quanto legate a tematiche quali salute, malattia, procreazione, morte, futuro, etc. che richiedono da parte del singolo, della coppia o di gruppi familiari una assoluta autonomia decisionale. La comunicazione вЂњnon direttivaвЂќ fa si che le scelte decisionali siano ad esclusivo appannaggio del soggetto e che il processo di decision making sia incentrato sui suoi reali bisogni e valori, cosГ¬ da essere il solo responsabile di sГЁ stesso e del proprio cambiamento. Lo psico-oncologo, tramite una comunicazione chiara ed aperta, prende in carico la persona, dГ informazioni sulle diverse e complesse implicazioni psicologiche della patologia, in modo da aiutarla a preservare la propria integritГ psichica, a ridefinire le proprie aspettative rispetto al nuovo percorso da affrontare e a perseguire tutti quegli obiettivi rilevanti per la propria salute. 244 La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche Un вЂњdisordine di natura geneticaвЂќ lacera lвЂ™individuo perchГЁ va a colpire il modo in cui percepisce se stesso, il suo senso di identitГ personale, il senso di appartenenza familiare, il desiderio interno di stabilitГ , di controllo ed il comune senso di onnipotenza; ГЁ comunque una ferita narcisistica che fa emergere sentimenti di rabbia, vergona, sensi di colpa e tristezza. Gli aspetti maggiormente critici sono: percezione del rischio, motivazioni speranze e paure prima del test genetico, vissuti psicoemozionali ad esso legati, modalitГ di adattamento al risultato, implicazioni psicosociali della comunicazione ai familiari; tutti questi elementi possono provocare ansia, depressione e paura ed ГЁ per questo che il CGO ГЁ un momento importante che serve sia per monitorare le modalitГ con le quali i familiari hanno vissuto le tappe della malattia del proprio congiunto sia per intervenire nella nuova situazione. Il malessere che puГІ accompagnare il test genetico diagnostico ГЁ legato allвЂ™incertezza circa la natura ereditaria della propria patologia e alla paura di avere trasmesso lвЂ™alterazione genetica ai figli ed ГЁ in questo delicato momento che il supporto psicologico si rivela particolarmente utile. Г€ compito di tutta lвЂ™equipe, ma in particolar modo dello psicooncologo fornire tutti gli strumenti psicologici utili al paziente per la gestione dello stress che puГІ derivare dal nuovo stato di вЂњmalato di rischioвЂќ. La comunicazione del rischio genetico comporta importanti cambiamenti nel modo in cui lвЂ™individuo percepisce se stesso, maggiormente quando lвЂ™esito del test risulta indeterminato o inconclusivo. Entrambi i risultati possono dar luogo ad un disagio psicoemotivo perchГЁ diventa piГ№ difficoltoso poter programmare un percorso di sorveglianza. Questo stato di incertezza puГІ generare ansia e determinare lвЂ™erronea percezione del proprio rischio con sovra- o sottostima del reale rischio di potersi ammalare. 4. Follow-up Il programma di counselling genetico non termina con la comunicazione del risultato del test al probando. Infatti, il consulente genetico dovrГ essere disponibile per rispondere ad ulteriori domande insorte nel probando dopo aver conosciuto il proprio esito, ma anche successivamente. SarГ inoltre necessario monitorare nel tempo il probando a rischio ed i suoi familiari poichГ© la storia familiare ГЁ continuamente in evoluzione. Infine, tenuto conto della rapida evoluzione della ricerca medica, i probandi devono essere incoraggiati a ricontattare periodicamente il consulente per ricevere le piГ№ recenti informazioni circa i progressi in campo preventivo e terapeutico di questo settore. 245 Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan Bibliografia [1] Augello C, Bruno L, Bazan V, Calo V, Agnese V, Corsale S, Cascio S, Gargano G, Terrasi M, Barbera F, Fricano S, Adamo B, Valerio MR, Colucci G, Sumarcz E, Russo A. 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[10] Ottini L, Palli D, Rizzo S, Federico M, Bazan V, Russo A. Male breast cancer. Crit Rev Oncol Hematol 2009. [11] Russo A, Agnese V, Rizzo S, La Paglia L, Bazan V. The genetics of breast cancer: application in clinical practice. In: Giordano A, Normanno N (Eds.). Breast Cancer in the Post-Genomic Era. New York, Humana Press, 2009, pp. 39-54. [12] Russo A, CalГІ V, Agnese V, Bruno L, Corsale S, Augello C, Gargano G, Barbera F, Cascio S, Intrivici C, Rinaldi G, Gulotta G, Macaluso M, Surmacz E, Giordano A, Gebbia N, Bazan V. BRCA1 genetic testing in 106 breast and ovarian cancer families from Southern Italy (Sicily): a mutation analyses. Breast Cancer Res Treat 2007;105:267-276. [13] Russo A, CalГІ V, Augello C, Bruno L, Agnese V, Schiro V, Barbera F, Cascio S, Foddai E, Badalamenti G, Intrivici C, Cajozzo M, Gulotta G, Surmacz E, Colucci G, Gebbia N, Bazan V. 4843delC of the BRCA1 gene is a possible founder mutation in Southern Italy (Sicily). Ann Oncol 2007;18 Suppl 6:vi99-102. 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[19] Smith A, Dougall A, Posluszny D, Somers T, Rubinstein W, Baum A. Psychological distress and quality of life associated with genetic testing for breast cancer risk. J Psycho- Oncology 2008;17:767-773. [20] Stormorken AT, Bowitz-Lothe IM, Noren T, Kure E, Aase S, Wijnen J, Apold J, Heimdal K, Moller P. Immunohistochemistry identifies carriers of mismatch repair gene defects causing hereditary nonpolyposis colorectal cancer. J Clin Oncol 2005;23:4705-4712. [21] Veschi S, Aceto G, Scioletti AP, Gatta V, Palka G, Cama A, Mariani-Costantini R, Battista P, Calo V, Barbera F, Bazan V, Russo A, Stuppia L. High prevalence of BRCA1 deletions in BRCAPRO-positive patients with high carrier probability. Ann Oncol 2007;18 Suppl 6:vi86-92. [22] Vogelstein B, Kinzler K. The Genetic Basis of Human Cancer, 2nd edition. New York, Mc-Graw-Hill, 2002. [23] Walsh T, Casadei S, Coats KH, Swisher E, Stray SM, Higgins J, Roach KC, Mandell J, Lee MK, Ciernikova S, Foretova L, Soucek P, King MC. 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Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 I tumori della mammella e/o dellвЂ™ovaio di tipo eredofamiliare: strategie di prevenzione nelle donne ad alto rischio [Heredo-familial breast and/or ovarian cancers: prevention strategies in high risk women] Sergio Rizzo1, Giuseppe Bronte1, Valentina CalГІ1, Loredana Bruno1, Nello Grassi2, Gianni Pantuso2, Luigi Sandonato2, Michele Frazzetta3, Giuseppe Cicero1, Girolamo Guarneri4, Giuseppe Lo Dico4, Massimo Cajozzo3, Nino Dispensa5, Aldo Gerbino6, Nicola Gebbia1, Viviana Bazan1 e Antonio Russo1 Sezione di Oncologia Medica e Centro di Riferimento Regionale per la caratterizzazione e lo Screening Genetico dei Tumori Ereditari, Dipartimento di Discipline Chirurgiche ed Oncologiche; 2Sezione di Oncologia Chirurgica, Dipartimento di Discipline Chirurgiche ed Oncologiche; 3Sezione di Chirurgia Generale, Dipartimento di Discipline Chirurgiche ed Oncologiche; 4Ginecologia ed Ostetricia, Dipartimento Materno Infantile; 6 Sezione di Urologia, Dipartimento di Medicina Interna, Malattie Cardiovascolari e Nefrourologiche; 6Sezione di Istologia ed Embriologia, Di.Me.S., UniversitГ degli Studi di Palermo (IT) 1 Parole chiave: Fattori di rischio, Carcinoma ereditario della mammella e Sidrome da Carcinoma ovarico, Screening, Prevenzione Key words: Risk factors, Hereditary Breast and Ovarian Cancer Syndrome, Screening, Prevention Riassunto. Numerosi fattori sono stati coinvolti nellвЂ™insorgenza del carcinoma mammario e dellвЂ™ovaio tra cui lвЂ™etГ , fattori ormonali e riproduttivi come lвЂ™etГ e il numero di gravidanze e lвЂ™utilizzo di contraccettivi orali e, in minor misura, fattori ambientali e stili di vita. Le possibilitГ per la gestione clinica delle pazienti considerate ad alto rischio di sviluppare carcinoma della mammella e/o dellвЂ™ovaio includono chirurgia profilattica (es. mastectomia bilaterale profilattica o ovarosalpingectomia bilaterale), chemioprevenzione (principalmente tamoxifene e raloxifene ma anche, piГ№ recentemente, inibitori dellвЂ™aromatasi nelle donne ad alto rischio in postmenopausa), cambiamenti nelle abitudini di vita e sorveglianza intensiva attraverso lвЂ™esame clinico periodico, la Risonanza Magnetica Mammaria, la Mammografia talvolta in associazione allвЂ™Ecografia e, per la prevenzione del carcinoma ovarico, lo screening combinato utilizzando il dosaggio del CA125 e lвЂ™ecografia transvaginale. Abstract. Several factors should be taken into account to estimate the risk for breast and ovarian cancer development such as age, reproductive and hormonal factors including parity and oral contraceptive use, strong family history of breast and ovarian cancer, and вЂ“ to a lesser extend вЂ“ environmental and lifestyle factors. Management options for the women at high-risk for breast and ovarian cancer development include prophylactic surgery (i.e. bilateral mastectomy and bilateral ovarosalpingectomy), chemoprevention (using mainly tamoxifen, but also raloxifen, and - recently - aromatase inhibitors in high-risk post-menopausal women), lifestyle changes and intensive surveillance which includes regular clinical examination, regular Magnetic Resonance Imaging, Mammography potentially in association with breast Ultrasonography, and ovarian cancer surveillance with transvaginal ultrasound and CA125 combined. 249 Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo Incidenza dei carcinomi della mammella e dellвЂ™ovaio Il carcinoma della mammella ГЁ frequente nelle popolazioni del mondo occidentale soprattutto in donne in post-menopausa. In Europa rappresenta il 22% dei tumori maligni femminili e colpisce il 27% delle donne affette da tumori. In Italia circa una donna su 11 si ammala di tumore della mammella entro i 70 anni; il nostro Paese, con una stima di circa 40.000 nuovi casi annui, si pone di poco al di sotto dei valori medi europei, con una incidenza del 30% inferiore nelle regioni del Sud rispetto alle regioni del Nord. Nel mondo occidentale il carcinoma dellвЂ™ovaio rappresenta una delle principali malattie ginecologiche ad alta mortalitГ , la quarta causa di morte per tumore nelle donne, con una incidenza di circa 192.000 nuovi casi ogni anno. Fattori di rischio per i carcinomi della mammella e dellвЂ™ovaio Carcinoma della mammella Ad oggi i fattori eziologici del carcinoma mammario sono in gran parte sconosciuti. Diversi studi epidemiologici hanno identificato alcuni fattori che possono aumentare il rischio di sviluppare un tumore della mammella. вЂў Fattori Familiari e Genetici: la presenza di un familiare di primo grado con carcinoma mammario ГЁ associata ad un rischio doppio di sviluppare la patologia nel corso della vita rispetto alla popolazione generale. La percentuale di rischio aumenta se i familiari affetti da carcinoma della mammella sono due o piГ№ (rischio fino al 40%). Si ritiene che circa il 10% di tutti i casi di tumore della mammella siano attribuibili a fattori genetici, in particolare a mutazioni allвЂ™interno dei geni di suscettibilitГ per questa neoplasia (BRCA1 e BRCA2) che conferiscono un rischio cumulativo (allвЂ™etГ di 80 anni) di sviluppare il carcinoma della mammella del 56-85% (Fig. 1). вЂў EtГ : le diagnosi di carcinoma della mammella prima dei 30 anni sono rare, di contro la frequenza di casi aumenta nel periodo della menopausa e successivamente. PiГ№ del 50% delle neoplasie alla mammella ГЁ infatti diagnosticato in donne di oltre 65 anni. вЂў Fattori Riproduttivi: le donne di etГ inferiore a 21 anni che hanno giГ sostenuto una gravidanza sviluppano un tumore al seno meno frequentemente rispetto a quelle che hanno il primo figlio dopo i 30 anni. Il rischio di insorgenza di un carcinoma mammario ГЁ dunque inversamente correlato con lвЂ™etГ della prima gravidanza. Si stima inoltre che esso ГЁ anche inversamente correlato al numero di gravidanze portate a termine. Sembra esserci poi una relazione diretta e lineare tra il numero di mesi di allattamento al seno ed il rischio relativo di insorgenza del tumore. Tra gli altri fattori che potrebbero influenzare lвЂ™insorgenza di un carcinoma della mammella rientrano anche lвЂ™etГ di comparsa di mestruazioni e la menopausa: la prima mestruazione (menarca) precoce e la menopausa tardiva sembra250 I tumori della mammella e/o dellвЂ™ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio no predisporre maggiormente allвЂ™insorgenza della neoplasia, infatti corrispondono ad una maggiore durata della vita riproduttiva ed una piГ№ lunga esposizione dellвЂ™epitelio ghiandolare agli stimoli proliferativi degli estrogeni ovarici. вЂў Fattori Ormonali: gli ormoni sessuali hanno un ruolo fondamentale nello sviluppo e nella progressione del carcinoma mammario. Evidenze suggeriscono che pazienti che assumono una terapia sostitutiva ormonale per prevenire i tipici disturbi della menopausa abbiano un incrementato rischio di neoplasia mammaria, ma solo per assunzioni prolungate (8-10 anni). Si ritiene dunque che per i primi anni di trattamento i benefici derivanti dalla terapia sostitutiva ormonale, tra cui la possibile riduzione di problemi cardiaci e dellвЂ™osteoporosi, compensino il rischio, per quanto aumentato, di sviluppare un tumore del seno. LвЂ™uso dei contraccettivi orali, soprattutto in caso di assunzioni prolungate con inizio in etГ molto giovane, puГІ incrementare il rischio di insorgenza di tumore della mammella e ridurre quello di tumore dellвЂ™ovaio. вЂў Fattori Dietetici: ГЁ stato rilevato che soggetti che hanno una dieta basata su farine eccessivamente raffinate e su alimenti di origine animale, come quella prevalente nei Paesi occidentali, sono maggiormente suscettibili allo sviluppo del tumore della mammella; inoltre, ГЁ stato evidenziato un legame tra lвЂ™obesitГ ed una maggiore suscettibilitГ a sviluppare un tumore al seno, sulla base del fatto che la principale fonte di estrogeni circolanti nella donna in post-menopausa ГЁ rappresentata dal tessuto adiposo. Carcinoma della mammella Figura 1. Rischio cumulativo di carcinoma della mammella e dellвЂ™ovaio allвЂ™etГ di 80 anni 251 Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo вЂў Radiazioni: sono rari i casi di carcinomi mammari causati da radiazioni. Sono considerate pericolose le radiazioni ionizzanti (raggi X) assunte con la radioterapia per la cura di tumori vicini alla mammella, ad esempio tiroide e linfomi; mentre, sono considerate meno dannose le radiazioni assorbite durante le mammografie o altri esami diagnostici. Carcinoma dellвЂ™ovaio вЂў Fattori Familiari e Genetici: Per il carcinoma ovarico lвЂ™unico fattore di rischio realmente significativo sembra, e una vasta rete bibliografica lo sottolinea (1-27), la storia familiare di questa malattia. Il rischio nel corso della vita di sviluppare un carcinoma ovarico aumenta dallвЂ™1.6% nella popolazione generale al 4% quando un parente di I grado ГЁ affetto da carcinoma ovarico e al 7% in presenza di due parenti di I grado affetti. Il rischio di neoplasia familiare diminuisce con lвЂ™incremento dellвЂ™etГ del familiare di I grado che ha sviluppato la malattia. Mutazioni nei geni BRCA predispongono anche al carcinoma ovarico con un rischio cumulativo pari al 20% per i portatori di mutazione nel gene BRCA2 e al 50% per i portatori di mutazione nel gene BRCA1 (Fig. 1). вЂў Fattori Riproduttivi: le donne che non hanno mai avuto una gravidanza sono piГ№ a rischio di sviluppare il carcinoma dellвЂ™ovaio. Anche un numero piГ№ basso di gravidanze correla con un aumentato rischio di ammalarsi di questa patologia, rispetto alle donne che hanno avuto un maggior numero di gravidanze. вЂў Fattori Ormonali: aumento del rischio ГЁ associato ai farmaci che inducono ovulazione incessante mediante stimolazione gonadotropinica continua, usati nel trattamento della sterilitГ . Questa associazione ГЁ stata spiegata con il verificarsi ad ogni ovulazione di microtraumi a carico dellвЂ™epitelio celomatico, che contribuirebbero allo sviluppo della neoplasia. Anche lвЂ™utilizzo in post-menopausa di terapie ormonali sostitutive ГЁ capace di aumentarne il rischio. Al contrario le donne che fanno uso di contraccettivi orali presentano un rischio inferiore di questa neoplasia. вЂў Fattori Dietetici: una dieta particolarmente ricca di grassi saturi e alcol e povera di frutta e verdura sarebbe associata con lвЂ™aumento del rischio. Carcinoma ereditario della mammella e/o dellвЂ™ovaio (HBOC) Principali geni di suscettibilitГ Sebbene il carcinoma della mammella sia piГ№ frequentemente sporadico, circa il 5-10% dei casi sono ereditari. Meno della metГ di questi sono associati a mutazioni germinali in geni di suscettibilitГ noti che conferiscono un alto rischio (BRCA1, BRCA2, TP53) o un rischio moderato (CHEK2, PTEN, ATM ed altri) di sviluppare la malattia nellвЂ™arco della vita. 252 I tumori della mammella e/o dellвЂ™ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio In oltre il 60% dei casi di carcinoma della mammella eredo-familiare sembrano essere coinvolte mutazioni in geni di suscettibilitГ ad alta penetranza non ancora identificati o polimorfismi in piГ№ loci a bassa penetranza, ciascuno dei quali conferisce un rischio di carcinoma della mammella lieve-moderato (suscettibilitГ poligenica). I due principali geni di suscettibilitГ sono Brca1 e BRCA2, entrambi geni oncosoppressori caretakers coinvolti nella riparazione del danno al DNA, le cui alterazioni determinano unвЂ™instabilitГ genetica che favorisce la comparsa di ulteriori mutazioni in altri geni importanti nel controllo del ciclo cellulare. Le mutazioni di questi geni sono ad alta penetranza e vengono trasmesse con modalitГ autosomica dominante (Fig. 2). Ma nella maggior parte dei casi la suscettibilitГ al carcinoma della mammella ГЁ soggetta a meccanismi poligenici, cioГЁ a combinazioni additive o moltiplicative di piГ№ varianti alleliche a bassa penetranza. Gli studi di associazione genomica che consentono di identificare caratteristiche genetiche comuni (GWA) relativi al carcinoma della mammella hanno consentito di identificare 8 varianti alleliche a bassa penetranza (FGFR2, TNCR9/ Tox3, MAP3K1, LSP1, H19, 8q, 2q35, ECHDC/RNF) che giustificano appena il 5% dei casi di carcinoma mammario eredo-familiare. Figura 2. Carcinoma ereditario della mammella e/o dellвЂ™ovaio (HBOC): principali geni di suscettibilitГ 253 Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo Sindromi ereditarie Le famiglie i cui membri sono portatori di mutazioni Brca1/BRCA2 possono presentare nellвЂ™albero genealogico esclusivamente casi di carcinoma della mammella e si parla allora di Carcinoma Ereditario della Mammella (Hereditary Breast Cancer, HBC), casi di carcinoma della mammella e carcinoma dellвЂ™ovaio e si parla di Carcinoma Ereditario della Mammella e dellвЂ™Ovaio (Hereditary Breast and Ovarian Cancer, HBOC) ovvero, meno frequentemente, soltanto casi di carcinoma dellвЂ™ovaio e si parla di Carcinoma Ereditario dellвЂ™Ovaio (Hereditary Ovarian Cancer, HOC) (Fig. 3). In particolare HBC, HOC e HBOC rappresentano lo spettro di una stessa patologia genetica con manifestazioni fenotipiche differenti. I criteri utilizzati per la selezione dei pazienti candidati alla consulenza oncogenetica e al test genetico allo scopo di identificare la sindrome di predisposizione al carcinoma ereditario della mammella e/o dellвЂ™ovaio includono una precoce etГ di insorgenza di carcinoma della mammella e/o carcinoma dellвЂ™ovaio, carcinoma della mammella bilaterale, carcinoma della mammella e/o carcinoma dellвЂ™ovaio nella stessa persona a qualsiasi etГ e carcinoma della mammella maschile (Male Breast Cancer, carcinoma della mammella maschile) (Fig. 4). Esistono casi di carcinoma ovarico familiare nellвЂ™ambito di altre sindomi ereditarie che Figura 3. Sindromi ereditarie di predisposizione al carcinoma della mammella e/o dellвЂ™ovaio 254 I tumori della mammella e/o dellвЂ™ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio Figura 4. Profili familiari dei portatori di mutazione nei geni BRCA coinvolgono il carcinoma del colon, in tal caso si parla di sindrome di Lynch II. I geni di suscettibilitГ coinvolti sono quelli del sistema del вЂњmismatch repairвЂќ (geni MMR). Rischio cumulativo Le mutazioni nel gene BRCA1 conferiscono nelle donne portatrici un rischio compreso tra il 50 e lвЂ™85% di sviluppare un carcinoma della mammella nellвЂ™arco della loro vita, un rischio del 40-60% di sviluppare un carcinoma della mammella controlaterale e un rischio compreso tra il 15 e il 45% di sviluppare un carcinoma dellвЂ™ovaio o un carcinoma tubarico. Anche le mutazioni nel gene BRCA2 conferiscono nelle donne portatrici un rischio compreso tra il 50 e lвЂ™85% di sviluppare un carcinoma della mammella e un rischio minore (20-30%) di sviluppare un carcinoma dellвЂ™ovaio. Alcuni ritengono che mutazioni nel gene BRCA1 nei soggetti maschili non comportino rischi di ammalarsi di CM ma piuttosto di carcinoma della prostata o del colon. Gli uomini che risultano portatori di mutazione del gene BRCA2 possono sviluppare un cancro alla mammella nel corso della vita con un rischio del 6%. Le diverse casistiche mostrano inoltre un leggero incremento (6-14%) del rischio di sviluppare carcinomi della prostata, del colon e del pancreas. 255 Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo I dati in letteratura suggeriscono che il rischio di sviluppare un carcinoma della mammella in donne portatrici di mutazioni Brca1/BRCA2 sia del 10-20% a 40 anni di etГ , del 30-50% a 50 anni e del 40-85% a 70 anni, rispetto a un rischio nella popolazione generale rispettivamente dello 0,5%, 2% e 8%. Le mutazioni in BRCA1 sono associate ad un rischio di carcinoma dellвЂ™ovaio nel corso della vita stimato tra il 15% e il 45%, in confronto al rischio dellвЂ™1.6% nella popolazione generale. Il rischio cumulativo di carcinoma dellвЂ™ovaio in famiglie che risultano portatrici di mutazione nel gene BRCA2 risulta compreso tra 0.4% e il 27%. Strategie di prevenzione nei gruppi ad alto rischio (Figg. 5,6) a. Chirurgia profilattica. Rappresenta la misura piГ№ efficace di prevenzione primaria. La mastectomia profilattica bilaterale con rimozione completa del parenchima mammario, dellвЂ™areola e del capezzolo, con o senza asportazione del rivestimento cutaneo delle mammelle (total o skin-sparing), dovrebbe quasi azzerare il rischio di Carcinoma o Cancro della Mammella (BC) ma, essendo una procedura drastica e traumatica, va considerata esclusivamente in donne portatrici di mutazioni BRCA1/BRCA2 o in altri geni di suscettibilitГ che conferiscono un alto rischio di BC; puГІ essere proposta inoltre a donne con anamnesi di carcinoma lobulare in situ poichГ© anchвЂ™esse ad alto rischio di carcinoma invasivo nella mammella inizialmente interessata e in quella controlaterale. In mancanza di diagnosi Figura 5. Strategie di prevenzione per BC in portatori di mutazioni BRCA e/o famiglie ad alto rischio 256 I tumori della mammella e/o dellвЂ™ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio istopatologica di BC nel tessuto asportato, non ГЁ indicata linfadenectomia ascellare. La mastectomia sottocutanea (nipple-sparing) consente di risparmiare lвЂ™areola e il capezzolo, con migliori risultati estetici, e dovrebbe ridurre il rischio di BC di almeno il 90%. Tale rischio non puГІ essere del tutto eliminato a causa della possibilitГ di insorgenza del BC a livello dei dotti galattofori del capezzolo, in prossimitГ dellвЂ™areola e nelle isole di parenchima mammario risparmiate dallвЂ™intervento chirurgico. Г€ stata anche impiegata lвЂ™asportazione del parenchima mammario e del capezzolo e non dellвЂ™areola (areola-sparing) ma manca un follow-up adeguato a supporto di questa procedura. Nonostante il rischio di Carcinoma Ovarico o Cancro dellвЂ™Ovaio (OC) sia decisamente inferiore rispetto al rischio di BC, la ovaro-salpingectomia bilaterale profilattica dovrebbe essere proposta a tutte le donne portatrici di mutazioni BRCA1/BRCA2 oppure ad alto rischio, in perimenopausa o al completamento del programma familiare, perchГЁ dimezza il rischio di BC, nonostante la maggior parte dei tumori in portatori di mutazione BRCA1 non esprimano recettori ormonali; nei confronti del carcinoma ovarico consente una riduzione del rischio di almeno il 95%, anche se tale rischio non puГІ essere com- Figura 6 257 Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo Figure 7a - 7b I tumori della mammella e/o dellвЂ™ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio pletamente eliminato per la possibile comparsa di carcinomi primitivi peritoneali, per la comune origine embriologica del peritoneo e dellвЂ™epitelio di rivestimento ovarico. Le salpingi possono rappresentare un sito elettivo di tumore nei portatori di mutazione BRCA ed ГЁ quindi indicata la contemporanea isterectomia per evitare la possibile insorgenza in corrispondenza dellвЂ™impianto delle tube nellвЂ™utero. b. Chemioprevenzione. La possibilitГ di ridurre il rischio cumulativo di sviluppare un tumore da parte di un soggetto predisposto mediante terapia ormonale, senza lвЂ™obbligo di ricorrere alla demolizione chirurgica ГЁ, data lвЂ™etГ delle pazienti interessate, un obiettivo di grande interesse. Tuttavia occorre considerare che i BC in portatori di mutazioni in BRCA1 sono per la maggior parte ER negativi (>80%) mentre i BC in portatori di mutazioni in BRCA2 sono prevalentemente ER positivi (75-80%). Di conseguenza, i portatori di mutazioni in BRCA2 potrebbero essere il gruppo che beneficia della chemioprevenzione con ormonoterapia; al contrario nei portatori di mutazione BRCA1, il ruolo ГЁ discutibile (Fig. 7a,7b). Nonostante lвЂ™approccio con SERMs (Selective Estrogen Receptor Modulators) come Tamoxifene e Raloxifene sia stato ampiamente studiato nei soggetti ad alto rischio familiare in pre- e post-menopausa (vedi NCCN Breast Cancer Risk Reduction Guidelines 2009), il loro utilizzo ГЁ comunque gravato da unвЂ™aumentata incidenza di tumori e sanguinamenti Figura 8 259 Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo endometriali cosГ¬ come di eventi tromboembolici. Un retinoide (Fenretide) ha mostrato di ridurre significativamente il rischio di recidiva locale nelle donne in pre-menopausa con anamnesi di BC in stadio I, indipendentemente dallo stato recettoriale del tumore primitivo. Tale risultato rappresenta la base degli studi di prevenzione con Fenretide in corso nelle donne giovani ad alto rischio. Sempre nellвЂ™ambito di studi clinici controllati, ГЁ in corso la valutazione dellвЂ™efficacia preventiva degli inibitori dellвЂ™aromatasi (anastrozolo ed exemestane) nelle donne in postmenopausa ad alto rischio (Fig. 8). c. Cambiamento dello stile di vita. Circa la possibilitГ di influire sui fattori di rischio non-genetici, poichГ© la produzione periferica di estrogeni aumenta con lвЂ™aumentare dellвЂ™etГ ed ГЁ correlata al grado di obesitГ , sono indicati la riduzione del peso, dellвЂ™assunzione di grassi e del consumo di alcool e lвЂ™incremento dellвЂ™esercizio fisico. Tuttavia la valutazione dei singoli interventi ГЁ difficile ed occorrono tempi lunghi per definire se le modifiche dello stile di vita possono davvero modificare il rischio. Г€ stata riportata unвЂ™associazione tra la Terapia Ormonale Sostitutiva (TOS) in postmenopausa e lo sviluppo di cancro ovarico, anche se la potenza statistica della maggior parte degli studi condotti a questo riguardo ГЁ assai limitata. AllвЂ™interno di una casistica inglese di ampie proporzioni, le donne classificate come utilizzatrici correnti (30%), pregresse (20%) o non utilizzatrici (50%) di TOS (intesa come assunzione di estrogeni con o senza progestinici) venivano seguite per lo sviluppo di carcinoma ovarico. Le donne utilizzatrici di TOS avevano unвЂ™incidenza significativamente maggiore di cancro ovarico rispetto alle non utilizzatrici (RR 1.20). Non ГЁ stato osservato alcun aumento significativo nelle donne appartenenti alla categoria delle utilizzatrici pregresse e nemmeno nelle donne che avevano utilizzato la TOS per un periodo inferiore ai 5 anni. Le utilizzatrici di TOS presentavano inoltre un aumento significativo del rischio di morte per carcinoma ovarico rispetto alle non utilizzatrici (RR 1.23). Di conseguenza, la terapia ormonale sostitutiva dovrebbe essere prescritta soltanto per brevi periodi. Il possibile effetto protettivo dellвЂ™assunzione protratta (6 anni) di contraccettivi orali nei confronti dellвЂ™ OC (riduzione del rischio del 60%) sembra bilanciato da un ulteriore incremento di rischio di BC tra le portatrici di mutazioni BRCA1/BRCA2 (RR = 1.2), di conseguenza lвЂ™utilizzo di contraccettivi orali non puГІ essere raccomandato tra le opzioni per ridurre il rischio di carcinoma ovarico nelle pazienti a rischio. d. Sorveglianza senologica. Le metodiche di sorveglianza convenzionalmente utilizzate nello screening per il BC nella popolazione generale comprendono autopalpazione, esame clinico della mammella, ecografia e mammografia. Soltanto la mammografia ha mostrato di ridurre la mortalitГ per BC del 20-35 % nelle donne di etГ 50-69 anni e di meno del 20% nelle donne di etГ 40-49 anni. Per delineare una strategia integrata di sorveglianza inten260 I tumori della mammella e/o dellвЂ™ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio siva da adottare nelle donne ad alto rischio di BC occorre valutare la precoce etГ di insorgenza (premenopausa), lвЂ™elevato rischio conseguente allвЂ™esposizione a radiazioni ionizzanti (associato alla possibile compromissione della capacitГ di riparazione dei danni cellulari radioindotti nei portatori di mutazione BRCA), la minore sensibilitГ della mammografia nei seni вЂњdensiвЂќ, lвЂ™alta velocitГ di crescita nei tumori BRCA che condiziona il maggior rischio di carcinomi dвЂ™intervallo, lвЂ™elevata percentuale di positivitГ linfonodale alla diagnosi. Nei soggetti ad alto rischio ГЁ quindi opportuno adottare programmi di sorveglianza con esordio precoce (25 anni) e brevi intervalli (6 mesi) tra due controlli successivi, prediligendo tecniche di imaging poco dipendenti dalle variazioni della densitГ ghiandolare. Tabella 1. Linee guida sulla sorveglianza nelle donne ad alto rischio e BRCA1/2 mut. F.O.N.Ca.M (Forza Operativa Nazionale sul Carcinoma Mammario) Auto-esame della mammella Esame clinico / EtГ > 25 в†’ ogni 6 mesi N.C.C.N. (National Comprehensive Cancer Network) EtГ > 18 в†’ ogni mese (dopo training adeguato) EtГ > 25 в†’ ogni 6 mesi EtГ > 30 в†’ ogni 12 mesi - Dose ghiandolare media complessiva (per mammelle standard) <4 mGy (= solo oblique) Mammografia (Mx) EtГ 35-50 в†’ ogni 12 mesi EtГ > 25 в†’ ogni 12 mesi Dose ghiandolare media complessiva (per mammella standard) <8 mGy Eta > 50 в†’ ogni 12 mesi Dose standard EtГ 25-35 в†’ ogni 6 mesi Ecografia della mammella EtГ 35-50 в†’ ogni 12 mesi (ogni 6 mesi se seniвЂќdensiвЂќ alla Mx) Solo a giudizio del Radiologo EtГ > 50 в†’ a giudizio del Radiologo Risonanza Magnetica EtГ > 25 в†’ ogni 12 mesi Ecografia TransvagiEtГ > 35 в†’ ogni 12 mesi nale + CA-125 261 Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo Numerosi studi hanno valutato lвЂ™impiego della risonanza magnetica (RM) con mezzo di contrasto paramagnetico nella sorveglianza delle donne ad alto rischio, concludendo che questa tecnica ГЁ la piГ№ sensibile ad ogni etГ per la diagnosi del BC (70-96%). Tuttavia tale metodica risulta sensibilmente meno specifica rispetto alla mammografia e puГІ comportare la ripetizione dellвЂ™esame prima del successivo passaggio di screening e agobiopsie non necessarie. Nonostante non sia stata ancora dimostrata una riduzione della mortalitГ mediante lo screening con RM nelle donne portatrici di mutazioni BRCA1/BRCA2 e ad alto rischio, le neoplasie cosГ¬ diagnosticate sono perГІ mediamente di minori dimensioni e meno frequentemente associate a coinvolgimento linfonodale. Il protocollo di sorveglianza proposto nel Maggio 2008 dalla Forza Operativa Nazionale per il Carcinoma della Mammella (FONCAM) ГЁ illustrato nella tabella (confronto con NCCN) (Tabella 1). e. Sorveglianza ginecologica. Una strategia di screening multimodale basata sulla combinazione di ecografia pelvica e dosaggio del CA125 sierico, non indicata nella popolazione generale, ГЁ suggerita nei pazienti ad alto rischio per la mutazione dei geni BRCA1/ BRCA2, con lвЂ™indicazione a ripetere semestralmente tali indagini. Implicazioni terapeutiche La maggior parte dei BC con mutazioni germinali o sporadiche di BRCA1, specialmente quelli diagnosticati al di sotto dei 50 anni, presentano le caratteristiche immunoistochimiche del fenotipo triplo-negativo e, per quanto riguarda lвЂ™espressione genica, appartengono alla classe molecolare basal-like. Nei tumori sporadici basal-like i livelli della proteina BRCA1 possono essere ridotti anche per fenomeni di tipo epigenetico, quali la metilazione del promotore del gene e il silenziamento dovuto alla espressione di repressori di BRCA1, ad esempio il fattore ID4. LвЂ™inattivazione delle proteine BRCA causerebbe una maggiore sensibilitГ delle cellule neoplastiche agli agenti citotossici che producono addotti al DNA come il cisplatino; viceversa proteine BRCA1 funzionalmente attive sarebbero necessarie affinchГЁ gli agenti antimicrotubuli come i taxani possano esercitare la loro azione citocida. Inoltre la selezione dei pazienti con neoplasia mammaria basal-like basata sullo stato mutazionale di BRCA1 potrebbe portare allвЂ™utilizzo di molecole target dirette contro lвЂ™enzima poli-ADPribosio-polimerasi (PARP). Le cellule tumorali che presentano la mutazione del gene BRCA dipendono dallвЂ™attivitГ di questo enzima per la riparazione del DNA danneggiato e quindi per la sopravvivenza. Con la nuova classe degli inibitori di PARP il bersaglio ГЁ costituito dalla riparazione del DNA danneggiato. Vari agenti (raggi UV), radiazioni ionizzanti, sostanze chimiche, chemiotertici come i derivati del platino, ecc.) possono produrre danni al DNA, con rottura della singola elica o di entrambi. Nel primo caso il proceso di riparazione ГЁ detto base excision repair ed ГЁ controlato dallвЂ™enzima PARP. Le cellule tumorali, anche 262 I tumori della mammella e/o dellвЂ™ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio a causa della rapida proliferazione, hanno difetti riparativi e molteplici danni da instabilitГ genetica. in caso di danno an un singolo filamento, PARP si lega ai punti di rottura, formando catene di poli-ADPribosio: cosГ¬ vengono recluatati specifici enzimi di riparazione. La mancata riparazione del singolo filamento, come conseguenza dellвЂ™esposizione ad un inibitore di PARP, determina una rottura della doppia elica nella fase S del ciclo cellulare. Le cellule con mutazioni di BRCA1/BRCA2 presentano difetti di riparazione delle rotture nella doppia elica. Di conseguenza il double stand break conseguente allвЂ™inibizione di PARP ed eventualmente occorso quando la cellula passa in fase S, non puГІ essere riparato. In effetti, le cellue con mutazioni o in attivazione di BRCA1/BRCA2 si mostrano particolarmente sensibili allвЂ™effetto cicotida degli inibitori di PARP e in alcuni studi clinici di fase II, gli inibitori di PARP hanno mostrato risultati incoraggianti in pazienti affette da neoplasie mammarie ed ovariche con mutazioni di BRCA1/BRCA2. 263 Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo Bibliografia [1] Augello C, Bruno L, Bazan V, et al. Y179C, F486L and N550H are BRCA1 variants that may be associated with breast cancer in a Sicilian family: results of a 5-year GOIM (Gruppo Oncologico dellвЂ™Italia Meridionale) prospective study. Ann Oncol 2006; 17 Suppl 7:vii30-3. [2] CalГІ V, Agnese V, Gargano G, et al. A new germline mutation in BRCA1 gene in a sicilian family with ovarian cancer. Breast Cancer Res Treat 2006; 96:97-100. [3] Chung DC, Yoon SS, Lauwers GY, et al. Case records of the Massachusetts General Hospital. Case 222007. A woman with a family history of gastric and breast cancer. N Engl J Med 2007; 357:283-91. [4] DeVita VT, Lawrence TS, Rosenberg SA. DeVita, Hellman, and RosenbergвЂ™s Cancer: Principles & Practice of Oncology, 8th edition. Philadelphia, Wolters Kluwer/Lippincott Williams & Wilkins, 2008. [5] Easton DF, Pooley KA, Dunning AM, et al. Genome-wide association study identifies novel breast cancer susceptibility loci. Nature 2007; 447:1087-93. [6] Falchetti M, Lupi R, Rizzolo P, et al. BRCA1/BRCA2 rearrangements and CHEK2 common mutations are infrequent in Italian male breast cancer cases. Breast Cancer Res Treat 2008; 110:161-7. [7] Ferla R, CalГІ V, Cascio S, et al. Founder mutations in BRCA1 and BRCA2 genes. Ann Oncol 2007; 18 Suppl 6:vi93-8. [8] Guillem JG, Wood WC, Moley JF, et al. ASCO/SSO review of current role of risk-reducing surgery in common hereditary cancer syndromes. Ann Surg Oncol 2006; 13:1296-321. [9] Hendriks YM, de Jong AE, Morreau H, et al. Diagnostic approach and management of Lynch syndrome (hereditary nonpolyposis colorectal carcinoma): a guide for clinicians. CA Cancer J Clin 2006; 56:21325. [10] Jorde LB, Carey JC, Bamshad MJ. Medical Genetics, 4th edition. Philadelphia, Mosby/Elsevier, 2009. [11] La Paglia L, Lauge A, Weber J, et al. ATM germline mutations in women with familial breast cancer and a relative with haematological malignancy. Breast Cancer Res Treat 2009. [12] Lynch HT, Lynch JF, Lynch PM, et al. Hereditary colorectal cancer syndromes: molecular genetics, genetic counseling, diagnosis and management. Fam Cancer 2008; 7:27-39. [13] Lynch HT, Shaw TG, Lynch JF. Inherited predisposition to cancer: a historical overview. Am J Med Genet C Semin Med Genet 2004; 129C:5-22. [14] Ottini L, Palli D, Rizzo S, et al. Male breast cancer. Crit Rev Oncol Hematol 2009. [15] Russo A, Agnese V, Rizzo S, La Paglia L, Bazan V. The genetics of breast cancer: application in clinical practice. In: Giordano A, Normanno N (Eds.). вЂњBreast Cancer in the Post-Genomic EraвЂќ. New York, Humana Press, 2009, pp. 39-54. [16] Russo A, CalГІ V, Agnese V, et al. BRCA1 genetic testing in 106 breast and ovarian cancer families from Southern Italy (Sicily): a mutation analyses. Breast Cancer Res Treat 2007; 105:267-76. [17] Russo A, CalГІ V, Augello C, et al. 4843delC of the BRCA1 gene is a possible founder mutation in Southern Italy (Sicily). Ann Oncol 2007; 18 Suppl 6:vi99-102. [18] Russo A, CalГІ V, Bruno L, et al. Hereditary ovarian cancer. Crit Rev Oncol Hematol 2009; 69:28-44. [19] Russo A, CalГІ V, Bruno L, et al. Is BRCA1-5083del19, identified in breast cancer patients of Sicilian origin, a Calabrian founder mutation? Breast Cancer Res Treat 2009; 113:67-70. [20] Russo A, Migliavacca M, Zanna I, Macaluso M, Gebbia N, Bazan V. Caretakers and Gatekeepers. In: вЂњEncyclopedia of the Human GenomaвЂќ. New York, Wiley, 2003. [21] Russo A, Zanna I, Tubiolo C, et al. Hereditary common cancers: molecular and clinical genetics. Anticancer Res 2000; 20:4841-51. [22] Schneider K. Counseling About Cancer: Strategies for Genetic Counseling, 2nd edition. New York, Wiley- 264 I tumori della mammella e/o dellвЂ™ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio Liss, 2002. [23] Stormorken AT, Bowitz-Lothe IM, Noren T, et al. Immunohistochemistry identifies carriers of mismatch repair gene defects causing hereditary nonpolyposis colorectal cancer. J Clin Oncol 2005; 23:470512. [24] Veschi S, Aceto G, Scioletti AP, et al. High prevalence of BRCA1 deletions in BRCAPRO-positive patients with high carrier probability. Ann Oncol 2007; 18 Suppl 6:vi86-92. [25] Vogelstein B, Kinzler K. вЂњThe Genetic Basis of Human CancerвЂќ, 2nd edition. New York, Mc-Graw-Hill, 2002. [26] Walsh T, Casadei S, Coats KH, et al. Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer. Jama 2006; 295:1379-88. [27] Welcsh PL, King MC. BRCA1 and BRCA2 and the genetics of breast and ovarian cancer. Hum Mol Genet 2001; 10:705-13. Oral Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 IL GRANULOMA CENTRALE A CELLULE GIGANTI DEI MASCELLARI [Central giant cell of the jaw] Silvia Tortorici1, Maria Lidia Buzzanca1, Francesco Burruano1, Aldo Gerbino2 e Maria Buscemi2 Dipartimento di Scienze Stomatologiche вЂњG. MessinaвЂќ Sezione di Chirurgia Speciale Odontostomatologica; Dipartimento di Medicina Sperimentale Sezione di Istologia ed Embriologia вЂњArcangelo Pasqualino di MarineoвЂќ, FacoltГ di Medicina e Chirurgia, UniversitГ degli Stuti di Palermo (IT) 1 2 Parole chiave: CGCG, Ossa mascellari, Osteoclasta; kB (RANK) Key words: CGCG, Jaws, Osteoclast, kB (RANK) Riassunto. Il granuloma centrale a cellule giganti (CGCG), ГЁ una lesione fibro-ossea. La maggiore frequenza ГЁ osservata nella mandibola e principalmente in bambini ed in giovani adulti, piГ№ spesso in femmine che in maschi. Questa lesione, la cui patogenesi rimane ancora oscura, si presenta come unвЂ™area di radiotrasparenza uni o multiloculare con trabecolature ossee. Lo studio istologico mostra cellule giganti, cellule fibroblastiche, focolai emorragici e tessuto osteoide. In base alle caratteristiche cliniche, radiologiche ed istologiche, il CGCG puГІ essere classificato come вЂњnonaggressivoвЂќ o вЂњaggressivoвЂќ; lвЂ™analisi istomorfometrica delle forme aggressive, mostra un aumento di volume delle cellule giganti e incremento dellвЂ™attivitГ mitotica. Invece lвЂ™immunoistochimica (Ki-67 e p53) non mostra differenze significative. Da un punto di vista clinico i pazienti affetti da forme aggressive riferiscono intenso dolore e, allвЂ™esame obiettivo, si evidenziano: tumefazione, riassorbimento degli apici radicolari, perforazione della corticale e/o ricorrenza della lesione dopo asportazione chirurgica. Le caratteristiche cliniche e istomorfometriche possono quindi essere considerate indicatori certi per la differenziazione fra CGCG aggressivo e non aggressivo, per il trattamento terapeutico e il follow-up. LвЂ™asportazione chirurgica ed un adeguato followup rappresentano il trattamento piГ№ opportuno nei pazienti affetti da CGCG. Abstract. Central giant cell granuloma (CGCG), is a fibro-osseous lesion. The majority of lesions are observed in the mandible and mainly in children and young adults, more often in females than in males. This lesion, which the pathogenesis still remains obscure, appears as radiolucency, well or ill-defined, uni or multilocular with trabeculations coursing through the lesion. Histologic study shows giant cells, fibroblastic cells, and foci of hemorrhage and osteoid tissue. On the basis of clinical, radiological and histologic features, central giant cell granulomas can be classified as вЂњnon-aggressiveвЂќ or вЂњaggressiveвЂќ; the histomorphometric analysis prove a significant increase in large giant cells, fractional surface area, and mitotic activity in aggressive CGCG lesions. Immunohistologic investigation (Ki-67 and p53 stain) reveal no significant differences. The patients with aggressive lesions show aggressive growth, pain, massive swelling, root resorption, cortical perforation, and/or recurrence. Clinical and histomorphometric features may be reliable indicators for the differentiation between aggressive and nonaggressive CGCG. This should be accounted for to improve the individual planning of the treatment and follow-up. Alternative treatments are worthy of consideration, although surgical excision remains the treatment of choice. 269 Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino, Maria Buscemi Introduzione In accordo col World Health Organization (W.H.O.) il granuloma centrale a cellule giganti (CGCG) ГЁ una lesione endo-ossea benigna costituita da tessuto cellulare fibroso nel cui contesto si trovano focolai multipli emorragici, cellule giganti multinucleate e trabecole di osso spugnoso. Г€ una lesione ossea non frequente che ha una percentuale di presentazione di meno del 7% rispetto alle altre lesioni ossee benigne dei mascellari [1]. Fu Jaffe [2] che, nel 1953, descrisse questa patologia come entitГ nosologica a sГ© stante e la definГ¬ col termine di вЂњgranuloma riparativo a cellule gigantiвЂќ (GRCG) per distinguerla dai tumori a cellule giganti che insorgevano nelle epifisi delle ossa lunghe; lвЂ™autore sosteneva che i GRCG dei mascellari non erano delle neoplasie ma costituivano una reazione riparativa locale conseguente a cause traumatiche di varia natura. Il granuloma centrale a cellule giganti (CGCG) ГЁ considerato una lesione osteolitica di variabile dimensione e con tendenza alla progressione la cui natura ed etiologia sono ancora controverse [3]. Il CGCG interessa quasi esclusivamente le ossa mascellari; raro ГЁ il suo riscontro nella restante parte dello scheletro cranio-facciale. In letteratura vengono riportati casi che interessano la base del cranio, lвЂ™osso sfenoidale, lвЂ™osso temporale, i seni paranasali ed etmoidali, lвЂ™osso occipitale, lвЂ™orbita; tali localizzazioni sono infrequenti e meritano una particolare attenzione viste le diverse modalitГ di approccio chirurgico che caratterizzano le varie forme. Nonostante la quasi esclusiva localizzazione alle ossa del massiccio facciale, il CGCG puГІ interessare anche le ossa extragnatiche e, in particolar modo, le ossa brevi di mani e piedi; rarissima ГЁ invece la presentazione nelle ossa lunghe. NellвЂ™ambito dei mascellari il CGCG ГЁ considerevolmente piГ№ frequente nella mandibola rispetto al mascellare superiore con una percentuale che raggiunge il 70% circa, soprattutto nellвЂ™area molare-premolare, estendendosi anche alla branca montante. Il condilo mandibolare rappresenta una sede piuttosto rara. Al mascellare superiore sono descritti casi in zona alveolare e al palato duro. Il CGCG dei mascellari interessa un ampio range di etГ con una prevalenza nella seconda decade di vita; si presenta quindi in adolescenti e giovani adulti approssimativamente nel 48% dei casi, mentre, nel 60% dei casi, le lesioni sono evidenziate prima dei 30 di etГ . Rara ГЁ la manifestazione in etГ infantile. Il sesso femminile ГЁ interessato nel 60% dei casi circa e predomina, con un rapporto di quasi 3 ad 1, nel gruppo di etГ tra gli 11 ed i 30 anni [4]. Sono riportati in letteratura casi di lesioni giganto-cellulari in soggetti affetti da sindromi quali Cherubismo, Sindrome di Noonan, Sindrome oculare ectodermica, sindrome descritta per la prima volta nel 1993, in soggetti con fibromatosi di tipo-1 e con iperparatiroidismo primario e feocromocitoma [5]. 270 Il granuloma centrale a cellule giganti dei mascellari Eziopatogenesi La patogenesi e la natura del CGCG dei mascellari sono incerte. Secondo alcuni autori [2] la lesione puГІ essere correlata ad un trauma, ad un processo riparativo o ad un difetto di sviluppo; secondo altri ad una cisti aneurismatica o ad una cisti traumatica [6]. Le cellule multinucleate esibiscono delle caratteristiche fenotipiche degli osteoclasti quali: positivitГ alla vitronectina (VNR) e alla fosfatasi acida tartrato-resistente (TRAP), risposte inibitorie alla calcitonina e capacitГ di determinare riassorbimento lacunare osseo. Le cellule mononucleate rappresentano invece una eterogenea popolazione di macrofagi e di cellule simili ai fibroblasti. Incerto ГЁ il ruolo che queste cellule fibroblastiche giocano nellвЂ™indurre la formazione degli osteoclasti e il riassorbimento osseo nel CGCG. Recente ГЁ la identificazione dei fattori cellulari ed umorali che inducono la formazione degli osteoclasti; essa richiede unвЂ™interazione tra le cellule stromali che esprimono il recettore attivatore del ligando del fattore nucleare kB (RANKL), e il recettore attivatore del fattore nucleare kB (RANK) espressione dei fagociti mononucleati precursori degli osteoclasti, trovati sia in circolo sia nei tessuti. Come simili fattori agiscano nellвЂ™indurre la formazione di cellule giganti osteoclastiche ГЁ ancora sconosciuto [7,8]. Queste ricerche sono importanti non solo per la determinazione della patogenesi ma anche per la valutazione dellвЂ™uso di strategie terapeutiche non chirurgiche, come lвЂ™uso di farmaci che inibiscono la formazione e la funzionalitГ degli osteoclasti, per esempio la calcitonina, e per il trattamento delle lesioni osteolitiche ricorrenti. Fig. 1 - Neoformazione a superficie irregolare, di consistenza duro-elastica, ricoperta da mucosa tesa che ne fa trasparire il colore rossastro; la massa ha dislocato posteriormente un elemento dentario. Fig.2 - La sezione istologica mostra numerose cellule di grandi dimensioni a contorni netti ma irregolari, contenenti molteplici nuclei. Tali elementi sono separati da stroma fusocellulare. Sono presenti stravasi emorragici interstiziali. 271 Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino, Maria Buscemi Aspetti clinici Г€ largamente diffuso il concetto che le lesioni a cellule giganti mostrano un comportamento clinico alquanto variabile; sebbene la maggior parte delle lesioni sia costituita da processi circoscritti a lenta crescita e che rispondono bene al semplice courettage, un certo numero di queste patologie sono caratterizzate da un atteggiamento aggressivo caratterizzato da dolori, parestesie, riassorbimento degli apici radicolari, da distruzione ossea, e tendenza alla recidiva dopo trattamento. LвЂ™incidenza delle recidive varia dallвЂ™11 al 49% [9]. Nella maggior parte dei casi, cвЂ™ГЁ una prima fase di latenza in cui la sintomatologia ГЁ muta e sono esami radiografici occasionali che evidenziano, nel contesto del mascellare colpito, unвЂ™area di rarefazione; a volte ГЁ lвЂ™elemento dentario che presenta mobilitГ e, una volta estratto, lвЂ™alveolo rimane beante e si va riempiendo di un tessuto rossastro, di aspetto cerebroide, molle elastico e indolente alla palpazione; il CGCG puГІ condurre ad unвЂ™espansione della corticale in maniera proporzionale alla sua crescita determinando mobilitГ e spostamento dei denti. Nella fase di deformazione compare una tumefazione evidente, e la concomitante erosione della corticale ossea esteriorizza una massa neoformata, ricoperta da mucosa tesa, ora normale ora iperemica, a consistenza duro-elastica; gli elementi dentari vengono spostati e si mobilizzano fino a cadere spontaneamente. La compressione di un tronco nervoso o una reazione infiammatoria possono dar luogo a sintomatologia piГ№ o meno intensa. La neoformazione puГІ raggiungere dimensioni notevoli. La sintomatologia assume, quindi, caratteristiche diverse secondo la localizzazione e lвЂ™entitГ della neoformazione. PoichГ© la manifestazione clinica di alcune di queste lesioni ГЁ una lieve reazione riparativa, oggi sono piГ№ comunemente accettati la designazione di вЂњgranuloma a cellule gigantiвЂќ o il piГ№ evasivo termine di вЂњlesione giganto-cellulareвЂќ. Г€ ancora oggetto di speculazione se il granuloma a cellule giganti delle ossa mascellari sia un tumore; secondo alcuni autori rappresenterebbe infatti la variante benigna del tumore a cellule giganti (osteoclastoma). Tuttavia, lвЂ™accettazione del termine granuloma a cellule giganti, indica come non si tratti di una neoplasia ma di unвЂ™iperplasia. Sebbene il granuloma a cellule giganti possa qualche volta presentare un accrescimento rapido e sia localmente distruttivo, il suo decorso ГЁ comunque benigno rispetto ai tumori a cellule giganti che hanno un comportamento locale piГ№ invasivo e tendono a metastatizzare nonostante lвЂ™aspetto istologico benigno. I tumori a cellule giganti si localizzano frequentemente a livello delle ossa lunghe ed alcune volte puГІ risultare molto difficile distinguerli dal granuloma a cellule giganti, sia dal comportamento clinico sia dal quadro istologico. Diversamente dal granuloma a cellule 272 Il granuloma centrale a cellule giganti dei mascellari giganti, i tumori a cellule giganti tendono a non avere predilezione per uno dei due sessi, ed a colpire pazienti piГ№ anziani. Inducono di solito, dolore e presentano un rapido accrescimento determinando, nel 10% dei casi, una frattura patologica che rappresenta il primo segno clinico. Anche i CGCG delle ossa mascellari con comportamento вЂњaggressivoвЂќ oppure con un aspetto istologico simile ai tumori a cellule giganti, sono molto meno distruttivi dei tumori a cellule giganti delle ossa lunghe (TGC) che hanno una percentuale di recidiva del 60% [10]. AllвЂ™ispezione il CGCG si presenta come una tumefazione rotondeggiante od ovoidale, a superficie liscia ovvero irregolarmente mammellonata; il volume varia da quello di un cece a quello di una grossa noce, raramente puГІ raggiungere diametri maggiori. Alla palpazione la consistenza va dal molliccio al duro elastico. Aspetti radiografici Da un punto di vista radiografico il CGCG si presenta come unвЂ™area di radiotrasparenza uniloculare o multiloculare. Non sempre i limiti con il tessuto osseo sono netti, a volte la radiotrasparenza sfuma verso i tessuti circostanti e non si riesce a delimitarne i contorni. Le forme localizzate al mascellare superiore si presentano sotto forma di molteplici aree osteolitiche a margini irregolari; nella mandibola invece il quadro radiografico ГЁ piГ№ netto, simil cistico. LвЂ™aspetto radiografico varia con le dimensioni delle lesioni. Le lesioni piccole di solito si presentano come aree di radiotrasparenza uniloculare e prive di setti ossei interni. Le lesioni di grandi dimensioni invece appaiono come aree di radiotrasparenza multiloculare [11]. Kaffe et al. [12] osservarono che, su 80 casi esaminati, il 51% delle lesioni erano multiloculari, il 44% uniloculari, il 5% non erano loculari, e il 68% delle lesioni multiloculari interessavano la mandibola. Essi evidenziarono anche che le lesioni uniloculari avevano una dimensione media di 4.05 cm, mentre le lesioni multiloculari di 7.38 cm. Essi stabilirono, quindi, una correlazione statisticamente significativa tra le dimensioni delle lesioni e la loro modalitГ di presentazione. Whitaker e Waldron [13], analogamente, trovarono che il 61% delle lesioni diagnosticate come CGCG erano multiloculari mentre il 39% erano uniloculari. Cohen e Hertzanu [14] stabilirono inoltre una correlazione tra la localizzazione delle lesioni e la loro locularitГ . Essi evidenziarono, infatti, che lвЂ™80% delle lesioni multiloculari erano mandibolari, il 75% delle lesioni uniloculari interessavano la mascella. Osservarono che le dimensioni medie delle lesioni uniloculari erano di 23.75 mm e di quelle multiloculari di 53.00 mm . Vi era quindi una differenza statisticamente significativa tra le dimensioni delle lesioni uniloculari e quelle multiloculari. Questi studi sono stati avvalorati recentemente da lavori 273 Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino, Maria Buscemi effettuati da Gungormus M. et al. [15]. LвЂ™esame radiografico fornirebbe delle indicazioni anche sul grado di maturazione della neoformazione: sembra che il CGCG piГ№ maturo abbia unвЂ™attivitГ osteoformatrice evidenziata dalla presenza di trabecole ossee; il CGCG non ancora maturo mostra, invece, una zona di osteocondensazione reattiva da parte del tessuto circostante e si presenta uniloculare [15]. Comunque lвЂ™immagine radiografica non ГЁ patognomonica. Numerose sono, infatti, le patologie che mostrano aree di osteorarefazione quali: вЂў Cisti odontogena вЂў Cisti non odontogene вЂў Tumori odontogeni a matrice differenziata вЂў Forme localizzate del mieloma multiplo o plasmocitoma solitario вЂў Reticolosarcoma primitivo dellвЂ™osso o linfoma primitivo dellвЂ™osso вЂў Carcinoma metastatico a partenza da parotide, retto, prostata, mammella, polmone вЂў Condroma nella sua forma mista вЂў Osteoma osteoide вЂў Osteosarcoma вЂў Condrosarcoma вЂў Emangioma centrale dei mascellari вЂў Lesioni similtumorali: il tumore melanotico neuroectodermico dellвЂ™infanzia, il granuloma eosinofilo, la displasia fibrosa (forma monostotica), il sarcoma di Ewing вЂў Osteiti rarefacenti вЂў Malattia di Paget вЂў Malattia di Recklinghausen PoichГ© il quadro radiologico non ГЁ patognomonico, fondamentale ГЁ il ruolo dellвЂ™indagine citologica per agoaspirazione e dellвЂ™indagine istologica per unвЂ™esatta diagnosi e per la programmazione di un appropriato approccio terapeutico. Macroscopicamente il CGCG si presenta come una massa rotondeggiante o ovoidale, a superficie liscia o mammellonata con aspetto cerebroide, di colore rosso o rosso-brunastro; la consistenza va dal molliccio al duro-elastico. La mancanza di una capsula fa sГ¬ che tale neoformazione non sia ben delimitata rispetto allвЂ™osso circostante. Il quadro microscopico classico (Fig. 1,2) mostra che il CGCG ГЁ costituito da tessuto mesenchimale. Gli elementi cellulari sono essenzialmente rappresentati da fibroblasti e macrofagi immersi in uno stroma costituito da scarse fibre connettive, vasi e cellule giganti; possono riscontrarsi anche leucociti, linfociti, plasmacellule ed eritrociti, espressione di episodi flogistici sovrapposti. 274 Il granuloma centrale a cellule giganti dei mascellari Le indagini ultrastrutturali al microscopio elettronico evidenziano che le cellule stromali piГ№ rappresentative sono i fibroblasti, elementi allungati ed ovali; il loro citoplasma ГЁ abbondante con reticolo endoplasmatico rugoso, alcune cisterne dilatate, parecchie chiazze di reticolo endoplasmatico liscio, numerosi poliribosomi liberi ed un esteso complesso di Golgi. Sono presenti inoltre cellule fagocitiche mononucleari, con superficie irregolare per la presenza di microvilli che possono dar luogo ad interdigitazione fra cellule contigue creando un quadro ritenuto tipico. Questi macrofagi contengono parecchie inclusioni anche a tipo fosfolipidico. Il tessuto vascolare, sempre rappresentato, puГІ essere tanto abbondante da conferire aspetto angiomatoso; sono presenti stravasi emorragici interstiziali depositi di emosiderina e zone di necrosi. Le cellule giganti sono gli elementi caratteristici della lesione; di forma rotondeggiante od ovalare, sono caratterizzate da una membrana cellulare irregolare per la presenza di microvilli ed invaginazioni. Le dimensioni sono varie e comunque di diametro compreso fra 10 e 150 micron. Il citoplasma ГЁ basofilo alla periferia ed acidofilo al centro, il numero dei nuclei ГЁ variabile e di solito non supera il numero di 20 essendo rari (6%) i casi con un numero maggiore. Le cellule giganti si localizzano vicino ai vasi sanguigni, a volte si dispongono a manicotto attorno ad essi o sporgono allвЂ™interno del lume vascolare; quando una cellula gigante sporge di poco nel lume vasale, ГЁ sempre ricoperta da un sottile strato di cellule endoteliali, quando sporge ampiamente tale continuitГ endoteliale ГЁ interrotta. Il citoplasma contiene numerosi mitocondri ed un reticolo endoplasmatico rugoso discretamente rappresentato. LвЂ™apparato di Golgi presenta vescicole ectasiche. I nuclei mostrano unвЂ™abbondanza in acidi nucleinici; ciГІ denuncia una notevole attivitГ metabolica. LвЂ™aspetto istologico del CGCG risulta molto simile a quello riscontrato nellвЂ™iperparatiroidismo. Le cellule giganti mostrano delle risposte molto simili agli osteoclasti per quanto riguarda lвЂ™utilizzo della calcitonina e dellвЂ™immunoistochimica . La natura e il significato delle cellule giganti sono stati indagati mediante indagini morfologiche ultrastrutturali, citometriche per stabilire lвЂ™attivitГ nucleinica; indagini immunoistochimiche in cui si esamina lвЂ™espressione di p53, MDM2, Ki-67, PCNA; tali studi convergono verso la considerazione che queste cellule sono osteoclasto-simili, e che hanno come precursori i monociti macrofagi [16]. PoichГ© lвЂ™evoluzione clinica di questa patologia ГЁ varia, alcuni autori hanno cercato di evidenziare delle correlazioni fra i quadri microscopici e il comportamento clinico delle lesioni. Su 142 casi di CGCG dei mascellari Whitaker et al. [13] osservarono che la maggior parte delle lesioni era asintomatica e relativamente innocua; tuttavia alcune mostravano un decorso clinico piГ№ aggressivo caratterizzato da riassorbimento radicolare, dolore o parestesia e perforazione corticale. In tutti i casi la percentuale di recidiva andava dallвЂ™11 275 Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino, Maria Buscemi al 49%. Gli autori supposero che quei quadri istologici caratterizzati da una regolare distribuzione di cellule giganti e dalla presenza di figure mitotiche riconoscibili rappresentavano processi neoplastici a crescita autonoma, invece, lГ dove le cellule giganti erano irregolarmente distribuite e dove erano presenti non poche figure mitotiche, si trattava di un processo reattivo. La presenza di tessuto osteoide alla periferia di lesioni non recidivanti suggeriva che queste erano state oggetto di un courettage piГ№ profondo o che un processo osteogenico reattivo le aveva delimitate. In realtГ non sono ancora ben chiari i rapporti tra CGCG e tumori gigantocellulari. Diagnosi differenziale Г€ opportuno menzionare altre lesioni delle ossa mascellari caratterizzate dalla presenza di cellule multinucleate simil-osteoclastiche; attualmente si riserva lвЂ™appellativo di neoformazioni вЂњa cellule gigantiвЂќ solo a quei quadri patologici dove lвЂ™espressione istologica fondamentale della lesione ГЁ costituita dalle suddette cellule. In tale gruppo troviamo: вЂў Il tumore bruno вЂў I tumori a cellule giganti вЂў Il granuloma a cellule giganti periferico вЂў La cisti ossea aneurismatica e la displasia ossea fibrosa вЂў Il cherubismo Terapia - note conclusive Il trattamento delle lesioni giganto-cellulari dei mascellari ГЁ sempre chirurgico. Il courettage ГЁ la prima scelta terapeutica e di solito ГЁ risolutiva. Vista la mancanza di capsula a volte residuano piccoli frammenti di tessuto che di solito non necessitano di ulteriore terapia e qualche volta vanno incontro a risoluzione spontanea. Altre volte invece queste formazioni tendono ad assumere un decorso locale piГ№ aggressivo se non sono completamente rimosse; le recidive necessitano di ulteriori interventi di courettage. Le lesioni ossee a cellule giganti localizzate al massiccio facciale comprendono una serie di quadri molto diversi fra loro per etiologia e comportamento clinico. La somiglianza istologica, clinica e radiologica di tali lesioni rende necessaria unвЂ™attenta valutazione prima di intraprendere un qualsiasi atto terapeutico. La piГ№ frequente delle lesioni giganto cellulari dei mascellari ГЁ rappresentata dal CGCG che, pur presentando invasivitГ locale e rapido accrescimento ha un comportamento clinico benigno. Contrapposto troviamo il вЂњtumore osseo a cellule gigantiвЂќ che ha un comportamento clinico piГ№ aggressivo; in realtГ dalla letteratura si evince come sia difficile una diagnosi differenziale ben definita tra le due forme. Alcuni autori [3,4,13] hanno distinto le lesioni giganto cellulari in forme non aggressive e forme aggressive in base allвЂ™aspetto 276 Il granuloma centrale a cellule giganti dei mascellari clinico, biologico ed istologico. Le lesioni non aggressive richiedono solo unвЂ™asportazione della neoformazione, insieme ad un accurato courettage della cavitГ , in quanto in questa categoria ГЁ molto rara lвЂ™insorgenza di recidive. Nelle lesioni aggressive il semplice courettage non ГЁ spesso sufficiente e la terapia piГ№ adeguata consiste invece nella resezione in blocco del segmento osseo interessato dalla lesione, con immediata ricostruzione. Alcune neoformazioni richiedono un approccio diverso; nonostante la scelta dellвЂ™intervento dipenda primariamente dallвЂ™aspetto clinico della lesione, occorre considerare la sede di interessamento delle ossa mascellari e lвЂ™etГ del paziente. In presenza di una forma aggressiva, se richiesta una resezione in blocco accompagnata alla perdita di strutture importanti, puГІ essere preferibile trattare tale lesione come non aggressiva e praticare un semplice courettage accettando il rischio di una recidiva e riservando ad un secondo tempo eventualmente la chirurgia resettiva. Nel mascellare superiore, a prescindere dallвЂ™aggressivitГ della lesione, il courettage puГІ costituire un ottimo compromesso come trattamento poichГ© le caratteristiche anatomiche di questa struttura ne rendono maggiormente complessa, rispetto alla mandibola, la ricostruzione dopo lвЂ™intervento di osteotomia [19]. Recenti studi propongono terapie alternative a quella chirurgica nel management del CGCG. Tali terapie si basano sulle caratteristiche biologiche di alcuni tipi di lesioni giganto cellulari. Sono state sperimentate iniezioni di calcitonina, iniezioni intralesionali di steroidi, e iniezioni subcutanee di alfa-interferone in soggetti con lesioni multiple, recidivanti o particolarmente aggressive [20-23]. In realtГ i meccanismi di azione non sono ancora definiti ed i risultati non sono uniformi anche perchГ© limitati a pochi casi, pertanto la terapia chirurgica in atto rimane quella elettiva. 277 Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino, Maria Buscemi Bibliografia [1] Kramer IRH, Pindborg JJ, Shear M. Histological Typing of Odontogenic Tumors. In: WHO International Histological Classification of Tumours, 2nd edn. Berlin, Germany, Springer-Verlag 1992. [2] Jaffe HL. Giant cell reparative granuloma, traumatic bone cyst and fibrous (fibro-osseus) dysplasia of the jaw bones. Oral Surg Oral Med Oral Pathol 1953; 6: 159-75. [3] Sapp P, Eversole CR, Wysocki GP. Contemporary oral pathology. St Louis: CV Mosby Co; 1997. [4] Waldrom CA, Shafer WG. The giant cell reparative granuloma of the jaws: an analysis of 38 cases. Am J Clin Pathol 1966; 45: 437-47. [5] Dahlin DC. Giant cell tumors of bone: highlights of 407 cases. AJR 1985; 144: 955-60. [6] Chiba I, Teh BG, Iizuka T, Fukuda H. Conversion of a Traumatic Bone Cyst Into Central Giant Cell Granuloma: Implications for Pathogenesis - A case report. J Oral Maxillofac Surg 2002; 60: 222-225. [7] Liu B, Yu SF, Li TJ. Multinucleated giant cells in various forms of giant cell containing lesions of the jaws express features of osteoclasts. J Oral Pathol Med 2003; 32(6): 367-75. [8] Itonaga I, Hussein I, Kudo O, Sabokbar A, Watt-Smith S, Ferguson D, Athanasou NA. Cellular mechanisms of osteoclast formation and lacunar resorption in giant cell granuloma of the jaw. J Oral Pathol Oral Med 2003; 32: 224-31. [9] Regezi JA, Sciubba JJ. Oral pathology: clinical pathologic correlations, 3rd edn. Philadelphia: WB Sauders 1993; 368-370. [10] Andersen L, Fejerskov O, Philipsen HP. Oral giant cell granulomas. A clinical and histological study of 129 new cases. Acta Pathol Microbiol Scand [ A] 1973; 81(5): 606-16. [11] Stavopoulos F, Katz J. Central giant cell granulomas: a systematic review of the radiographic characteristics with the addition of 20 new cases. Dentomaxillofac Radiol 2002; 31(4): 213-7. [12] Kaffe I, Ardekian L, Taicher S, Littner MM, Buchner A. Radiologic features of central giant cell granuloma of the jaws. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996; 81: 720-6. [13] Whitaker SB, Waldron CA. Central giant cell lesion of the jaws: A clinical, radiological, and histopathologic study. Oral Surg Oral Med Oral Pathol 1993; 75(2): 199-208. [14] Cohen MA, Hertzanu Y. Radiologic features, including those seen with computed tomography, of central giant cell granuloma of the jaws. Oral Surg Oral Med Oral Pathol 1988; 65(2): 255-61. [15] Gungormus M, Akgul M. Central Giant cell Granuloma of the Jaws: A Clinical and Radiologic Study. The Journal of Contemporary Dental Practice 2003; 4(3), 1-8. [16] Souza PE, Mesquita RA, Gomez RS. Evaluation of p53, PCNA, Ki-67, MDM2 and AgNOR in oral peripheral and central giant cell lesions. Oral Dis 2000; 6 (1): 35-9. [17] Chiba I, Teh BG, Iizuka T, Fukuda H. Conversion of a Traumatic Bone Cyst Into Central Giant Cell Granuloma: Implications for Pathogenesis - A case report. J Oral Maxillofac Surg 2002; 60: 222-225. [18] Sapp P, Eversole CR, Wysocki GP. Contemporary oral pathology. St Louis: CV Mosby Co; 1997. [19] Cannistra C, Fadda T, Guerrieri L, Vero S, Della Rocca C, Iannetti G. Central giant cell granuloma of the palate: unusual localization in a five year old child. Bull Group Int Rech Sci Stomatol Odontol 1999; 41 (4): 112-4. [20] Kaban LB, Mulliken JB, Ezekowitz A. Antiangiogenic therapy of a recurrent giant cell tumor of the mandible with interferon alpha-2a. Pediatrics 1999; 103: 1145-419. [21] OвЂ™Regan EM, Gibb DH, Odell EW. Rapid growth of giant cell granuloma in pregnancy treated with calcitonin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001; 92(5): 532-8. [22] Pogrel MA. Calcitonin therapy for central giant cell granuloma. J Oral Maxillofac Surg 2003; 61(6): 649-53. [23] Khafif A, Krempl G, Medina JE. Treatment of giant cell granuloma of the maxilla with intralesional injection of steroids. Head Neck 2000; 22(8): 822-5. 278 Methodology Physical activity Clinical applications Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 MAGNETIC RESONANCE SPECTROSCOPY: PRINCIPLES AND CLINICAL APPLICATIONS [Spettroscopia di risonanza magnetica: principi ed applicazioni cliniche] Giulia Bivona1, Maria Buscemi2, Aldo Gerbino2 and Marcello Ciaccio1 1 Chair of Clinical Biochemistry, 2Chair of Histology and Embryology (Di.Me.S.), School of Medicine, University of Palermo (IT) Key words: Magnetic Resonance Spectroscopy, Neurodegeneration, Mitochondrial Diseases Parole chiave: Spettroscopia di Risonanza Magnetica, Neurodegenerazione, Malattie Mitocondriali Abstract. While Magnetic Resonance Imaging (MRI) provides anatomic images and morphometric characterization of disease, magnetic resonance spectroscopy (MRS) provides metabolite/biochemical information about tissues non-invasively in vivo. MRS has been used clinically for more than two decades in the investigation of neurological dysfunction and in the evaluation of the prostate gland (1H MRS) and muscle tissue (31P MRS). in vivo proton MRS allows the presence of certain metabolites in brain tissue which present clinical importance, including: N-acetyl aspartate (Naa), Creatine (Cr), Choline (Cho) and Myoinositol (mI). Other metabolites that can be detected via proton MRS include lactate and amino acids. This technique is useful whenever biochemical or metabolic assessment may be necessary, such as in differential diagnosis of focal brain lesions (neoplastic and non-neoplastic diseases); diagnosis of neurodegenerative diseases; assessment of epilepsy; biochemical alterations in hepatic encephalopathies and inborn errors of metabolism and hypoxic encephalopathy. 31 P MRS can be used to investigate energy metabolism in several tissues, but muscle is in many ways an ideal tissue for MRS studies; 31P MRS spectra metabolites from muscle contain the following peaks: О±, ОІ and Оі ATP, phosphocreatine (PCr) inorganic phosphates (Pi); phosphomonoesters (PME) and phosphodiesters (PDE). The technique provides information about intracellular pH and intracellular magnesium concentration. 31 P MRS can be used for diagnostic purposes in metabolic myopathies and mitochondrial diseases, in which such an information can be obtained to correctly identify the pathology. Riassunto. La Spettroscopia di Risonanza Magnetica del Protone, differentemente rispetto allвЂ™Imaging Morfologico Standard, consente di ottenere informazioni di natura biochimica e metabolica sul distretto indagato, che puГІ essere rappresentato dal Sistema Nervoso Centrale, dal muscolo scheletrico o dalla ghiandola prostatica. Mediante Spettroscopia del protone ГЁ possibile valutare i seguenti metaboliti indicativi dello stato metabolico- funzionale del Sistema Nervoso Centrale: N- acetil aspartato, Creatina, Colina e Mioinositolo; altri metaboliti rilevabili includono il Lattato ed alcuni aminoacidi. LвЂ™indagine biochimica puГІ essere molto utile ai fini della diagnosi differenziale fra patologie neurologiche di natura neoplastica e non neoplastica, nella diagnosi delle patologie neurodegenerative e nelle epilessie, nelle encefalopatie epatica ed ipossico- ischemica. La spettroscopia del fosforo ГЁ utile nellвЂ™indagine metabolico-funzionale di molti tessuti fra i quali in particolare emerge in termini di applicazioni cliniche quello muscolo-scheletrico. I metaboliti rintracciabili sono i seg- 281 Giulia Bivona, Aldo Gerbino, Maria Buscemi, Marcello Ciaccio uenti: О±, ОІ e Оі ATP, fosfocreatina (PCr) fosfato inorganico (Pi); fosfomonoesteri (PME) e fosfodiesteri (PDE). Г€ possibile inoltre, contestualmente allвЂ™indagine, ottenere delle informazioni riguardanti le concentrazioni intracellulari di pH e di Magnesio. Le applicazioni principali in biomedicina della tecnica includono la diagnosi ed il monitoraggio delle malattie mitocondriali e delle miopatie metaboliche. The first in vivo magnetic resonance spectroscopy was performed at the beginning of the 1980s and was done using resonance of the phosphorus nucleus (31P), thereby revealing the energy metabolism of the tissue cells studied. However, this method had the disadvantages of low magnetic sensitivity, low concentration of 31P atoms and also inadequate spatial resolution in focal brain lesions with small and mild dimensions, and it has been replaced by proton (hydrogen) spectroscopy, especially in brain studies. Proton (1H) resonance is nowadays the method most frequently used in neurospectroscopy, because hydrogen is the most abundant atom in the human body and its nucleus emits the most intense radiofrequency signal, when in an external magnetic field, in relation to other nuclei. Moreover, proton magnetic resonance spectroscopy is more quickly accomplished and easily interpreted [1]. While Magnetic Resonance Imaging (MRI) provides anatomic images and morphometric characterization of disease, magnetic resonance spectroscopy (MRS) provides metabolite/biochemical information about tissues non-invasively in vivo. MRS has been used clinically for more than two decades in the investigation of neurological dysfunction and in the evaluation of the prostate gland (1H MRS, figures 1-2-3) and muscle tissue (31P MRS, figures 4-5). The main difference between standard MRI and MRS is that the frequency of the MR signal is used to encode different types of information: while MRI uses high spatial resolution to generate anatomical images, MRS, based on the principle of chemical shift, provides chemical information about the tissues presented as line spectra. This technique has provided a useful bridge between metabolism and the anatomic and physiological studies available from MRI, and represents a versatile diagnostic tool in biomedicine. Biochemical informations in vivo are provided by MRS selecting volumes of interest (voxel or VOI) on the standard imaging sequence and the peaks on the spectra obtained correspond with various metabolites which are typical for every tissue examined and may be identified precisely. There are two methods of magnetic resonance spectroscopy: single voxel and multivoxel, with or without spectroscopic imaging. Single voxel proton magnetic resonance spectroscopy provides a rapid biochemical profile of a localized volume within a region of interest that may be determined, especially in brain studies. Spectroscopic imaging provides biochemical information about multiple, small and contiguous volumes focalized on a particular region of interest that may allow the mapping of metabolic tissue distribution. By 282 Magnetic Resonance Spectroscopy: Principles and Clinical Applications using this method, the data obtained may be manipulated by computer and superimposed on the image of an abnormality, thereby illustrating the distribution of such metabolites within that area. In vivo proton MRS allows the presence of certain metabolites in brain tissue to be detected if the minimum concentrations are between 0.5 and 1.0 mM. Some of these present clinical importance, such as: N-acetyl aspartate (Naa), which is a neuronal marker that is present in neuron bodies and axons and indicates their density and viability; the Naa peak in proton spectroscopy will be decreased whenever there is neuronal loss, such as in glioma, ischemia and degenerative diseases; Creatine (Cr) is a marker of the aerobic energy metabolism, its peak is practically constant and may be used as a control value in relation to other metabolites. Choline (Cho), which is a constituent molecule of the phospholipid metabolism of cell membranes and reflects membrane turnover. Increased choline indicates greater membrane synthesis and cell proliferation. Its concentration is normally greatly increased in cases of brain neoplastic expansible processes. Lactate is not commonly detected in proton spectroscopy of brain tissue: its presence indicates a pathological condition with regard to the final products of anaerobic metabolism. Lactate can be identified in cysts, hypoxic/ischemic tissues and some neoplasms. Lipids are usually not detected by proton spectroscopy, either. In pathological situations in which necrosis occurs, such as in malignant neoplasms and inflammatory/ infectious processes, there is an accentuated lipid peak, signifying cell membrane degradation; Myoinositol, which is considered to be a glial function marker, generally presents reduction in hepatic encephalopathy and elevation in AlzheimerвЂ™s disease. Other metabolites that can be detected via proton MRS include the following amino acids: alanine, acetates and succinates [2-3]. Every metabolite has a normal concentration that generates a pattern of peaks that is the same from person to person unless there is an underlying pathology. Diagnosis with MRS can therefore be made by either comparing the numeric values of metabolite concentrations or by recognizing abnormal patterns of peaks in the spectra (Figg. 2-3). Proton magnetic resonance spectroscopy of the brain is useful whenever biochemical or metabolic assessment may be necessary, such as in differential diagnosis of focal brain lesions (neoplastic and non-neoplastic diseases); diagnosis of dementia and other degenerative diseases; follow-up radiation therapy for patients with brain neoplasms; demyelinating diseases such as multiple sclerosis and leukodystrophy; diagnosis and prognosis of brain ischemic and traumatic lesions; assessment of epilepsy; biochemical alterations in hepatic encephalopathies; and neuropediatric affections such as brain tumors, inborn errors of metabolism and hypoxic encephalopathy [2-4]. 31 P MRS can be used to investigate energy metabolism in several tissues, but muscle is in many ways an ideal tissue for MRS studies because it can be stressed in the magnet 283 Giulia Bivona, Aldo Gerbino, Maria Buscemi, Marcello Ciaccio with exercise while the biochemical response is monitored. 31P MRS records signals from high energy phosphate compounds which are central to energy metabolism in vivo. Phosphorus MR spectra from muscle contain seven resonances: three arise from the phosphate groups of adenosine triphosphate (О±, ОІ and Оі ATP), one from phosphocreatine (PCr) and one from inorganic phosphates (Pi); two additional smaller resonances can be observed from phosphomonoesters (PME) and phosphodiesters (PDE). The spectral distance between Pi and PCr provides information about intracellular pH while the chemical shift of ATP can be used to calculate intracellular magnesium concentration. The concentration of the metabolically active adenosine diphosphate (ADP), which is important in the regulation of rates of mitochondrial ATP synthesis, does not produce a signal visible in the spectra, but can be calculated indirectly using the creatine kinase (CK) equilibrium equation. One of the most important features of 31P MRS is its ability to monitor time-dependent changes of metabolites non invasively. In normal exercising muscle, one can observe in the MR spectra a PCr decrease stoechiometrically linked to Pi accumulation, while the ATP signal remains unchanged at least when exercise intensity is light or moderate. During recovery, [PCr] gradually increases, [Pi] and [ADP] decrease, and pH returns to its rest level (Figg. 4-5) . 31 P MRS can be used for diagnostic purposes in metabolic myopathies and mitochondrial diseases, in which such an information can be obtained to correctly identify the pathology. Particularly regarding on mitochondrial diseases, the clinical spectrum of these disorders is large, due in part to their genetic and biochemical heterogeneity [5]. A majority of these diseases include neurological symptoms besides muscle symptoms and can be suspected when an increase in serum of lactate and pyruvate is found, even at rest. The proportion of mutant versus wild-type mitochondrial DNA determines the biochemical phenotype; however, the link between the biochemical phenotype and the clinical phenotype is not always clear making the diagnosis difficult. Defects of respiratory chain enzymes are the most prevalent types of mitochondrial diseases and their common endpoint is a deficit in ATP production leading to a decreased exercise tolerance and a shared pattern of detectable abnormalities. Among syndromic mitochondrial diseases at least two encephalomyopathies are greatly known and studied: Leber Hereditary Optic Neuropathy (LHON) and Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS); LHON is characterized by visual loss resulting from retinal ganglion cell degeneration; despite the important role of respiratory chain deficiency and oxidative stress induced by mtDNA point mutations affecting complex I, excitotoxic injury has been postulated as a concurrent pathogenic factor. MELAS syndrome is the most common syndromic manifestation of mitochondrial encephalomyopathies that occur in all age groups. The clinical course of MELAS is slowly progressive with stepwise deterioration; the severity of lactic acidosis correlates with the severity of neurologic deficit. 284 Magnetic Resonance Spectroscopy: Principles and Clinical Applications Fig 1. 1HMRS multivoxel spectra of prostate gland. Fig 2. 1HMRS voxel localization and correspondent spectrum. Giulia Bivona, Aldo Gerbino, Maria Buscemi, Marcello Ciaccio Fig 3. Differences between 1HMRS spectra peaks and correspondent metabolite concentrations. Fig 4. 31 PMRS spectrum obtained during rest. Fig 5. 31 PMRS spectrum obtained during exercise and recovery after exercise. Magnetic Resonance Spectroscopy: Principles and Clinical Applications Deficits in carbohydrate metabolism can present either with exercise-induced symptoms such as painful cramps, contractures, and rhabdomyolysis or with progressive weakness. Muscles with impaired glycolysis or glycogenolysis lack the ability to produce lactic acid during exercise, deficiency of glicogenolytic and glycolytic enzymes leads to impaired anaerobic synthesis of ATP in association with reduced production of lactate; the insufficient production of reducing equivalents (nicotinamide adenine dinucleotide, NADH) and pyruvate during aerobic exercise can also result in impaired mitochondrial ATP production. This represents the biochemical basis for a distinctive muscle 31PMRS pattern in glycogenolytic enzyme defects, characterized by a rapid depletion of PCr, appearance of cytosolic alkalosis or reduced acidification during muscle exercise and slow rate of PCr resynthesis during recovery after exercise. One of the most studied myopathy in this group is the myophosphorylase deficiency, known as McArdle disease, in which 31P MRS findings is PCr rapid depletion and paradoxical increase in muscle pH, due to proton consumption by PCr hydrolysis during exercise [6]. 287 Giulia Bivona, Aldo Gerbino, Maria Buscemi, Marcello Ciaccio References [1] Van der Knaap MS, Van der Grond J, Van Rijen PC, Faber JA, Valk J, Willemse K. Age-dependent changes in localized proton and phosphorus MR spectroscopy of the brain. Radiology 1990; 176: 509-15. [2] Lin A, Ross BD, Harris K, Wong W. Efficacy of Proton Magnetic Resonance Spectroscopy in Neurological Diagnosis and Neurotherapeutic Decision Making. NeuroRx 2005; 2: 197-214. [3] Kantarci K. 1H Magnetic Resonance Spectroscopy in Dementia. Br J Radiol, 2007; 80(2): S146-52. [4] Soares DP, Law M. Magnetic resonance spectroscopy of the brain: review of metabolites and clinical applications. Clinical Radiology 2009; 64: 12-21. [5] Kemp GJ, Meyerspeer M, Moser E. Absolute quantification of phosphorus metabolite concentrations in human muscle in vivo by 31P MRS: a quantitative review. NMR Biomed 2007; 20(6): 555-65. [6] Prompers JJ, Jeneson JA, Drost MR, Oomens CC, Strijkers GJ, Nicolay K. Dynamic MRS and MRI of skeletal muscle function and biomechanics. NMR Biomed 2006; 19(7): 927-53. 288 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 Immunoistochimica e analisi dвЂ™immagine multi spettrale [Immunohistochemistry and multispectral image analisys] Giovanni Francesco Spatola Di.Me.S. Sezione di Istologia ed Embriologia вЂ“ FacoltГ di Medicina - UniversitГ degli Studi di Palermo (IT) Parole chiave: Analisi dвЂ™immagine, Immagini multispettrali, Immunoistochimica Key words: Image analisys, Multispectral, Immunohistochemistry Sommario. LвЂ™uso di metodiche dвЂ™analisi dвЂ™immagine ha permesso di rendere piГ№ affidabili e facilmente riproducibili i risultati ottenuti mediante tecniche di immunoistochimica (IHC). La maggiore diffusione e la semplificazione dei software che permettono uno studio colorimetrico ha fatto sГ¬ che tali metodiche siano alla portata di tutti e ha permesso di standardizzare la tecnica mediante dei sistemi di вЂњscoreвЂќ affidabili. Inoltre, lвЂ™introduzione negli ultimi anni di metodiche di acquisizione dвЂ™immagine mediante sistemi multispettrali ha affinato ulteriormente tali tecniche permettendo di evitare artefatti e svincolare la valutazione dellвЂ™operatore dai dati oggettivi. Abstract. The use of methods of image analysis has allowed us to make more reliable and reproducible results obtained by immunohistochemistry (IHC). Wider use and simplification of software allowing a colorimetric study has meant that these methods are available to everyone and made it possible to standardize the technique by reliable systems score. Moreover, the introduction in recent years of multispectral image acquisition systems methods has further refined these techniques allowing to avoid artifacts and release the evaluation of the data by the observer. Premessa La tecnica immunistochimica viene impiegata sia nel campo della ricerca per evidenziare antigeni specifici sia dagli anatomo patologi per evidenziare marker tipici di svariate patologie. Sin dalla sua messa a punto essa ha sofferto di alcune gravi limitazioni intrinseche allвЂ™esecuzione della tecnica stessa. Se i passaggi che precedono la valutazione dei risultati possono comunque essere standardizzati [1,2], molto piГ№ difficile ГЁ ottenere una ripetitivitГ e una corretta valutazione dei dati ottenuti. Infatti la lettura di un preparato IHC dipende strettamente dallвЂ™operatore e dalla sua esperienza. Vi sono molteplici fattori che influenzano lвЂ™osservazione a partire dal tipo di microscopio, alla temperatura di colore della luce utilizzata, al tipo di colorante usato per la rivelazione, alla capacitГ visiva dellвЂ™operatore stesso ed ultimo, ma non ultimo per importanza, dalla non linearitГ ottica delle reazioni. Difatti se per basse concentrazioni di antigene si puГІ affermare che ad un aumento della concentrazione aumenta anche lвЂ™intensitГ della colorazione questo dato si perde a maggiori concentrazioni di antigene. Pertanto la nostra percezione ottica non permette una valutazione corretta della effettiva reattivitГ . 289 Giovanni Francesco Spatola Al fine di superare questi limiti sopra esposti negli ultimi anni si ГЁ cercato di standardizzare al massimo la tecnica IHC sia nelle fasi di preparazione, che nella vera e propria reazione e nella successiva lettura dei dati [1,2]. A tal fine un importante aiuto ГЁ stato fornito dai sistemi di imaging digitale che da circa 15 anni vengono affiancati da software sempre piГ№ perfezionati di analisi dвЂ™immagine. LвЂ™analisi dвЂ™immagine ГЁ una metodica informatica che ci permette mediante software piГ№ o meno dedicati di avere una valutazione sia del numero di cellule positive sia dellвЂ™intensitГ della reazione intesa come parametri colorimetrici derivanti da un immagine digitale. Analisi dвЂ™immagine I maggiori problemi legati alla valutazione delle reazioni immunoistochimiche dipendono, come precedentemente sottolineato, da diversi parametri prevalentemente soggettivi. Quello che per un operatore ГЁ una positivitГ del test per un altro puГІ essere invece assolutamente negativo e ciГІ dipende dalla percezione personale dei colori, dalle condizioni di osservazione, dalla propria capacitГ visiva etc. Bisogna, inoltre, tenere conto del fatto che le reazioni IHC non hanno una resa totalmente lineare dal punto di vista cromogenico. Esse difatti risultano effettivamente lineari a bassa concentrazione dellвЂ™anticorpo nella cellula ma se la sostanza ricercata risulta essere presente in maggiori concentrazioni la risposta cromogenica risulta essere non lineare ossia dopo una certa soglia (per altro molto bassa) non risulta piГ№ possibile discriminare in maniera attendibile la quantitГ di sostanza presente allвЂ™interno della cellula. Diverso ГЁ il caso della immunofluorescenza in quanto essa tende a dare una risposta quasi del tutto lineare ossia a maggiore concentrazione della sostanza corrisponde un effettivo aumento della fluorescenza. Per tale motivo oggi si tende ad utilizzare sempre piГ№ questa ultima tecnica. A partire dai primi anni novanta lвЂ™uso di immagini digitali in microscopia si ГЁ sempre piГ№ diffuso e ha permesso di ottenere un sempre crescente numero di informazioni dai campioni studiati. Chiaramente in sistemi pioneristici come i primi metodi basati sullвЂ™acquisizione di immagini video e non fotografiche la bassa risoluzione dei frames acquisiti non permetteva di potere effettuare valutazioni colorimetriche accurate. Nonostante ciГІ lвЂ™introduzione di alcuni software come il Lucia M della Nikon sono da considerare come una vera rivoluzione nel campo della valutazione IHC in quanto hanno permesso di semplificare sia il conteggio di cellule positive sia la valutazione dei risultati introducendo il concetto di quantificazione colorimetrica. Tale tipo di valutazione permetteva di avere, sia pure in maniera approssimativa, una idea della quantitГ di sostanza presente allвЂ™interno di vari citotipi. A causa della bassa risoluzione si era convenuto che bisognava utilizzare immagini di tipo binario (bianco e nero a 2 bit) oppure immagini in scala di grigio che 290 Immunoistochimica e analisi dвЂ™immagine multi spettrale Fig.1 Profili CMYK per i piГ№ comuni cromogeni risultano essere piГ№ ricche di informazioni in quanto a 8 bit o 256 toni di grigio. Г€ importante considerare che il nostro occhio riesce a distinguere circa 70 toni di grigio quindi un uso di immagini a 256 toni necessita di un software che effettui una valutazione corretta dellвЂ™intensitГ di reazione. LвЂ™uso del b&w evitava le differenze legate ai diversi tipi di cromogeni usati e permetteva una maggiore standardizzazione delle tecniche di analisi dвЂ™immagine [3,4]. Per ovviare alla predetta non linearitГ delle reazioni IHC e alla impossibilitГ di valutazione oggettiva del singolo operatore (frasi del tipo вЂњdiffusamente reattivoвЂќ, oppure вЂњmolto presenteвЂќ o ancora вЂњscarsamente immunoreattivoвЂќ risultano essere assolutamente inutili e imprecise) e al fine di individuare correttamente la presenza e la distribuzione di una sostanza risulta sempre piГ№ di fondamentale importanza lвЂ™uso di un sistema di analisi dвЂ™immagine che provveda a quantificare mediante uno score la reattivitГ permettendoci di rispondere alle domande base che sono: вЂў La reazione ГЁ positiva in rapporto ai controlli ? вЂў Se ГЁ positiva che intensitГ presenta e questa ГЁ correlata alla quantitГ di antigene ? вЂў Dove ГЁ localizzata la reattivitГ ? LвЂ™evoluzione dei sistemi di acquisizione di immagine ha modificato alcuni parametri per cui se fino a qualche anno fa risultava indispensabile utilizzare le immagini binarie o al massimo ad 8 bit, come prima chiarito, oggi la maggiore risoluzione permette anche un uso di immagini a colori. Le immagini digitali a colori si basano su alcuni parametri fondamentali ed in particolare su degli istogrammi legati alla combinazione dei colori di base siano essi RGB (rosso, giallo, blu) oppure CMYK (ciano, magenta, giallo, e nero). I profili di questi istogrammi variano in maniera sensibile in base al colore che andiamo a prendere in considerazione. In particolare si ГЁ visto [5] che il profilo CMYK ГЁ quello che maggiormente ci permette una valutazione progressiva ed in particolare il canale del giallo ГЁ indipendente dallвЂ™osservatore e applicabile a diversi tipi di cromogeno in quanto sensibile a minime variazioni di intensitГ 291 Giovanni Francesco Spatola IHC con una scala lineare che permette di ottenere un corretto score riproducibile con facilitГ . Teniamo conto che ogni canale viene suddiviso in 250 toni quindi basta associare secondo una progressione lineare a un determinato valore un punteggio variabile da 1 a 7, dove 1 corrisponde allвЂ™assenza di colore o 0 e 7 al massimo valore ottenibile in base al cromogeno, ossia 250 (Fig.1). Parallelamente allвЂ™evoluzione dei sistemi di acquisizione di immagini la semplificazione dei software di analisi ha permesso una diffusione della metodica che risulta oggi alla portata di chiunque abbia un minimo di esperienza nellвЂ™uso di comuni programmi di fotoritocco come ad esempio Photoshop [6,7] che nelle sue ultime versioni implementa anche un menГ№ di analisi dвЂ™immagine dove con pochi semplici comandi ГЁ possibile effettuare una serie di rilevazioni che vanno dal conteggio di cellule positive fino alla quantificazione di valori colorimetrici a cui assegnare uno score [4,8,9]. Analisi dвЂ™immagine multi spettrale Recentemente alla valutazione basata su uno score derivante dal profilo CMYK si ГЁ affiancata la metodica di acquisizione di immagini multi spettrali. Tale metodica, utilizzata inizialmente per migliorare la qualitГ di immagini acquisite dai satelliti e applicata in diversi campi di studio, dalla valutazione geologica del suolo sino a studi di archeologia dove le immagini multispettrali permettono di valutare la presenza di insediamenti nel sottosuolo, si basa sulla possibilitГ mediante particolari espedienti tecnici di superare i limiti di visione dellвЂ™occhio umano che ha una percezione del colore dipendente dalla luce e dalle condizioni di osservazione. In fisica uno stimolo colore ГЁ definito univocamente dallвЂ™вЂќintensitГ вЂќ che esso assume nello spettro di luce visibile. Pertanto ad ogni colore si deve associare una вЂњcurva spettraleвЂќ costruita in base allвЂ™energia irradiata dalle singole lunghezze dвЂ™onda [10]. Per evidenziare tali curve ГЁ necessario acquisire delle immagini che tengano conto di quella che in fisica viene definita come вЂњriflettanzaвЂќ ossia la proporzione di luce incidente che una data superficie ГЁ in grado di riflettere. Un sistema di imaging multi spettrale pertanto permette di determinare la riflettanza nello spettro del visibile e di ricostruire fedelmente i colori della stessa immagine. I sistemi multi spettrali utilizzano particolari filtri ottici sensibili alle diverse lunghezze dвЂ™onda nello spettro del visibile e successivamente dei sistemi software capaci di ricostruire le curve di riflettanza in ogni punto della scena restituendoci una immagine che puГІ essere studiata nelle sue varie componenti colorimetriche. In pratica i sistemi di analisi multi spettrali sono composti da un sistema di filtri che ci permettono, guardando attraverso uno di essi settato per una certa lunghezza dвЂ™onda nel range visibile, di distinguere il singolo colore (Fig.2). Un вЂњcuboвЂќ di dati spettrali ГЁ ottenuto per selezione di una lunghezza dвЂ™onda dopo lвЂ™altra, acquisendo unвЂ™immagine per ciascuna 292 Immunoistochimica e analisi dвЂ™immagine multi spettrale Fig. 2 Curve spettrali e filtri di essa e ricostruendola mediante appositi software. La possibilitГ di acquisire immagini a diverse lunghezze dвЂ™onda fa si che si possa anche sottrarre alcune di esse dallвЂ™immagine finale e quindi mettere in evidenza soltanto quelle lunghezze dвЂ™onda specifiche per un determinato cromogeno o fluorocromo. Le applicazioni risultano essere quindi molteplici. In particolare nel caso di immagini in fluorescenza la possibilitГ di separare le varie lunghezze dвЂ™onda permette di eliminare tutta o in massima parte lвЂ™autofluorescenza dei tessuti con indubbio vantaggio per la valutazione di risultati legati ad una determinata reattivitГ specifica. NellвЂ™immunoistochimica classica lвЂ™uso di tale metodica permette di isolare ed evidenziare, mediante una elaborazione definita pseudo fluorescenza (immagini trasformate in negativo e ben contrastate), la presenza di un determinato cromogeno, la sua localizzazione e successivamente anche la sua intensitГ (Fig.3-4-5). Inoltre ГЁ possibile separare ed isolare diversi cromogeni utilizzati per valutare una colocalizzazione di antigeni diversi allвЂ™interno della stessa cellula o dello stesso tessuto [11,12,13]. Successivamente alla fase di acquisizione applicando una separazione selettiva dei vari spettri mediante una eliminazione progressiva di quelli che non corrispondono ai cromogeni in esame ГЁ possibile anche qui trasformare lвЂ™immagine secondo il profilo CMYK e valutare il canale del giallo ottenendo come giГ precedentemente esposto una curva colorimetrica lineare a cui applicare uno score per la valutazione dellвЂ™intensitГ della reazione e quindi indirettamente quantificare la presenza del nostro antigene. 293 Giovanni Francesco Spatola Conclusione La metodica IHC ancora oggi risulta essere utile a visualizzare la presenza allвЂ™interno di cellule di determinati antigeni. Associare le metodiche di analisi dвЂ™immagine e di analisi multi spettrale permette inoltre di ottenere risultati riproducibili e separati dalle limitazioni oggettive determinate dai vari osservatori. La possibilitГ inoltre di quantificare mediante uno score la quantitГ di antigene presente nel nostro campo microscopico ci permette in associazione a metodiche di biologia molecolare di avere una visione piГ№ completa e sicuramente piГ№ corrispondente alla realtГ rispetto allвЂ™utilizzo delle singole metodiche. 294 Immunoistochimica e analisi dвЂ™immagine multi spettrale Fig.3 Acquisizione multi spettrale - positivitГ leptinergica (cromogeno AEC) in alcuni epiteliociti duttali вЂ“ immagine multispettrale - Ob. 10x Fig.4 Acquisizione multi spettrale - positivitГ MMP9 (cromogeno AEC) in endoteliociti di aneurisma aortico dilatativo umano вЂ“ immagine multispettrale - Ob. 40x Fig.5 Confronto tra microscopia classica (A) e immagine multi spettrale (B) - positivitГ MMP9 (cromogeno AEC) in cellule di polpa dentale вЂ“ immagine multispettrale - Ob. 40x Giovanni Francesco Spatola Bibliografia [1] Walker RA. Quantification of immunohistochemistry issues concerning methods, utility and semiquantitative assessment. I; Histopathology 2006; 49: 406-410. [2] Taylor CR, Levenson RM, Quantification of immunohistochemistry issues concerning methods, utility and semiquantitative assessment. II; Histopathology 2006; 49: 411-424. [3] Oberholzer M, Ostreicher M, Christen H, BrГјhlmann M. Methods in quantitative image analysis. Histochem Cell Biol 1996; May 105(5):333-55. [4] Kaczmarek E et al, Techniques of image analysis for quantitative immunohistochemistry. Annales Academiae Medicae Bialostocensis Vol. 49, 2004; Suppl. 1, Proceedings. [5] Nhu-An Pham et al, Quantitative image analysis of immunohistochemical stains using a CMYK color model. Diagnostic Pathology 2007; 2:8. [6] Lehr HA, Mankoff DA, Corwin D, Santeusanio G, Gown AM. Application of photoshop-based image analysis to quantification of hormone receptor expression in breast cancer. J Histochem Cytochem 1997; 45(11): 1559-65. [7] Kirkeby S, Thomsen CE. Quantitative immunohistochemistry of fluorescence labelled probes using lowcost software. J Immunol Methods 2005; Jun;301(1-2):102-13. [8] Skaland I, Ovestad I, Janssen EA, Klos J, Kjellevold KH, Helliesen T, Baak JP. Digital image analysis improves the quality of subjective HER-2 expression scoring in breast cancer. Appl Immunohistochem Mol Morphol 2008; Mar;16(2):185-90. [9] Skaland I, Г�vestad I, Janssen EA, Klos J, Kjellevold KH, Helliesen T, Baak JP. Comparing subjective and digital image analysis HER2/neu expression scores with conventional and modified FISH scores in breast cancer. J Clin Pathol 2008; Jan;61(1):68-71. Epub 2007 Apr 5. [10] G. Novati et al, Acquisizione di immagini multispettrali - www.ivl.disco.unimib.it 2003. [11] Gavrilovic M, WГ¤hlby C. Quantification of colocalization and cross-talk based on spectral angles. J Microsc 2009; Jun; 234(3):311-24. [12] Jaskolski F, Mulle C, Manzoni OJ. An automated method to quantify and visualize colocalized fluorescent signals. J Neurosci Methods 2005; Jul 15;146(1):42-9. [13] Landmann L, Marbet P. Colocalization analysis yields superior results after image restoration. Microsc Res Tech 2004; Jun 1;64(2):103-12. 296 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 EFFECTS OF SPECIFIC TRAINING ON HORMONE LEVELS IN FEMALE ATHLETES [Effetti dellвЂ™allenamento specifico sui livelli ormonali nelle atlete] Arcangelo Benigno, Attilio Licciardi and Giuseppe Morici Department of Experimental Medicine (Di.Me.S), University of Palermo (IT) Key words: Exercise, Menstrual disorders, Soccer, Gonadotropins, Training Parole chiave: Esercizio, Alterazioni mestruali, Calcio, Gonadotropine, Allenamento Abstract. Intensity of exercise, specific type and amount of training, reduced caloric intake, low body mass index (BMI) or body fat may contribute to cause various forms of reproductive dysfunctions including oligomenorrhea, amenorrhea and luteal phase defects. To explore the possible relationships between hormone levels and specific training programmes we determined plasma levels of ОІ-endorphin, prolactin (PRL), follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) in 15 female soccer players (SP) of the same team (6 eumenorrheic, 9 oligomenorrheic) at rest and after an individualized role-related training session (IT), and in 10 sedentary eumenorrheic controls (C) at rest. At rest, FSH, LH and E2 levels were lower, and ОІ-endorphin level higher, in SP than in C. ОІ-endorphin level was unrelated to hormone levels in either SP or C. IT increased hormone concentrations, with higher values of E2 and LH in eu- compared to oligomenorrheic SP. ОІ-endorphin increased less in eu- than in oligomenorrheic SP. In the entire SP group after IT, we found that a higher increase in ОІ-endorphin resulted in a lower change in LH (r= -0.59, p<0.05) and E2 (r= -0.58, p<0.05). As for positional roles, most forwards were eumenorrheic. Conversely, all midfielders were anovulatory oligomenorrheic, and their LH response to IT correlated with the ОІ-endorphin response (r=-0.99, p=0.01). Our data suggest an interaction between specific training, hormone levels and menstrual status in female SP. Riassunto. LвЂ™intensitГ dellвЂ™esercizio, la specificitГ e la quantitГ dellвЂ™allenamento, la riduzione dellвЂ™apporto calorico, un basso indice di massa corporea (IMC) o una ridotta percentuale di grasso corporeo possono contribuire a causare diverse forme di disfunzioni della riproduzione come oligomenorrea, amenorrea e difetti della fase luteinica. Per esaminare la possibile relazione tra livelli ormonali e programmi di allenamento specifici abbiamo determinato i livelli plasmatici di: ОІ-endorfina, prolattina (PRL), ormone follicolostimolante (FSH), ormone luteinizzante (LH) e estradiolo (E2) in 15 giocatrici di calcio (SP) della stessa squadra (6 eumenorroiche, 9 oligomenorroiche) a riposo e dopo una seduta di allenamento personalizzata per il ruolo (IT). Dieci donne sedentarie eumenorroiche a riposo (C) sono state studiate come controlli. A riposo, i livelli di FSH, LH e E2 erano piГ№ bassi, ed i livelli di ОІ-endorfina piГ№ alti, nelle SP che nei C; i livelli di ОІ-endorfina non erano correlati con i livelli ormonali nelle SP o nei C. LвЂ™IT incrementava le concentrazioni ormonali, con valori piГ№ alti di E2 e LH nelle SP eu- rispetto alle oligomenorroiche. LвЂ™incremento di ОІ-endorfina era inferiore nelle SP eu- che nelle oligomenorroiche. NellвЂ™intero gruppo delle SP dopo IT, lвЂ™incremento della ОІ-endorfina era inversamente correlato alle variazioni di E2 (r=-0.58, p<0.05) e di LH (r=-0.59, p<0.05). In considerazione dei ruoli, la maggior parte delle attaccanti erano eumenorroiche. Viceversa, tutte le centrocampiste erano oligomenorroiche anovulari e la risposta allвЂ™IT dellвЂ™ormone LH correlava con la risposta della ОІ-endorfina (r=0.99, p<0.01). I nostri dati suggeriscono unвЂ™interazione tra allenamento specifico, livelli ormonali e funzione mestruale nelle calciatrici. 297 Arcangelo Benigno, Attilio Licciardi, Giuseppe Morici Introduction Paragraph 1: Intensity of exercise, specific type (endurance vs. power) and amount of training [1-4], reduced caloric intake, low body mass index (BMI) or body fat [4, 5] may partially contribute to cause various forms of menstrual cycle disorders (MCDs). Evidence accumulated to date indicates that low energy availability plays a causal role in the development of exercise induced amenorrhea [5]. Disordered eating in combination with high energy expenditure from exercise can lead to energy deprivation [6]. Paragraph 2: The most frequent alterations of the menstrual cycle in female athletes are delayed puberty if agonistic training begins before puberty, and hypothalamic chronic oligo-/amenorrhea if agonistic training begins in post-menarcheal age [7-9]. The trainingassociated changes in the menstrual cycle are reversible, with return to eumenorrhea upon cessation of training [10]. Paragraph 3: The aetiology of exercise-associated menstrual disorders is not completely understood. Plasma levels of endogenous opioid hormones like ОІ-endorphin, prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol (E2) are believed to reflect an alteration in the hypothalamic control of gonadotropin release [1113]. The pulsatility of gonadotropins is also reduced in athletes with anovulatory oligomenorrhea [14,15]. The decrease of estrogenic hormone concentrations is proportional to training intensity [16,17], and plasma levels of ОІ-endorphin increase after short-term anaerobic exercise [18]. However, there is a lack of information regarding the effects of exhaustive aerobic endurance exercise and of aerobic-anaerobic alternated sports, like soccer, on sex steroids and ОІ-endorphin [19,20]. Paragraph 4:Soccer players need a high aerobic power in order to sustain a high workrate for 90 minutes. But, according to their position in the team, they need high anaerobic or explosive power, too. The greatest running distance is covered by midfielders, while the frequency of jumps is greater among forwards than fullbacks or midfielders. Therefore, the alactacid anaerobic power of forwards should be higher than that of players in other positions. For these reasons, training programmes for soccer players have been developed in accordance with their positional roles in the team [21]. Paragraph 5: The aim of this study in eumenorrheic and anovulatory oligomenorrheic female soccer players was to determine the acute effects of an individualized role-related training session on plasmatic levels of ОІ-endorphin, PRL and gonadotropins. Methods Subjects Paragraph 6: Fifteen female soccer players (SP) from the same team, and ten healthy 298 Effects of specific training on hormone levels in female athletes sedentary women of similar age (C) were recruited for the study. All experiments were in agreement with the ethical standards of the local University Ethical Committee. All subjects gave written informed consent to the study. No use of tobacco, alcohol, cannabis or other drugs was allowed by the study protocol. No subject was on oral contraceptives or other exogenous hormones, or had a history of eating disorders (anorexia, bulimia). Paragraph 7: The anthropometric characteristics of SP and C were similar (age: 20.4В±6.4 yr in SP, 20.6В±3.4 yr in C; body weight: 54.0В±7.9 kg in SP, 59.3В±9.7 kg in C; height: 157.2В±5.5 cm in SP, 157.5В±6.2 cm in C; percentage of body fat: 26.5В±4.1% in SP, 27.5В±6.7% in C). All C women had cyclic menstrual cycles (11-13 menses/year) and their menarcheal age was 12.5В±1.1 yr. The SP group comprised 6 eumenorrheic (11-13 menses/year), and 9 oligomenorrheic (5-7 menses/year) women. In the SP group, menarcheal age was 13.4В±1.0 yr, slightly higher than in C (p<0.05 by unpaired t-test), but similar in eu- (13.7В±1.1 yr) and oligomenorrheic SP (13.2В±0.9 yr). The oligomenorrheic SP were studied on the 3rd day of the cycle to assess basal gonadotropin pattern, and by basal body temperature to ascertain oligomenorrheic anovulation, leading to the diagnosis of stressrelated hypothalamic chronic anovulation [22]. Paragraph 8: All SP had been playing agonistic soccer for at least 6 years. The team included 4 forwards, 4 midfielders and 7 fullbacks. Each training session lasted approximately 2 hrs, and included skills, tactics and physical conditioning exercises. Their training programme (4 sessions/week) was individualized according to positional roles in the team: most work carried out by midfielders and fullbacks was to develop aerobic capacity, whereas training in forwards aimed at improving strength and alactacid anaerobic capacity and power. Protocol Paragraph 9: Data in SP were collected between 4 and 6 p.m. towards the end of a 14-week training period (23). Data in controls were collected in the same month and hour of the day. The percentage of body fat was determined by using a bioelectrical impedance apparatus (Human - IM Scan, DS MediGroup, Milan, Italy). Paragraph 10: In all subjects, blood sampling was scheduled during the follicular phase of the menstrual cycle between days 10 and 14 (to emphasize the possible different conditions between eumenorrheic and anovulatory oligomenorrheic groups). In SP, blood samples were obtained 29 В± 4 min before, and 28 В± 5 min after the training session. During the training session, water drinking was allowed at pleasure. Paragraph 11: Blood was drawn from the subjectвЂ™s antecubital vein into sterile tubes containing EDTA (7,2 mg/5 ml) at 4вЂўC, and centrifuged for 15 min at 2000 rpm. Aliquots of plasma were frozen and kept at - 20вЂўC in polycarbonate tubes and subsequently analysed 299 Arcangelo Benigno, Attilio Licciardi, Giuseppe Morici in duplicate for ОІ-endorphin (Incaster RIA Kit, Stillwater, Minnesota, USA), LH and FSH (Cis Bio-International RIA Kit, Gif-Sur-Yvette Cedex, France), and E2 and PRL (Biodata RIA Kit, Rome, Italy). Statistics Paragraph 12: Results are presented as means В± standard deviation (SD). Data from SP and C, and from eumenorrheic and anovulatory oligomenorrheic SP, were compared by two-tailed unpaired t-test. Statistical evaluation in the SP group according to positional roles was by analysis of variance, with Bonferroni multiple-range test. Association between variables was assessed by simple linear regression. The level of significance was set at 0.05. Results Resting conditions Paragraph 13: Table 1 reports plasma hormone concentrations at rest in SP and C. Compared to C, both eu- and oligomenorrheic SP showed decreased plasma levels of LH and E2. Both subgroups showed increased plasma level of ОІв€’EP, while plasma FSH was significantly lower than normal in oligomenorrheic SP only. Eumenorrheic SP showed significantly lower plasma PRL values than C. As expected, there was a difference between eu- and oligomenorrheic SP at rest regarding the plasma level of E2, which was significantly lower in the latter than in the former group (p<0.005 by unpaired t-test). Paragraph 14: No significant relationship was found at rest between plasma hormones and PRL or ОІ-endorphin concentration in any group (C, all SP, eu- or oligomenorrheic SP). Table 1. - Hormones values at rest in controls and soccer players FSH (mUI/ml) LH (mUI/ml) PRL (ng/ml) E2 (pg/ml) ОІ-Endorphin (pmol/L) Sedentary controls (n=10) 9.3В±1.1 20.6В±1.2 12.7В±2.3 147.0В±12.1 5.8В±0.4 Soccer players Eumenorrheic (n=6) 7.9В±1.7 12.9В±8.4* 7.1В±2.5* 83.2В±9.3* 6.5В±0.5* Oligomenorrheic (n=9) 6.5В±2.0* 7.0В±4.4* 13.9В±12.1 48.3В±23.7* 6.8В±0.5* Values are meanВ± S.D. * indicates p<0.05 between soccer players and controls 300 Effects of specific training on hormone levels in female athletes Fig. 1. Summary data of hormonal levels at rest (empty bars) and after exercise (hatched bars) in eumenorrheic and anovulatory oligomenorrheic SP (upper and lower panel, respectively). * indicates significant difference between resting and post-exercise values. Fig. 2. Hormonal responses to exercise in eumenorrheic (empty bars) and oligomenorrheic (filled bars) SP expressed as percentage change from baseline. * indicates significant difference between eumenorrheic and oligomenorrheic SP. Arcangelo Benigno, Attilio Licciardi, Giuseppe Morici fullbacks, eumenorrheic (n=3) fullbacks, oligomenorrheic (n=4) midfielders, oligomenorrheic (n=4) forwards, eumenorrheic (n=3) forwards, oligomenorrheic (n=1) Fig. 3. Analysis of exercise-associated average hormonal changes according to positional role in the team and to menstrual status. Effects of specific training on hormone levels in female athletes Response to exercise according to menstrual status paragraph 15: Post-exercise values of all hormones and ОІ-endorphin were higher than pre-exercise values in both eu- and oligomenorrheic SP (Figure 1), but the E2 level after exercise was much higher in eumenorrheic SP. Paragraph 16: Figure 2 illustrates exercise-induced changes in hormonal levels expressed as percentage increase over baseline: the three-fold increase in plasma LH found in eumenorrheic SP was blunted in oligomenorrheic SP (p=0.001 by unpaired t-test), while ОІ-endorphin showed a slightly larger increase in oligo- than in eumenorrheic SP (p<0.05, Figure 2). The increase in FSH, PRL and E2 were not statistically different in the two groups. Paragraph 17: In the whole SP group (eu- + oligomenorrheic SP), exercise-induced changes in ОІ-endorphin correlated inversely with changes in LH (r=-0.59, p<0.05) and E2 (r=-0.58, p<0.05). No significant relationship was found between exercise-induced changes in ОІ-endorphin and PRL or FSH, either in the whole SP group or in the two subgroups. Response to exercise according to positional role in the team Paragraph 18: Both 3 out of 4 forwards and 3 out of 7 fullbacks were eumenorrheic. All 4 midfielders were oligomenorrheic. The average trends in hormonal responses to exercise in SP, according to both the menstrual status and the role in the team are reported in figure 3. ОІ-endorphin increased similarly after exercise irrespective of positional roles or menstrual status. No significant difference among positional roles was evident for any hormone. Eumenorrheic SP showed a significant increase in plasma LH and E2 after exercise, which was blunted in oligomenorrheic SP (Figure 3). The LH response to exercise correlated negatively to the ОІ-endorphin response in midfielders (r=-0.99, p=0.01). Discussion Paragraph 19: Exercise-induced menstrual irregularities appear related to the intensity and duration of training [1,2]. Menstrual disturbances, such as hypothalamic chronic anovulation, have been associated with significant weight loss, decreased body fat, stress, intensity of athletic training, disordered eating in combination with high energy expenditure from exercise [6,14,24]. Still, the mechanism by which strenuous exercise might lead to decreased reproductive function has not been elucidated [22]. Paragraph 20: High plasma levels of ОІ-endorphin have been shown to decrease the basal levels of LH and FSH. ОІ-endorphin has been implicated in the regulation of circulating gonadotropin-releasing hormone (GnRH) from the median eminence of the hypothalamus. Furthermore, the pulsatile secretion of GnRH may be inhibited by the increased level of 303 Arcangelo Benigno, Attilio Licciardi, Giuseppe Morici ОІ-endorphin, which binds to norepinephrine receptors in the hypothalamus leading to the suppression of LH and FSH release. These hormonal changes result in hypothalamic chronic anovulation [14,25,26]. Paragraph 21: In our sample, ОІ-endorphin at rest was higher, and FSH, LH, and E2 lower, in SP than in controls. In addition, a similar percentage of body fat in SP and controls ruled out any possible effect of altered body composition on hormonal levels at rest. Our data do not support an exclusive role of ОІ-endorphin in reproductive hormone regulation, since no significant difference in ОІ-endorphin was found between eu- and oligomenorrheic SP at rest. These data suggest that other factors, including the individual sensitivity to hormones and possibly the type of training, may modulate the relationship between ОІ-endorphin and reproductive hormone levels. Paragraph 22: The effect of acute strenuous exercise on plasma hormone concentrations has also been the object of much controversy. Serum FSH, LH and E2 concentrations were reported to be increased, decreased or unchanged with both acute and chronic exercise [14]. Our study provides evidence that when SP undergo intense exercise, FSH, LH, E2 and PRL all increase significantly. Furthermore, despite different basal levels, the percentage increase over baseline after exercise was not different for PRL, FSH and E2 in eu- and oligomenorrheic SP. However, anovulatory oligomenorrheic SP displayed higher ОІ-endorphin and lower LH increase after the training session, in agreement with the known reduced variation of gonadotropin secretion in response to different stress in this group. In the whole SP sample, we found that a higher exercise-induced increase in ОІ-endorphin resulted in a lower increase in LH. These data suggest that the acute exercise-induced alterations in ОІ-endorphin and LH are tightly associated. Paragraph 23: As the possible role of PRL is concerned, lower resting levels of PRL were found in eu- than in oligomenorrheic SP. Since PRL was not increased over baseline in oligomenorrheic SP, our data suggest a minor role of PRL in causing chronic hypothalamic anovulation in athletes. In addition, the PRL response to exercise was similar in eu- and oligomenorrheic SP. Paragraph 24: Little information is presently available regarding the effects of aerobic exhaustive endurance exercise and aerobic-anaerobic alternated sports like soccer on sex steroids and ОІ-endorphin [27,28]. Ideally, the evaluation of the long-term effects of training on reproductive hormone concentrations and menstrual cycle would require a long-term follow-up of a large sample of women along many seasons of training and agonistic practice. In our study, we compared the FSH, LH, E2, PRL and ОІ-endorphin responses to exercise in forwards, midfielders and fullbacks. Although no statistical difference in hormonal levels was found among positional roles in the team, one interesting observation was that all midfielders in our study were affected by hypothalamic chronic anovulation. A possible 304 Effects of specific training on hormone levels in female athletes explanation for this particular endocrine pattern is that, as already shown in long-distance runners [12], chronic aerobic stress may modify the pulsatile secretion of GnRH and determine hypothalamic chronic anovulation. Conversely, forwards who perform an intermittent anaerobic stress, were mostly eumenorrheic. Therefore, positional roles seem to affect the endocrine pattern possibly through different effects of role-specific training. Midfielders and fullbacks perform a more constant and continuous physical aerobic-anaerobic alternated exercise than forwards, who perform a strenuous and fast but discontinuous anaerobic exercise with several pauses. The hypothesis of a modulating effect of the type of exercise is supported by the findings of correlation analysis in fullbacks indicating that exercise-induced changes in LH are linked to changes in ОІ-endorphin. Paragraph 25: In conclusion, a low LH or E2 response to exercise in female soccer players was associated with a large increase in ОІ-endorphin concentration. On the other hand, intense exercise increased reproductive hormones and ОІ-endorphin concentrations in both eumenorrheic and anovulatory oligomenorrheic athletes. It is possible that anovulatory oligomenorrheic soccer players are more susceptible to exercise-related increase in ОІ-endorphin concentration. The large post-stress increase in hormone concentrations noted in eumenorrheic SP supports that the integrity of hypothamic-pituitary-gonadal axis is necessary for a ready response to exercise-induced stress. Paragraph 26: Our results in soccer players confirm the known effects of chronic exercise on menstrual irregularities. Because no significant differences in body composition were found between soccer players and controls, the data suggests that the type of training (aerobic versus aerobic/anaerobic) can significantly affect the menstrual status of women involved in competitive sports. Soccer appears as a very useful model to study exercise-associated menstrual dysfunction, because of the marked differences among training programmes according to positional roles. The size of soccer teams (at least 15 subjects) may allow assessing the effects of different training patterns in a relatively homogeneous context (i.e same trainer and frequency of training sessions, etc). Longitudinal studies are warranted to better define soccer-induced menstrual abnormalities and the impact of progressive specific training according to positional roles in the team. Acknowledgments The Authors gratefully thank: Dr. Rosario Giuliano, Specialist in Sport Medicine for his help in data collection and analysis. 305 Arcangelo Benigno, Attilio Licciardi, Giuseppe Morici References [1] Bonen A. Recreational exercise does not impair menstrual cycle: a prospective study. Int J Sports Med 1992; 13: 100-120. [2] Lehmann M, Knizia K, Gastmann U. Influence of 6 week, 6 days per week, training on pituitary function in recreational athletes. Br J Sports Med 1993; 27: 186-192. [3] Warren MP, Perlroth NE. The effects of intense exercise on the female reproductive System. 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J Sports Med Phys Fitness 1998; 38: 294-7. 306 Effects of specific training on hormone levels in female athletes [22] Chen EC, Brzyski RG. Exercise and reproductive dysfunction. Fertil Steril 1999; 71:1-6. [23] Dent RRM, Guilleminault C, Albert LH, Posner BI, Cox BM, Goldstein A. Diurnal rhythm of plasma immunoreactive ОІ-endorphin and its relationship to sleep stages and plasma rhythms of cortisol and prolactin. J Clin Endocrinol Metab 1982; 52: 942-7. [24] Bonen A. Exercise-induced menstrual cycle changes: a functional temporary adaptation to metabolic stress. Sports Med 1994; 17: 373-392. [25] De Cree C. Endogenous opioid peptides in the control of normal menstrual cycle and their possible role in athletic menstrual irregulation. Obstet Gynecol 1989; 44: 720-728. [26] Loucks AB, Heath EM, Law T. Dietary restriction reduces luteinizing hormone (LH) pulse frequency, during waking hours and increase LH pulse amplitude during sleep in young menstruating women. J Clin Endocrinol Metab 1994; 78: 910-915. [27] Hakkinen K, Pakarinen A. Acute hormonal responses to heavy resistance exercise in men and women at different age. Int J Sports Med 1995; 16: 507-513. [28] Raynaud E, Mannier JF, Brun JF. Biochimie et hormonologie de lвЂ™exercise submaximal: standardisation dвЂ™un test dвЂ™effort chez le sportif. Science & Sports 1997; 12: 72-74. 307 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 BENEFICIAL EFFECTS OF LOW INTENSITY ENDURANCE TRAINING IN ADULT MDX MICE [Effetti positivi dellвЂ™allenamento dвЂ™endurance leggero in topi adulti mdx] Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno and Attilio Licciardi Department of Experimental Medicine (Di.Me.S), University of Palermo (IT) Key words: Training, Strength, Degree of fatigue, Body weight, Muscular dystrophy Parole chiave: Allenamento, Forza, Grado di fatica, Peso corporeo, Distrofia muscolare Abstract. High intensity exercise induces muscle damage in mdx mice; however, the effects of low intensity training on the contractile properties of mdx mice muscles are still controversial. The aim of this study was to examine whether low intensity endurance training had beneficial effects on strength, fatigue and body weight in adult mdx mice. Wild-type (C57BL/10) and mdx mice (C57BL/10Sc-Dmdmdx/J) ran daily on a motorized Rota-Rod under standard laboratory conditions (temperature: 20В° C, humidity: 50%). Exercise bouts were performed daily five times per week for a total time period of 4 weeks at progressively increasing loads. To study the recovery effects, mice were also observed after two weeks of cessation of training. At week 3, sedentary mdx mice (MDX-SD) showed a lower grip strength fatigue (-2.8 В± 33.5 %) compared to both exercised mdx mice (MDX-EX) (23.2 В± 13.6 %, p<0.05) and exercised wild-type mice (WT-EX) (30.3 В± 18.4 %, p<0.05). Conversely, after recovery MDX-SD group showed a grip strength fatigue (39.9 В± 7.3 %) significantly greater than MDX-EX (11.1 В± 1.1 %, p<0.05), WT-EX (19.2 В± 15.5 %, p<0.05) and sedentary wild-type mice (WT-SD) groups (6.9 В± 15.2 %, p<0.05). In conclusion, low intensity endurance training in mdx mice induced physiological adaptations resulting in a trend to lower body weight gain and in a positive effect on the degree of fatigue. These preliminary data provided evidence for beneficial effects of low intensity endurance training in adult mdx mice. Riassunto: LвЂ™esercizio molto intenso determina danno muscolare nei topi mdx; comunque, gli effetti di un allenamento di bassa intensitГ sulle proprietГ contrattili dei muscoli di topi mdx sono ancora controversi. Lo scopo di questo studio ГЁ stato quello di osservare se lвЂ™allenamento di resistenza leggero avesse effetti positivi sulla forza, la fatica e il peso corporeo in topi mdx adulti. Topi allenati wild-type (C57BL/10) e mdx (C57BL/10Sc-Dmdmdx/J) correvano giornalmente su un Rota-Rod motorizzato in condizioni di laboratorio standard ( temperatura 20В°C, umiditГ 50%). Gli allenamenti sono stati effettuati giornalmente cinque volte la settimana per un periodo totale di 4 settimane aumentando il carico in modo progressivo. Per osservare gli effetti del recupero, i topi sono stati studiati dopo due settimane di cessazione dellвЂ™allenamento. Alla terza settimana di allenamento, il gruppo dei topi mdx sedentari (MDX-SD) mostrava una ridotta fatica al grip strength (-2.8 В± 33.5 %) sia rispetto al gruppo dei topi mdx allenati (MDX-EX) (23.2 В± 13.6 %, p<0.05) che al gruppo dei topi wild-type allenati (WT-EX) (30.3 В± 18.4 %, p<0.05). Invece, dopo il recupero, il gruppo MDX-SD mostrava una maggiore fatica al grip strength (39.9 В± 7.3 %) rispetto ai gruppi MDX-EX (11.1 В± 1.1 %, p<0.05), WT-EX (19.2 В± 15.5 %, p<0.05) e wild-type sedentari (WT-SD) (6.9 В± 15.2 %, p<0.05). In conclusione, lвЂ™allenamento di resistenza leggero ha indotto nei topi mdx adattamenti fisiologici quali una tendenza ad un ridotto accrescimento ponderale e un effetto positivo sul grado di fatica. Questi dati preliminari hanno evidenziato degli effetti benefici dellвЂ™allenamento di resistenza leggero nel topo adulto mdx. 309 Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno, Attilio Licciardi Introduction Paragraph 1: Dystrophin-deficient (mdx) mice are the classical models for Duchenne muscular dystrophy (DMD). The mdx mice have a mutated dystrophin gene and are therefore genetically valid models of DMD. Although these animals have been useful for genetic and pharmacologic treatment manipulations nevertheless they have not been considered an ideal clinical model [1]. Paragraph 2: Young mdx mice (3-4 weeeks of age) show muscle weakness and appear more susceptible to fatigue (increased myofibre necrosis) than control mice, in mdx adult mice (over 8 weeks of age) this acute phase decreases significantly to a chronic low level of damage, <10% muscle affected, which persists throughout life but is further decreased by 1 year of age [2]. The onset of progressive replacement of muscle by fibrous connective tissue is reported in limb muscle of mdx mice from 10-13 weeks with extensive fibrosis from 16 to 20 months of age [3]. No obvious clinical weakness is apparent until 18 months. Paragraph 3: As DMD results in progressive skeletal muscle atrophy and weakness, appropriate exercise may be a suitable therapy. A number of studies (Table 1) have shown that muscle from mdx mice adapts differently to exercise. These discrepancies in the literature may be explained by differences in experimental procedures: duration, intensity and type of exercise, variations in age of mdx mice, different environmental conditions. However, whether exercise therapy is beneficial or detrimental to dystrophic skeletal muscle needs to be further documented. Paragraph 4: Moreover, Connolly et al. showed that mdx mice at 8-10 weeks of age weighed significantly more than age-matched normal mice. This excess weight-gain was related to chronic under-activity in mdx mice [4]. Paragraph 5: The aim of this study was to examine whether regular low intensity endurance training had beneficial effects on strength, fatigue and body weight in adult mdx mice. Table 1. Studies on the effects of different training protocols in mdx mice Methods Author, year [ref] Starting age (week) Exercise Time Weeks of Exercise (min per day) training Main results Training sessions per week Intensity Granchelli JA et al. 2000 [5] 4 running 30 6 2 12 m/min 19 % decrease in strength Kaczor JJ et al. 2007 [6] 4 running 30 8 2 9 m/min Decreased lipid and protein oxidative damage 310 Beneficial effects of low intensity endurance training in adult mdx mice De Luca A et al. 2003 [7] 3-4 running 30 4-8 2 12 m/min Muscular damage. Mdx mice significantly weaker than wilde-type Dupont-Versteegden EE et al., 1994 [8] 3 running voluntary - - 5 km/day (approximately) No deleterious effects on soleus muscle function Call JA et al. 2008 [9] 3 running voluntary 3 - 1-2 km/day Increased running distance Hayes A and Williams DA, 1996 [10] 4 running voluntary 16 - Hayes A and Williams DA, 1998 [11] 40-96 swimming until exhaustion 10 7 Faist V et al. 2001 [12] 4 (young) and 16 (adult) Wineinger MA et al. 1998 [13] 8-62 running running - voluntary 6 52 7 7 Force output improvement of 4 km/day at soleus muscle and greater 1.6 km/h (ap- fatigue resistance of soleus proximately) muscle and of the extensor digitorum longus until exhaustion No detrimental effects 480 m/day Mitochondrial oxygen consumption was significantly lower in skeletal muscle of exercised young mdx mice - Beneficial effects on both the old mdx mice and their controls. 9% reduction in the fatigability of the extensor digitorum longus of the old mdx mice after the exercise Methods Mice Paragraph 6: Experiments were carried out in our laboratory (temperature: 20В°C, humidity: 50%) using 31 male dystrophic mdx mice (C57BL/10ScSn-Dmdmdx/J from Jackson Laboratories) and 35 male wild type mice (C57BL/10ScSn from Harlan Italy). All mice were housed under a 12h day-night cycle and allowed access to food and water ad libitum. Experiments were carried out in agreement with the European Communities Counsil Directive (86/609/EEC) regarding care and use of animals for experimental procedures. Exercise training protocol Paragraph 7: At 10 weeks of age mdx mice were randomly assigned to sedentary (n=17; MDX-SD) or trained (n=14; MDX-EX) groups. At 14 weeks of age wild type mice were randomly assigned to sedentary (n=19; WT-SD) or trained (n=16; WT-EX) groups 311 Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno, Attilio Licciardi too. Preliminarily all mice underwent a 2-week period of acclimatization trial at very low speed. Paragraph 8: To avoid the confounding effects of the accelerated necrotic stage of the disease present in young mdx mice (3-4 weeks of age) and the hormone effects on skeletal muscle typical of pre-adolescence and puberty in mice (5-7 weeks of age) [1] we tested the effects of low intensity endurance training in adult mice (> 8 weeks of age). Paragraph 9: Training was performed in the same room where the mice were housed using a motorized rotor (Rota-Rod; Ugo Basile, Biological Research Apparatus, Comerio Varese, Italy). Mice ran 5 days/week for 4 weeks at progressively increasing loads. In order to study the recovery effects, mice were housed for more 2 weeks after completion of the exercise training protocol described above (Table 2). Weight Paragraph 10: Wild-type and mdx mice were weighed at the start time of exercise training protocol and after 2, 3 and 4 weeks of training. Mice were still weighed after the recovery period. Forelimb strength and fatigue Paragraph 11: All mice groups were examined for forelimb strength at 1, 3 and 4 weeks of training and after recovery. Briefly, the mouse was placed on a metal grid attached to a force transducer (Grip strength meter; Ugo Basile, Biological Research Apparatus, Comerio Varese, Italy), and pulled gently from the base of the tail until release. Because the increase in strength paralleled somatic growth in normal animals, we analyzed the strength of the mdx and controls as a function of weight. Each mouse underwent five trials and peak grip strength was normalized for body weight (force/BW) and recorded as the average of the three best efforts divided by the mouseвЂ™s body weight. Grip strength fatigue was calculated by comparing the first two pulls to the last two pulls [4]. The decrement between pulls 1 + 2 and pulls 4 + 5 gives a measure of fatigue. In the formula (4 + 5)/(1 + 2), animals with no fatigue have a value of 1. For convenience, the value resulted from the formula above was subtracted from the value of вЂњ1вЂќ so that an animal without fatigue has a calculated fatigue value of 0, and an animal that can only complete three pulls, because completely fatigued, has a value of вЂњ1вЂќ. The fatigue was expressed as percentage so that a value of 0% fatigue would be assigned to mice without fatigue and a value of 100% fatigue to mice with complete fatigue. Statistical analysis Paragraph 12: Results are reported as mean В± SD. Analysis of variance (ANOVA) and FisherвЂ™s PLDS correction for post hoc comparison were used to test for differences between sedentary wild type (WT-SD), trained wild type (WT-EX), sedentary dystrophic mice (MDX-SD) and trained dystrophic mice (MDX-EX) at different time points. Time trends in 312 Beneficial effects of low intensity endurance training in adult mdx mice each group were assessed by paired t-test (Statview 5.0.1; SAS Institute, Inc., Cary, NC). Significance was at p<0.05. Table 2. Training protocol on the rotarod apparatus. Week Session time (min) Rotations/min (RPM) Length (m) Speed (m/min) 1 30 20 120 4 2 45 20 180 4 3 60 20 240 4 4 60 24 288 4,8 5 recovery recovery recovery recovery 6 recovery recovery recovery recovery Results Body weight and exercise Paragraph 13: As shown in Figure 1, at the beginning of exercise training protocol, body weight in MDX-EX (26.2 В± 2.2 g, p<0.05) and MDX-SD (24.8 В± 1.9 g, p<0.001) was lower compared to WT-EX (27.9 В± 1.8 g) and WT-SD (28.0 В± 2.0 g). After 2 weeks of training, body weight was lower in MDX-SD (26.8 В± 2.0 g) than in WT-SD (28.8 В± 2.3 g, p<0.05). After 3 and 4 weeks of training and after recovery no significant differences were found between groups; however, in MDX-EX body weight tended to be lower than in MDX-SD. Forelimb strength Paragraph 14: After 1, 3 and 4 weeks of training and after recovery there was no significant difference in forelimb strength (FS) between all groups. The time course of forelimb strength was analysed for each group of mice. The main variations were found between week 1 and 3, and reported as follows. In MDX-EX, FS was 63.9 В± 14.3 g at week 1 and 51.6 В± 8.3 g at week 3 (p<0.05); in WT-EX, FS was 64.2 В± 15.7 g at week 1 and 49.5 В± 16.6 g at week 3 (p<0.0001); in WT-SD, FS was 61.2 В± 13.5 g at week 1 and 47.4 В± 9.8 g at week 3 (p<0.05); in MDX-SD, FS did not change at any time point. Forelimb strength normalized for weight Paragraph 15: Forelimb strength normalized for body weight (FSW, gram strength per gram of body weight) did not show significant difference at any time point of the training protocol and after recovery between all groups. The time course of FSW was analysed for each group of mice. The main variations were found between week 1 and 3, and reported as follows. In MDX-EX, FSW was 2.40 В± 0.61 g/g at week 1 and 1.88 В± 0.34 g/g at week 3 (p<0.05); in WT-EX, FSW was 2.29 В± 0.57 g/g at week 1 and 1.76 В± 0.57 g/g at week 3 313 Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno, Attilio Licciardi (p<0.0001); in WT-SD, FSW was 2.15 В± 0.46 g/g at week 1 and 1.64 В± 0.28 g/g at week 3 (p<0.05). In MDX-SD, FSW did not change at any time point. Fatigue Paragraph 16: Grip strength fatigue is reported in Figure 2. At week 3, MDX-SD showed a lower grip strength fatigue (-2.8 В± 33.5 %) than both MDX-EX (23.2 В± 13.6 %, p<0.05) and WT-EX (30.3 В± 18.4 %, p<0.05). Conversely, after recovery, grip strength fatigue was significantly greater in MDX-SD compared to all groups. In particular, MDX-SD showed a greater fatigue (39.9 В± 7.3 %) than MDX-EX (11.1 В± 1.1 %, p<0.05), WT-EX (19.2 В± 15.5 %, p<0.05) and WT-SD (6.9 В± 15.2 %, p<0.05) (Fig. 3). In addition, the time course of fatigue was analyzed for each group of mice. A reduced grip strength fatigue was observed in MDX-EX after recovery compared with week 3 (11.1 В± 1.1 % vs 23.2 В± 13.6 %, respectively; p<0.05). Discussion Paragraph 17: The main finding of this study is that forced low intensity endurance training, without any corticosteroid and/or nutritional therapies may have beneficial effects in adult mdx mice. Paragraph 18: It has been previously reported that voluntary endurance exercise in mdx mice induced greater force-generating capacity of soleus muscles and more fatigue resistance in extensor digitorum longus [10,13]. Other studies showed that voluntary wheel running produced no effects on soleus or extensor digitorum longus muscle function in mdx mice [8,14]. In a recent study Landisch and co-workers [15] hypothesized that a вЂњvoluntary, endurance type of exercise training would improve mdx mouse muscle to the same extent that exercise benefited muscle from healthy control miceвЂќ. In contrast, high intensity forced treadmill running, especially with episodes of eccentric contractions, induced or accelerate muscle damage in mdx mice [7,16,17,18]. Paragraph 19: Moreover, an excess of weight-gain and obesity in mdx mice at 8-10 weeks of age was related to their chronic under-activity [4]. Paragraph 20: However, it is not currently known whether exercise therapy in mdx mice is beneficial or detrimental to dystrophic skeletal muscle. Muscle from mdx mice adapts differently to exercise, depending on the exercise protocol and age. At present, exercise parameters (type, duration, intensity, frequency) need to be defined. Paragraph 21: To fill this gap, we have trained adult mdx mice according to a low intensity endurance protocol to determine whether beneficial physiological adaptation may be induced. Moreover, we tested the effects of low intensity endurance training in adult mice (> 8 weeks of age) to avoid the confounding effects of acute skeletal muscle necrosis present in young mdx mice (3-4 weeks of age) and the hormone effects on skeletal muscle 314 Beneficial effects of low intensity endurance training in adult mdx mice typical of pre-adolescence and puberty in mice (5-7 weeks of age). Paragraph 22: The novel finding of our study in mdx mice is that the low intensity endurance training protocol proposed had not detrimental effects on forelimb strength. Indeed, in exercised mdx mice we found: a) lower grip-strength fatigue after recovery compared to sedentary mdx mice; b) a trend toward an increased forelimb strength after 4 weeks of low intensity endurance training; c) a body weight similar to that of exercised wild-type mice with no evidence of obesity. Paragraph 23: In conclusion, our preliminary data suggest that low intensity endurance training had beneficial effects on skeletal muscle of adult mdx mice. Therefore, remediation of skeletal muscle weakness and fatigue changes may represent a physiologic adaptive response to specific training in mdx mice. These results may be relevant for therapeutic exercise in patients with Duchenne Muscular Dystrophy but further studies are necessary to determine the intensity and/or duration of exercise in humans. В 315 Beneficial effects of low intensity endurance training in adult mdx mice Figure 1. Body weight changes in sedentary and exercised mdx and wild type mice during training and recovery periods. Data are expressed as means. MDX-SD: sedentary mdx mice; MDX-EX: exercised mdx mice; WT-SD: sedentary wild type mice; WT-EX: exercised wild type mice. Figure 2. Grip strength fatigue in sedentary and exercised mdx and wild type mice during training and recovery periods. Data are expressed as means. MDX-SD: sedentary mdx mice; MDX-EX: exercised mdx mice; WT-SD: sedentary wild type mice; WT-EX: exercised wild type mice. Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno, Attilio Licciardi Figure 3. Fatigue of forelimb strength in sedentary and exercised mdx and wild type mice after 3 weeks of training (upper panel; * p<0.05 vs MDX-EX and WT-EX) and after recovery (lower panel; * p<0.05 vs all groups). Data are expressed as means В± SD. MDX-SD: sedentary mdx mice; MDX-EX: exercised mdx mice; WT-SD: sedentary wild type mice; WT-EX: exercised wild type mice. Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno, Attilio Licciardi References [1] Grounds MD, Radley HG, Lynch GS, Nagaraju K, De Luca A. Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy. Neurobiol Dis 2008 Jul; 31(1):1-19. [2] McGeachie JK, Grounds MD, Partridge TA, Morgan JE. Age-related changes in replication of myogenic cells in mdx mice: quantitative autoradiographic studies. J Neurol Sci 1993; 119:169-79. [3] Lefaucheur JP, Pastoret C, Sebille A. Phenotype of dystrophinopathy in old mdx mice. Anatom Rec 1995; 242:70-76. [4] Connolly AM, Keeling RM, Mehta S, Pestronk A, Sanes JR. Three mouse models of muscular dystrophy: the natural hystory of strength and fatigue in dystrophin-, dystrophin/utrophin-, and laminin О± 2-deficient mice. Neuromuscul Disord 2001; 11:703-12. [5] Granchelli JA, Pollina C, Hudecki MS. Pre-clinical screening of drugs using the mdx mouse. Neuromuscul Disord 2000; 10 (4-5):235-9. [6] Kaczor JJ, Hall JE, Payne E, Tarnopolsky MA. Low intensity training decreases markers of oxidative stress in skeletal muscle of mdx mice. Free Radic Biol Med 2007; 43 (1):145-54. [7] De Luca A, Pierno S, Liantonio A, Cetrone M, Camerino C, Fraysse B, Mirabella M, Servidei S, Ruegg UT, Conte Camerino D. Enhanced dystrophic progression in mdx mice by exercise and beneficial effects of taurine and insulin-like growth factor-1. J Pharmacol Exp Ther 2003; 304 (1):453-63. [8] Dupont-Versteegden EE, McCarter RJ, Katz MS. Voluntary exercise decreases progression of muscular dystrophy in diaphragm of mdx mice. J Appl Physiol, 1994; 77 (4):1736-41. [9] Call JA, Voelker KA, Wolff AV, McMillan RP, Evans NP, Hulver MW, Talmadge RJ, Grange RW. Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract. J Appl Physiol 2008; 105 (3):923-32. [10] Hayes A, Williams DA. Beneficial effects of voluntary wheel running on the properties of dystrophic mouse muscle. J Appl Physiol 1996; 80 (2):670-9. [11] Hayes A , Williams DA. Contractile function and low-intensity exercise effects of old dystrophic ( mdx) mice. Am J Physiol 1998; 274 (4 Pt 1):C1138-44. [12] Faist V, Konig J, Hoger H, Elmadfa I. Decreased mitochondrial oxygen consumption and antioxidant enzyme activities in skeletal muscle of dystrophic mice after low-intensity exercise. Ann Nutr Metab 2001; 45 (2):58-66. [13] Wineinger MA, Abresch RT, Walsh SA, Carter GT. Effects of aging and voluntary exercise on the function of dystrophic muscle from mdx mice. Am J Phys Med Rehabil 1998; 77 (1):20-7. [14] Carter GT, Wineinger MA, Walsh SA, Horasek SJ, Abresch RT, Fowler WM Jr. Effect of voluntary wheelrunning exercise on muscles of the mdx mouse. Neuromusc Disord 1995; 5 (4):323-32. [15] Landisch RM, Kosir AM, Nelson SA, Baltgalvis KA, Lowe DA. Adaptive and nonadaptive responses to voluntary wheel running by mdx mice. Muscle Nerve 2008; 38:1290-303. [16] Vilquin JT, Brussee V, Asselin I, Kinoshita I, Gingras M, Tremblay JP. Evidence of mdx mouse skeletal muscle fragility in vivo by eccentric running exercise. Muscle Nerve 1998; 21:567-76. [17] Ridgley JA, Pinniger GJ, Hamer PW, Grounds MD. The physiological effects of IGF-1 (class 1:Ea transgene) over-expression on exercise-induced damage and adaptation in dystrophic muscles of mdx mice. Pflugers Arch 2008. DOI 10.1007/s00424-008-0568-4. [18] Payne ET, Yasuda N, Bourgeois JM, Devries MC, Rodriguez MC, Yousuf J, Tarnopolsky MA. Nutritional therapy improves function and complements corticosteroid intervention in mdx mice. Muscle Nerve 2006; 33:66-77. 318 Invited article and report Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis [Epigenetica della Galectina-3. Un nuovo meccanismo regolatorio della sua trascrizione] Hafiz Ahmed University of Maryland, Biotechnology Institute, Baltimore (USA) Key words: Epigenetics, Galectin-3, Carcinogenesis, Promoter methylation, Prostate cancer Parole chiave: Epigenetica, Galectina-3, Carcinogenesi, Promoter della metilazione, Carcinoma prostatico Abstract. Protein-carbohydrate interactions play significant role in modulating cell-cell and cell-extracellular matrix interactions, which, in turn, mediate various biological processes such as growth regulation, immune function, cancer metastasis, and apoptosis. Galectin-3, a member of a b-galactoside-binding protein family, is found multifunctional and is involved in normal growth development as well as cancer progression and metastasis, but the detailed mechanisms of its functions or its transcriptional regulations are not well understood. Besides, several regulatory elements such as GC box, CRE motif, AP-1 site, and NF-ОєB sites, the promoter of galectin-3 gene contains several CpG islands that can be methylated during tumorigenesis leading to the gene silencing. This review discusses the galectin-3 epigenetics, which represents a novel regulatory mechanism of its transcription. Riassunto. Le interazioni proteine-carboidrato giocano un ruolo significativo nella modulazione delle interazioni cellula-cellula e cellula-matrice extracellulare, che, in definitiva, mediano i vari processi biologici quali la regolazione della crescita, la funzione immunitaria, la metastatizzazione tumorale e lвЂ™apoptosi. La galectina-3, un membro della famiglia delle proteine leganti il b-galactoside, ГЁ una molecola multifunzionale ed ГЁ coinvolta nei processi fisiologici di sviluppo cosГ¬ come nella progrressione tumorale e nella metastatizzazione, ma i meccanismi dettagliati delle sue funzioni o delle sue regolazioni trascrizionali non sono stati ancora ben compresi. Oltre a diversi elementi regolatori come il box CG, il motivo CRE, il sito per AP-1 e i siti per NF-kB, il promotore del gene per la galectina-3 contiene diverse isole CpG che possono essere mutilate durante la tomorigenesi, portando al silenziamento del gene. Questa review tratta lвЂ™epigenetica della galectina-3, che rappresenta un nuovo meccanismo regolatorio della sua trascrizione. Introduction Interactions between cells, and between cells and the extracellular matrix (ECM) are pivotal for proper cellular function. In recent years, protein-carbohydrate interactions have been considered as very important for modulation cell-cell and cell-ECM interactions (Fig. 1), which, in turn, mediate various biological processes such as cell activation, growth regulation, cancer metastasis, and apoptosis. Thus, the identification of carbohydratebinding proteins (lectins) and their partners (carbohydrate ligands), and the detailed un321 Hafiz Ahmed derstanding of the molecular mechanisms and downstream effects of these protein-carbohydrate interactions are subjects of current intense research. Galectins (gal), a family of b-galactoside-binding proteins, are involved in growth development as well as cancer progression and metastasis [1-5]. However, the detailed mechanisms of these functions remain largely unknown. Of the fifteen members of the galectin family identified so far, gal1, 2, 5, 7, 10, 11, 13, 14, and 15 are examples of the вЂњprotoвЂќ type galectins (one carbohydrate-recognition domain [CRD] per subunit), while Gal4, 6, 8, 9, and 12 are вЂњtandemrepeatвЂќ type galectins, which contain two CRDs [6]. Gal3 is the only representative of the вЂњchimeraвЂќ galectin type, containing one CRD connected to a collagen-like sequence rich in proline and glycine [7]. Gal3, previously known as Mac-2, L-29, L-31, L-34, IgE binding-protein, CBP35, and CBP30, is a very interesting multifunctional molecule and probably the most studied member of the galectin family [2,8,9]. Gal3 plays an important role in normal development and tumorigenesis through regulating cell proliferation, apoptosis, cell adhesion, invasion, angiogenesis and metastasis by binding to the cell surface b-galactose-containing glycoconjugates or glycolipids. Gal3 may exert its multiple biological roles intracellularly within the nucleus or the cytoplasm, or after its secretion, at the cell surface and/or the extracellular space, mediating interactions between cells and the extracellular matrix [3,4,6]. Its expression in normal and tumor tissues and regulation of its expression including the newly discovered epigenetic regulation in some tumor tissues have been discussed in this review. Gal3 expression in normal tissues- role in growth development Gal3 is developmentally regulated and expressed in many tissues of adults [10,11]. During mouse embryogenesis, gal3 first appears at fourth day of gestation in the trophectoderm of blastocyst, followed by its expression in the notochord cells between 8.5 and 11.5 days of gestation [10]. In later stages of mouse development, gal3 is expressed in the cartilage, ribs, facial bones, the suprabasal layer of epidermis, the endodermal lining of the bladder, larynx and oesophagus [9]. In adult, gal3 is mainly expressed in the epithelial cells such as small intestine, colon, cornea, kidney, lung, thymus, breast, and prostate [9]. The expression of gal3 is also detected in ductal cells of salivary glands, pancreas, kidney, and eye and in intrahepatic bile ducts [9]. Regarding cell type, gal3 expression is observed in fibroblasts, chondrocytes and osteoblasts, osteoclasts, keratinocytes, Schwann cells and gastric mucosa, endothelial cells, and also immune related cells such as neutrophils, eosinophils, basophils and mast cells, Langerhans cells, dendritic cells, as well as monocytes and macrophages from different tissues [3,9,12]. 322 Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis Gal3 expression (increased or decreased) in tumors- role in tumor progression and metastasis Gal3 mediates homotypic and heterotypic aggregation and promotes tumor cells endothelial interactions, angiogenesis, and tumor metastasis. It was shown that cell surface gal3 mediates homotypic cell adhesion by binding to soluble complementary glycoconjugates [13]. Interactions of metastatic cancer cells with vasculatory endothelium are critical during early stages of cancer metastasis. Recent studies demonstrate that the endothelium gal3 participates in docking of cancer cells including breast and prostate cancers on capillary endothelium by specifically interacting with cancer cells-associated ThomsenFriedenreich disaccharide (Gal b1,3GalNAc) [14]. Intracelluar gal3 in cancer cells confers anti-apoptotic activity. For example, gal3 acts as a specific binding partner of activated K-Ras and this interaction promotes strong K-Ras activation of PI3K (phosphoinositide 3-kinase) [15]. Gal3 is the only member of the galectin family that contains the NWGR antideath domain of the Bcl-2 family. Bcl-2 translocation to the mitochondrial membrane leads to anti-apoptosis activity resulting from blocking cytochrome c release [8]. In human breast carcinoma BT547 cells, gal3 can also inhibit cytochrome c release thereby protecting cells against nitric oxide-induced apoptosis [8]. Moreover, gal3 binds Bcl-2 protein in vitro to form heterodimers and thus behaves as a mitochondria-associated apoptotic regulator in the cytoplasm. Interestingly, synexin (annexin 7, a Ca2+- and phospholipid-binding protein), is required for gal3 prevention of mitochondrial damage [8]. Extracellular gal3 secreted from tumor cells induces apoptosis of cancer-infiltrating T-cells suggesting its role in the immune escape mechanism during tumor progression [16]. Expression of gal3 is observed in many tumors. However, the intensity of the gal3 expression in tumors depends on the type of tumor, its invasiveness and metastatic potential [17,18]. For example, increased expression of gal3 is observed in colon, head and neck, gastric, endometrial, thyroid, liver, bladder cancers and breast carcinomas [17,18]. Gal3 plays an important role in tumor progression and metastasis in many cancers such as breast cancer, colon cancer, and human brain tumors [2,4,8,12,17,18]. Gal3 transfected human breast cancer cells BT549, which is gal3 null, after intrasplenic injection, formed metastatic colonies in the liver, while gal3 null BT549 cells did not [19]. In colorectal cancer, gal3 expression in the primary lesions of the cancer was significantly increased compared to the normal tissue, correlating with the progression of clinical stage, liver metastasis, venous invasion, and lymph node metastasis [17,18]. The expression of the endogenous gal3 also correlates with the malignant potential and metastasis of tumors in the central nervous system. Change in cellular localization of gal3 is observed during progression of various cancers. Down-regulation of gal3 expression has been demonstrated in colorectal cancer, with increased cytoplasmic expression of gal3 at more advanced stages [17,18]. 323 Hafiz Ahmed In tongue cancer, nuclear gal3 is decreased, but cytoplasmic gal3 is increased during progression from normal to cancer [17,18]. The decreased expression of galectin-3 is known so far in prostate [20-22], kidney [23], and pituitary cancers [24]. Recent data by us and others indicated that decreased expression of gal3 in pituitary and prostate tumors is, in part, due to its gal3 promoter methylation [22,24,25, please see described later]. Transcriptional regulation of gal3 expression Although a large body of data about gal3 expression is available in the literature, the mechanisms of regulation of gal3 expression are not well understood. However, the expression of gal3 depends on cell type, external stimuli and environmental conditions and involves numerous transcription factors and signaling pathways. Gal3 expression may serve as differentiation marker for certain cell types. For example, the differentiation of the human monocytes or promyelocytic cell line HL-60 to macrophage-like cells induced by phorbol ester is accompanied by increased expression of gal3 [26]. Gal3 expression is upregulated in phagocytic macrophages and thus considered as a вЂњmacrophage activation markerвЂќ [9]. Gal3 expression is also elevated in microglia and macrophages activated by phagocytosis of myelin or when exposed to granulocyte-macrophage colony-stimulating factor [9]. In contrast, activation of human monocots by lipopolysaccharide and interferon-Оі is accompanied by decrease of gal3 expression [9]. The reduced expression of gal3 was also observed in monocytic THP-1 cells treated with non-steroidal or corticosteroidal antiinflammatory drugs [9]. Interestingly, gal3 expression is absent or barely detected in the resting lymphocytes, but the activated B and T cells induce gal3 expression [9]. In the promoter region of the gal3 gene, several regulatory elements such as five putative Sp1 binding sites (GC boxes), five cAMP-dependent response element (CRE) motifs, four AP-1- and one AP-4-like sites, two NF-ОєB-like sites, one sis-inducible element (SIE) and a consensus basic helixвЂ“loopвЂ“helix (bHLH) core sequence are found [27]. The presence of multiple GC box motifs for binding ubiquitous expressed Sp1 transcription factor is a characteristic of constitutively expressed вЂњhousekeepingвЂќ genes. The activation of the Sp1 binding transcription factor is responsible for gal3 induction by Tat protein of HIV [9]. The SIE that binds sisinducible factors was suggested to be a possible candidate for the growth-induced activation of gal3 gene expression, caused by the addition of serum. The presence of CRE and NF-ОєB-like site in the gal3 promoter suggests that the activation of gal3 expression could be regulated through the signaling pathways involving the cAMPresponse element-binding protein (CREB) or the NF-ОєB transcription factor. The CREB/ATF and the NF-ОєB/Rel transcription factors pathways may be involved in the regulation of 324 Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis gal3 expression by the Tax protein during HTLV-I infection of T cells [9]. The involvement of the NF-ОєB transcription factor in regulation of gal3 expression, as well as the Jun protein, a component of AP-1 transcription factor has recently been confirmed [9]. The regulation of gal3 expression through the NF-ОєB transcription factor was shown to be mediated by nucling, a novel apoptosis-associated protein, which interferes with NF-ОєB via the nuclear translocation process of NF-ОєB/p65, thus inhibiting galectin-3 expression on both protein and mRNA level [9]. In skeletal tissues, the regulation of gal3 expression is mediated by the transcription factor Runx2 [9]. Very recently, gal3 expression is found to be regulated in pituitary and prostate tumors by methylation of CpG islands in promoter region [22, 24, 25], which is described later in a greater detail. Epigenetic events- DNA methylation in the promoter results in transcriptional inactivation Epigenetic mechanisms such as DNA methylation and histone modification play essential roles in many molecular and cellular alterations associated with the development and progression of cancer [28]. DNA methylation (the addition of a methyl group to the carbon-5 position of cytosine residues) refers to the covalent binding of a methyl group specifically to the dinucleotide CpG (Fig. 2). This is catalyzed by a family of enzymes, the DNA methyltransferases (DNMTs) [29]. Two types of DNA methylation alterations have been demonstrated in human cancers. The first refers to global hypomethylation where the genomes of cancer cells show decreased methylation compared to normal cells [30]. This hypomethylation is primarily due to the loss of methylation in repetitive elements and other non-transcribed regions of the genome, which results in genomic instability. The second type of methylation alteration in cancer cells is the methylation of CpG islands that lie in promoter regions of tumor suppressor and other regulatory genes that are normally unmethylated. The promoter regions of these genes are inactivated by methylation and their gene expression silenced. This is referred to as gene hypermethylation. It is thought that DNA methylation alters chromosome structure and defines regions for transcriptional regulation. Clusters of CpG sites are found dispersed around the genome and are referred to as CpG islands [31]. These islands are found in the promoter region of about 60% of genes, in exons and introns, and in repetitive elements. In normal cells, most CpG islands in the promoter regions are unmethylated whereas CpG islands in intronic regions and repetitive sequences are heavily methylated, perhaps to help the cell identify regions for gene transcription. The suppression of transcription by DNA methylation is probably due to direct inhibition of transcription factor binding [32]. However, some transcription factors are insensitive to methylation [32], and many gene promoters do not have CpG sites within their recognition sequences. Recently, a more generally applicable mechanism by which 325 Hafiz Ahmed DNA methylation can maintain transcriptional repression has been proposed. A family of proteins known as methylbinding domain (MBD) proteins specifically binds to DNA containing methylated CpG sites [33]. At least three of the five known members of this family (MeCP2, MBD2 and MBD3) have been shown to be associated with large protein complexes containing histone deacetylase (HDAC1 and HDAC2) and chromatin-remodeling (Sin3a and mi-2) activities [34]. A large number of genes (such as DNA repair and tumor suppressor genes) have been found hypermethylated in cancer. These genes are correlated with pathological grade or clinical stage, and thought to contribute to initiation and progression of the disease [35]. Expression of gal3 gene is transcriptionally regulated by DNA methylation. Gal3 was shown highly expressed in androgen independent PC-3 and DU-145 cells, but weakly expressed in androgen dependent LNCaP cells [25]. Treatment of LNCaP cells with azacytidine (DNA methyltransferase inhibitor) showed restored expression of gal3 indicating that the promoter methylation is responsible for gal3 gene silencing [25] (Fig. 3). We have also demonstrated DNA methylation on the gal3 promoter in LNCaP cells. For this purpose, DNA was treated with sodium bisulfite and subsequently amplified by PCR [25, 36] with primer pairs located outside the CpG sites. This method allows precise analysis of methylation in a selected region by converting all non-methylated cytosines (C) into uracil (U), while methylated cytosines remain unchanged. All cytosines of CpG sites (about 50) in the gal3 promoter from LNCaP DNA were methylated in the 384 bp PCR product, but no gal3 promoter methylation was observed in azacytidine-treated LNCaP. Similarly, no methylation in the gal3 promoter from either PC-3 or DU-145 DNA was observed [22, 25]. Gal3 silencing by promoter methylation is not specific to prostate cancer cells, rather it is a common mechanism for gal3 gene silencing in cancer, as treatment of two gal3 null breast cancer cell lines BT549 [25] and SKBR3 [24] with azacytidine induced expression of gal3. Expression of gal1 was also shown to be regulated by promoter methylation [37]. A small genomic region of approximately 100 base pairs surrounding the transcriptional start site (-50/+50) accounts for most transcriptional activity of gal1 [37]. Decreased expression of gal3 in pituitary and prostate tumors is associated with increased methylation in the gal3 promoter. Expression of gal3 in conjunction with its promoter methylation has been recently been investigated in pituitary [24] and prostate [22, 25] tumors. Among pituitary tumors, mainly in follicle-stimulating hormone/luteinizing hormone-producing (38%) and null cell (57%) adenomas, the gal3 promoter was found to be methylated and silenced [24]. Interestingly, the prolactin- and adrenocorticotropic hormone-producing tumors were unmethylated and 326 Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis expressed gal3 [24]. We investigated gal3 expression in normal, benign prostatic hyperplasia (BPH), and various stages of prostate adenocarcinoma [22]. Gal3 was found strongly expressed in normal, BPH, and high-grade prostatic intraepithelial neoplasia (HGPIN, a precursor lesion to development of invasive prostatic adenocarcinoma) tissues in both the nucleus and the cytoplasm. However, gal3 showed decreasing immunopositivity during stage evolution. Moreover, localization of gal3 is interesting during stage evolution. In particular, stage I tumors showed a strong immunopositivity both in nucleus and cytoplasm, while in more advanced stages immunostaining was less intense and localized mainly in cytoplasm, with rare, occasional nucleus positivity. However, two stage I specimens (out of 10) showed little or no gal3 immunopositivity [22]. Similar observation on gal3 expression in prostate tumor was made by others [20,38]. The role of cytoplasmic and nuclear gal3 in cancer progression was examined by specifically expressing gal3 in either cytoplasm or nucleus of LNCaP, a gal3-negative human prostate cancer cell line [18]. Cytoplasmic gal3 was anti-apoptotic and promoted tumor growth and angiogenesis. Interestingly, nuclear gal3 affected these parameters in an opposite fashion with an overall antitumoral activity [18]. The role of decreased expression of gal3 in early stage of prostate tumor is not known. However, in normal cells gal3 is believed to interact to the members of Nkx homeodomain family [18], especially to a prostate tumor suppressor, Nkx3.1 [39] and decrease the expression of the cancer phenotype [18]. Therefore, the silencing of gal3 gene during the development of prostate tumor could be necessary to suppress the influence of Nkx3.1 gene and thereby to help tumor cells to proliferate. Since gal3 expression is decreased in stage evolution of prostate adenocarcinoma as evidenced from our studies [22] and others [20], we hypothesized that DNA isolated from early stages of prostate cancer would be methylated in the gal3 promoter region. Results revealed that the gal3 promoter from multiple specimens of stage II tumor is heavily methylated throughout its entire length, but that from multiple specimens of stage III and IV tumor is lightly methylated. Whereas gal3 promoter in stage III showed few methylation sites, mostly between -199 to -252 nt, the gal3 promoter from stage IV tumor specimens was methylated between -112 to -227 nt. In stage I prostate cancer, however, both light and heavy methylation is evident in the gal3 promoter. In multiple normal prostate and BPH samples, the gal3 promoter was almost unmethylated. Overall, results indicated that the decreased expression of gal3 in tumor prostate is associated with the hypermethylation of its promoter. Concluding remarks Recent studies by us and others have shown that the expression of gal3 in prostate and breast cancer cell lines is regulated by the DNA methylation in its promoter and is 327 Hafiz Ahmed negatively correlated [22,24,25]. Moreover, decreased expression of gal3 in pituitary and prostate tumors was found associated with hypermethylation in the gal3 promoter [22,24]. The gal3 promoter showed almost complete methylation in all CpG sites in early stages of prostate cancer, but is lightly methylated in later stages. The methylation pattern of the gal3 promoter at various stages of prostate cancer, and in particular its complete methylation in early stages, is unique, because in other genes such as GSTP1, CpG methylation correlates positively with tumor grade and stage (i.e. low methylation in early stages and high methylation in late stages) [40]. However, the degree of gal3 promoter methylation and its expression at various stages of prostate cancer cannot be correlated from this study [22]. This is because the tumor tissue is heterogeneous and so DNA population extracted from the tumor tissue may not necessarily represent single tumor stage. To answer this question, however, further studies are needed from the micro-dissected tissues such as those obtained from laser capture micro-dissection approach. Acknowledgments I apologize to the authors whose works are not cited due to the limited space. The work carried out in the authorвЂ™s laboratory has been supported by the UMBI Presidential Proof of Concept Award and the National Institute of Health Grant RO3 CA133935-01. 328 Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis Fig. 1 - Schematic representation of lectin-mediated cell-cell and cell-extracellular matrix (ECM) interactions. Galectins mediate interactions between cells or between cells and ECM by binding to laminin, integrin and other cell surface glycoconjugates A representative of each type of galectins, proto (gal1), chimera (gal3), and tandem-repeat (gal8) is shown. Fig. 2 - DNA methylation catalyzed by DNA methyltransferase. DNA methyltransferase transfers methyl group from S-adenosyl methionine (SAM-CH3) to cytosine yielding S-adenosyl homocysteine (SAH) and 5-methylcytosine. Hafiz Ahmed Fig. 3 - Simplified cartoon showing gal3 transcription by unmethylated promoter (A) and gal3 gene silencing by the methylated promoter (B). A. In normal prostate and pituitary tissues, gal3 promoter is unmethylated and accessible to binding to the transcription factors such as AP-1 and Sp-1 for stimulation of gal3 transcription. B. In prostate and pituitary tumors, gal3 promoter is methylated and therefore bound by the methyl binding proteins (MBD) and histone deacetylase (HDAC). Thus the methylated promoter is not accessible to binding to the transcription factors and inactive. Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis References [1] Ahmed H, Du SJ, OвЂ™Leary N, Vasta GR. Biochemical and molecular characterization of galectins from zebrafish (Danio rerio): notochord-specific expression of a prototype galectin during early embryogenesis. Glycobiology. 2004; 14:219-32. [2] Nakahara S, Raz A. Regulation of cancer-related gene expression by galectin-3 and the molecular mechanism of its nuclear import pathway. Cancer Metastasis Rev 2007; 26:605-10. [3] Rabinovich GA, Liu FT, Hirashima M, Anderson A. An emerging role for galectins in tuning the immune response: lessons from experimental models of inflammatory disease, autoimmunity and cancer. Scand J Immunol 2007; 66:143-58. [4] Nakahara S, Raz A. Biological modulation by lectins and their ligands in tumor progression and metastasis. Anticancer Agents Med Chem 2008; 8:22-36. [5] Vasta GR.Roles of galectins in infection. Nat Rev Microbiol 2009; 7:424-38. [6] Elola MT, Wolfenstein-Todel C, Troncoso MF, Vasta GR, Rabinovich GA. Galectins: matricellular glycanbinding proteins linking cell adhesion, migration, and survival. Cell Mol Life Sci 2007; 64:1679-700. [7] Gong HC, Honjo Y, Nangia-Makker P, Hogan V, Mazurak N, Bresalier RS, Raz A. The NH2 terminus of galectin-3 governs cellular compartmentalization and functions in cancer cells, Cancer Res 1999; 59:6239-45. [8] Nakahara S, Oka N, Raz A. On the role of galectin-3 in cancer apoptosis. Apoptosis 2005; 10: 267-75. [9] Dumic J, Dabelic S, FlГ¶gel M. Galectin-3: an open-ended story. Biochim Biophys Acta 2006; 1760:61635. [10] Colnot C, Ripoche MA, Scaerou F, Foulis D, Poirier F. Galectins in mouse embryogenesis. Biochem Soc Trans 1996; 24:141-6. [11] Poirier F. Roles of galectins in vivo. Biochem Soc Symp 2002; 69:95-103. [12] Liu FT, Rabinovich GA. Galectins as modulators of tumour progression. Nat Rev Cancer 2005; 5:2941. [13] Inohara, H., et al., Interactions between galectin-3 and Mac-2-binding protein mediate cell-cell adhesion. Cancer Res 1996; 56:4530-4. [14] Glinsky VV, Glinsky GV, Rittenhouse-Olson K, Huflejt ME, Glinskii OV, Deutscher SL, Quinn TP. The role of Thomsen-Friedenreich antigen in adhesion of human breast and prostate cancer cells to the endothelium. Cancer Res 2001; 61:4851-4857. [15] Elad-Sfadia G, Haklai R, Balan E, Kloog Y. Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity. J Biol Chem 2004; 279:3492230. [16] Fukumori T, Takenaka Y, Yoshii T, Kim HR, Hogan V, Inohara H, Kagawa S, Raz A. CD29 and CD7 mediate galectin-3-induced type II T-cell apoptosis. Cancer Res 2003; 63:8302-11. [17] Danguy A, Camby I, Kiss R. Galectins and cancer. Biochim Biophys Acta 2002; 1572:285-93. [18] Califice S, Castronovo V, Van Den BrГ»le F. Galectin-3 and cancer. In: J Oncol 2004; 25:983-92. [19] Song YK, Billiar TR, Lee YJ. Role of galectin-3 in breast cancer metastasis: involvement of nitric oxide. Am J Pathol 2002; 160:1069-75. [20] Pacis RA, Pilat MJ, Pienta KJ, Wojno K, Raz A, Hogan V, Cooper CR. Decreased galectin-3 expression in prostate cancer. Prostate 2000; 44:118-23. [21] Merseburger AS, Kramer MW, Hennenlotter J, Simon P, Knapp J, Hartmann JT, Stenzl A, Serth J, Kuczyk MA. Involvement of decreased Galectin-3 expression in the pathogenesis and progression of prostate cancer. Prostate 2008; 68:72-7. 331 Hafiz Ahmed [22] Ahmed H, Cappello F, Rodolico V, Vasta GR. Evidence of heavy methylation in the galectin-3 promoter in early stages of prostate adenocarcinoma: Development and validation of a methylated marker for early diagnosis of prostate cancer. Translational Oncology 2009 (In press). [23] Merseburger AS, Kramer MW, Hennenlotter J, Serth J, Kruck S, Gracia A, Stenzl A, Kuczyk MA. Loss of galectin-3 expression correlates with clear cell renal carcinoma progression and reduced survival. World J Urol 2008; 26:637-42. [24] Ruebel KH, Jin L, Qian X, Scheithauer BW, Kovacs K, Nakamura N, Zhang H, Raz A, Lloyd RV. Effects of DNA methylation on galectin-3 expression in pituitary tumors. Cancer Res 2005; 65:1136-40. [25] Ahmed H, Banerjee PB, Vasta GR. Differential expression of galectins in normal, benign and malignant prostate epithelial cells: Silencing of galectin-3 expression in prostate cancer by its promoter methylation. Biochem Biophys Res Commun 2007; 358: 241-6. [26] Abedin MJ, Kashio Y, Seki M, Nakamura K, Hirashima M. Potential roles of galectins in myeloid differentiation into three different lineages. J Leukoc Biol 2003; 73:650-6. [27] Kadrofske MM, Openo KP, Wang JL. The human LGALS3 (galectin-3) gene: determination of the gene structure and functional characterization of the promoter. Arch Biochem Biophys 1998; 349:7-20. [28] McKenna ES, Roberts CW. Epigenetics and cancer without genomic instability. Cell Cycle 2009; 8:23-6. [29] Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999; 99:247-57. [30] Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene 2002; 21:5400-13. [31] Cross SH, and Bird AP. CpG islands and genes. Curr Opin Genet Dev 1995; 5:309-14. [32] Tate PH, Bird AP. Effects of DNA methylation on DNA-binding proteins and gene expression. Curr Opin Genet Dev 1993; 3:226-31. [33] Hendrich B, Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol Cell Biol 1998; 18:6538-47. [34] Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 1998; 393:386-9. [35] Das PM, Singhal R. DNA methylation and cancer. J Clin Oncol 2004; 22:4632-42. [36] Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, and Paul CL. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci US. 1992; 89:1827-31. [37] Salvatore P, Benvenuto G, Caporaso M, Bruni CB, Chiariotti L. High resolution methylation analysis of the galectin-1 gene promoter region in expressing and nonexpressing tissues. FEBS Lett 1998; 421:152-8. [38] van den BrГ»le FA, Waltregny D, Liu FT, Castronovo V. Alteration of the cytoplasmic/nuclear expression pattern of galectin-3 correlates with prostate carcinoma progression. Int J Cancer 2000; 89:361-7. [39] Kim MJ, Bhatia-Gaur R, Banach-Petrosky WA, Desai N, Wang Y, Hayward SW, Cunha GR, Cardiff RD, Shen MM, Abate-Shen C. Nkx3.1 mutant mice recapitulate early stages of prostate carcinogenesis. Cancer Res 2002; 62:2999-3004. [40] JerГіnimo C, Henrique R, Hoque MO, Mambo E, Ribeiro FR, Varzim G, Oliveira J, Teixeira MR, Lopes C, and Sidransky D. A quantitative promoter methylation profile of prostate cancer. Clin Cancer Res 2004; 10:8472-8. 332 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 1859: Naturalismo. Motivi evoluzionistici e anatomia cellulare [1859: Naturalism. Motifs for the Evolution and Cellular Anatomy] Aldo Gerbino Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia вЂњArcangelo Pasqualino di MarineoвЂќ, FacoltГ di Medicina e Chirurgia, UniversitГ degli Studi di Palermo (IT) Parole chiave: Evoluzione, Radiolari, Peduncolo, Flagello, FC, LCA Key words: Evolution, Radiolarians, Peduncle, Flagellum, First Cell, Last Common Ancestor *** Relazione su invito per: вЂњIncontri di Morfologia FunzionaleвЂќ, Dipartimento di Anatomia Umana, Farmacologia e Scienze Medico Forensi di Parma, nellвЂ™ambito del Forum: вЂњLa morfologia delle costituzioni nella tradizione anatomica dello Studium di Parma: Lorenzo Tenchini (18521906), Cesare Lombroso (1835-1909)вЂќ; a cura di Roberto Toni, Marco Vitale e Guglielmo Masotti (Parma, 25 marzo 2009 Aula Canuto-Ospedale Maggiore. FacoltГ di Medicina e Chirurgia, UniversitГ degli Studi di Parma; SocietГ di Medicina e Scienze Naturali di Parma). 27 dicembre 1831. Il brigantino вЂ�BeagleвЂ™, armato di dieci cannoni, salpa da Devonport al comando del capitano Fitzroy; a bordo, un giovane intellettuale dвЂ™eccezione, Charles Darwin, ГЁ pronto ad intraprendere un viaggio che avrГ la durata di ben cinque anni (1836). Un lustro per unвЂ™incubazione e un fermento speculativo che lo trasformeranno, da curioso osservatore della natura, in uno scienziato sorretto da una pertinace idea del mondo, portatore, al fine, di un metodo, dвЂ™una rivoluzionaria poetica della natura. Il 13 aprile del 1834, il вЂ�BeagleвЂ™, ancorato alla foce del Santa Cruz, fiume a 100 Km a Sud di Porto San Giuliano, in Patagonia, segna la dimensione assoluta della sua ricerca estrema, in un territorio compreso tra valli di basalto ed una inquietante omogeneitГ geologica, ma, allo stesso tempo, stimolatrice di suggerimenti speculativi (Fig. 1). CiГІ che in natura appare, a prima vista, poco significativo, nasconde molto spesso tra le sue pieghe un mondo variegato, tutto da rivelare e decifrare, in cui ГЁ possibile avviare nuove comparazioni, focalizzare insospettati comportamenti. Una regione che, se in apparenza si mostra В«completamente priva dвЂ™interesseВ», disegna invece un vivido scenario metafisico. Darwin annoterГ nel suo вЂ�DiarioвЂ™, tra i tanti aspetti etologici che lo attraggono, quello del cannibalismo: В«Per quanto povera sia sotto certi aspetti, la Patagonia puГІ vantare il maggior numero di piccoli roditori di quello che possa farlo qualsiasi altra parte del mondo. Parecchie specie di topi sono caratterizzate esternamente da orecchie grandi e sottili e da una bellissima pelliccia. Questi animali 333 Aldo Gerbino Fig. 1. Charles Darwin. Il вЂ�BeagleвЂ™ ancorato alla foce del Santa Cruz. LвЂ™Origine della specie per elezione naturale, pubblicata a Londra nel 1859, nella sua prima traduzione italiana del 1864. abbondano fra i boschetti nelle valli, dove per mesi di seguito non possono assaggiare una sola goccia dвЂ™acqua, tranne la rugiada. Sembrano tutti cannibali perchГ© non appena un topo era preso in una delle mie trappole, veniva divorato dagli altri.В» [1] Scorrendo lвЂ™enorme mole di notizie raccolte in questo esaltante viaggio, possono di continuo essere percepiti le compenetrazioni esistenti tra вЂ�generaleвЂ™ e вЂ�particolareвЂ™, tra ambiente e animali che lo popolano, tra animali e piante, tra macro e microstrutture, tutti nellвЂ™accezione posta nella celebre frase di Goethe: В«Il generale e il particolare coincidono; il particolare ГЁ il generale che appare sotto condizioni diverseВ» [2]. UnвЂ™espressione che, per camminamenti diversi, ritrova le sue coincidenze nel naturalista inglese, per quellвЂ™essere portata, sottolinea Giuseppe Zamboni scrivendo sulle вЂ�riflessioniвЂ™ goethiane, В«a stabilire leggi organiche nella vita dellвЂ™uomo e della naturaВ», tendendo, cosГ¬, a В«sollevarsi dallвЂ™individuale e particolare allвЂ™idea e alla regolaВ» [3]. Anche Darwin troverГ la sua regola, quella regola che detiene il governo del mondo, portatrice di una palmare potenzialitГ trasformativa: lвЂ™вЂњevoluzioneвЂќ. 1859: Naturalismo. Motivi evoluzionistici e anatomia cellulare Fig. 2. a. Ernst Haeckel e lвЂ™acquerello raffigurante lo Stretto di Messina. b. Tavola XXXIII del Die Radiolarien (1862). Tale strettissimo vincolo in cui il particolare ГЁ visto quale modello dellвЂ™interezza biologica e in cui la cellula (lвЂ™unicellulare) si esprime in forma di marchio arcaico, evolutivamente disposto alla mutazione, lo ritroviamo nella misura culturale di un intellettuale e morfologo dвЂ™eccezione: Ernst Haeckel (1835-1919), lвЂ™autore del celebre Die Radiolarien (1862), della notissima NatГјrliche SchГ¶pfungsgeschichte (вЂњLa storia della creazione naturaleвЂќ, 1868) la quale, con le sue ben dodici ristampe, rappresenta lвЂ™opera che chiama a raccolta В«tutte le letture sulla teoria dellвЂ™evoluzione in generale e quelle di Darwin, Goethe e Lamark in particolare, sullвЂ™applicazione di queste teorie allвЂ™origine dellвЂ™uomo e ad altre questioni fondamentali della scienzaВ» [4], contribuendo, cosГ¬, alla comprensione e conoscenza della ricerca darwiniana. Г€ del 1874 la monografia sulle вЂ�spugne calcareeвЂ™ per le quali postula come В«lвЂ™evoluzione ontogenetica ricapitola la filogenesiВ», e, infine, nel 1870, la stampa della laboriosa Antrhopogenie oder Entwickelungsgeschichte des Menschen (вЂњAntropogenia o la storia dello sviluppo del genere umanoвЂќ). Haeckel si era formato alla universitГ di Wurzburg seguendo il chimico analitico Scherer, lвЂ™istologo Rudolf Albert von KГ¶lliker (1817-1905), il patologo Rudolf Ludwig K. Virchow (1821-1902), ma, pur studiando medicina, dichiara con convinzione di vedere В«lвЂ™anatomia esclusivamente dal punto di vista della storia naturale dellвЂ™uomo, non dal punto di vista medico!В». LвЂ™anatomia cellulare pervade la mente di Haeckel interrelando le sue conoscenze biologiche e fisiologiche con i dati mineralogici di Christian Samuel Weiss (1780-1856), colui il quale definГ¬, in maniera inoppugnabile, la simmetria dei cristalli. Allora, i radiolari non sono soltanto organismi unicellulari che vivono nelle grandi vertigini dei mari sin dal periodo precambriano, ma, come intuito da Haeckel, Aldo Gerbino Fig. 3. Dendrogrammi di linee filetiche. Da Martino Rizzotti, 1998 (mod.). rappresentano unвЂ™estetica, una вЂ�cristallografia organicaвЂ™, una filosofia, una scienza ed anche unвЂ™arte, tanto da imprimere nellвЂ™immaginario artistico di RenГ© Binet la possibilitГ di ricreare forme e contenuti ideali nelle versioni architettoniche destinate alla вЂ�EsposizioneвЂ™ parigina del 1900; una estetica che trovГІ fertile substrato creativo nel tessuto espressivo dello Jugendstil [5, 6]. Con Haeckel sopravvivono le Nereidi dello Stretto di Messina, lвЂ™antica Zancle, per quelle migliaia di forme in migrazione capaci di alimentare il sospiro del mondo, lвЂ™anima stessa delle nostre ombre. Dello stretto messinese esiste un lontano acquerello che ci narra delle sue ricerche operate in questo luogo del mito. Stimolato dal pittore Hermann Allmers (1821-1902), il suo percorso scientifico sui Radiolari restituisce, proprio attraverso i capillari disegni, la meraviglia degli вЂњSphaerozoumвЂќ nelle varietГ вЂ�italicumвЂ™, вЂ�spinosumвЂ™, вЂ�ovodimareвЂ™, вЂ�punctatumвЂ™, o quelle della вЂњAulacantha scolymanthaвЂќ, e della вЂњThalassicolla ZancleaвЂќ, o del вЂњDictyophimus TripusвЂќ, e, ancora, delle eleganti geometrie delle вЂњHeliosphaereвЂќ (вЂ�inermisвЂ™, вЂ�tenuissimaвЂ™, вЂ�actinotaвЂ™, вЂ�echinoidesвЂ™, вЂ�elegansвЂ™) oppure delle stellari iridescenze che circondano il pulviscolo costituito dalle catenarie di вЂњDictyocha MessanensisвЂќ [7] (Fig.2, a, b) . 336 1859: Naturalismo. Motivi evoluzionistici e anatomia cellulare Fig. 4. a. Cellula di lievito. b. Flagello batterico (Gram-). Da Vera Bianchi, Pietro Amodeo, 1983 (mod.). Г€ di estremo interesse comprendere, nellвЂ™ecosistema cellulare, come i vari compartimenti, embricati nellвЂ™ambiente cellulare, entrino nella sostanza del processo filogenetico in rapporto sinergico, ora attraverso il comparto endocellulare (вЂ�locus genicoвЂ™), ora attraverso quello extracellulare. Sappiamo, inoltre, quale grande importanza rivestano, ad esempio, le parti mobili delle cellule; cioГЁ quelle specializzazioni che sembrano essere alla base delle primitive tappe filogenetiche, capostipiti per nuovi filomeri, attori nella confezione futura di una cronospecie. CiГІ in virtГ№ dello sviluppo di quelle cellule evolutive identificate come First Cell (FC, prima cellula capace di traduzione), ed LCA (Last Common Ancestor o Cenancestor), lвЂ™ultimo progenitore comune dellвЂ™attuale biosfera (3,5в†’1,8 mld anni; Fig. 3). Un esempio di compartecipazione biologica tra le varie classi di cellule (procarioti ed eucarioti) puГІ essere alimentato dalla вЂњcellula del lievitoвЂќ (eucariotica/ancestrale), tradizionalmente ascritta ai funghi ascomiceti o lieviti (saccaromiceti). Durante la sua divisione, da corpiccioli posti in prossimitГ del nucleolemma, si formano microtubuli che entrano in rapporto con i cromatidi. In essa coesistono la вЂ�pareteвЂ™ polisaccaridica di stampo procariotico e gli organelli citoplasmatici, una novitГ evoluzionistica (mitocondri, complesso del Golgi, reticolo endoplasmatico granulare), inoltre: un distinto compartimento nucleare delimitato da una membrana (costituita da due membranelle unitarie di 2x6 nm con una intercapedine di 20 nm) attraversata da pori, mentre, nella cariolinfa, sono presenti alcuni cromosomi, un nucleolo e delle vescicole con materiale di riserva. La вЂњOchromonas danicaвЂќ (una cloroficea delle alghe verdi, eucariota) mostra, giГ organizzato, un flagello ed un corpuscolo basale; inoltre, un cloroplasto (marchio delle cellule vegetali) con uno stigma a forma di sferule, e, al di sotto, 337 Aldo Gerbino Fig. 5. Ipotesi del peduncolo in un eucariote unicellulare. Da Martino Rizzotti, 1998 (mod.). un fotorecettore. LвЂ™ вЂњEuglena gracilisвЂќ (unвЂ™altra alga verde eucariota, fornita di flagelli simmetrici) offre allвЂ™osservazione cloroplasti multipli ricchissimi di clorofilla, mitocondri, e varie figure organulari. Quadri biologici, questi, interpretati attraverso fenomeni endosimbiontici e inseriti in un ventaglio polifiletico dove coesistono, pur avendo avuto origine da uno stesso philum, forme arcaiche e forme evolutivamente avanzate. Anche gli apparati mobili cellulari mostrano similitudini e divergenze sul piano della complessitГ molecolare tra procarioti ed eucarioti. Nelle procellule (batteri) troviamo un meccano-enzima formato da 6 subunitГ (giГ presente in LCA), innestato in una sorta di motore rotante destinato a imprimere al flagello un movimento passivo. Nelle eucellule (dalla rodoficee alle cellule dei primati) le macchine proteiche, legate alla genesi del movimento, mostrano complessi di actina e miosina o di tubulina e dineina, e, nel ciglio vibratile della cellula eucariotica, troviamo un codificato apparato mobile espresso in una articolazione biologica in cui agiscono 600 proteine (Fig. 4, a, b) [8]. Appare particolarmente interessante, quanto suggestiva, nella genesi del movimento, lвЂ™ipotesi del peduncolo [9], in cui il вЂњsistema mobile ciglioвЂќ sembra, infatti, trarre origine da 338 1859: Naturalismo. Motivi evoluzionistici e anatomia cellulare un вЂ�peduncoloвЂ™ sorretto dai microtubuli presenti nellвЂ™eucariote unicellulare (per specializzazione del citoscheletro); oppure, come ГЁ dвЂ™altre fonti sperimentali sostenuto, le componenti microtubulari verrebbero assunte, per simbiosi extracellulare, da altri microrganismi (Spirocheta) [10]. La formazione originaria di un peduncolo emesso da un corpo cellulare, sospinto da necessitГ alimentari, organizzerebbe i microtubuli disponendoli in una architettura ordinata nella esemplare ultrastruttura dellвЂ™assonema (9+2), consentendo unвЂ™interazione con bracci di una dineina primitiva, proteina ad alta attivitГ ATPasica (Fig. 5). Sollevarsi da un substrato, con una rete di microtubuli allineati in parallelo, consentirebbe alle cellule ancestrali di acquisire una maggiore flessibilitГ ed autonomia dal substrato originario, e, quindi, essere oggetto di una probabile selezione filogeneticamente piГ№ vantaggiosa. Un vantaggio che potrebbe essere non esclusivamente ricondotto alla individualitГ cellulare ma, secondo la вЂњregola di HamiltonвЂќ, al gene вЂњaltruisticoвЂќ (1964), a quei geni comuni, giГ osservati da J. B. S. Haldane [11] nel 1955, condivisi da una molteplicitГ di familiari, e che verrebbero altruisticamente preservati durante il processo evolutivo. Durante tale processo si assiste, comunque, ad un incremento della complessitГ (in senso вЂ�epigeneticoвЂ™: domini degli aggregati biologici agenti nella categoria spazio/tempo) rispetto alla вЂ�semplicitГ вЂ™ operativa genica; la definizione di cellula, allora, letta piГ№ comunemente come un sistema dualistico (geni/proteine: genotipo/fenotipo), dovrebbe essere interpretata, secondo i suggerimenti di Barbieri, anche come entitГ triadica (geni/codemaker/proteine: genotipo/fenotipo/ribotipo), dove il вЂ�codemakerвЂ™ (il В«fabbricante di codici organiciВ») ГЁ caratterizzato dal ribotipo, cioГЁ dal sistema delle ribonucleoproteine impegnato a leggere e costruire i materiali proteici [12]. Dopo i tempi prebiotici, caratterizzati da una evoluzione della materia inanimata (evoluzione conformazionale), lвЂ™evoluzione adattativa (darwiniana) trova una sua specificitГ con la costruzione di linee filetiche adattabili alle varie esigenze ambientali le quali, allontanandosi sempre piГ№ dalla condizione di immobilitГ e fissitГ propria della materia inanimata [13], conserverebbero nel grande bagaglio della memoria chimica progetti che saranno ritrovati nella loro futura biologia. Le cellule riprodurranno e rifletteranno forme e significati di transizione, traducendo, trasformando e facendo proprie nuove orme вЂ�realiвЂ™ di appartenenza. I cambiamenti evolutivi identificano nuovi filomeri e aggregano citotipi in quellвЂ™ammodernato вЂњdarwinismo di EdelmanвЂќ [14] che inevitabilmente conduce ad una revisione epistemologica del concetto di вЂ�formaвЂ™ [15], per una morfologia cellulare, specchio primario della corporeitГ futura, sollecitata, soprattutto nella sua fase di sviluppo, nel delicato momento della aggregazione e della selezione cellulare. CiГІ ГЁ quanto fu notato anche da Haeckel, quando, nel lontanissimo settembre del 1869, al largo dellвЂ™isola norvegese di Gisoe, descrivendo il minuscolo flagellato dalla forma sferica, la Magosphera planula, caratterizzГІ il suo plastico ciclo vitale, ponendolo quale anello di transizione tra la fase unicellulare e quella pluricellulare [16]. Dai protozoi ai metazoi; dunque, verso le scandite bioarchitetture delle вЂњcittГ cellulariвЂќ. 339 Aldo Gerbino Bibliografia [1] Charles Darwin. Viaggio di un naturalista intorno al mondo (Trad. di M. Magistretti). Firenze, Giunti-Martello, 1982. [2] Wolfgang Goethe. Aus den maximen und reflexionen ein brevier (Trad. It., вЂњBreviario dalle Massime e RiflessioniвЂќ). Firenze, Fussi, 1950. [3] Giuseppe Zamboni, in Goethe, op. cit., p. 11. [4] Olaf Breidbach, in: Ernst Haeckel, The Radiolarian Atlas of 1862. London, Prestel, 2005. [5] Sylvain Ageorges. Sur les traces des Expositions universelles, Paris, Parigramme, 2007. [6] AA.VV. Paris 1900. Paris, MusГ©e Carnavalet, 2001. [7] Aldo Gerbino. 1859: Naturalism. Motivs for the Evolution and Cellular Anatomy. В«Acta Biomed.В», in press. [8] Vera Bianchi, Pietro Amodeo (a cura). Anatomia della cellula. Milano, В«Le ScienzeВ» (quaderni), 1983. [9] Martino Rizzotti. Prime tappe dellвЂ™evoluzione cellulare. Bologna, Decibel/Zanichelli, 1998. [10] Lynn Margulis. Archaeal-eubacterial mergers in the origin of Eukarya. В«Proc. Natl. Acad. Sci.В», USA, 93:1071-1073, 1996; Id.: Symbiosis in cell evolution. New York, Freeman, 1993. [11] William Donald Hamilton. The collected papers of W.D. Hamilton, Narrow Road of Gene.New York, LH Freeman, 1996. [12] Marcello Barbieri. I codici organici: metafore o realtГ ? В«Systema NaturaeВ», 5: 99-113, 2003. [13] Nikolay P. Kolomiytsev, Nadezhda Ya. Poddubnaya. LвЂ™origine della vita come risultato del cambiamento del meccanismo evolutivo. В«Biology ForumВ», 100: 11-26, 2007. [14] Gerald M. Edelman. Topobiology, an Introduction to Molecular Embryology. New York, В«Basic BooksВ», 1988; Id.: Topobiology. В«Scientific AmericanВ», May 76-88, 1989; Id.: CAMs and Igs: Cell Adhesion and the Evolutionary Origins of Immunity. В«Immunol. Rev.В», 100: 9-43, 1987b. [15] Roberto Toni, Epistemologia della forma corporea. В«Systema Naturae, Annali di Biologia TeoricaВ», 2: 200-333, 1999a; Id.: Topobiology: Epistemological Implication of an Ontic Theory in Biomorphology. В«EpistemologiaВ», XXVII: 83-106, 2004. [16] Andrew Reynolds, Norbert HГјlsmann. Ernst HaeckelвЂ™s Discovery of Magosphera planula: A Vestige of Metazoan Origins? В«History and Philosophy of the Life SciencesВ», 30: Вѕ, 2008. 340 Proceedings & Meetings Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 Ambiente staminale e prospettive di transdifferenziamento verso il fenotipo osteoblastico [Staminal environment and transdifferentiation perspectives towards the osteoblastic phenotype] Annamaria Mauro, Luana Lipari, Maria Buscemi e Aldo Gerbino Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia вЂњArcangelo Pasqualino di MarineoвЂќ, FacoltГ di Medicina e Chirurgia, UniversitГ degli Studi di Palermo (IT) Key words: MSCs, Transdiffentiation, Wharton Jelly Parole chiave: Cellule Staminali Mesenchimali, Transdifferenziazione, Gelatina del Wharton Abstract. In the last years there is a growing interest in the use of adult stem cells for the treatment of many pathologies. Our group has focused its attention on the study of вЂњmature mucous tissueвЂќ and on the stem cells that can be isolated from it; Mesenchimal Stem Cells (MSCs) derived from the WhartonвЂ™s jelly of umbilical cord and stem cells from dental pulp. The isolation of these types of cells is obtained through Fluorescence Activated Cell Sorter (FACS) targeting specific stem cell markers (surface antigens). The differentiation towards the phenotypes of interest is induced exposing the stem cells to specific differentiation media; in particular the osteogenic differentiation is induced with a medium containing Dexamethasone, Ascorbic acid and ОІ-Glycerophosphate, while the condrogenic differentiation can be induced with a medium containing Transforming Growth Factor (TGF), Dexamethasone, Ascorbic acid, Sodium Pyruvate, Insulin, Transferrin and Linoleic acid. *** Negli ultimi anni la ricerca e la medicina stanno tentando sempre di piГ№ di avvalersi dellвЂ™uso delle cellule staminali come strumento per la cura di molte patologie. Le cellule staminali adulte o somatiche suscitano un particolare interesse in quanto piГ№ facili da reperire e sottoposte a minori restrizioni di carattere legislativo o etico. Una caratteristica delle cellule staminali adulte di fondamentale importanza per le applicazioni cliniche, ГЁ la loro вЂњplasticitГ вЂќ o capacitГ di вЂњtransdifferenziareвЂќ, cioГЁ la possibilitГ di differenziarsi in tipi cellulari caratteristici di altri tessuti. Il nostro gruppo ha da diversi anni rivolto la sua attenzione allo studio del tessuto mucoso maturo e delle cellule staminali che da esso possono essere isolate: 343 Annamaria Mauro, Luana Lipari, Maria Buscemi, Aldo Gerbino cellule staminali da polpa dentale e cellule staminali mesenchimali (MSCs) derivate dalla gelatina di Wharton del cordone ombelicale. *** LвЂ™isolamento delle cellule staminali dalla gelatina di Wharton viene ottenuta con la citofluorimetria di flusso (FACS) utilizzando markers specifici per cellule staminali mesenchimali (CD105, CD44 e CD73) [1]. Il differenziamento in senso condrogenico viene indotto con un mezzo contenente: TGF (transforming growth factor), 100 nM dexamethasone, acido ascorbico, sodio piruvato, insulina, trasferrina e acido linoleico. Il differenziamento in senso osteogenico viene indotto con un mezzo contente 100 nM dexamethasone, acido ascorbico e 10 mM glicerofosfato per 4 settimane [1], oppure con lвЂ™esposizione alla Bone Morphogenetic Protein 2 (BMP2) [2]. Per confermare il potenziale osteogenico delle cellule sottoposte ad induzione vengono ricercati alcuni markers specifici come la fosfatasi alcalina, sialoproteina ossea, il collagene tipo I, lвЂ™osteocalcina e lвЂ™osteopontina. Viene valutata anche la risposta a fattori osteoinduttivi come lвЂ™ormone paratiroideo, IGF1, BMP2 e TGF-ОІ1. Da qualche tempo cellule staminali sono state isolate anche dalla polpa dentale, ciГІ ГЁ di grande interesse in quanto il rapporto cellule staminali/massa di questo tessuto ГЁ uno dei piГ№ elevati ed inoltre sia il livello proliferativo di queste cellule che la loro вЂњcapacitГ di transdifferenziamentoвЂќ sono ragguardevoli. Le cellule staminali estratte dalla polpa dentale vengono separate mediante FACS in base allвЂ™espressione di specifici markers staminali quale ad es. STRO-1. Il differenziamento in senso osteogenico viene indotto utilizzando dexamethasone, acido ascorbico e beta-glicerofosfato, mentre il differenziamento in senso condrogenico con acido ascorbico, sodio piruvato e TGF-ОІ [3]. Le MSCs sono state utilizzate in vari trattamenti: difetti ossei in modelli animali sperimentali e in piccoli gruppi di pazienti. I fattori cruciali nel controllo della proliferazione e differenziazione non sono del tutto noti. Tra di essi il TGF-ОІ che media le funzioni di osteoblasti/osteoclasti e la LTBP che media lвЂ™accumulo di TGF-ОІ nellвЂ™osso e lвЂ™accumulo di matrice extracellulare [4]. Topi knockout per LTBP-3 mostravano una ridotta proliferazione cellulare e una ridotta differenziazione osteogenica. Referenze bibliografiche [1] [2] [3] [4] 344 Karahuseyinoglu S et al. Stem Cells. 2007 Feb; 25(2):319-31. Hou T et al. Tissue Eng (Part A). 2009 Feb 18. Lindroos B et al. Biochem Biophys Res Commun. 2008 Apr 4;368(2):329-35. Koli et al. Bone. 43(2008) 679-688. Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 CAUSES OF ALLERGIC RHINITIS AND ASTHMA [Cause della rinite allergica e dellвЂ™asma] Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone and Vincenzo Bellia Dipartimento di Medicina, Pneumologia, Fisiologia e Nutrizione Umana (DIMPEFINU), University of Palermo, Palermo (IT) Key words: Allergic Rhinitis, Asthma Parole chiave: Rinite Allergica, Asma Abstract. Many allergic diseases may involve the respiratory tract: the most common are rhinitis and asthma. These are linked one to the other, according to the concept of вЂњone airway, one diseaseвЂќ. These two diseases are common worldwide and there is evidence that their prevalence has been increasing in the last decades. Several studies have clearly shown that modifications of the pattern of microbial exposure of children associated with social and economic development represent a critical factor underlying the rising severity and prevalence of atopic disorders. These observations are the basis for the so-called вЂњhygiene hypothesisвЂќ, that could be a reasonable explanation for the allergy epidemics. The purpose of the present review is to sum up how allergy is linked to the development of rhinitis and asthma. Riassunto. Numerose malattie allergiche possono coinvolgere lвЂ™apparato respiratorio: le piГ№ comuni sono lвЂ™asma e la rinite allergica, che sono anche legate lвЂ™una allвЂ™altra secondo il concetto вЂњuna via aerea, unвЂ™unica malattiaвЂќ. Queste due malattie sono molto comuni in tutto il mondo e la loro prevalenza ГЁ aumentata negli ultimi anni. Numerosi studi hanno chiaramente dimostrato che le modificazioni nel tipo di esposizione microbica dei bambini associato con lo sviluppo socio-economico rappresenta un fattore critico che sta alla base dellвЂ™aumento di gravitГ e di prevalenza delle malattie atopiche. Queste osservazioni sono il fondamento della cosiddetta вЂњipotesi igienicaвЂќ, che potrebbe essere una ragionevole spiegazione dellвЂ™epidemia allergica. Lo scopo della presente revisione ГЁ quello di riassumere come lвЂ™allergia sia legata allo sviluppo della rinite e dellвЂ™asma. Introduction Allergic rhinitis is defined as a symptomatic disorder of the nose, induced after allergen exposure, by an IgEmediated inflammation of the nasal membranes [1]. It is characterized by symptoms of nasal congestion, rhinorrhea, sneezing, and itching, and it is often associated with 345 Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone, Vincenzo Bellia ocular symptoms [2,3]. The first description of вЂњhay feverвЂќ was formulate by John Bostock [4] who observed a patients suffered form allergy with both rhinitis and asthmatic manifestations. He wrote: вЂњAbout the beginning or middle of June in every yearвЂ¦ a sensation of heat and fulness is experienced in the eyes... to this succeeds irritation of the nose producing sneezingвЂ¦ to the sneezings are added a further sensation of tightness of the chest, and a difficulty of breathingвЂќ On the basis of the timing of exposure allergic rhinitis was previously distinguished into seasonal, perennial, and occupational. This subdivision is not entirely satisfactory. A novel classification of allergic rhinitis based on duration: subdivides into вЂњintermittentвЂќ and вЂњpersistentвЂќ disease. Depending on severity of symptoms and quality of life impairment, the disease is further subdivided into вЂњmildвЂќ or вЂњmoderate-severeвЂќ forms,. The counterpart of rhinitis at the level of intrapulmonary airways is asthma: it is defined as a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role [5]. The chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. These episodes are usually associated with widespread, but variable, airflow obstruction of airways that is often reversible either spontaneously or with treatment. Rhinitis and asthma are pathogenetically linked one to the other, suggesting the concept of вЂњone airway, one diseaseвЂќ [1]. Indeed, many cross-sectional studies have demonstrated that rhinitis and asthma occur commonly together. Moreover, patients with allergic rhinitis and no clinical evidence of asthma frequently exhibit bronchial hyperresponsiveness to bronchoconstrictor agents such as methacholine or histamine. For these reasons, some investigators have postulated that bronchial hyperreactivity may represent an intermediate phase along the route leading from nasal allergy to symptomatic asthma [6]. It is well demonstrated not only that the rhinitis impacts on asthma but also that the severity of the two conditions are correlated [7,8]. Epidemiology Allergic rhinitis is a common disease worldwide, affecting at least 10 to 25 % of the population and its prevalence is increasing. Although it is not usually a severe disease, it affects health-related quality of life, and it is a risk factor for asthma [1]. Asthma is a worldwide problem as well, with an estimated population of 300 million affected individuals [9]. Asthma is the third leading cause of hospitalization among persons under 18 years of age in the United States [12]. There is evidence that asthma prevalence has been increasing in the last decades in some countries, including Italy [10,11]. According to the Centers for Disease Control and Prevention, the prevalence of asthma among U.S. 346 Causes of allergic rhinitis and asthma children increased from 3.6% in 1980 to 5.8% in 2003. There is widespread concern about such a rising in prevalence in developed world: in these countries the economic and humanitarian effects of asthma are probably greater and the prevalence is also rising [13]. Analyses of the cost of asthma lead to conclude that the burden of the disease depends on on the effectiveness of prevention and early therapy of exacerbations since emergency treatment is far more expensive than regular treatment [5]. In Italy, it has been calculated that direct costs account for 47.3% of the total economic burden of the disease, medications being the largest component [14]. In 2001 direct medical costs for asthma were above в‚¬ 800.000.000, i.e. 1% of the entire Italian health care system expenses, asthma medications being responsible for в‚¬ 650.000.000 [15]. A number of risk factors are associated with the development of asthma, including (but not limited to) sensitisation to aeroallergens, maternal diet during pregnancy and/or lactation, pollutants (particularly environmental tobacco smoke), microbes and their products, and psychosocial factors. However, evidence for effectiveness of avoidance measures in preventing asthma is lacking in many cases. Factors that influence the development and the expression of asthma include host factors (which are primarily genetic, i.e. atopy) [16] and environmental factors (including viruses) [17], allergens and occupational exposures [18]. However, the mechanisms that influence the development and expression of asthma are complex. Epidemiological studies [19-25] have clearly shown that modifications of the pattern of microbial exposure of children associated with Westernisation represent a critical factor underlying the rising severity and prevalence of atopic disorders: the so-called вЂњhygiene hypothesisвЂќ will be discussed below. This mechanism is a reasonable explanation for the вЂњallergy epidemicsвЂќ [13]. Although allergy is closely linked to asthma, a large proportion of asthmatics are not allergic. Therefore, the mechanisms reviewed hereafter may be not valid for such patients. Asthma pathogenesis: allergic inflammation in asthma Airway inflammation in asthma is a multicellular process involving mainly eosinophils, neutrophils, lymphocytes and mast cells, with eosinophilic infiltration being the most peculiar feature [26]. The inflammatory process involves central airways and spreads distally to include the small airways [27,28]. A fundamental feature of asthma associated with allergic sensitisation is the ability of the airways to recognize common environmental allergens and to generate a Th2 cytokine response to them. IgE antibodies have been linked to the severity of asthma [29] and the early and late phase responses of the airway to allergens. To initiate the synthesis of IgE, inhaled allergens must encounter dendritic cells situated in the airway epithelium and submucosa and extending their processes to the airway surface [30,31]. These dendritic cells uptake and process the inhaled allergens and then 347 Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone, Vincenzo Bellia they migrate to draining lymph nodes, where they present processed antigen to T and B cells [32]. Interactions among these cells in presence of specific cytokines (such as interleukin-4 or IL-13) and other mediators prompt B cells to the production of IgE antibodies. After their release by B cells, IgE antibodies bind to high-affinity IgE receptors on the surface of tissue mast cells or peripheral-blood basophils [32]. The interaction of allergen with receptor-bound IgE molecules causes activation of the cell and the release of preformed and newly generated mediators (such as IL-4 or IL-13). In addition uptake of allergen is enhanced by IgE bound to high affinity receptors on dendritic cells that facilitate allergen internalisation [33]. These mechanism contribute to amplify the synthesis of IgE. Several data demonstrate that about 40% of the Western population is atopic and have elevated IgE antibodies to common environmental allergens, only about 7% express their atopy in the form of asthma [34]. On this basis, it has been proposed that the immune response to allergens is regulated at the surface of the airways: this mechanism should explain why only a small part of patients with atopy show a specific expression in the conducting airways [34]. The role of the type 2 T helper (Th2) cell-mediated immune response against вЂњinnocuousвЂќ environmental antigens (allergens) in the immunopathogenesis of allergic atopy is well documented. It has been proposed that the immune system of the newborn infant is skewed toward Th2 cells and needs timely and appropriate environmental stimuli to create a balanced immune response. in this contest, infections have been identified as factors that enhance Th1-mediated responses and that are associated with a reduced incidence of allergy and asthma [32]. Several data suggest that allergen-specific T-cell response can be shifted from Th2 to Th1 by microbial products that favour the production of IL-12 and interferons by cells of the innate immunity, thus strongly supporting the concept that many bacterial and viral infections that stimulate dendritic cells and natural killer cells may not only promote Th1 polarization but also inhibit the development of Th2 cells. The effect of reduced microbial stimulation on the Th2вЃ„Th1 balance (missing immune deviation) should lead to the over expression of Th2 cells-mediated immune and in turn it should favour the development of allergy [35]. The link between the Th1/Th2 balance and allergic diseases is mediated in part by IgE: Th2 cells, by secreting IL-4 and IL-13, promote immunoglobulin class-switch recombination to IgE [36]. Hygiene hypothesis As it has been presented above, the hygiene hypothesis links epidemiological observations on the rising in prevalence of atopic disorders associated with the Westernisation [21-24] to experimental evidence of Th2вЃ„Th1 imbalance due to reduced microbial stimula348 Causes of allergic rhinitis and asthma tion (37-40). In addition, the importance of reduced activity of T regulatory cells (reduced immune suppression) has been emphasized [41-43]. In discussing the hygiene hypothesis at least three aspects can be considered: i) bacterial, viral and parasites infections in early life; ii) exposure to microbial compounds, such as endotoxins, during childhood; iii) interaction of the first two conditions with the innate and adaptive immune response [44]. Bacterial, viral and parasites infections in early life: childвЂ™s increased exposure to viral infections through day care or contact with other children can influence the phenotypic expression. In cross-sectional surveys recurrent respiratory tract infections during the first 3 years of life have been shown to be negatively associated with atopy among asthmatic children at school age (45, 46). In a study involving 1035 children followed since birth as a part of the Tucson ChildrenвЂ™s Respiratory Study, exposure of young children to older children at home or to other children at day care protects against the development of asthma and frequent wheezing later in childhood [47]: the editorial article of this paper had a provocative and appealing title: вЂњplease, sneeze on my childвЂќ [48]. There is good evidence to suggest that in endemic areas, such as in Africa or Latin America, parasitic infections are strongly inversely related to the development of atopy [41,49]. The helminthic infections favour a Th2 type of immune response and this condition should protected from allergic diseases [50]. Attention should be paid in generalising these observations. Indeed, among respiratory viruses, some might exert more deleterious effects than others and infections of the lower respiratory tract early in life have been identified as risk factors for persistent wheeze and asthma [51,52]. In this perspective, two viruses have been associated with asthma and wheeze in a large number of studies: rhinovirus and respiratory syncytial virus (RSV). Rhinovirus has been detected in 80% of nasal aspirates of school-age asthmatic children within 4 days after parents reported episodes of wheezing [53]. A birth cohort of high-risk infants (Childhood Origins of ASThma [COAST] Study) has extended this work to younger age groups and confirmed that also in infants and toddlers up to the age of 3 years rhinovirus was the main isolate from nasal lavage specimens taken during symptomatic periods [54]. Infantile rhinovirus illnesses were the ones most significantly associated with the prevalence of wheezing in the third year of life. In older children viral infections might interact with airway inflammation in several ways. Damage to the airway epithelium and consequently enhanced absorption of aeroallergens might lead to increased airway inflammation (55). Furthermore, induction of pro-inflammatory cytokines (IL-6, IL-1b, and TNFa), chemokines (IL-8), adhesion molecules and leukotrienes enhance cellular recruitment, activation and inflammatory responses [56, 57]. Other data suggest that the site of infection could be important [58]. In the prospective 349 Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone, Vincenzo Bellia Multicentre Allergy Study (MAS) cohort, that involved 1314 children followed from birth to the age of 7 years, it was found that repeated viral infections of the upper respiratory tract early in life may reduce the risk of developing asthma up to school age, whereas repeated lower respiratory tract infections in the first 3 years of life showed a positive association with wheeze up to the age of 7 years [58]. Exposure to microbial compounds during childhood: a large number of studies has documented that children raised in farms have a lower prevalence of hay fever and atopic sensitisation in childhood, as well as in adulthood, whereas effects on asthma are more consistent for the atopic than the non-atopic phenotype [23,24,59,60]. Higher concentrations of bacterial products were found in the mattresses of farm children compared with concentrations found in the mattresses of non-farm children [25,61] and higher levels of fungal exposures were found in these environments [22]. Timing of the exposure played a crucial role. In fact, the protection was strongest when the exposures occurred in the first years of life compared with in later years [62]. A paradox has been observed in the hygiene hypothesis in US inner cities, where asthma is rising, despite unclean living conditions. Indeed, asthma prevalence is higher for those living in USA urban versus rural communities, but it is not significantly influenced by ethnicity or poverty [63]. However, for hygiene hypothesis considerations, unclean innercity living conditions might not be the equivalent of farm and rural living. In this context, endotoxin levels in homes of low-income metropolitan Denver families were much lower than from farm homes or rural homes and similar to those of nonвЂ“low-income families [64]. Finally, it might be considered that the role of infections could be different in relation to the timing of the exposure: early exposure might prevent subsequent allergic disease development, whereas exposure worsens asthma once it is established. Aeroallergens Atopic sensitization to common aeroallergens, especially perennial inhalant allergens, is an important risk factor associated with asthma [38]. Several types of aeroallergens are particularly important in relation to asthma, including house dust mites, animal allergens (including cockroaches) and molds. A Cochrane analysis questioned the effectiveness of house dust mite avoidance for the treatment of established asthma [65]. Moreover, there is no evidence that anti-house dust mite measures prevent the onset of asthma [66-68]. The relationship between exposure and sensitization to allergens from companion animals is not clear, and there are insufficient data to recommend for, or against, the presence of a pet in the house unless the child has become sensitized to the pet species [38,69-71]. Exposure to cockroach allergen in 350 Causes of allergic rhinitis and asthma the living quarters is associated with the development of sensitization, and this, in turn, is associated with an increased risk of developing asthma [72]. Sensitization to alternaria is a risk factor not only for the development of asthma in children, but also for its severity [73,74]. Alternaria is usually considered an outdoor aeroallergen, but outdoor and indoor concentrations may be similar [75]. Conclusions On the basis of epidemiological and experimental studies the вЂњhygiene hypothesisвЂќ has been developed and has received much attention. 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J Allergy Clin Immunol 2006;118:892-8. [75] OвЂ™Connor G T, Walter M, Mitchell H, Kattan M, Morgan WJ, Gruchalla RS, Pongracic JA, Smartt E, Stout JW, Evans R, Crain EF, Burge HA. Airborne fungi in the homes of children with asthma in low-income urban communities: The Inner-City Asthma Study. J Allergy Clin Immunol 2004;114:599-606. 355 Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009 Celiac Disease: a Widespread Emerging Condition in Mediterranean Countries [Celiachia: Una diffusa patologia emergente nei Paesi del Mediterraneo] Salvatore Accomando, Veronica Pellitteri e Giovanni Corsello Department of Pediatrics; UniversitГ degli Studi di Palermo (IT) Key words: Celiac Disease, Gluten Enteropathy, Upper Endoscopy, Histological Features, Gluten Free Diet Parole Chiave: Celiachia, Enteropatia da Glutine, EGDS, Istologia, Dieta aglutinata Abstract. Celiac Disease is up to date the most well known example of autoimmune disease. It is largely widespread all over the world and its incidence of 0,5-1% is similar in different countries. Gluten intake is necessary for triggering disease in genetically predisposed subjects. Genetic factors and environmental ones are embedded in determining gluten intolerance. Diagnosis of celiac disease is combined and based on clinical features, serology, histology and genetics. In this review we consider clinical ways of presentation, several serological markers available at the moment, endoscopical patterns, and histological features following duodenal biopsy. Up to date Gluten Free Diet remains the only safe and effective therapy provided that is well conducted and long-life. Riassunto. Ad oggi la celiachia ГЁ lвЂ™esempio piГ№ conosciuto di patologia autoimmunitaria. Essa ГЁ largamente diffusa in tutto il mondo e la sua incidenza si aggira tra 0,5 ed 1% in tutti i paesi. LвЂ™introito di glutine ГЁ necessario per innescare la patologia in soggetti geneticamente predisposti. Fattori genetici ed ambientali si embricano nel determinare lвЂ™intolleranza al glutine. La diagnosi di malattia celiaca ГЁ combinata e si basa su segni clinici, markers sierologici, aspetti endoscopici e quadri istologici successivi alla biopsia duodenale. Ad oggi la dieta priva di glutine ГЁ lвЂ™unica terapia codificata: efficace e scevra di effetti collaterali. Introduction Celiac Disease (CD) is a permanent, gluten dependent, autoimmune enteropathy. Ingested gluten is responsible of intestinal damage triggered by T lymphocytes and magnified by tissue trans-glutaminase and pro-inflammatory cytokines. Starting in the gut the physio-pathological process may extend involving other organs. Clinical features and mucosal intestinal damage are reversible provided that patients start a gluten free diet (GFD) [1,2]. 357 Salvatore Accomando, Veronica Pellitteri e Giovanni Corsello Epidemiology CD incidence is increased in the last decade. Some years ago several reports showed different incidence rates also among near countries. This finding was understood as an epidemiological paradox. Nowadays the incidence of disease is about 1:100-1:180, also in people coming from developing countries, thanks to higher sensitivity and specificity of new serological screening tests such as Anti Endomysium Antibodies (AEA) and anti human trans glutaminase (hTTG) antibodies [3-6]. In Mediterranean countries the larger consumption of wheat and other gluten containing cereals (barley, rye) contribute to increase incidence of disease. In particular, modern grain cultivars are esaploid ones, such as Triticum Aestivum, in which gluten derived proteins may exceed 50%. Gluten derived proteins are useful to make dough, bread and other baker products because of their glue properties. This is the reason why polyploid wheat cultivars were selected across centuries [3,7,8]. Amongst the world affected population Mossi people, living in Burkina Faso, seem to be free from CD in spite of gluten presence in their diet regimen. On the other side Saharawi people, living in Tindourf oasis in West Algeria, have the highest incidence of disease in the world reaching 5.6%. In fact long lasting endogamic tradition is responsible of enormous diffusion of CD predisposing genes, first of all DQ2. The association with large gluten consumption in their daily diet rich of cous-cous and other wheat derived foodstuffs also contribute to CD development [9]. Pathogenesis In determining CD the above mentioned environmental factor is embedded with genetic ones [10]. HLA related and HLA unrelated genes confer susceptibility to disease. 90% of celiac affected people carry II Class HLA heterodimer DQ2 and almost the remaining CD patients who, are DQ2 negative, carry DQ8 aplotype. Only a small amount of CD patients is both DQ2 and DQ8 negative [7,11,12]. In fact non-HLA genes also matter in determining predisposition to develop CD [10,13-15]. Antigen Presenting Cells of intestinal Lamina Propria, containing DQ2 or DQ8 aplotype, interact with TCR of CD4+ cells triggering a Th1 inflammatory response. Gliadin peptides rich of prolyn, lisin and glutamine in the lamina propria undergo a transamination process by TTG conferring them a negative electrical charge. This kind of biochemical change is responsible of increased interaction between HLA DQ2 aplotype present in APC and the TCR of CD4+ lamina propria cells. Enterocytes themselves act as APC towards CD8+ intra epithelial lymphocytes. IL-15 produced by NK cells and entero358 Celiac Disease: a Widespread Emerging Condition in Mediterranean Countries cytes amplifies innate immune response contributing to tissue destruction [15-21]. Cooperation between T and B cells results in antibodies production against Gliadin (AGA), Endomysium (AEA) and Tissue Transglutaminase (TTG) [22]. Diagnosis Clinical features Clinical features in CD may be distinct in typical and atypical ones. The first are directly gut related and constitute the classical presentation way of disease in younger children. In deed failure to thrive, abdominal distension, chronic diarrhea, vomiting, bloating, malabsorption with fat in stools, represent the clinical features of overt forms of CD [2,3,10]. The atypical ones are more common in the elder child and in adulthood and consist of extra intestinal features giving proof of a systemic disease [23,24]. (Table 1). CD is often associated to other clinical conditions such as Type 1 Mellitus Diabetes, thyroditis, IgA deficiency or genetic syndromes like DownвЂ™s Syndrome or WilliamвЂ™s Syndrome [25]. These syndromes confer to affected individuals an additional, sometimes HLA independent, risk of doing CD. In these last years risk of malignancy in CD and in particularly of enteropathy associated T Lymphoma, was undersized. In fact it seems more related to Refractory Celiac Sprue, that probably is a pre-neoplastic pathological condition different than CD [16]. Serology Up to date serology consists of three different kind of antibodies against Gliadin, Endomysium and Transglutaminase respectively. AGA are those with best sensitivity and specificity under two years of age, reflecting the first immunological response towards heterologous proteins of wheat gliadin. They are the first kind of antibodies detected in serum samples of celiac patients. In early 90вЂ™s AEA were also available by Indirect Immuno-Fluorescence method conferring to serologic suspicion more reliability also for the follow up of the disease. In fact this new serologic finding allowed the ESPGHAN committee to revise the diagnostic criteria for CD, reducing the number of duodenal biopsies needed to diagnosis, from three (Interlaken 1970) to only one [26]. In 1997 the last and more accurate category of serologic antibodies was discovered by Dieterich e Coll. and provided a new useful tool for screening and monitoring the disease without undergoing duodenal biopsy: anti TTG antibodies in deed are the highest specific and sensitive class of serologic markers for CD suspected diagnosis [4]. First line generation of TTG substrate was extracted by guinea pig while the more recent ones by human recombinant TTG. Also for AEA the first used substrate was monkey esophagus in recent times substituted by human umbilical cord. All kind of serologic an359 Salvatore Accomando, Veronica Pellitteri e Giovanni Corsello tibodies may be performed using IgA or IgG circulating classes. IgG response ought to be considered in IgA deficient patients. Endoscopy It is the most common way to perform duodenal biopsy for diagnostic purpose. Upper endoscopy allows multiple bioptic samples and the direct visualization of duodenum giving to the performer possibility of choosing bioptic location. The endoscopic pattern of duodenal damage may be largely variable going from absolutely normal to several grades of endoscopical damage: scalloping, hyperemia, decreased number and height of Kerckring folds, vascular reticule evidence [27]. Histology Histological damage in CD consists of three different periods. The infiltration phase characterized by intraepithelial lymphocytes (IEL) infiltration, the hyperplastic phase with decrease of villous/crypt ratio and the destructive one consisting of villous atrophy and mucosal flattening. The early period of CD is linked to intraepithelial infiltration by CD3+ CD8+ lymphocytes. More than 25 IEL per cent enterocytes are suggestive of CD in its infiltrative form. Diagnosis may be possible only with immunohistochemical staining for CD3+ cells. The hyperplastic phase is the result of over expressed bowel cellular mitosis at the crypt level associated to reduced villous height (Vh/CrD ratio). Total mucosal atrophy is typical of the last destructive phase. Up to date diagnosis of CD rests upon this histological findings of increased intra epithelial lymphocyte infiltration, decreased villous/crypt ratio and mucosal flattening according to Marsh-Oberhuber stadiation [28,29]. Conclusions Diagnosis of CD depends on clinical, serological, genetic, endoscopic, and histological findings. Anyway duodenal biopsy remains the gold standard. Long life Gluten Free Diet is at the moment the only effective and safe therapy although new ones are under experimental evaluation [30]. 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Capsula Eburnea 2006; 1:1 1-5. 363 Analitical index and Contributors Analitical index and Contributors Abdominal Aortic Aneurysm, 99 Accomando S., 357 Ahmed Hafiz., 321 Allergic Rhinitis, 345 Angiogenesis, 81 Anphibians, 167 Anthropoids, 167 Anzalone R., 25, 149 Asthma, 345 Atherosclerosis, 99 Badalamenti G., 197 Bajardi G., 99 Baldi B., 25 Basile I., 67 Battaglia G., 81 Battaglia S., 345 Bazan V., 35, 197, 213, 223, 249 Bellafiore M., 81 Bellia V., 345 Benigno A., 297, 309 Bivona G., 281 Bonaventura G., 57, 181, 189 Bracale UM., 99 Breast cancer, 213, 223, 249 Bronte G., 213, 249 Bruno L., 223, 249 Burruano F., 269 Buscemi M., 157, 213, 223, 269, 281, 343 Buzzanca ML., 269 Caiozzo M., 197, 213, 223, 249 CalГІ V., 197, 213, 249 Capillaroscopy, 91 Cappello F., 71, 149, 167 Carini F., 91 Casarrubea M., 137 CB1 receptor, 189 Celiac disease, 357 Central giant cell granuloma, 269 Chaperoning system, 9 Chimenti L., 309 Chronic obstructive pulmonary disease, 25 Ciaccio M., 281 Cicero G., 197, 249 Cicero MV., 213 Cocorullo G., 91 Conway de Macario E., 9, 149 Corrao S., 25, 149 Corsello G., 357 Crescimanno G., 137 Cucco D., 57, 181 Cytokeratins, 157 DвЂ™Angelo L., 67 De Luca A., 71 Di Felice V., 71 Di Stefano A., 25 Dispensa N., 249 Dopamin, 137 Evolution, 333 Fanale D., 197 Farina F., 81, 149 Fazio G., 67 Fibrosis, 67 First Cell, 333 Foddai E., 213 Frazzetta M., 197, 223, 249 Galectin-3, 321 Gastrointestinal stromal tumors, 197 Gebbia N., 197, 223, 249 Genetic counselling, 223 GEP system, 181 Gerbino A., 35, 67, 157, 197, 249, 269, 281, 333 Grassi N., 197, 223, 249 Guadagna FP., 213 Guarneri D., 223 Guarneri G., 249 Gullo S., 213 Heat shock proteins, 9, 125 Human umbilical cord, 157 Image analysis, 289 La Manna M., 35 La Paglia L., 223 La Rocca G., 25, 149 Langerhans cells, 35 Last Common Cell, 333 Leone A., 91 Leptin, 57 Licciardi A., 297, 309 Lipari E.F., 167 Lipari L., 125, 343 Lo Coco G., 213 367 Lo Dico G., 223, 249 Lo Iacono M., 25, 149 Lo Piccolo C., 91 Loria T., 25, 149 Lunetta M., 67 Macaluso F., 71 Macario AJL., 9, 149 Magnetic resonance spectroscopy, 281 Magno F., 25, 149 Manna G., 213 MarcianГІ V., 167 Maresi E., 67 Marino Gammazza A., 71 Matrix Metalloproteinases, 99 Mauro A., 99, 157, 343 Mesenchimal Stem Cells, 343 Mesolimbic-mesocortical dopaminergic neurons, 137 Messina P., 91 Montalbano A., 71 Morici G., 297, 309 Muscular dystrophy, 309 Naturalism, 333 Nigrostriatal dopaminergic neurons, 137 Non-compaction, 67 Novo G., 67 Novo S., 67 Orexin, 57, 181 Orphanin, 57, 181 Ovarian cancer, 223, 249 Paglino G., 345 Pantuso G., 197, 223, 249 Pecoraro F., 99 368 Peduncle, 333 Pellitteri V., 357 Placenta, 149 Primates, 167 Prostate cancer, 321 Radiolarians, 333 Reptiles, 167 Rheumatoid arthritis, 125 Ridola CG., 167 Rimonabant, 189 Rizzo S., 197, 213, 223, 249 Role tolerogenic, 35 Russo A., 197, 213, 223, 249 Salerno A., 35 Sandonato L., 197, 249 Scardina GA., 91 Scichilone N., 345 Smads, 25 Sorbera F., 137 Spagnolo G., 25 Spatola GF., 57, 181, 189, 289 Sutera L., 67 Tessitore V., 57, 181, 189 Tortrici S., 269 Training, 297, 309 Transdifferentiation, 343 Uzzo ML., 57, 181, 189 Valenza V., 91 Varia P., 91 Vasostatin I, II, 71 WhartonвЂ™s Jelly, 157, 343 Zummo G., 25, 71 Contents 7 Guest contribution 9THE CHAPERONING SYSTEM: PHYSIOLOGY AND PATHOLOGY Alberto J. L. Macario and Everly Conway de Macario 23 Airway cells,epidermis, signaling 25TGFb signaling: Roles of Smads Francesca Magno, Giampiero La Rocca, Rita Anzalone, Simona Corrao, Tiziana Loria, Melania Lo Iacono, Gaetana Spagnolo, Antonino Di Stefano, Bruno Balbi and Giovanni Zummo 35 New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role Marco La Manna, Viviana Bazan, Aldo Gerbino and Alfredo Salerno 57 Espressione immunoistochimica della orexina A, della orfanina FQ e della leptina nellвЂ™apparato respiratorio del ratto Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo e Vincenzo Tessitore 65 Heart, circulation 67ISOLATED Left ventricular NON-COMPACTION, STROKE AND trabecular meshwork FIBROSIS Giovanni Fazio, Ivana Basile, Monica Lunetta, Luciana DвЂ™Angelo, Loredana Sutera, Emiliano Maresi, Giuseppina Novo, Aldo Gerbino and Salvatore Novo 71The Chromogranin A-derived peptides: structural and functional features in heart biology Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso, Antonella Marino Gammazza, Francesco Cappello and Giovanni Zummo 81 New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina 91ORAL CAPILLAROSCOPY: OUR EXPERIENCE Francesco Carini, Giuseppe Alessandro Scardina, Gianfranco Cocorullo, Pierluigi Varia, Chiara Lo Piccolo, Angelo Leone, Pietro Messina and Vincenzo Valenza 99 GLI ANEURISMI DELLвЂ™AORTA ADDOMINALE Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale 369 123 Cellular stress 125 HEAT SHOCK PROTEINS: KEY PLAYERS IN PATHOGENESIS AND THERAPY OF RHEUMATOID ARTHRITIS Luana Lipari 135 Brain, behavior 137 EFFECTS OF APOMORPHINE ON ORIENTING REACTION IN THE RAT Maurizio Casarrubea, Filippina Sorbera and Giuseppe Crescimanno 147 Organogenesis, Philogenesis, Clinical Embryology 149 The balance of Hsps expression during pregnancy: the role of placenta Simona Corrao, Giampiero La Rocca, Rita Anzalone, Francesca Magno, Tiziana Loria, Melania Lo Iacono, Everly Conway de Macario, Alberto JL Macario, Francesco Cappello and Felicia Farina 157 Expression of cytokeratins in human umbilical cord at term Annamaria Mauro, Maria Buscemi and Aldo Gerbino 167The evolutionary course of Vertebrate foot, from Fish to Man Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello and Elvira Farina Lipari 179 Gastrointestinal tract 181Orexina e Orfanina FQ nuovi neuropeptidi regolatori delle funzioni del Sistema Gastroenteropancreatico. Uno studio immunoistochimico Giuseppe Bonaventura, Giovanni Francesco Spatola, Maria Laura Uzzo e Vincenzo Tessitore 189 EFFETTI DEL TRATTAMENTO CRONICO CON UN ANTAGONISTA DEL RECETTORE CB1 (RIMONABANT) SULlвЂ™APOPTOsi pancreatica DI RATTI ZUCKER OBESI E DIABETICI. UNO STUDIO IMMUNOISTOCHIMICO Vincenzo Tessitore, Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola e Maria Laura Uzzo 195 Tumor process 197 GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia e Viviana Bazan 213The impact on the patientвЂ™s adjustment to breast cancer of the caregiverвЂ™s burden and distress Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna, Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo and Antonio Russo 370 223 LA CONSULENZA GENETICA IN ONCOLOGIA: IMPLICAZIONI BIOMOLECOLARI E CLINICHE Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan 249I tumori della mammella e/o dellвЂ™ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan e Antonio Russo 267 Oral 269IL GRANULOMA CENTRALE A CELLULE GIGANTI DEI MASCELLARI Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino e Maria Buscemi 279 Methodology, Physical activity, Clinical applications 281 MAGNETIC RESONANCE SPECTROSCOPY: PRINCIPLES AND CLINICAL APPLICATIONS Giulia Bivona, Maria Buscemi, Aldo Gerbino and Marcello Ciaccio 289Immunoistochimica e analisi dвЂ™immagine multi spettrale Giovanni Francesco Spatola 297 EFFECTS OF SPECIFIC TRAINING ON HORMONE LEVELS IN FEMALE ATHLETES Arcangelo Benigno, Attilio Licciardi and Giuseppe Morici 309 BENEFICIAL EFFECTS OF LOW INTENSITY ENDURANCE TRAINING IN ADULT MDX MICE Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno and Attilio Licciardi 319 Invited article and report 321 Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis Hafiz Ahmed 333 1859: Naturalismo. Motivi evoluzionistici e anatomia cellulare Aldo Gerbino 341 Proceedings & Meetings 343Ambiente staminale e prospettive di transdifferenziamento verso il fenotipo osteoblastico Annamaria Mauro, Luana Lipari, Maria Buscemi e Aldo Gerbino 345 CAUSES OF ALLERGIC RHINITIS AND ASTHMA Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone and Vincenzo Bellia 371 357 Celiac Disease: a Widespread Emerging Condition in Mediterranean Countries Salvatore Accomando, Veronica Pellitteri e Giovanni Corsello 365 Analitical index and Contributors 372 Annotazioni Annotazioni Annotazioni Finito di stampare nel mese di dicembre 2009 presso le Officine Tipografiche Aiello & Provenzano Bagheria, Palermo