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plumelia

ricerca

E xperimental M edicine R eviews


Morphophysiological Remarks
in english and italian

Edited by Aldo Gerbino Giovanni Zummo Giuseppe Crescimanno

plumelia
edizioni

Dipartimento di Medicina Sperimentale

Di.Me.S.

Universit degli Studi di Palermo Facolt di Medicina e Chirurgia Di.Me.S. Dipartimento di Medicina Sperimentale
Sezioni: Anatomia Umana Emerico Luna (via del Vespro, 129) Istologia ed Embriologia Arcangelo Pasqualino di Marineo (via del Vespro, 129) Fisiologia Umana G. Pagano (corso Tukory, 129)

Experimental Medicine Reviews Morphophysiological Remarks


in English and Italian

Palermo, Italy - vol 1, 2007 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 Graphic design Rosario Notaro Plumelia edizioni Collana Ricerca ISBN 978-88-89876-08-4 This volume can be found on line at the following address: www.unipa.it/dimes Printed by Officine Tipografiche Aiello & Provenzano, Bagheria (Palermo)

Preface Il Dipartimento di Medicina Sperimentale dellUniversit degli Studi di Palermo stato istituito con Decreto Rettorale n 1446 del 01.12.2000, con decorrenza dal 01 gennaio 2001, dalla volont dei Docenti di Anatomia Umana, Fisiologia Umana e di Istologia ed Embriologia generale di costituire un centro di ricerca coordinata tra discipline di base che si riconoscono in una matrice culturale comune, la conoscenza del corpo umano e delle sue funzioni, sia pure con approcci metodologici propri. Le attivit didattiche e scientifiche dei Docenti del Dipartimento di Medicina Sperimentale, DiMeS, sono rappresentate nel sito online www.unipa. it/dimes/ Dallepoca della sua costituzione il Dipartimento ha sperimentato molteplici modalit di cooperazione tra i settori disciplinari che lo costituiscono e con centri di ricerca italiani, europei ed extraeuropei tanto da includere ben due Dottorati di ricerca, quello di Fisiopatologia Neurosensoriale e quello di Scienze delle Attivit Motorie. Fin dallepoca della sua costituzione, il Dipartimento di Medicina Sperimentale ha svolto il suo compito istituzionale sia nellattivit di ricerca che in quella di didattica in tutte le Facolt in cui sono presenti gli insegnamenti che vi insistono. Lattivit didattica stata svolta in coerenza con gli ordinamenti didattici dei Corsi di Laurea delle Facolt di Medicina e Chirurgia, Scienze Matematiche, Fisiche e Naturali, Farmacia e Scienze Motorie. Lattivit di ricerca stata di interesse The Department of Experimental Medicine, University of Palermo was created on 01.12.2000 by the Deans note n 1446. This Department, operative from 01.01.2001, was shaped as a cross-disciplinary centre where Human Anatomy, Physiology and Histology and Embryology combine their cultural bases, their different side of knowledge on the human body and its functions. Both the scientific and didactic activieties of the DiMeS (standing for Dipartimento di Medicina Sperimentale), are found in the website www.unipa.it/dimes/ From its constitution the Department has been working cooperating with Italian and internation research centres, so that today it produces two Phd courses, in Neurosensorial Fisiopathology and in Movement Science. The Department has also run courses and research activities in any Faculty where its topics are taught, adapting the courses according to the main strain of the specific Faculty (Faculty of Medicine and Surgery, Mathematics Phisics and Natural Sciences and Movement Science). The scientific research has been in accordance with the CIVR evaluation standards and with the cultural mission given since the department origin. Conferences, Workshops, International congresses have collaborated to feed the cultural debate, born from the local and international collaborations led by the Department. After seven years since its constitution, the Scientific Board has therefore felt the need
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scientifico, conforme sia alla valutazione del CIVR sia agli obiettivi culturali che erano stati prefissati alla costituzione del Dipartimento. Il dibattito culturale stato intenso e stimolante tanto che, dallepoca della sua costituzione, sono state svolte numerose conferenze, workshop e simposi internazionali, frutto delle numerose collaborazioni locali, nazionali ed internazionali e dei diversi temi scientifici svolti nei laboratori del Dipartimento. Pertanto, dopo sette anni dalla sua fondazione, il Consiglio ha sentito la necessit di consolidare la decisione di mantenere stabili le fondamenta culturali del Dipartimento promuovendo il presente volume che vuole essere non solo lo strumento dellinformazione ma anche lavvio di un confronto sia interno tra i ricercatori del Dipartimento di Medicina Sperimentale sia tra le componenti dellAteneo palermitano, che attraverso il riconoscimento dei temi e delle metodiche possano stabilire interrelazioni e possibili collaborazioni. Come gi evidente dal sommario sono riportati la maggior parte dei temi di ricerca che vengono svolte nei laboratori scientifici del DiMeS e si evidenziano i gruppi e le collaborazioni consolidate. Sono convinto che sia impossibile parlare in modo compiuto e definito della ricerca. Di essa sono state date numerose definizioni, che tuttavia ne descrivono solo la soggettivit storica o gli interessi commerciali. In questultimo trentennio frequentemente abbiamo sentito distinguere la ricerca di base da quella applicata, la prima producendo mera conoscenza, la seconda, invece, tecnologia,
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of a volume representing not only a functional cultural instrument but also a virtual dialogue between researches to create new paths of cultural interrelations and collaborations. In the summary, the main themes, working teams and collaborations among scientific labs are highlighted. lm strongly convinced that a definite and pro per way to talk about research is possible. Many definitions have been given but they describe only commerciai aspects or historical monodisciplinary aspects. During these last thirty years two different kinds of research

D.i.Me.S - Anatomia Umana e Istologia ed Embriologia, Palermo

D.i.Me.S - Fisiologia Umana, Palermo

innovazione, ricchezza. In questi tempi di ristrettezze finanziarie, la ricerca di base o ricerca pura viene considerata un accessorio, un lusso da sacrificare perch la congiuntura economica poco favorevole agli sprechi. Questo concetto stato esasperato in modo strumentale in questi ultimi anni, in particolare considerando lo slittamento degli obiettivi della politica a favore dei valori economici, esaltati dallindustria. AI valore delle idee si contrappone quello del profitto. Permettetemi una provocazione: la ricerca applicata non esiste. Esiste solo la ricerca ed esistono poi le sue applicazioni che possono essere immediate o futuribili. A nessuno sfugge che quando qualcuno osserv che un cerchio ruotava su se stesso, ancora non erano noti n i raggi n gli assali n alcun sistema di ammortizzamento n alcun strumento da utilizzare per il trasporto. La ruota ed il carro sono nati non da un bisogno immediato ma da singole e conseguenti osservazioni (ricerche, conoscenze, competenze) cui sono conseguite le applicazione tecniche. Non vanno confuse la cultura e le sue applicazioni tecniche. Non va confusa la sperimentazione con le esigenze del commercio. Da qui il tentativo ancora limitato di dimostrare come ricerca ed applicazioni possano convivere e come la sperimentazione di laboratorio possa rappresentare obiettivi culturali attuali e non necessariamente indirizzati allimmediatezza del profitto. La ricerca non pu che essere anarchica. La ricerca certezza del dubbio e dellignoranza. So che non so quel che non so; invidio coloro che ne sa-

have been outlined, the first producing me re knowledge, the second producing innovations, technology and wealth through its applicability. We are living n an age where the pure knowledge is considered a waste of money because it cant produce money itself, an idea that has been pushed by the political and industriai world. The idea of culture is set against the value of wealth, and it looses. Let me share a provocation: the applied research does not exist. Reserch means pure cultural speculation, and only in a second moment its application in immediate or programmed technologies. It is infact undoubt that the wheel wasnt born because of an immediate desire of a means of transportation but because someone realised that something with a circle form could move for its own. I mean that the wheel, the carriage, and our car are originated by our observation and speculations, followed by their applications. Culture and its applications are not the same thing. Research must not be confused with commerciai needs. This volume intends to show how research and its applicability can both represent current cultural goals not necessarily addressed to an immediate economic advantage. Research must be anarchic, it is the knowledge of doubt and ones ignorance I know that I dont know what I know not. I envy those who will know more that I know that like me, they will need to measure, assume, hypothesize, mistrust their own deductions. They will need to tell the truth from the falsehood and consider there is always a part of falsehood in truth. (M.
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pranno di pi, ma so che anchessi: come me, avranno da misurare, pesare, dedurre e diffidare delle deduzioni ottenute, stabilire nellerrore qual la parte del vero e tener conto nel vero delleterna presenza del falso (M. Yourcenar: Lopera al nero). Noi speriamo che lo notte della ricerca finisca presto e che si torni a riconoscere alla ricerca scientifica il ruolo di motore indispensabile per lo crescita e il rilancio della cultura e dei valori etici che essa rappresenta. Palermo Dicembre 2007 Giovanni Zummo

Yourcenar: The Work in the Black Phase). We do hope it could be recognised the role of the scientific research as the cultural engine that is essential for the growth and the acceleration of those ethical and cultural values it represents.

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 perphas useful that rise some differences about impostation, that not seem damage editings quality. The Editors
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Guest contribution

Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

GASTRULATION IN SEA URCHIN AND AMPHIBIAN EMBRYOS


[Gastrulazione nel Riccio di mare e negli Anfibi] Giuseppina Turturici, Fabiana Geraci, Gabriella Sconzo and Giovanni Giudice
Dipartimento di Biologia Cellulare e dello Sviluppo Alberto Monroy, Facolt di Scienze Matematiche, Fisiche e Naturali, Universit degli Studi di Palermo (I)

Key words: Gastrulation, Paracentrotus lividus, Xenopus, Xbra, BENI Parole chiave: Gastrulazione, Paracentrotus lividus, Xenopus, Xbra, BENI Abstract. The molecular aspects of the sea urchin Paracentrotus lividus and Strongylocentrotus purpuratus and of the amphibian Xenopus are reviewed. Particular emphasis is given to the gene activity, as for example that of Xbra for Xenopus and genes active before gastrulation for P. lividus. Riassunto. In questa review sono trattati gli aspetti molecolari della gastrulazione del riccio di mare Paracentrotus lividus e dello Strongylocentrotus purpuratus e dellanfibio Xenopus. Si d speciale enfasi allattivit genica, cos come ad esempio quella di Xbra per lo Xenopus e a quella dei geni attivi prima della gastrulazione per il P. lividus.

Gastrulation in sea urchin embryos In this short review we will deal with three most significant examples of gastrulation i.e. that of sea urchins and that of amphibians, seen from a cytological and from a molecular bioological point of view (fig. 1,2,3). In sea urchins, as thorougly described by Hardin [1], the all process begins with the invagination of few cells at the vegetal pole of the blastula embryo, .i.e. the primary mesenchyme cells, followed by invagination of other neighbouring cells, so that a primary intestine starts to be formed. At the same time an active elongation of the intestinal cells occurs, so that gatrulation proceeds. This process involves many sulfate containing macromolecules and can be inhibited by a variety of means, as e.g. treatment with valproate, [2] treatment with Cadmium [3, 4] (or inhibition of RNA synthesis at selected times before blastula) [5]. The process of gastrulation advances with the aid of some philopodia emanating form the secondary mesenchyme cells and [6] explore the walls of the blastocoel, in a process of touching and detouching trough a selective adhesion, till they find a place where to selectively attacch, i.e. where the future mouth will beopened, and from where they pull the other cells to invaginate, thus completing sea urchin gastrulation.

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Giuseppina Turturici, Fabiana Geraci, Gabriella Sconzo and Giovanni Giudice

Gastrulation in amphibians In Xenopus different cell behaviours have been described by Wilson et al [7-10]. In any case, cells from different prospective germ layers are brought to their proper locations by concerted cell movements, so that at the end of the process the ectoderm forms the external part of the embryo, while mesoderm is the middle part of it and endoderm is inside. As described by Nie and Chang [11] several signals influence Xenopus gastrulation, all related to the Erb B tyrosine Kinase receptor. Homma et al. [12] found that a novel gene, which they called BENI (from Brachiury Expression Nuclear Inhibitor) is required for the phase of convergent extension of Xenopus gastrulation, as shown by experimental inhibition with morpholinos or by experiments of BENI [1] iperexpression followed by whole mounts observations. The conclusion of the authors is that the results suggest that BENI expression is regulated by activin-like signaling and that this regulation is crucial for Xbra expression. Ito et al. [13] suggest that SU(H)2 (CBF-1 Suppressor od Hairless, Lag1) is an essential factor for gene expression and morphogenesis of the Xenopus gastrula embryos. They base their conclusions a on experiments of inhibitions with morpholinos and of rescue with the appropriate mRNAs. Chung et al. [14] reported about the role of the protein ANR5, a target of FGF, in regulating Xenopus gastrulation, as shown by experiments of morholinos inhibition and of rescue, by its mRNA. This ankinin repeat domain, called xANR5 acts through Rho and interacts with PAPC, a paraxial protocadherin, to regulate cell protrusion formation and tissue separation in Xenopus gastrulaion. Yun et al. [15] showed that SRF, (Serum Responsive Factor) negatively regulates the Activin/Nodal signal, wich is important for Xenopus gastrulation, because it is crucial for ectoderm specification and for correct positioning of mesoderm and endoderm. Its transcription is restricted to the animal pole ectoderm of the early embryos. The experimental ectopic expression of SRF suppresses mesoderm induction in both the marginal zone in
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Gastrulation in sea urchin and amphibian embryosb

[2,3]

vivo and that caused by Activin/Nodal signal in the animal caps. The inhibition of SRF transcription by antisense or morpholino expands the expression of mesentoderma genes toward the ectodermal territory and enhances the activity id activin, SRFyntercts withh Smad-2 and Fast-1. The authors therefore suggest that XRF might act to ensure proper mesoderm induction in the appropriate region by inhibiting the mesoderm inducing signals during early embryogenesis. Suzawa et al. [16] said that the glucose transporter (xGLUT1) is required for Xenopus gastrulation. Actually the gene GLUT1 is the most important among the thirteen GLUT genes described in animal tissues. Loss of function of GLUT1 experimentally caused in Xenopus brings about microcefaly and axis elongation error. The authors show by whole mount analyses that this gene is expressed in the dorsal region of the embryos especialy in dorsal blastopore lip at the gastrula stage. The authors conclusion is that GLUT1 is an important player in Xenopus gastrulation. Nie and Chang [11] showed that PI3K and Erk MAPK mediate Erb signalings in Xenopus gastrulation. This conclusion is supported by data on rescue of gastrulation defects, by activation of MAPK or P13K in ErbB4 morphant embryos. The authors conclusion is that PI3K Erk and MAPK act downstream of ErbB to participate in gastrulaion morphogenesis. Finally Wallingford and Harland [17] comment on the importance of BMPs in vertebrate gastrulation, through their control of cell adhesion.
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Giuseppina Turturici, Fabiana Geraci, Gabriella Sconzo and Giovanni Giudice

References [1] [2] [3] [4] Hardin J. The cellular basis of sea urchin gastrulation. Current topics in developmental biology vol 33 159-262. Ed. Pedersen and Schatten. Academic press (1996). Sconzo G, Fasulo G, Romancino D, Cascino D, Giudice G. Effect of retinoic acid and valproate on sea urchin development. Pharmazie 1996 51: 175-80. Roccheri MC, Agnello M, Bonaventura R, Matranga V. Cadmium induces the expression of specific stress proteins in sea urchin embryos. Biochem Biophys Res Commun 2004 321: 80-7. Luparello C., Sirchia R., Paci L., Miceli V., Vella R., Scudiero R., Trinchella F. Response to cadmium stress by neoplastic and immortalized human breast cells: evidence for different modulation of gene expression. In Corvin A.J. (ed.) New Developments in Cell Apoptosis Research, pp. 213-239. Happauge, NY (USA): Nova Science Publishers, Inc. (2007). Giudice G, Mutolo V, Donatuti G. Gene expression in sea urchin development. Wilhelm Roux Archiv 1968 161: 118-128. Wu SY, McClay DR. The Snail repressor is required for PMC ingression in the sea urchin embryo. Development 2007 134: 1061-70. Wilson P, Keller R. Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants. Development 1991 112: 289-300. Winklbauer R,, Nagel M, Selchow A and Wacker S. Mesoderm migration in the Xenopus gastrula. Int J Dev Biol 1996 40: 305311. Keller R, Davidson LA, Shook DR. How we are shaped: the biomechanics of gastrulation. Differentiation 2003 71: 171-205. L. Solnica-Krezel, Conserved patterns of cell movements during vertebrate gastrulation, Curr. Biol 2005 15: R213R228. Nie S, Chang C. Regulation of Xenopus gastrulation by ErbB signaling. Dev Biol 2007 303: 93-107. Homma M, Inui M, Fukui A, Michiue T, Okabayashi K, Asashima M. A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation. Dev Biol 2007 303: 270-80. Ito M, Katada T, Miyatani S, Kinoshita T. XSu (H)2 is an essential factor for gene expression and morphogenesis of the Xenopus gastrula embryo. Int J Dev Biol 2007 51: 27-36. Chung HA, Yamamoto TS, Ueno N. ANR5, an FGF target gene product, regulates gastrulation in Xenopus. Curr Biol 2007 17: 932-9. Yun CH, Choi SC, Park E, Kim SJ, Chung AS, Lee HK, Lee HJ, Han JK. Negative regulation of Activin/ Nodal signaling by SRF during Xenopus gastrulation. Development 2007 134: 769-77. Suzawa K, Yukita A, Hayata T, Goto T, Danno H, Michiue T, Cho KW, Asashima M. Xenopus glucose transporter 1 (xGLUT1) is required for gastrulation movement in Xenopus laevis. Int J Dev Biol 2007 51: 183-90. Wallingford JB, Harland RM. Vertebrate gastrulation: the BMP sticker shock. Curr Biol 2007 17: R206-9.

[5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]

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Airway cells

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

EXERCISE-INDUCED CHANGES IN AIRWAY CELLS


[Variazioni delle cellule delle vie aeree indotte dallesercizio fisico] Laura Chimenti1, Giuseppe Morici2,3, Alessandra Patern1, Anna Bonanno3, Vincenzo Bellia1, and Maria R. Bonsignore1,3
Department of Medicine, Pneumology, Physiology and Nutrition (DIMPEFINU), 2Department of Experimental Medicine (DIMES) and 3Institute of Biomedicine and Molecular Immunology (IBIM), National Council of Research (CNR), Palermo (I)
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Key words: Airway inflammation, Bronchial epithelial damage, Remodelling, Induced sputum, Endurance training Parole chiave: Infiammazione delle vie aeree, Danno dellepitelio bronchiale, Rimodellamento, Espettorato indotto, Allenamento di endurance Abstract. Background. Exercise-induced changes in airway cells are largely documented in athletes and animal models. Increased inflammatory cells have been reported in the airways of non-asthmatic endurance athletes independently of symptoms or spirometric alterations, but the functional significance of such findings is still uncertain. Aim. The purpose of this review is to summarize the current state of knowledge about the physiological changes in airway cells induced by acute exercise and training. Results. Runners and athletes of different endurance activities carried out under moderate environmental conditions showed increased airway neutrophils at rest, which tended to further increase after exercise. Skiers and swimmers also showed large increases in airway lymphocytes and eosinophils, possibly related to chronic exposure to cold and dry air or irritants, respectively. In endurance trained mice we found increased inflammatory cells and damaged bronchial epithelium in small airways. Nevertheless, the increase in airway inflammatory cells observed in athletes and mice was not associated with inflammatory activation, and the airway inflammation of athletes did not correlate with bronchial hyperreactivity or post exercise respiratory symptoms. Conclusions. Training-induced airway changes may represent adaptive responses to exercise hyperventilation. However, further studies are necessary to understand the mechanisms responsible for control of inflammatory activation, and assess the relationship between amount/intensity of training and morphologic/ functional changes in airways. Riassunto. Premessa. Variazioni delle cellule delle vie aeree indotte dallesercizio sono ampiamente documentate negli atleti e nei modelli animali. Nelle vie aeree di atleti di endurance non asmatici stato descritto un incremento delle cellule infiammatorie indipendente da sintomi o alterazioni spirometriche, ma il significato funzionale di queste osservazioni resta incerto. Scopo. Lo scopo di questa review di riassumere lo stato attuale delle conoscenze sulle variazioni fisiologiche delle cellule delle vie aeree indotte dallesercizio acuto e dallallenamento. Risultati. Podisti ed atleti di altre attivit dendurance praticate in condizioni ambientali moderate mostravano, a riposo, un aumento dei neutrofili nelle vie aeree che tendevano ad aumentare dopo esercizio. Sciatori

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Laura Chimenti, Giuseppe Morici, Alessandra Patern, Anna Bonanno, Vincenzo Bellia, and Maria R. Bonsignore

di fondo e nuotatori, inoltre, mostravano un notevole aumento di linfociti ed eosinofili, possibilmente correlati, rispettivamente, ad una esposizione cronica ad aria fredda e secca o irritanti. In topi sottoposti ad allenamento dendurance abbiamo trovato un aumento delle cellule infiammatorie e danno allepitelio bronchiale nelle piccole vie aeree. Tuttavia, laumento delle cellule infiammatorie delle vie aeree osservato negli atleti e nei topi non era necessariamente associato ad attivazione infiammatoria e linfiammazione delle vie aeree non era sempre correlata ad iperreattivit bronchiale o a sintomi respiratori post-esercizio. Conclusioni. I cambiamenti delle vie aeree indotte dallallenamento potrebbero rappresentare risposte adattative alliperventilazione durante lesercizio. Ulteriori studi sono necessari, tuttavia, per capire i meccanismi responsabili del controllo dellattivazione infiammatoria, e per valutare le relazioni tra quantit/intensit dellallenamento e cambiamenti morfo/funzionali nelle vie aeree.

Exercise-induced bronchoconstriction and airway inflammation Paragraph 1: Exercise-induced changes in airways cells were initially studied in relation to occurrence of exercise-induced bronchoconstriction (EIB). It was hypothesized that athletes developing EIB after intense exercise might show a background of inflammatory activation in their airways. However, changes in airways cells were shown to commonly occur in athletes independent of concomitant symptoms or spirometric alterations. Paragraph 2: EIB is defined as a decrease in forced expiratory volume of 1s (FEV1) 10% from the baseline value after appropriate exercise provocation [1], and describes the acute, transient airway narrowing that usually occurs typically 5 to 15 min after cessation of exercise. In some instances, a late-phase response (LPR) can also occur 3 to 13 h after completing exercise [2]. Exercise is the most common trigger of bronchospasm in those who are known to be asthmatic, and 50% to 90% of all individuals with asthma have airways that are hyperreactive to exercise [3]. However, EIB also occurs in up to 10% of subjects who are not known to be atopic or asthmatic [4]. Paragraph 3: The pathogenesis of EIB is likely multifactorial and is not completely understood. The mechanism of EIB is believed to relate to the consequences of insufficient heating and humidification of large volumes of air during exercise. The thermal hypothesis proposed that cooling of the airways needed to be followed by rapid re-warming and that these two events caused vasocostriction followed by reactive hyperemia of the bronchial microcirculation, together with edema of the airway wall [5]. Nevertheless, neither airway cooling or re-warming appeared to be necessary for EIB to occur [6]. Paragraph 4: The main theory on EIB pathophysiology is that exercise hyperventilation causes drying of the epithelial surface, thus increasing osmolarity of the airway surface lining fluid [3, 6, 7]. As water evaporates, the airway surface liquid becomes hyperosmolar and provides an osmotic stimulus for water to move from any cell nearby, resulting in cell

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Exercise-induced changes in airway cells

volume loss. Therefore, the increase in regulatory volume after cell shrinkage is believed to be the key event, which results in release of inflammatory mediators that cause airway smooth muscle to contract and the airways to narrow [6]. Paragraph 5: The functional and cellular events triggered by exercise hyperventilation have been studied in experimental models. In anesthetized dogs challenged with high flows of air into a lung segment during bronchoscopy, hyperventilation with dry air caused hyperosmolarity of airway surface lining [8] and bronchoconstriction [9]. Repeated dry air challenges (DACs), mimicking chronic exposure such as in athletes during training, caused epithelial damage with eosinophil and neutrophil influx and increased peptidoleukotriene concentrations in bronchoalveolar lavage fluid (BALF) [10]. In cultured human bronchial epithelial cells, exposure to a hyperosmolar medium or cooling-rewarming triggered an inflammatory cascade by increasing the expression of IL-8 and RANTES partly through the activation of p38 MAP Kinase [11, 12]. Therefore both hyperventilation and airway hyperosmolarity appear capable to cause bronchoconstriction and inflammatory response. Paragraph 6: Athletes who compete in endurance sports such as cross-country skiing, swimming and long-distance running are more likely to experience symptoms of EIB as ventilation is increased for long periods of time during training and competitions, allowing for relatively more evaporative water loss and subsequent airway narrowing [13]. The increased prevalence of EIB in winter sports athletes (cross-country skiing, skating, hockey) is believed to be linked to enhanced exposure to dry and cold air and the relative increase in the reactive hyperemia in the pulmonary vasculature [14]. Finally, athletes who train under environmental conditions of pollutant exposure are at increased risk for the development of EIB. Chlorine compounds in swimming pools and chemicals related to ice-resurfacing machinery in ice rinks may be additional risks for certain populations of athletes [15, 16]. Particulate matter and gases such as carbon monoxide and nitrogen dioxide, which are abundant in indoor ice arenas, and chlorine from swimming pools may trigger and/or exacerbate bronchospasm in athletes who are predisposed to EIB. Paragraph 7: This hypothesis is supported by the finding that 78% of athletes who were EIB-positive after a sport- and environment-specific exercise test turned negative when the test was repeated in the laboratory [17]. However, clinical occurrence of EIB is variable in EIB-positive athletes, since exercise-induced respiratory symptoms poorly predicted EIB [14, 18]. Paragraph 8: In summary, both EIB and airway inflammation may result from exposure to different environmental factors. However, a causal association between inflammation and EIB is likely to occur only in a limited set of conditions. Moreover, inflammatory cells in the airways are increased in well-trained athletes independent of EIB.
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Laura Chimenti, Giuseppe Morici, Alessandra Patern, Anna Bonanno, Vincenzo Bellia, and Maria R. Bonsignore

Airway cells after training Paragraph 9: Airway inflammation has been largely described in athletes who strenuously exercise at low temperatures as skaters and cross-country skiers [19, 20]. Crosscountry skiers studied at rest showed increased lymphocytes in BALF (19) and evidence of airway remodelling in endobronchial biopsies of proximal airways [21]. Airway damage was also observed in sled dogs after intense and prolonged exercise in extreme cold and dry environment [22]. Skiers showed neutrophils infiltration, which is not a typical finding in either atopic or non-atopic asthma, and relatively mild infiltration with eosinophils, mast cells, and macrophages. Moreover, bronchial biopsy findings did not correlate with bronchial hyperresponsiveness (BHR), atopy, or symptoms of asthma [21]. These results suggested the hypothesis that repeated cold weather hyperpnea can predispose these athletes to chronic airway disease different from classic asthma. The term ski-asthma, in fact, describes the syndrome of non-atopic airway inflammation and hyper-reactivity in elite winter sport athletes [19-22]. The peculiar features of ski asthma are further underlined by the lack of changes in airway cells after administration of inhaled steroids [23], a finding opposite to the response of classic asthma to steroid treatment [24]. Paragraph 10: In non-asthmatic amateur runners exercising in a temperate environment, the percentage of neutrophils in induced sputum at rest was higher than in sedentary controls, suggesting a chronic increase in neutrophils in the airways possibly related to habitual training [25]. Neutrophil counts in induced sputum further increased after a marathon race, in the absence of post-race respiratory symptoms or spirometric changes. Because the subjects were not asthmatic, these data suggest that endurance exercise may cause airway inflammation independent of associated asthma or BHR [21]. Paragraph 11: Similar findings were obtained in athletes of other sports. Well-trained young competitive rowers with normal bronchial reactivity to methacholine showed predominance of neutrophils in induced sputum both at rest and after exercise [26]. In swimmers, airway neutrophil differential counts at baseline were higher than in sedentary subjects but cell counts did not change significantly after a 5-km trial in an outdoor pool [27]. After a 5-km race in the sea (hypertonic airway exposure), the same swimmers showed slightly increased eosinophils and lymphocyte differential counts in induced sputum [27]. Paragraph 12: Data obtained in an experimental model in mice support the interpretation that exercise causes limited inflammation in the airways of athletes. Mice were kept sedentary or underwent mild intensity training for 6 weeks under standard laboratory conditions, in order to avoid the potential effect of environmental factors such as air temperature or humidity. Increased leukocyte infiltrate was observed in bronchiolar walls and lumen of endurance trained mice (Figure 1) [28]. Paragraph 13: Inflammatory cell recruitment into the airways could be triggered by da20

Exercise-induced changes in airway cells

mage of airway epithelium, but data obtained in vivo are variable. In large airways of skiers, no clear evidence of epithelial damage was reported despite evidence of remodelling [20]. In marathon runners and swimmers, BEC counts in induced sputum were low at rest and after exercise [25, 26], but their apoptosis increased after a race. Only in rowers after an all-out test did BEC counts in induced sputum tend to increase [26]. Epithelial shear stress caused by very high ventilation observed during supramaximal exercise may account for this result [29]. Conversely, BEC damage occurred in horses after exercise while breathing cold air [30], and repeated dry and cold air challenges in dogs caused loss of ciliated epithelium and airway remodeling [31]. In endurance-trained mice bronchiolar epithelium showed progressive changes during training. After 45-days, the number of ciliated epithelial cells was four-fold lower in trained compared to sedentary mice, and apoptosis of bronchiolar epithelial cells almost doubled in trained mice (Figure 2, panels A and B). Epithelial thickness was 56% higher in trained than in sedentary mice, and bronchiolar epithelium showed a five-fold increase in the number of proliferating cells in trained mice. (Figure 2, panels C and D). The changes observed in trained mice were similar to the epithelial damage described in horses [30] and in dogs [31]. However, the evidence of active repair, indicated by the increase in epithelial thickness and proliferation, suggested that habitual exercise may increase epithelial turnover in bronchioles. In summary, the hypothesis that epithelial damage may trigger influx of inflammatory cells into the airways is supported by some pieces of experimental evidence, although it is hard to test in human studies. Markers of airway inflammation Paragraph 14: To assess whether the increased inflammatory cells in the airways were activated, markers of inflammation were analysed in endurance athletes at rest or after exercise. The available data agree that the increase of airway inflammatory cells in athletes is not usually associated with major evidence of activation, either in BALF and induced sputum in cross-country skiers studied at rest [23] or in induced sputum of runners studied at rest and after a marathon race [25]. Increased concentrations of inflammatory markers (eosinophil peroxidase, neutrophil lipocalin) in induced sputum were observed only in elite swimmers of the Finnish National team [15], while in amateur swimmers training outdoor throughout the year, there was no evidence of inflammatory cell activation at rest or after exercise in outdoor pool or sea [27]. Therefore, data from athletes studies do not provide clear evidence of significant inflammatory activation in the airways induced by acute exercise or prolonged training. Paragraph 15: Other data, obtained in a murine model of allergic asthma, suggest that inflammatory activation in the airways may actually be inhibited by exercise training. In
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Laura Chimenti, Giuseppe Morici, Alessandra Patern, Anna Bonanno, Vincenzo Bellia, and Maria R. Bonsignore

ovalbumin-sensitized mice, nuclear translocation of nuclear-factor-kB (NF-kB) in airway cells was lower in trained than in sedentary animals [32]. Furthermore, in small airways of endurance trained nonasthmatic mice, NF-kB translocation and inhibitor-alpha of NF-kB (IkBa) phosphorylation were not affected and goblet cells in bronchioles were negative at Alcian-PAS staining, indicating that training did not cause excess mucus production [28]. Expression of adhesion molecules by inflammatory cells in the airways Paragraph 16: Expression of adhesion molecules by inflammatory cells provides information on their activation state. In runners or swimmers, airway neutrophils after exercise were shown to express low levels of adhesion molecules, suggesting a possible mechanism for the frustrated airway inflammation found in endurance athletes [7]. L-selectin and b2-integrins on the surface of leukocytes play an important role in leukocyte-endothelial cell interaction. These molecules undergo quantitative and qualitative changes in response to various stimuli. Exercise is known to mobilize neutrophils in peripheral blood, an event associated with shedding of L-selectin [33]. In both runners [25] and swimmers [27], expression of L-selectin by airway neutrophils decreased after exercise, while CD11b/CD18 decreased in runners but was unaffected in swimmers. Paragraph 17: A low level of expression of adhesion molecules, however, does not account for the mechanism of inflammatory cell recruitment at the airway levels in athletes, since a stimulus is necessary for chemoattraction of neutrophils from the bloodstream into the airways. As reported above, bronchial epithelial cells were shown to release IL-8 and RANTES [11, 12] upon exposure to a hyperosmolar medium or cooling-rewarming, suggesting a possible mechanism for exercise-induced leukocyte migration into airways. However, besides recruiting inflammatory cells, hyperosmolar exposure could also decrease their activation state [7]. Therefore, the same stimulus could account for the apparent discrepancy between presence of inflammatory cells in the airways of athletes and lack of inflammatory activation (Figure 3). Discussion Paragraph 18: Studies in trained athletes have shown increased inflammatory cells in the airways with some variability among sports possibly explained by different environmental exposures during exercise. A frustrated inflammatory process possibly related to modulation of adhesion molecules was also proposed. In animal models endurance training caused: decrease in the number of ciliated cells in bronchiolar epithelium; epithelial damage, increased epithelial thickness and proliferation suggesting active repair processes; leukocytes infiltrate in bronchiolar walls and lumen with blunted or absent activation. Paragraph 18: The present state of knowledge only allows to hypothesize the sequence
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Exercise-induced changes in airway cells

of events leading to increased inflammatory cells in the airways (Figure 3). The airway influx of neutrophils is likely driven by chemoattractant mediators released by BECs. Trained mice showed increased leukocyte infiltrate in bronchiolar walls, in line with the findings obtained in samples from large airways in human athletes of different disciplines [19-23]. We speculate that exercise may cause influx of inflammatory cells into the airways secondary to epithelial changes. BECs might be affected by osmotic changes or cooling-rewarming during exercise, and trigger an inflammatory cascade [11, 12]. However, the increase in inflammatory cells in the lumen could also represent a mechanism limiting inflammation. During an inflammatory event, excessive accumulation of leukocytes can be prevented by a fine balance of immune cell recruitment and removal in the damaged tissue. Apoptosis is a mechanism potentially useful to remove granulocytes during inflammation [34], but apoptosis of leukocytes was similar in trained and sedentary mice [28]. Therefore, transepithelial migration might be considered as a highly efficient mechanism, alternative to apoptosis, for clearance of mucosal granulocytes [35]. Paragraph 19: As for the mechanism(s) involved in the pathogenesis of epithelial changes, intense exercise hyperpnea can affect airway epithelium by causing changes in viscosity, tonicity, or amount of the airway lining fluid [6]. It is also possible that high airflows during exercise may cause epithelial shear stress, although this hypothesis is little considered in the current literature on the effects of exercise on airway cells [29]. After all-out rowing, which requires a very high ventilation, BECs in induced sputum tended to increase, suggesting a potential link between maximal airflows during exercise, and epithelial damage [27]. Paragraph 20: Increased inflammatory cells in induced sputum of athletes showed no evidence of activation at rest or after exercise [7]. Markers of inflammation were not found to be increased in BALF or induced sputum in cross-country skiers at rest [23] and in runners at rest and after a marathon race [25]. In trained mice, goblet cells in bronchioles were negative at Alcian-PAS staining. In addition, training did not increase translocation of the NF-kB subunit p65 or IkBa phosphorylation, suggesting blunted or absent activation of inflammatory cells in small airways of trained mice [28]. Overall, an increase in the number of inflammatory cells without activation might represent an adaptive response to increased ventilation during exercise. Conclusions Paragraph 22: Long-term consequences of habitual training are unknown, but available evidence supports that exercise-induced changes do not necessarily mean that exercise is harmful to the lungs. The data collected through these studies suggest that inflammatory activation in the airways is blunted after exercise, and imply that athletes are not inevitably
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Laura Chimenti, Giuseppe Morici, Alessandra Patern, Anna Bonanno, Vincenzo Bellia, and Maria R. Bonsignore

exposed to significant risk in terms of detrimental effects on respiratory health. Exercise per se might cause physiological adaptive responses rather than airway damage. However, further studies are necessary to ascertain the effects of acute exercise and regular training on airway cell pathophysiology in athletes.

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Exercise-induced changes in airway cells

Figure 1. Immunohistochemistry for inflammatory (CD45+) cell infiltration in small airways of endurance-trained mice. In lung sections from sedentary (Panels A and C, original magnification 20x and 40x, respectively) and trained mice (Panels B and D, original magnification 20x and 40x, respectively), CD45+ cells can be identified in the bronchiolar walls (B, large arrow) and the luminal side (D, small arrows).

Figure 2. Immunohistochemical staining (original magnification: 40x) of bronchiolar epithelial cells for apoptosis (TUNEL, panels A and B) and proliferation (PCNA, panels C and D).

Figure 3. Cellular events possibly triggered by exercise hyperventilation.

Laura Chimenti, Giuseppe Morici, Alessandra Patern, Anna Bonanno, Vincenzo Bellia, and Maria R. Bonsignore

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] Kyle JM, Walzer RB, Hanshaw SL, Leaman JR, Frobase JK. Exercise-induced bronchospasm in the young athlete: guidelines for routine screening and initial management. Med Sci Sports Exerc 1992; 24:856-859. Speelberg B, van der Berg NJ, Oosthoek CHA, Verhoeff NPLG, van der Brick WTJ. Immediate and late asthmatic response induced by exercise in patients with reversible airflow limitation. Eur Respir J 1989; 2: 402-408. Rundell KW, Jenkinson DM. Execise-induced bronchospasm in the elite atlete. Sports Med 2002; 32: 583-600. Gotshall RW. Exercise-induced bronchoconstriction. Drugs 2002; 62: 1725-1739. McFadden ER Jr. Hypothesis: exercise-induced asthma as a vascular phenomenon. Lancet 1990; 335: 880-3. Anderson SD, Daviskas E. The mechanism of exercise-induced asthma is. J Allergy Clin Immunol 2000; 106: 453-459. Bonsignore MR, Morici G, Vignola AM, Riccobono L, Bonanno A, Profita M, Abate P, Scichilone N, Amato G, Bellia V, Bonsignore G. Increased airway inflammatory cells in athletes: what do they mean? (Review) Clin Exper Allergy 2003; 33: 14-21. Freed AN, Davis MS. Hyperventilation with dry air increases airway surface fluid osmolarity in canine peripheral airways. Am J Respir Crit Care Med 1999; 159: 1101-7. Freed AN, Bromberger-Barnea B, Menkes HA. Dry air-induced constriction in lung periphery: a canine model of exercise-induced asthma. J Appl Physiol 1985; 59: 1986-90. Davis MS, Freed AN. Repeated hyperventilation causes peripheral airways inflammation, hyper-reactivity, and impaired bronchodilation in dogs. Am J Respir Crit Care Med 2001; 164: 785-9. Hashimoto S, Matsumoto K, Gon Y, Nakayama T, Takeshita I, Horie T. Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase. Am J Respir Crit Care Med 1999; 159: 634-640. Hashimoto S, Gon Y, Matsumoto K, Takeshita I, Maruoka S, Horie T. Inhalant corticosteroids inhibit hyperosmolarity-induced, and cooling and rewarming-induced interleukin-8 and RANTES production by human bronchial epithelial cells. Am J Respir Crit Care Med 2000; 162: 1075-1080.. Holzer K, Brukner P. Screening of athletes for exercise-induced bronchoconstriction. Clin J Sport Med 2004; 14: 134-138. Rundell KW, Im J, Mayers LB, Wilber RL, Szmedra L, Schimtz HR. Self-reported symptoms and exerciseinduced asthma in the elite athletes. Med Sci Sports Exerc 2001; 33:208-213. Helenius IJ, Rytila P, Metso T, Haahtela T, Venge P, Tikkanen HO. Respiratory symptoms, bronchial responsiveness, and cellular characteristics of induced sputum in elite swimmers. Allergy 53: 346-352, 1998. Brauer M, Spengler JD. Nitrogen dioxide exposures inside ice skating rinks. Am J Public Health 1994; 84: 429-433. Rundell KW, Wilber RL, Szmedra L, jenkinson DM, Mayers LB. Exercise-induced asthma screening of elite athletes: field versus laboratory exercise challenge. Med Sci Sports Exerc 2000; 32: 309-316. De Baets F, Bodart E, Dramaix-Wilmet M, Van Daele S, De Bilderling G, Masset S, Velmeire P, Michel O. Exercise-induced respiratory symptoms are poor predictors of bronchoconstriction. Pediatr Pulmonol 2005; 39: 301-305. Sue-Chu M, Larsson L, Moen T, Rennard SI, Bjermer L. Bronchoscopy and bronchoalveolar lavage find-

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ings in cross-country skiers with and without ski asthma. Eur Respir J 1999; 13: 626-32. [20] Provost-Craig MA, Arbour KS, Sestili DC, Chabalko JJ, Ekinci E. The incidence of exercise-induced bronchospasm in competitive figure skaters. J Asthma 1996; 33: 67-71. [21] Karjalainen EM, Laitinen A, Sue-Chu M, Altraja A, Bjermer L, Laitinen LA. Evidence of airway inflammation and remodeling in ski athletes with and without bronchial hyperresponsiveness to methacholine. Am J Respir Crit Care Med 2000; 161: 2086-2091. [22] Davis MS, McKiernan B, McCullough S, Nelson S Jr, Mandsager RE, Willard M, Dorsey K. Racing Alaskan sled dogs as a model of ski asthma. Am J Respir Crit Care Med 2002; 166: 878-882. [23] Sue-Chu M, Karjalainen EM, Laitinen A, Larsson L, Laitinen LA, Bjermer L. Placebo-controlled study of inhaled budesonide on indices of airway inflammation in bronchoalveolar fluid and bronchial biopsies in cross-country skiers. Respiration 2000; 67: 417-425. [24] National Asthma Education and Prevention Program. Expert panel report 2: guidelines for the diagnosis and management of asthma. Bethesda, MD: National Institute of Health, April 1997:97-4051. [25] Bonsignore MR, Morici G, Riccobono L, Insalaco G, Bonanno A, Profita M, Paterno A, Mirabella A, Vassalle C, Vignola AM. Airway inflammation in non-asthmatic amateur runners. Am J Physiol 2001; 281: 668-676. [26] Morici G, Bonsignore MR, Zangla D, Riccobono L, Profita M, Bonanno A, Patern A, Di Giorgi R, Mirabella F, Chimenti L, Benigno A, Vignola AM, Bellia V, Amato G, Bonsignore G. Airway cell composition at rest and after an all-out test in competitive rowers. Med Sci Sports Exerc 2004; 1723-1729. [27] Bonsignore MR, Morici G, Riccobono L, Profita M, Bonanno A, Paterno A, Di Giorgi R, Chimenti L, Insalaco G, Cuttitta G, Abate P, Mirabella F, Vignola AM, Bonsignore G. Airway cells after swimming outdoors or in the sea in nonasthmatic athletes. Med Sci Sports Exerc 2003; 35: 1146-1152. [28] Chimenti L, Morici G, Paterno A, Bonanno A, Siena L, Licciardi A, Veca M, Guccione W, Macaluso F, Bonsignore G, Bonsignore MR. Endurance training damages small airway epithelium in mice. Am J Respir Crit Care Med 2007; 175(5): 442-449. [29] Nucci G, Suki B, Lutchen K. Modeling airflow-related shear stress during heterogeneous constriction and mechanical ventilation. J Appl Physiol 2003; 95: 348-356. [30] Davis MS, Lockard AJ, Marlin DJ, Freed AN. Airway cooling and mucosal injury during cold weather exercise. Equine Vet J Suppl 2002; 34: 413-416. [31] Davis MS, Schoefield B, Freed AN. Repeated peripheral airway hyperpnea causes inflammation and remodelling in dogs. Med Sci Sports Exerc 2003; 35: 608-616. [32] Pastva A, Estell K, Schoeb TR, Atkinson P, Schwiebert LM. Aerobic exercise attenuates airway inflammatory responses in a mouse model of atopic asthma. J Immunol 2004; 172: 4520-4526. [33] Van Eeden SF, Granton J, Hards JM, Moore B, Hogg JC. Expression of the cell adhesion molecules on leukocytes that demarginate during acute maximal exercise. J Appl Physiol 1999; 86: 970-976. [34] Bratton DL, Fadok VA. Theirs but to do and dye. J Allergy Clin Immunol 1999; 103: 555-558. [35] Erjefalt JS, Uller L, Malm-Erjefalt M, Persson CG. Rapid and efficient clearance of airway tissue granulocytes through transepithelial migration. Thorax 2004; 59: 136-143.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

NEW PERSPECTIvES ON THE ROLES Of PROTEINASES AND LUNG STRUCTURAL CELLS IN THE PATHOGENESIS Of CHRONIC OBSTRUCTIvE PULMONARY DISEASE
[Nuove prospettive sui ruoli delle proteasi e delle cellule strutturali polmonari nella patogenesi della bronchite cronica ostruttiva]

Giampiero La Rocca*, Rita Anzalone, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Marco Gervasi, Melania Lo Iacono, Giovanni Zummo and Felicia Farina
Sezione di Anatomia Umana, Dipartimento di Medicina Sperimentale, Universit degli Studi di Palermo, Italy * Corresponding author - The two authors contributed equally to this work

Key words: Metalloproteinases, Cigarette smoke, COPD, Fibroblasts, Proteinase inhibitors Parole chiave: Metalloproteasi, Fumo di sigaretta, BPCO, Fibroblasti, Inibitori delle proteasi Abstract. Cigarette smoke is among the major risk factors for the development of chronic lung diseases such as COPD. In the last years, a number of reports investigated the multiple roles of proteolytic enzymes in lung pathophysiology. These molecules are expressed at various levels since the beginnings of lung development, and their expression should be restored during injury repair as well as inflammatory processes. Recent literature reports have enlightened the role of fibroblasts as key cells in mediating not only the composition of the extracellular matrix in the lungs, but also as possible regulators of the inflammatory processes following the exposure to environmental toxic stimuli as cigarette smoke. These cells constitutively produce MMP-2 as well as other proteolytic enzymes, and the expression of these molecules is regulated by cigarette smoke ad different extents. Since inhibition of proteolytic enzymes is being viewed as a promising therapeutic strategy for lung diseases, it is important to know which enzymes should be inhibited for their pro-inflammatory action, and which ones should not, since it has been demonstrated their anti-inflammatory role. Riassunto. Il fumo di sigaretta uno dei principali fattori di rischio per lo sviluppo di malattie polmonari croniche come la BPCO. Negli ultimi anni, diversi gruppi hanno studiato i molteplici ruoli degli enzimi proteolitici nella fisiopatologia polmonare. Queste molecole sono espresse a vari livelli sin dalle fasi iniziali dello sviluppo polmonare, e possono essere riespresse durante i processi di riparo delle ferite e infiammazione. Diversi articoli recenti hanno messo in luce il ruolo centrale dei fibroblasti nel mediare non solo la composizione della matrice extracellulare polmonare, ma anche nella regolazione dei processi infiammatori innescati dallesposizione a stimoli ambientali tossici come il fumo di sigaretta. I fibroblasti producono costitutivamente la MMP-2, insieme ad altri enzimi litici, e lespressione di tali molecole regolata a diversi livelli dal fumo di sigaretta. Poich linibizione dellattivit degli enzimi proteolitici della matrice vista come una strategia terapeutica promettente per le malattie polmonari, importante conoscere quali enzimi debbano essere inibiti a causa del loro ruolo pro-infiammatorio, e quali invece debbano essere preservati per il loro dimostrato ruolo anti-infiammatorio.

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Giampiero La Rocca, Rita Anzalone, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Marco Gervasi, Melania Lo Iacono, Giovanni Zummo, Felicia Farina

1. Introduction Chronic lung inflammatory diseases constitute a broad group of pulmonary pathologies, with a growing number of affected subjects and an increase in the sanitary expense worldwide. New pharmacological tools are being developed to fight these diseases, which include anti-inflammatory drugs and molecule-aimed drugs to interfere with intracellular an extracellular mechanisms involved in the pathogenetic process. Chronic Obstructive Pulmonary Disease (COPD) is a progressive and death-causing disease, which progression from the mild to severe grade is little affected by drug administration, therefore being viewed as a leading cause of death in western countries. The disruption of the airway wall organisation is one of the key features of such pathologies, followed by an increase in collagen deposition which leads to a progressive loss of lung function [1]. Cigarette smoke is among the major risk factors for the development of chronic lung diseases such as COPD and emphysema [2]. This mixture of as many as 3000 different chemical species, most of which possess a high reactivity towards biological molecules, may interact with cellular populations of the upper as well as lower airways. Between the most striking phenomena caused by smoke exposure, there is the accumulation of macrophages and neutrophils, observed in pulmonary emphysema, and, as demonstrated for COPD, the inflammatory state is maintained, even following the removal of the initial causing stimulus (e.g. for smoke cessation after the diagnosis) [2,3]. 2. The role of extracellular matrix in lung pathophysiology The extracellular matrix represents a complex mixture of macromolecules which provide a stable substrate for cells and, as shown by different reports in recent years, is able to provide morphogenetic signals as well as serving as a reservoir of matrix-bound growth factors [4]. Therefore, it is now accepted that there is a delicate balance between growth factors production and their interaction with molecules bound to the extracellular matrix, therefore regulating growth factors function itself [5,6]. Apart from the known instructive role in lung morphogenesis and repair events and from serving as a molecular scaffold for tissue organisation, cell-extracellular matrix interactions are believed to play a role in lung pathology. For instance, in the development of inflammatory processes, one of the potential mechanisms for the perpetuation of the inflamed state in airways may involve the control of extracellular matrix (ECM) turnover [7]. In the case of COPD, the activation of inflammatory cells by cigarette smoke results also in the production of large amount of proteinases, as well as the decrease of their tissue inhibitors levels. Therefore, the global effect is an imbalance of tissue homeostasis which favours a pathogenetic remodelling of the matrix [8,9]. The production of matrix-degrading enzymes goes more far than the simple degradation of molecules to favour inflammatory cells migration in response to chemotactic stimuli. In
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New perspectives on the roles of proteinases and lung structural cells in the pathogenesis of chronic obstructive pulmonary disease

fact, the effects of inflammation may be prolonged also indirectly, for example by the generation of proteolytic fragments (matrikins) of ECM molecules by the proteolytic enzymes secreted by different cell types, even after the cessation of the causative stimulus (cigarette smoke, oxidative stress, air pollution). This process may take place by the recruiting activity of ECM fragments towards neutrophils and monocytes, but also by the activation of growth/survival factors (or their mobilisation from their extracellular matrix storage sites); all of these processes may contribute to trigger inflammation [10,11]. Therefore in airways, as well as in other body systems, the delicate balance between production, activation and inhibition of proteinases should stringently regulate the tissutal responses to external injuries. The alteration of this equilibrium should favour the development of pathologies which reside in the inability to repair properly the injuries. 3. Matrix metalloproteinases and their roles in lung pathophysiology Matrix degrading proteinases belong to different classes, grouped on the basis of their structural and catalytic features. In particular, matrix metalloproteinases (MMPs) constitute a broad family of 25 members, which share a significant structural homology and domain organisation and feature a zinc ion binding site into their catalytic domain [12,13]. Different subgroups of MMPs have been characterised, on the basis of their substrate specificity (e.g. collagenases, elastases and gelatinases). As a key feature of these molecules, the same substrate may be cleaved by different enzymatic species. This overlap of target molecules involves both ECM structural proteins and regulatory ones, therefore reflecting the intricate organisation of matrix microenvironmental regulation. Between MMPs, gelatinases, also named Type IV collagenases, are two enzymes (MMP-2 or gelatinase A and MMP-9 or gelatinase B) which play a key role in a number of physiological processes. These enzymes are able to degrade the type IV collagen, which is abundant in the basement membranes, as well as gelatin derived from collagenases-dependent cleavage of the fibrillar collagen types. It is well demonstrated that in lung ECM biology these molecules are involved in developmental processes, as well as in tissue repair and fibrosis [14]. An increased knowledge on the function and spatiotemporal regulation of these enzymes during pulmonary development will be of central importance to understand lung repairing processes. Various enzymes participate in the development of lung from the earliest stages of lung buds formation through the final development of the bronchial tree, with a strict regulation both in terms of places and length of expression, with some MMPs considered as key players in these processes. As reviewed recently by Greenlee and coworkers (15 and refs therein), there are multiple experimental works on the differential roles of MMPs during the development of mice lungs, showing that while some MMPs were absent (as demonstrated for MMP-3 and MMP-9), MMP-2 and its main activator, MMP-14, decreased
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Giampiero La Rocca, Rita Anzalone, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Marco Gervasi, Melania Lo Iacono, Giovanni Zummo, Felicia Farina

with the progression of lung development. Therefore, two key enzymes are of central importance during development: MMP-2 and MMP-14. In particular, some reports indicated that gelatinase A expression persists during lung development [16]. Although not all members of the MMP family are found within the adult lung tissue, some enzymatic activities are upregulated during the acute and chronic phases of pulmonary diseases. In fact, their role has been highlighted in several pathological conditions, as asthma, COPD and lung cancer. Although small amounts of MMP2 and MMP14 are present in the lining fluid of the lungs under normal conditions, other MMPs such as MMP-9, and MMP-12 are significantly upregulated under such pathological conditions, as shown for macrophage-derived enzymes. Therefore, a wrong pathway of proteolytic signaling may involve, as suggested by different reports, the cleavage of a-1 proteinase inhibitor. The decrease of this molecule is associated with smoke-induced COPD development and the cleavage by MMP-9 is likely to enhance the activity of neutrophil elastases, therefore resulting in elastin degradation [17,18]. Furthermore, it must be considered that damaging external stimuli (as airborne pollutants or smoke components inhaled with respiration) can provide a variety of signals which can induce an inflammatory response. The presence of reactive oxygen species as well as other highly reactive chemical species, favours the interactions with cellular populations of the respiratory tract, in both upper and lower airways. In recent years, the attention of researchers has been focused on the contribution of structural cells of the lungs, as fibroblasts, to the development and the maintenance of chronic lung diseases following their exposure to noxious agents. The use of cigarette smoke (and in particular its soluble extracts, named CSE) as source of damage for lung cells allows mimicking the effects that may take place in vivo as a consequence of smoking and, as demonstrated in several studies on animal models, cigarette smoke exposure is currently considered the best model for COPD development [19]. Indeed, CSE-treated lung fibroblasts are able to secrete chemotactic molecules for both neutrophils and macrophages [20,21]. In addition, fibroblasts exposed to cigarette smoke may produce prostaglandin, thereby contributing to the creation of a proinflammatory microenvironment [22]. Moreover, gelatinases produced by structural cells may play a key role in the pathogenesis of COPD, as demonstrated for mouse lung fibroblasts. Indeed, this process appeared to be regulated by CSE exposure [23]. Therefore it is apparent that the pulmonary fibroblast should no more be viewed as a mere structural cell of the respiratory apparatus. In fact, fibroblasts regulate the composition of the ECM scaffold, by deposing new collagen and elastin molecules, both in the physiological turnover of matrix and in the reparative processes following injuries in the lungs. Instead, when a persistent inflammatory state is triggered by cigarette smoke, the general mechanisms of tissue repair fail, and matrix alterations may accumulate, leading to disease development.
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New perspectives on the roles of proteinases and lung structural cells in the pathogenesis of chronic obstructive pulmonary disease

In this process, the correct balance between proteinases and inhibitors is critical for tissue repair and remodelling [24]. The potential contribution of fibroblasts as active players in chronic lung diseases has been further elucidated in a recent study from our group. By performing several experiments, we determined the variations in the gelatinolytic pattern of cultured human lung fibroblasts exposed to increasing concentrations of CSE. Interestingly, the most significant variations were observed for the extracellular MMP-2 levels, which were shown to decrease following exposure to growing doses of cigarette smoke. In addition, we demonstrated a decrease in MMP-2 mRNA and protein, which suggested a transcriptional modulation due to cigarette smoke exposure [25]. Moreover, the mRNA levels of TIMP-2, the main inhibitor of MMP-2, conversely increased following exposure to cigarette smoke, thus reinforcing the global negative effect on MMP-2 enzyme levels. The observed modulator effect of CSE on fibroblast-secreted gelatinases and inhibitors may in part explain the effects due to cigarette smoke exposure in vivo, and confirm the recent hypotheses on the central role of fibroblasts in the development of chronic lung diseases. Since the anti-inflammatory properties of enzymes as MMP-2, we argued that in this case, an inhibition of the enzyme should just worsen the clinical features of the patients. Recent experimental evidences from different groups support our hypothesis. First of all, some members of the metalloproteinases family, and in particular gelatinase A, have been reported to have a limiting role in inflammatory processes [26,27]. In fact, some chemokines may be inactivated by MMP-2, thereby restricting monocyte migration towards sites of injury. Therefore, it should be suggested that in a normal inflammatory process some enzymes (as macrophage-derived MMP-9) may be initially required to promote cell influx to damaged areas, while when inflammation must be dampened, other enzymatic activities belonging to the same family may participate degrading proinflammatory mediators to stop the process. Indeed, when the inflammatory process persists, as in COPD, the cellular interplay, as well as cell-matrix interactions are severely affected. In addition, in a more recent work which described a novel functional proteomics approach, the authors identified several proteins in the BAL fluid that should be cleaved by gelatinases A and B, and are essential for regulating inflammatory pathways in experimental asthma, as S100A8 and S100A9 [28]. These proteins all have chemotactic properties and are upregulated in allergic lung inflammation. 4. Therapeutic approaches of proteases inhibition: are they always beneficial? Given the importance of cell-ECM interactions for the development of inflammatory lung diseases, the possibility to use natural or synthetic inhibitors of metalloproteinases for the clinical management of COPD patients is being investigated by various groups [29]. One point in favour of this option is the clear relationship between increased expression of
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Giampiero La Rocca, Rita Anzalone, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Marco Gervasi, Melania Lo Iacono, Giovanni Zummo, Felicia Farina

some enzymes (MMP-8, MMP-9, and MMP-12) and the progression of the disease. Therefore, the use of specific inhibitors designed to inhibit these enzymatic activities should be beneficial for the treatment of patients. On the other hand, given their high structural similarity, it is a very difficult task to obtain a molecule with single enzymatic specificity; therefore it is likely that an inhibitor should affect a number of enzymes, and therefore blocking several different extracellular signalling pathways. Indeed, currently it is not clear whether MMP inhibition should be beneficial or harmful for patients treatment, and in which situations it would really be of clinical usefulness. The complexity of the pattern is increased by the evidence that different activation pathways are known for the different forms of metalloproteinases, and these mechanisms should be varied under different pathological conditions. A second aspect to be considered is the possible but not sure redundancy in function between similar MMPs. In this case, the two gelatinases, even if they share a great number of substrate molecules between the metalloproteinase families, should play non-redundant roles in lung pathologies. For example, it has been demonstrated that MMP-2 and MMP-9 should play a nonredundant role in the pathogenesis of obliterative airway disease (OAD), a known complication of chronic lung allograft rejection. Given the experimental results obtained, it has been proposed that inhibition of MMP-9, rather than MMP-2, may represent a useful therapeutic opportunity for chronic lung allograft rejection [30]. In conclusion, despite the current efforts to use proteinase inhibitors in the clinical practice for the treatment of inflammatory lung diseases, a lot of work is still to be performed. Understanding the single roles of the different enzymes, in physiology and pathology, will give us the opportunity to design better drugs to improve the patients condition.

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New perspectives on the roles of proteinases and lung structural cells in the pathogenesis of chronic obstructive pulmonary disease

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] Di Stefano A, Caramori G, Ricciardolo FLM, Capelli A, Adcock IM, Donner CF. Cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview. Clin. Exp. Allergy 2004; 34: 1156-1167. Shapiro SD. End stage chronic obstructive pulmonary disease: The cigarette is burned out but inflammation rages on. Am J Respir Crit Care Med 2001; 164: 339-340. Shapiro SD. The macrophage in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160: S29-S32. Ramirez F, Rifkin DB. Cell signalling events: a view from the matrix. Matrix Biol 2003; 22: 101-107. Corbel M, Belleguic C, Boichot E, Lagente V. Involvement of gelatinases (MMP-2 and MMP-9) in the development of airway inflammation and pulmonary fibrosis. Cell Biol Toxicol 2002; 18: 51-61. Aumailley M, Gayraud B. Structure and biological activity of the extracellular matrix. J Mol Med 1998; 76: 253-265. Kleinmann HK, Philp D, Hoffman MP. Role of the extracellular matrix in morphogenesis. Curr Opin Biotechnol 2003; 14: 526-532. Ten Hacken NHT, Postma DS, Tiemens W. (2003). Airway remodeling and long-term decline in lung function in asthma. Curr Opin Pulmon Med 2003; 9 :9-14. Shapiro SD. Proteolysis in the lung. Eur. Respir. J. 2003; 22 (Suppl. 44): 30s-32s. Fowlkes JL, Winkler MK. Exploring the interface between metallo-proteinase activity and growth factor and cytokine bioavailability. Cytokine Growth Factor Rev 2002; 13: 277-287. Schenk S, Quaranta V. (2003). Tales from the crypt[ic] sites of the extracellular matrix. TRENDS Cell Biol 2003; 13: 366-375. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behaviour. Annu Rev Cell Dev Biol 2001; 17: 463-516. Somerville RPT, Oblander SA, Apte SS. Matrix metalloproteinases: old dogs with new tricks. Genome Biol 2003; 4: 216. Chua F, Sly PD, Laurent GJ. Pediatric lung disease: from proteinases to pulmonary fibrosis. Pediatr Pulmonol 2005; 39: 392-401. Sato E, Koyama S, Takamizawa A, Masubuchi T, Kubo K, Robbins RA, Nagai S, IzumiT. Smoke extract stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activities. A JP Lung 1999; 277: 1149-1157. Greenlee KJ, Werb Z, Kheradmand F. Matrix metalloproteinases in lung: multiple, multifarious and multifaceted. Physiol Rev 2007; 87: 69-98. Masumoto K, de Rooij JD, Suita S, Rottier R, Tibboel D, de Krijger RR. Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases during normal human pulmonary development. Histopathology 2005; 47: 410419. Liu Z, Zhou X, Shapiro SD, Shipley JM, Twining SS, Diaz LA, Senior RM, Werb Z. The serpin alpha1proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo. Cell 2000; 102: 647655. Senior RM. Mechanisms of COPD: conference summary. Chest 2000; 117: 320S323S. Numanami H, Koyama S, Nelson DK, Hoyt JC, Freels JL; Habib MP, Amano J, Haniuda M, Sato E, Robbins RA. Serine protease inhibitors modulate smoke-induced chemokine release from human lung fibroblasts. Am J Respir Cell Mol Biol 2003; 29: 613-619. Martey CA, Pollock SJ, Turner CK, OReilly KM, Baglole CJ, Phipps RP, Sime PJ. Cigarette smoke induces cyclooxygenase-2 and microsomal prostaglandin E2 synthase in human lung fibroblasts: implications

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for lung inflammation and cancer. Am J Physiol Lung Cell Mol Physiol 2004; 287: L981-L991. [22] Ning W, Li C-J, Kaminski N, Feghali-Bostwick CA, Alber SM, Di YP, Otterbein SL, Song R, Hayashi S, Zhou Z, Pinsky DJ, Watkins SC, Pilewski JM, Sciurba FC, Peters DG, Hogg JC, Choi AM. Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease. PNAS 2004; 101: 14895-14900. [23] Mott JD, Werb Z. Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 2004; 16: 558-564. [24] La Rocca G, Anzalone R, Magno F, Farina F, Cappello F, Zummo G. Cigarette smoke exposure inhibits extracellular MMP-2 (gelatinase A) activity in human lung fibroblasts. Respir Res 2007; 8: 23. [25] McQuibban GA, Gong JH, Tam EM, McCulloch CA, Clark-Lewis I, Overall CM. Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science 2000; 289: 1202-1206. [26] McQuibban GA, Gong JH, Wong JP, Wallace JL, Clark-Lewis I, Overall CM. Matrix metalloproteinase processing of monocyte chemoattractant proteins generates CC chemokine receptor antagonists with anti-inflammatory properties in vivo. Blood 2002; 100: 1160-1167. [27] Greenlee KJ, Corry DB, Engler DA, Matsunami RK, Tessier P, Cook RG, Werb Z, Kheradmand F. Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation. J Immunol 2006; 177: 7312-7321. [28] Belvisi MG, Bottomley KM. The role of matrix metalloproteinases (MMPs) in the pathophysiology of chronic obstructive pulmonary disease (COPD): a therapeutic role for inhibitors of MMPs? Inflamm Res 2003; 52: 95-100. [29] Fernandez FG, Campbell LG, Liu W, Shipley JM, Itohara S, Patterson GA, Senior RM, Mohanakumar T, Jaramillo A. Inhibition of obliterative airway disease development in murine tracheal allografts by matrix metalloproteinase-9 deficiency. Am J Transplant 2005; 5: 671683.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

AIRWAY CELLS IN SWIMMERS: A CASE REPORT AND A REvIEW Of THE LITERATURE


[Cellule delle vie aeree nei nuotatori: case report e rassegna della letteratura] Giuseppe Morici1,3, Laura Chimenti2, Anna Bonanno3, Loredana Riccobono3, Mirella Profita3, Alessandra Patern3 and Maria R. Bonsignore2,3
Department of Experimental Medicine (DIMES)1, and Department of Medicine, Pneumology, Physiology and Nutrition (DIMPEFINU)2; Institute of Biomedicine and Molecular Immunology (IBIM), National Council of Research (CNR)3, Palermo (I)

Key words: Endurance training, Marathon swimming, Induced sputum, Adhesion molecules Parole chiave: Allenamento dendurance, Nuoto di gran fondo, Espettorato indotto, Molecole di adesione Abstract. Background. Inflammatory cells are increased in the airways of non-asthmatic endurance athletes without clear evidence of activation. Their role and the mechanisms involved in their recruitment into the airways, however, are still poorly understood. Previous studies suggest that habitual training and exercise duration may be critical factors in determining airway cell counts and composition. Aim. To assess the effects of very prolonged endurance exercise on airway cells we studied a well-trained non-asthmatic amateur swimmer who covered the distance of 34.6 km between Lipari (Eolian Islands) and Capo dOrlando (Northern coast of Sicily) in 15 hours. Results. Induced sputum, collected at baseline and 12 hours after the trial, showed a very high percentage of neutrophils (98% and 82.5%, respectively). Moreover, eosinophils were slightly increased after the trial, similar to previous data collected in a group of swimmers after a competition in the sea. In peripheral blood, leukocytosis and neutrophilia, release of muscle enzymes and evidence of systemic inflammatory activation were also found. Conclusions. These data confirm that endurance exercise affects both airway and peripheral blood cells, and suggest a modulating role of exercise duration in neutrophil recruitment into the airways. Riassunto. Premessa. Le vie aeree di atleti endurance a riposo mostrano un aumento delle cellule infiammatorie, senza tuttavia evidenza di attivazione. Il significato fisiologico e i meccanismi responsabili del reclutamento delle cellule infiammatorie nelle vie aeree sono ancora poco chiari. Studi precedenti suggeriscono che lallenamento abituale e la durata dellesercizio possono modulare la composizione e la quantit di cellule presenti nelle vie aeree. Scopo. Gli effetti di un esercizio dendurance molto prolungato sulle cellule delle vie aeree sono stati valutati in un nuotatore amatoriale ben allenato, non-asmatico, che ha percorso la distanza di 34,6 km tra Lipari (Isole Eolie) e Capo DOrlando (Costa Nord della Sicilia) in 15 ore. Risultati. Lespettorato indotto mostrava una percentuale di neutrofili molto alta sia in condizioni di base che 12 ore dopo la prova (98% e 82.5%, rispettivamente). Inoltre, gli eosinofili erano leggermente aumentati dopo la prova, confermando dati precedenti ottenuti in un gruppo di nuotatori dopo una gara a mare. Il nuoto molto prolungato era anche associato a leucocitosi e neutrofilia del sangue periferico, rilascio di enzimi muscolari ed evidenza di attivazione infiammatoria sistemica.

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Giuseppe Morici, Laura Chimenti, Anna Bonanno, Loredana Riccobono, Mirella Profita, Alessandra Patern and Maria R. Bonsignore

Conclusioni. Questi dati confermano che lesercizio dendurance influenza le cellule delle vie aeree e del sangue periferico, e suggeriscono che la durata dellesercizio possa modulare il reclutamento dei neutrofili nelle vie aeree.

Background Paragraph 1: Several studies have shown increased inflammatory cells in the airways of endurance athletes exercising in a cold [1-6] or moderate [7-9] environment. The degree and type of airway inflammation under resting conditions was variable in athletes of different sports, and the role of inflammatory cells in the airways is currently unclear. In cross-country skiers studied at rest, lymphocytes were increased in bronchoalveolar lavage fluid [1], and endobronchial biopsies showed increased inflammatory cells (lymphocytes, but also neutrophils and eosinophils) and evidence of airway remodeling [3] suggesting chronic airway disease (ski asthma) [1-6]. Paragraph 2: We have previously reported that in non-asthmatic amateur runners exercising under moderate environmental conditions neutrophil counts in induced sputum were increased after a marathon race over the baseline level, in the absence of post-race respiratory symptoms or spirometric changes [7]. Under baseline conditions, the percentage of neutrophils in induced sputum of runners was higher than in sedentary controls, suggesting a chronic increase in neutrophils in the airways, possibly related to habitual training [7]. Similar to runners, well-trained, non-asthmatic young competitive rowers showed predominance of neutrophils in induced sputum both at rest and after exercise [9]. In swimmers, airway neutrophil differential counts at baseline were higher than in sedentary controls but cell counts did not change significantly after a 5-km trial in an outdoor pool [8]. After a 5-km race in the sea, a condition of hypertonic airway exposure during exercise, the same swimmers showed slightly increased eosinophils and lymphocyte differential counts in induced sputum [8]. Overall, 5-km swimming exerted smaller effects on airway cells than running a marathon. As for the mechanism of cell recruitment, it is possible that changes in epithelial cells associated with hyperosmolarity of the airway surface lining fluid or with cooling-rewarming of the airways, may drive inflammatory cells into the airways, as suggested by in vitro studies [10, 11]. Paragraph 3: The increase in inflammatory cells in the airways of athletes was not associated with evidence of inflammatory activation at rest or after exercise [7, 8, 12] as assessed by markers of inflammation in BALF or induced sputum in cross-country skiers studied at rest [13] and runners studied at rest and after a marathon race [7]. Furthermore, in both runners [7] and swimmers [8], expression of L-selectin by airway neutrophils decreased after exercise, while CD11b/CD18 decreased in runners but was unaffected in

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Airway cells in swimmers: a case report and a review of the literature

swimmers, confirming lack of inflammatory activation in the airways of endurance athletes. Similarly, intracellular markers of inflammation such as nuclear-factor-kB (NF-kB) translocation and inhibitor-alpha of NF-kB (IkBa) phosphorylation were not increased in endurance trained compared to sedentary mice [14]. Elite swimmers of the Finnish National team were the only athletes where increased concentrations of inflammatory markers (eosinophil peroxidase, neutrophil lipocalin) were found in induced sputum at rest [15]. Instead, in amateur swimmers training outdoor throughout the year, analysis of induced sputum showed no evidence of inflammatory cell activation at rest or after exercise either in outdoor pool or sea, in fact, neutrophil elastase concentration was low at all times [8]. It is likely that exposure to high chlorine compound concentrations in indoor pool may account for the inflammatory pattern found in the Finniah athletes, confirming the importance of environmental factors in exercise-induced airway changes. Paragraph 4: Estimating the respective role of endurance exercise and airway exposure to environmental factors in increasing airway inflammatory cells in athletes is difficult. Increased neutrophils in the airways may be a direct consequence of endurance training, as they were found in all athletes tested, irrespective of practised sport. Conversely, increased eosinophils and lymphocytes may depend on environmental exposure to factors such as chlorine compounds in swimmers, or dry and cold air in skiers. We report the results of a study on an amateur long-distance swimmer who completed a 35-km trial between Eolian Islands and Sicily. These data may shed some light on the effects of very prolonged endurance exercise on airway cells, extending our previous data collected in swimmers after 5-km races. Case report Paragraph 5: We studied an amateur non-asthmatic swimmer who covered the 35-Km distance between Lipari (Eolian Islands, Mediterranean Sea) and Capo dOrlando (Northern coast of Sicily) in 15 hours. Paragraph 6: The athlete (age: 30 yr; body weight: 67 kg; height: 169 cm) had been swimming at amateur level for ten years. To prepare for this trial, he swam 48 km/week. He was not taking any drug. Spirometric values at rest were: FEV1 4.15 L (104% predicted), FVC 5.32 L (113% predicted). The protocol was approved by the Ethical Committee, University of Palermo, and written informed consent was obtained before the study. Paragraph 7: The trial started at 07:00 a.m., at the Faraglioni of Lipari, Eolian Islands, Italy. Arrival at Capo dOrlando, on the Northern coast of Sicily (linear distance: 34.6 Km) was at 10:00 p.m. of the same day. The weather was good (barometric pressure: 1017-1019 mbar), with no or light wind. Air temperature increased from 25.5C at 07:00 a.m to 37.5C at 03:00 p.m; then, it decreased gradually to 26.5C at 10:00 p.m. The
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Giuseppe Morici, Laura Chimenti, Anna Bonanno, Loredana Riccobono, Mirella Profita, Alessandra Patern and Maria R. Bonsignore

athlete did not stop or mount on boat; he only briefly interrupted swimming to drink or eat small snacks. Body weight at arrival was 64 kg. The trial was regular. The athlete referred no major complaint during or after the trial. Paragraph 8: Methods. Data were collected over four weeks: at rest, two weeks before the trial (baseline-intensive training); at rest, in the pre-trial week during progressive decrease in training intensity and duration (baseline-tapering); at the end of the trial; and 12 and 36 hours post-trial. Samples of induced sputum were obtained at baseline-tapering, and 12 hours post-trial. Samples were processed as previously reported [7, 8], and analyzed for cell counts and composition. Briefly, the subject underwent hypertonic saline (3%) aerosol for 20-min, and expectorated into sterile ampoules. After addition of an equal volume of 0.1% dithiothreitol saline solution (Sigma Chemical Co, St Louis, MO, USA), each sample was mixed (37C, 15 min), and centrifuged (800g, 10 min). The supernatant was frozen at 20C, for subsequent analysis of albumin and neutrophil elastase concentration. Cell pellets were resuspended in saline, and cell count (standard hemocytometer) and viability (Trypan blue exclusion) assessed in 10-l aliquots. Cells were cytocentrifuged (Cytospin 2, Shandon Instruments, Runcorn, UK) and stained (DiffQuick, Merz-Dade, Dudingen, Switzerland). Slides were blindly read (at least 400 cells/slide), and differential counts expressed as corrected percentage after subtracting squamous cells. Paragraph 9: Expression of adhesion molecules was analyzed in cells of induced sputum and peripheral blood neutrophils (Ficoll-Hypaque gradient separation), as previously reported [7,8]. Slides were prepared (acetone/methanol, 4C, 10 min), and incubated (37C, 1 h) with monoclonal antibodies (DAKO A/S, Denmark) anti-L-selectin, CD11a/CD18 (LFA-1) and CD11b/CD18 (MAC-1). Immunoreactivity was revealed by the labeled streptavidin biotin (LSAB) alkaline-phosphatase technique. Four hundred cells/slide were counted, and results were expressed as percentage of positive cells. Paragraph 10: Blood was drawn from the antecubital vein into sterile tubes containing EDTA (Vacutainer, Becton Dickinson) for complete blood cell and reticulocyte counts (ADVIA counter, Bayer). Aliquots of plasma and serum were collected and stored at 80C. Muscle enzymes (lactic dehydrogenase or LDH and creatine kinase or CK) and plasma neutrophil elastase concentrations were measured by enzymatic assays (Olympus Diagnostica, GmbH, and Ecoline, Kit Merck, Germany). Serum cortisol was assessed by radioimmunoassay (Immunotech, SA, Marseille, France). Paragraph 11: Results. In induced sputum (Table 1): total corrected cell counts were in the normal range, but increased at 12 hours post-trial compared to baseline; neutrophils accounted for >90% of cells in induced sputum at baseline, and for a slightly lower percentage (82.5%) after the trial; absolute neutrophil counts post-trial increased by 54% over baseline; neutrophil elastase concentration did not differ between samples; eosinophils
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Airway cells in swimmers: a case report and a review of the literature

were absent at baseline, and accounted for about 1% of total cells post-trial. Expression of adhesion molecules by neutrophils in induced sputum was low. L-selectin was expressed by 14.8 and 18.3% of neutrophils, before and after the trial respectively; corresponding figures were 27.6 and 19.0% for LFA-1, and 13.7 and 18.0% for MAC-1 expression. Paragraph 12: In peripheral blood (Table 2): red blood cell counts did not change, while reticulocytes increased slightly at 12h post-trial; prolonged swimming doubled WBC counts, largely due to increased neutrophil counts; peripheral blood leukocytosis and neutrophilia were still present at 12 hours, and returned to baseline by 36 h after the trial; the percentage of circulating neutrophils expressing adhesion molecules (L-selectin, LFA-1, MAC-1) did not differ among time points (data not shown). Paragraph 13: Markers of muscle damage, stress, and systemic inflammation (Table 3): after the trial, serum creatine phosphokinase (CK) and interleukin-6 (IL-6) levels showed a fifteen- and a seven-fold increase, respectively. Neutrophil elastase and TGF-1beta concentrations also increased, while lactic dehydrogenase (LDH) and cortisol levels did not show major changes compared to baseline values. Discussion Paragraph 14: Despite the obvious limitation of studying a single athlete, analysis of the effects of 35-km swimming may allow some insight into the effects of very prolonged exercise on airway cells. Based on previous work from our laboratory [7, 8], we hypothesized that exercise duration might be important in modulating airway cells. Neutrophil counts in induced sputum were much higher in runners after a marathon race (exercise lasting 3 to 4 h) than in swimmers after a 5-km race (lasting about 1 h). In the present study, the induced sputum sample obtained at rest after intensive training showed that almost all cells were neutrophils; a similar pattern was observed in the post-trial sample. Therefore, the data support the interpretation that intense endurance training and prolonged exercise increase airway neutrophils. However, no evidence of active inflammation in the airways was found after 35-km swimming. Neutrophil elastase concentration did not increase in induced sputum, and expression of adhesion molecules by airway neutrophils was low, in agreement with previous findings in runners and swimmers [7, 8]. Therefore, airway inflammation in athletes appears under tight control even after very prolonged exercise [12]. Paragraph 15: Our previous study in swimmers showed that eosinophils in induced sputum slightly increased after 5-km swimming in the sea [8]; because eosinophils did not increase after swimming in an outdoor pool, we interpreted their change as related to exposure to sea water. The development of hyperosmolarity of the airway surface layer during exercise is a highly debated issue [16, 17], and swimming in the sea might be a model of airway exposure to a hypertonic environment during exercise. After 35-km swimming,
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Giuseppe Morici, Laura Chimenti, Anna Bonanno, Loredana Riccobono, Mirella Profita, Alessandra Patern and Maria R. Bonsignore

eosinophils in induced sputum increased very slightly, suggesting that very prolonged exposure to sea water did not exert deleterious effects. Swimming in the sea for prolonged time seems safe for healthy airways. Paragraph 16: Muscle enzymes were elevated after the trial, suggesting significant muscle damage. The changes in peripheral blood cells and mediators after 35-km swimming confirm that endurance exercise causes systemic inflammatory activation [18,19]. Plasma cortisol and IL-6 concentration, however, were much lower in this swimmer compared to values found after a marathon race [20], possibly because exercise intensity was lower than during a marathon race [21]. Conclusions Paragraph 17: Our athlete showed intense airway neutrophilia at rest and after prolonged swimming in the sea, but no active inflammation in the airways. Changes in peripheral blood cells and mediators were in line with the known systemic inflammatory activation induced by endurance exercise. Our findings suggest that exercise duration is involved in the modulation of the effects of endurance exercise at both airway and peripheral blood levels. However, very prolonged exposure to sea water did not appear to exert harmful effects to the airways. Acknowledgments We thank the athlete Mauro Giaconia who repeatedly came to the laboratory for data collection. We also thank the dedicated people who made this study possible, in particular Dr. Pietro Miraglia, Laboratory of Clinical Pathology Delta, Brolo (Messina), for his help in sample processing at night after end of the trial.

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Airway cells in swimmers: a case report and a review of the literature

Giuseppe Morici, Laura Chimenti, Anna Bonanno, Loredana Riccobono, Mirella Profita, Alessandra Patern and Maria R. Bonsignore

Airway cells in swimmers: a case report and a review of the literature

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] Sue-Chu M, Larsson L, Moen T, Rennard SI, Bjermer L. Bronchoscopy and bronchoalveolar lavage findings in cross-country skiers with and without ski asthma. Eur Respir J 1999; 13: 626-32. Provost-Craig MA, Arbour KS, Sestili DC, Chabalko JJ, Ekinci E. The incidence of exercise-induced bronchospasm in competitive figure skaters. J Asthma 1996; 33: 67-71. Karjalainen EM, Laitinen A, Sue-Chu M, Altraja A, Bjermer L, Laitinen LA. Evidence of airway inflammation and remodeling in ski athletes with and without bronchial hyperresponsiveness to methacholine. Am J Respir Crit Care Med 2000; 161: 2086-2091. Sue-Chu M, Karjalainen EM, Altraja A, Laitinen A, Laitinen LA, Noess AB, Larsson L, Bjermer L. Lymphoid aggregates in endobronchial biopsies from young elite cross-country skiers. Am J Respir Crit Care Med 1998; 158: 597-601. Larsson K, Ohlsen P, Larsson L, Malmberg P, Rydstrom PO, Ulriksen H. High prevalence of asthma in cross country skiers. BMJ 1993; 307: 1326-1329. Davis MS, McKiernan B, McCullough S, Nelson S Jr, Mandsager RE, Willard M, Dorsey K. Racing Alaskan sled dogs as a model of ski asthma. Am J Respir Crit Care Med 2002;166: 878-882. Bonsignore MR, Morici G, Riccobono L, Insalaco G, Bonanno A, Profita M, Paterno A, Mirabella A, Vassalle C, Vignola AM. Airway inflammation in non-asthmatic amateur runners. Am J Physiol 2001; 281: L668-L676. Bonsignore MR, Morici G, Riccobono L, Profita M, Bonanno A, Paterno A, Di Giorgi R, Chimenti L, Insalaco G, Cuttitta G, Abate P, Mirabella F, Vignola AM, Bonsignore G. Airway cells after swimming outdoors or in the sea in nonasthmatic athletes. Med Sci Sports Exerc 2003; 35: 1146-1152. Morici G, Bonsignore MR, Zangla D, Riccobono L, Profita M, Bonanno A, Patern A, Di Giorgi R, Mirabella F, Chimenti L, Benigno A, Vignola AM, Bellia V, Amato G, Bonsignore G. Airway cell composition at rest and after an all-out test in competitive rowers. Med Sci Sports Exerc 2004; 1723-1729. Hashimoto S, Matsumoto K, Gon Y, Nakayama T, Takeshita I, Horie T. Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase. Am J Respir Crit Care Med 1999; 159: 634-640. Hashimoto S, Gon Y, Matsumoto K, Takeshita I, Maruoka S, Horie T. Inhalant corticosteroids inhibit hyperosmolarity-induced, and cooling and rewarming-induced interleukin-8 and RANTES production by human bronchial epithelial cells. Am J Respir Crit Care Med 2000; 162: 1075-1080. Bonsignore MR, Morici G, Vignola AM, Riccobono L, Bonanno A, Profita M, Abate P, Scichilone N, Amato G, Bellia V, Bonsignore G. Increased airway inflammatory cells in athletes: what do they mean? Clin Exper Allergy 2003, 33: 14-21. Sue-Chu M, Karjalainen EM, Laitinen A, Larsson L, Laitinen LA, Bjermer L. Placebo-controlled study of inhaled budesonide on indices of airway inflammation in bronchoalveolar fluid and bronchial biopsies in cross-country skiers. Respiration 2000; 67: 417-425. Chimenti L, Morici G, Patern A, Bonanno A, Siena L, Licciardi A, Veca M, Guccione W, Macaluso F, Bonsignore G, Bonsignore MR. Endurance training damages small airway epithelium in mice. Am J Respir Crit Care Med 2007; 175(5): 442-449. Helenius IJ, Rytila P, Metso T, Haahtela T, Venge P, Tikkanen HO. Respiratory symptoms, bronchial responsiveness, and cellular characteristics of induced sputum in elite swimmers. Allergy 1998; 53: 346-352. Kotaru C, Hejal RB, Finigan JH, Coreno AJ, Skowronski ME, Brianas LJ, McFadden ER Jr. Influence of hyperpnea on airway surface fluid volume and osmolarity in normal humans. J Appl Physiol 2002;

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93:154-160. [17] Freed AN, Davis MS. Hyperventilation with dry air increases airway surface fluid osmolality in canine peripheral airways. Am J Respir Crit Care Med 1999; 159: 1101-1107. [18] Shek PN, Shephard RJ. Physical exercise as a human model of limited inflammatory response. Can J Phyiol Phramacol 1998; 76: 589-97. [19] Jordan J, Beneke R, Hutler M, Veith A, Luft FC, Haller H. Regulation of MAC-1 (CD11b/CD18) expression on circulating granulocytes in endurance runners. Med Sci Sports Exerc 1999; 31: 362-367. [20] Bonsignore MR, Morici G, Santoro A, Pagano M, Cascio L, Bonanno A, Insalaco G, Abate P, Mirabella F, Profita M, Gioia M, Vignola AM, Majolino I, Testa U, Hogg JC. Circulating hematopoietic precursor cells (HPCs) in well-trained runners. J Appl Physiol 2002, 93: 1691-97. [21] Dwyer J. Marathon swimmers: physiologic characteristics. J Sports Med Phys Fitness 1983; 23: 263-272.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

PRESENZA DEL fATTORE NATRIURETICO ATRIALE NELLAPPARATO RESPIRATORIO


[Presence of Atrial Natriuretic Peptides in the Respiratory System] Biagio Valentino, Diego Lipari, Giovanni Peri e Elvira Farina Lipari
Dipartimento di Medicina Sperimentale, Sezione Anatomia Umana, Facolt di Medicina e Chirurgia, Universit degli Studi di Palermo (I)

Parole chiave: ANP, Vie aerifere, Polmone Key words: ANP, Airways, Lung Riassunto. LANP un neuropeptide con propriet natriuretiche diuretiche e vasoattive. Tali propriet sono state indagate con metodiche immunoistochimiche e PCR analisi. Abbiamo indagato con tali metodiche la presenza di ANP nelle cavit nasali, trachea polmone e pleura per studiare il ruolo dellANP nei diversi organi deputati al trasporto dellaria ed alla ematosi area-sangue. Abstract. ANP is a neuropeptide that plays natriuretic, diuretic and vasoactive actions. These activities had been by immunohistochemical methods and PCR analysis in the nasal cavity, trachea, lung and pleura to study the ANP role in different organs that are involved in the air-transport and in the airblood haemostasis.

Introduzione Il fattore natriuretico atriale (ANP) un neuropeptide costituito da 28 amminoacidi ottenuto per attivit proteolitica da un precursore costituito da 126 amminoacidi C- terminale con attivit natriuretica, diuretica e vasoattiva primariamente trovato nel cuore di diversi mammiferi compreso luomo. In questi ultimi anni sono stati trovati altri in altri organi altri neuropeptidi BNP, CNP legati allANP con propriet natriuretiche diuretiche e vasoattive. Lespressione dei neuropeptidi appartenenti alla famiglia di ANP stata indagata con tecniche immunoistochimiche usando anticorpi anti-ANP o anticorpi anti-proANP e per mezzo di ibridizzazione in situ e PCR amplificazione.Lazione dellANP comporta il rilascio della muscolatura liscia dei vasi, lincremento della diuresi e natriuresi che ha come effetto la regolazione della distribuzione dei fluidi tra i compartimenti extra e intravasasali. Da diversi anni il nostro gruppo di lavoro si interessato allo studio e alla classificazione dellANP e degli altri neuropeptidi legati allANP in diversi organi ed apparati [2,3,4,5]. Fra gli apparati abbiamo preso in esame lapparato respiratorio che per la sua organizzazione morfologica e per la sua funzione dipende molto dal sistema nervoso e da diversi neurotrasmettitori. Sono stati indagati con metodiche immunoistochimiche e amplificazione c-DNA per ANP, le cavit nasali la trachea, il polmone e la pleura di diversi mammiferi e delluomo.
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Biagio Valentino, Diego Lipari, Giovanni Peri, Elvira Farina Lipari

Analisi immunotopografica Le cavit nasali Lorganizzazione strutturale e langioarchitettura dei turbinati delluomo sono atti a modificare alcune caratteristiche fisico-chimiche dellaria inspirata. stato osservato da diversi AA. che la termoregolazione dellaria inspirata dipende da diversi neurotrasmettitori quali VIP (vasoactive intestinal peptide) NPT (neuropeptide Y) somatostatina e ANP che producono effetti diversi sui vasi sanguiferi. Infatti il neurotrasmettitore ANP agendo sulla muscolatura liscia dei vasi sanguiferi e sulla attivit secretoria degli acini ghiandolari partecipa alla variazione di alcuni parametri fisici dellaria inspirata. I turbinati presentano una superficie molto estesa che agevola i contatti fra aria inspirata e mucosa e una tonaca propria infarcita di numerose ghiandole sieromucose e vasi sanguiferi. Le nostre indagini immunoistochimiche per ANP hanno messo in evidenza che lANP presente nelle cellule acinari delle ghiandole, nelle cellule epiteliali sierose ed in alcune cellule della tonaca propria poste in vicinanza ai sinusoidi e shunts artero-venosi. Tale disposizione spaziale dei siti reattivi per ANP permette di supporre che lANP, per azione paracrina, possa agire sulla muscolatura liscia dei sinusoidi inducendo vasodilatazione e aumentandone la capacitanza mentre agendo sugli shunts artero-venosi ne diminuisce la resistenza. Inoltre la presenza di ANP nelle cellule acinari modificherebbe la composizione e la viscosit del muco. Trachea e Polmone Nella trachea e nel polmone la distribuzione dellANP stato studiato con metodiche immunoistochimiche e PCR amplificazione. I risultati immunoistochimici su sezioni di trachea, confermati dai risultati di amplificazione PCR di ANP c-Dna, indicano che le cellule mucose,le cellule basali e le cellule ciliate sono immunopositive per ANP. Tali risultati concordano con quelli di Lofton (ottenuti con cellule isolate di trachea. Questi risultati indicano che nella trachea lepitelio della mucosa respiratoria sede di sintesi di ANP che potrebbe essere coinvolto nel regolare il battito ciliare e parteciperebbe alla stratificazione del muco sulla mucosa stessa. Nel polmone i risultati immunoistochimici mettono in evidenza pneumociti di 2 ordine risultano immunopositivi per ANP in accordo con i dati di Perrault e Gutkwoska [1]. La presenza di ANP nei pneumociti di 2 ordine interviene nel regolare la secrezione e la composizione del surfactant alveolare (fig. 1). Pleura La ricerca di ANP nelle cellule pleurali stata eseguita con metodiche immunoistochimiche e amplificazione PCR ANPc-DNA. I risultati hanno evidenziato cellule mesoteliali positive per ANP con granuli disposti nello spazio perinucleare (fig. 2). Le cellule mesoteliali svolgono
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Presenza del fattore natriuretico atriale nellapparato respiratorio

un ruolo fondamentale nella produzione del liquido pleurico per cui lANP presente su tali cellule svolgerebbe un ruolo determinante sulla sintesi e omeostasi del fluido pleurico. Da queste osservazioni risulta che lapparato respiratorio sede di sintesi di ANP che regolando con gli altri neuropeptidi lattivit specifica dei diversi organi che lo compongono permette lo scambio gassoso fra laria inspirata e il sangue.

Fig. 1 - Polmone di coniglio. Cellule immunopositive per ANP.

Fig. 2 - Pleura parietale di coniglio. Cellule mesotelieli positive per ANP. 49

Biagio Valentino, Diego Lipari, Giovanni Peri, Elvira Farina Lipari

Bibliografia [1] [2] [3] [4] [5] Perrault T, Gutkowska J. Role of atrial natriuretic factor in lung physiology and pathology. Am. J. Resp. Crit. Care Med. 1995; 151:226-42. Valentino B, Farina Lipari E, Carini F, Valenza V. Immunohistochemical localization of atrial natriuretic factor (ANP) in the excretory of the rabbit parotid gland. Eur. J. Histochem. 1999; 43:241-5. Farina Lipari E, Valentino B, Lipari D, Dieli F. ANF-immunolocalization in the rabbit airways and lung. It. J. Anat. Embryol. 1999; 104 (suppl.1) :85. Valentino B, Lipari D, Dieli F, Leone A, Farina Lipari E. Distribution of Atrial Natriuretic Factor (ANF) in the Respiratory Tract and Lung showed by Immunohistochemical and PCR Methods. Dent. Med. Probl. 2003; 40:17-22. Farina Lipari E, Lipari D, Leone A, Dieli F, Valentino B. Localization of Atrial Natriuretic Factor (ANF) in the Normal Parietal Pleura in Rabbit. Immunohistochemical Studies and PCR Analysis. Dent. Med. Probl. 2004; 41:209-212.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

IMMUNOHISTOCHEMICAL EXPRESSION Of CB1 CANNABINOIDS RECEPTOR IN RAT LUNG


[Espressione immunoistochimica dei recettori CB1 cannabinoidi del polmone di ratto] Giuseppe Bonaventura, Daniela Cucco, Diego Lipari1, Giovanni Francesco Spatola, Maria Laura Uzzo e Vincenzo Tessitore
DI.ME.S. Dipartimento di medicina sperimentale - Sezione di Istologia ed Embriologia Arcangelo Pasqualino di Marineo. (1) e Sezione di Anatomia umana Emerico Luna, Facolt di Medicina e Chirurgia, Universit degli Studi di Palermo (I)

Key words: CB1 receptor, Endocannabinoids, Lung, Immunohistochemistry Parole chiave: Recettore CB1, Endocannabinoidi, Polmone, Immunoistochimica Abstract. Recent studies document that the CB1 receptor considered since its first identification as brain specific could be expressed by different pheripheral tissues (adipose organ, enteric nervous system, skeletal muscles, hepatocytes, salivary glands). Following this method we have considered interesting to carry out a study on the CB1 receptor immunohistochemical expression and distribution in rat lung. Our results indicate that CB1 receptor are expressed with different intensity in the various cytotypes of respiratory pseudostratified epithelium, in neuroepithelial bodies (NEBs) in nerve bundles distribute to peribronchial myocytes, and in the type II pneumocytes. The findings provide a morphohistochemical basis to speculate novel pulmonary peripheral role played by endocannabinoids with neurocrine, paracrine, endocrine mechanism. Riassunto. Studi recenti documentano che il recettore CB1, considerato in passato come brain specific, pu essere espresso in altri tessuti periferici (organo adiposo, sistema nervoso enterico, muscolo scheletrico, epatociti, ghiandole salivari). Ci sembrato interessante, pertanto, estendere lo studio dellespressione immunoistochimica e della distribuzione del recettore CB1 nel polmone di ratto. I nostri reperti documentano lespressione del CB1 nellepitelio respiratorio pseudostratificato, nei corpi neuroepiteliali (NEBs), nei tronchi nervosi distribuiti alla muscolatura peribronchiale e nei pneumociti di II tipo. I nostri risultati forniscono la base morfoistochimica per prospettare un nuovo ruolo periferico polmonare svolto dagli endocannabinoidi con meccanismo neurocrino, paracrino ed endocrino.

Introduction The toxicological study of the inhalation effects of the marijuana smoke on the lung functions has aroused great interest at the end of the 70s [1,2,3,4]; as regards, the bibliography of those years is full of contributions above all regarding the investigations done on the respiratory effects of the major psychoactive component in cannabis: the delta9-tetraidrocannabinol (THC). Pulmonary studies with inhaled delta-9-tetraidrocannabinol demonstrated a bronchodilating activity in normal human volunteers and reversal of exer-

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Giuseppe Bonaventura, Daniela Cucco, Daniela Lipari, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore

cise-and methacholine-induced bronchospasm and hyperinflation in asthmatics [5,6]. The identification of the receptors CB1 and CB2 [7,8,9] for the cannabinoids and then of the respective endogenous ligands (endocannabinoids) [10,11,12,13,14] has risen a new interest in front of this theme in the 90s again; some outcome of previous investigations have been revised and the recent studies have acquired a more accurate scientific value concentrating the attention on receptorial molecules and on their endogenous ligands to cannibimimetic activity that together constitute the endocannabinoid system. The observations given by recent studies document that the CB1 receptor, considered since its first identification as brain specific, that is expressed exclusively by the central nervous system neurons, could be expressed by different peripheral tissues (adipose organ, enteric nervous system, striated muscles, hepatocytes, salivary glands) [15,16,17] and that the endocannabinoid system could have other perypheral targets outside of the central nervous system. Our previous studies document that CB1 receptor is widely distributed in many structures of the gastroenteropancreatic system (GEP) and in the rat salivary glands [18,19,20]; following this method on the exam of the immunohistochemical distribution of CB1 receptor in peripheral tissues, we have considered interesting to carry out a study on the CB1 receptor expression in the rat lung. Materials and methods Specimens of rat trachea, primary bronchi and lung were fixed in Bouins mixture and embedded in paraffin; obtained sections were processed with anti CB1 (Biosource Europe S.A.) by En-Vision + System HRP (AEC). Negative controls were performed by omission of primary antibody, and by absorption control on the primary antibody with the purified antigen. Other sections near by those processed have been submitted at the histological stain using the method hematoxylin-eosin. All the samples have been studied with microscope Leica DMLB. Results Intense immunohistochemical staining for CB1 receptor was found in the entire pseudostratified columnar epithelium of trachea, large and small bronchi, and bronchioles. Immunoreactivity appears more often diffuse in cytoplasm or distributed in thin and spread granula and was absent in the cell membranes. (Fig.1,2,3) The various epithelial cytotypes express the CB1 immunoreactivity not uniformly but with different intensity: it is absent in the goblet mucous cells, strong in ciliated cells and in Clara cells (Fig.4) very strong in the so-called neuroepithelial bodies (NEBs) (Fig. 5a-5b) that are corpuscular organoid structures within the bronchial epithelium, composed of pulmonary neuroendocrine cells (PNECS) which are part of the diffused neuroendocrine system (DNES), distributed through the body
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Immunohistochemical expression of CB1 cannabinoids receptor in rat lung

(cardiovascular system, GEP system, genital male and female tracts). NEB cells appears as tall columnar cells extending from the basement membrane to the airway lumen (Fig.6). In tangential cut, they usually appear as distinct ovoid corpuscles which are predominantly innervated by afferent sensory fibres of vagal origin with cell bodies residing in the nodose ganglion [21, 22]. Remarkable CB1 immunoreactivity appears in the nerve bundles that pass in the bronchial submucosal layer (Fig. 7a-7b) whose axon terminals distribute themselves to peribronchial miocytes, that are poorly immunoreactive (Fig. 8). In the alveolus strong CB1 immunoreactivity, was observed exclusively in the type II pneumocytes (Fig. 9a-b). Conclusions Our findings justify some clarifications. The CB1 immunoreactivity expressed by citotypes of bronchial epithelium authorizes to believe that the endocannabinoids, via CB1 activation, influence the secretory function of Clara cells, the cleansing function of ciliated cells and the general epithelial trophism during the development. Another important aspect of pulmonary CB1 immunoreactivity, that our research put in evidence for the first time, its a very strong immunopositivity in all cells of NEBs, that demonstrate that the endocannabinoids, activating the CB1 receptors of the neuroepithelial bodies, may modulate the NEB functions. Physiologic studies have affirmed that the NEBs represent intrapulmonary hypoxia sensory chemioreceptors, regulating local ventilation/ perfusion, ratio in the lung lobule [23-24-25]. These function is particularly important during intrauterine life, and during the birth, for the control of pulmonary circulation, in which could be implicated the endocannabinoids. The big CB1 immunoreactivity that appears in the nerve fibres distributed among bronchial and bronchiolar smooth muscles, and with low intensity in the same miocytes surrouding the lamina mucosa, suggests that endocannabinoids influence the bronchial muscle contractility. Previous biochemical analyses indicate that anandamide is synthesized in lung tissues on calcium-ion stimulation and this suggests that locally generated anandamide partecipates in the intrinsic control of airway responsiveness, as well as a prejunctional mechanism, as directly interacting with CB1 receptors expressed by smooth muscles [26]. Our findings agrees with previous studies of Calignano et al. [26] that have studied the ultrastructural localization of CB1 receptor in rat lung, demonstrating that CB1 receptor are present in axon terminals in close proximity to smooth muscle cells. Previous study by Rice et al. demonstrate that CB1 receptor mRNA is expressed in alveolar type II cells [27]. Our findings document, for the first time, that the CB1 immu53

Giuseppe Bonaventura, Daniela Cucco, Daniela Lipari, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore

noreactivity in the alveoli is just present in the alveolar type II pneumocytes, and suggest that endocannabinoids regulate surfactant production from alveolar type II cells that is responsible of successful adaptation to air-breathing at birth in the perinatal period. Cerlet and Scott have recently investigated the potential health risks of marijuana use studying the effects of delta-9-tetraidrocannabinol on foetal lung development specifically related to surfactant production by alveolar type II cells isolated from foetal rabbit lungs. The result obtained suggests that THC significantly reduced formation of surfactant [28]. In conclusion, our findings demonstrated that CB1 receptor is expressed in selected structures of rat lung and provide the morphohistochemical basis to display that endocannabinoids, acting through the CB1 receptor, affect several lung function, carrying on new perypherical functions done with different mechanisms: neurocrine, autocrine-paracrine and endocrine. In addiction, the lung represents the birthplace of endocannabinoids (anandamide). This checking further emphasizes the endocannabinoids role in the control of the pulmonary functions. Consequently the lung can be now added rightfully to the list of sites of peripherical tissues that express the CB1 receptor outside the central nervous system.

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Immunohistochemical expression of CB1 cannabinoids receptor in rat lung

[1]

[2]

[3]

[4]

[5a]

[5b]

Figures 1-5b: [1] Intense but not uniform CB1 immunoreactivity in bronchiolar pseudostratified epithelium. Ob. 40x - [2] CB1 immunoreactivity diffuse in bronchiolar epithelium. Ob. 40x - [3] CB1 immunoreactiviy: microgranular staining in the bronchiolar epithelium. Ob. 20x - [4] Strong CB1 immunoreactivity in bronchiolar Clara cells. Ob. 20x - [5a-b] Very strong CB1 immunoreactivity in all neuroendocrine cells of NEB. Ob. 40x.

[6]

[7a]

[7b]

[8]

[9a]

[9b]

Figures 6-9b: [6] A neuroepithelial body stained with hematoxylin eosin . Ob. 40x - [7a-b] CB1 immunoreactivity in nerve bundle of submucosal layer Ob. 20x (a) Ob. 40x (b) - [8] CB1 immunoreactivity in bronchiolar mucosal epithelium, in peribronchial smooth muscle and in type 2 pneumocytes. Ob. 40x - [9a-b] Remarkable CB1 Immunoreactivity is observed exclusively in alveolar type 2 pneumocytes. Ob. 40x.

Giuseppe Bonaventura, Daniela Cucco, Daniela Lipari, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] Rosenkrantz H, Fleischman RW. - Effects of cannabis on lungs. Adv Bio Sci 1978 Jul 22-23; 22-23: 279-99. Fleischman RW, Baker JR, Rosenkrantz H. Pulmonary pathologic changes in rats exposed to marihuana smoke for 1 year. Toxicol Appl Pharmacol 1979 Mar 15;47(3):557-66. Vachon L, Fitzgerald MX , Solliday NH, Gould IA & Gaensler EA. Single-dose effects of marihuana smoke. Bronchial dynamics and respiratory-center sensitivity in normal subjects. New Engl J Med 1973 288, 985 989. Tashkin DP, Shapiro BJ, Lee YE & Harper CE. Effects of smoked marijuana in experimentally induced asthma. Am Rev Respir Dis 1975 112, 377386. Tashkin DP, Reiss S, Shapiro BJ, Calvarese B, Olsen JL, Lodge JW. Bronchial effects of aerosolized delta 9-tetrahydrocannabinol in healthy and asthmatic subjects. Am Rev Respir Dis 1977 Jan;115(1):57-65. Abboud RT, Sanders HD. Effect of oral administration of delta-9-tetrahydrocannabinol on airway mechanics in normal and asthmatic subjects. Chest 1976 70, 480 485. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC and Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature, 1990 346:561-564. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature, 1993 365:61-65. Herkenham M. in: Cannabinoid Receptors (Pertwee RG, ed. Academic Press) 1995 145-166. Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A, Etinger A, Mechoulam R. Isolation and structure of a brain costituent that binds to the cannabinoid receptor. Science, 1992 258:1946-1949. Hanus L, Gopher A, Almog S, Mechoulam R. Two new unsaturated fatty acid ethanolamides in brain that bind to the cannabinoid receptor. J Med Chem, 1993 36:3032-3034 Bayewitch M, Avidor-Reiss T, Levy R, Barg J, Mechoulam R, Vogel Z. The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling. FEBS Lett, 1995 375:143-147. Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K, Yamashita A, Waku K. 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Comm, 1995 215:89-97. Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE, Schatz AR, Gopher A, Almog S, Martin BR, Compton DR. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol, 1995 50:83-90. Pertwee RG. Evidence for the presence of CB1 cannabinoid receptors on peripheral neurons and for the existence of neuronal non CB1 cannabinoid receptors. Life Sci 1999 Vol65, Nos. 6/7, pp597-605. Pinto L, Capasso R, Di Carlo G, Izzo AA. Endocannabinoids and the gut. Prostaglandins Leukot Essent Fatty Acids 2002 Feb-Mar; 66(2-3):333-41A. Storr M, Sibaev A, Marsicano G, Lutz B, Schusdziarra V, Timmermans JP, Allescher HD. Cannabinoid receptor type 1 modulates excitatory and inhibitory neurotransmission in mouse colon. Am J Physiol Gastrointest Liver Physiol 2004 Jan;286(1). Tessitore V, Uzzo ML, Spatola G, Cucco D, Bonaventura G. Immunohistochemical studies on leptin, grelin, orexin-A, orphanin FQ and endocannabinoids expression in the stomach of lean and obese (fa/fa) Zucker rats. First World Congress on Therapies against Obesity Paris 18-19 2006. Tessitore V, Uzzo ML, Spatola G, Cucco D, Bonaventura G. Immunhistochemical study and densitometric comparison on CB1 receptor expression in the gastrointestinal tract and pancreas of obese (fa/fa)and

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lean Zucker rats. It J Anat Embryol 2006 vol. 111 suppl.2, p. 256. [20] Bonaventura G, Mandracchia R, Spatola G, Uzzo ML. Immunohistochemical evidence for the presence of CB1 cannabinoid receptor in rat salivary glands. It J Anat Embryol 2006 vol. 112 (1), p. 43. [21] Cutz E. Neuro-endocrine (APUD-type) cells of the lung. In: Motta PM, Ultrastructure of endocrine cells and tissues, 1984. [22] Lauweryns JM, Cokelaere M, Theunynck P. Neuro-epithelial bodies in the respiratory mucosa of various mammals. Z Zellforsch 1972; 135: 569592. [23] Linnoila RI. Functional facets of the pulmonary neuroendocrine system. Lab Invest 2006 May; 86(5): 425-44. [24] Cutz E, Jackson A. Neuroepithelial bodies as airway oxygen sensors. Respir Physiol 1999; 115: 201214. [25] Adriaensen D, Brouns I, Van Genechten J, Timmermans JP. Functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors. Anat Rec A Discov Mol Cell Evol Biol 2003 Jan; 270(1): 25-40. [26] Calignano A, Ktona I, Dsarnaud F, Giuffrida A, La Rana G, Mackie K, Freund TF, Piomelli D. Bidirectional control of airway responsiveness by endogenous cannabinoids. Nature 2000 Nov 2; 408(6808): 96-101. [27] Rice W, Shannon JM, Burton F, Fiedeldey D. Expression of a brain-type cannabinoid receptor (CB1) in alveolar Type II cells in the lung: regulation by hydrocortisone. Eur J Pharmacol 1997 May 30; 327(2-3): 227-32. [28] Cherlet T, Scott JE. Tetrahydrocannabinol (THC) alters synthesis and release of surfactant-related material in isolated fetal rabbit type II cells. Drug Chem Toxicol 2002 May; 25(2): 171-90.

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Heart, microcirculation

Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

MORPHOLOGICAL fEATURES Of THE IDIOPATHIC DILATED CARDIOMYOPATHY


[Aspetti morfologici della Cardiomiopatia Dilatativa Idiopatica] Consolato Sergi, Francesco Cappello*, Hans-Joerg Steiner, Valentina Di Felice*, Giovanni Zummo* and Gregor Mikuz
Institute of Pathology, Medical University of Innsbruck, A-6020 Innsbruck, Austria, and *Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana, Universit di Palermo (I)

Key words: Cardiomyopathies, Idiopathic Dilated Cardiomyopathy, Genetic diseases Parole chiave: Cardiomiopatie, Cardiomiopatia Dilatativa Idiopatica, Malattie genetiche Abstract. Cardiomyopathies are a group of myocardial diseases that are characterized by not always defined etiologies, with a mixture of genetic and acquired contributing factors, by the frequent coexistence of distinctive pathologic traits and non-specific clinical and pathological features, and finally by an almost consistent overlap of natural history and outcome. In this paper, we review the main morphological features of Idiopathic Dilated Cardiomyopathy. Riassunto. Le cardiomiopatie sono un groppo di malattie del miocardio caratterizzate da una non sempre definibile e chiara eziologia, derivando da un insieme di fattori contribuenti genetici e acquisiti, e mostrando la coesistenza di caratteristiche patologiche specifiche e non, oltre a una storia naturale non patognomonica. In questo lavoro riportiamo le principali caratteristiche morfologiche della Cardiomiopatia Dilatativa Idiopatica.

Definition Dilated cardiomyopathy is defined as a heart muscle disorder defined by the presence of a dilated and poorly functioning left ventricle in the absence of abnormal loading conditions (hypertension, valve disease) or ischaemic heart disease sufficient to cause global systolic impairment [1]. It is important to stress that the presence of coronary atherosclerosis does not rule out a diagnosis of dilated cardiomyopathy. However, a conditio sine qua non is that the degree of myocardial dysfunction is not explained by the extent of ischemic damage. In a minority of cases with dilated cardiomyopathy genetic factors, viruses, toxins, catabolytes of an altered metabolism or immunological causes are found. However, the majority of cases with of dilated cardiomyopathy remains idiopathic (IDCM) and therefore it remains the reference standard in books and medical journals [2]. Clinical Diagnosis Diagnostic criteria for IDC are an ejection fraction < 0.45 and/or a fractional shorten61

Consolato Sergi, Francesco Cappello, Hans-Joerg Steiner, Valentina Di Felice, Giovanni Zummo and Gregor Mikuz

ing of < 25%, and a left ventricular end diastolic dimension of > 112% predicted value corrected for age and body surface area. Exclusion criteria include absence of systemic hypertension (> 160/100 mm Hg), coronary artery disease (> 50% in one or more major branches), chronic excess alcohol (> 40 g/day female, > 80 g/day male for more than five years after six month abstinence), systemic disease known to cause dilated cardiomyopathy, pericardial diseases, congenital heart disease, cor pulmonale. The criteria for left ventricular enlargement are based on data of Henry et al. [3]. Macroscopy Grossly, hearts harboring IDCM show a massive increase in mass in both pediatric and adult age. In adults, heart weight often exceeds 500 g and the heading of cor bovinum is used in these circumstances. The survival of patients affected with IDCM influences the heart weight. In fact, long-term survivors have significantly heavier hearts than those who die in the short term. In this case, the functionality plays a major role. Moreover, hearts of patients affected with IDCM show an apical rounding other than ventricular dilation (Roman arch). In the usual and complete configuration with four-chamber dilation, the heart shows an almost spherical appearance with disappearance of a proper apex. The epicardium is usually normal and coronary arteries do not usually show high-grade atherosclerosis. On cut surface, the myocardium is flaccid and there is eccentric hypertrophy. Heart hypertrophy is shown by cardiac weight increase, although this finding is not always evident because of dilation. The ratio of parietal thickness to cavity diameter drops from 0.48 (normal adult value) to 0.17-0.21, according to the survival time. Fibrosis is also seen with areas of replacement scarring. At this time, the pathologist needs to make a differential diagnosis with ischemic heart disease (coronary artery check) and myocarditis (virus real-time PCR). Other findings include endocardial fibrosis and mural thrombi. These latter are due to the reduced ejection fraction (a pattern of the cardiac apex that is also called: the change from caput vivum to caput mortuum). Cross-section planes may also show signs of secondary valvular incompetence with mitral valve leaflets harboring rolled edges. Light Microscopy Light microscopy plays an important role in the normal routine of a pathologist and IDCM is characterized by a particular complex of nonspecific features, that are probably due to the parietal stress rather than etiological features. IDCM change involves all myocardial components, including myocytes, interstitium, small vessels, and endocardium. Myocytes show cell hypertrophy defined by a global enlargement of hyperchromatic, often bizarrely shaped nuclei. Moreover, there are two other characteristics associated with dilation and increased lengthening, including thinning, waving, and side-to-side slippage of
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myocytes within muscular bundles. Myocytes may assume the form of attenuated ones, showing an enlarged, rectangular and hyperchromatic nucleus that is transversely arranged and occupies the entire cross-sectional area. Thus, there is an increase of variability in the myocellular area. Another histologic feature is constituted by the myofibril loss, which causes hydropic change of the myocyte. Hydrops of the myocyte is often localized in the subendocardial layer and ranges from a perinuclear halo to a pattern of colliquative myocytolysis. A disordered arrangement of desmin intermediate filaments also characterizes a myocellular derangement of the myocytes as detected by immunohistochemistry. In particular, an immunohistochemical procedure is also needed to demonstrate an abnormal type I/type III fibrillar collagen ratio. In a recent study, atrial natriuretic protein (ANP) and CD34 (an endothelial and stem cell marker) were significantly over-expressed in IDCM compared to normal heart (NH). Patients with a difference of more than 20 myocardial fibers in the compared expression between CD34 and troponin T were associated with a quite less favorable survival although the difference was not significant [4]. The increase in ANP positive cells in IDCM could be a consequence of neuro-hormonal activation due to a decline of the impaired myocyte contractility. Further, since it was already shown that ANP could be important in the control of vascular remodeling, we postulated that the increment of CD34 positive cells could be functionally correlated to the increase of ANP production. Differential expression of CD34 and troponin T might be used for future studies to evaluate their prognostic value. Four types of fibrosis are commonly seen in IDCM hearts. Interstitial (interfiber) and perivascular fibroses prevail, whereas replacement and endocardial fibroses are evident focally. A number of inflammatory cells may be found in the myocardium, mostly close to areas of replacement fibrosis. These cells are almost exclusively lymphocytes, but no diagnosis of myocarditis should be made. The paucity of the infiltration and the topographic distribution help in distinguishing these two diseases. This can, however, become a challenge if limited sampling, e.g. endomyocardial biopsy, is the material to work on. Finally, intramyocardial branches of the coronary arteries may show a proliferation of smooth muscle cells of the tunica media that can evolve to a luminal narrowing. Morphometric analysis is an important help in light microscopy. The potential advantages of measurement in histopathology have been recognized for many years. The need for measurement arises from our realization that some of the diagnostic decisions that we make are poorly reproducible and the incorporation of computers in photo-microscopy and morphometry has opened the possibility of better evaluating remodeling structures during development [5]. A morphometric system has been used for the development of the biliary tract that has proved to be useful for cardiac morphometry as well [6]. We used a set-up that consisted of the following components: a light microscope (Zeiss, Jena, Germany),
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CCD grayscale video camera (Sony CCD-IRIS, Sony Corp., Cologne, Germany), BNC cable hookup, an Intel-based personal computer equipped with video frame grabber card, a Windows 3.11 operating system, Bioscan Optimas version 5.2 software (Bioscan, Edmonds, WA, U.S.A.) running in interlaced mode, a 200 color monitor (GDM, 2063 MS, Stimmler, Munich, Germany), and a drawing pad (WacomTM digitizer II, equipped with an induction mouse). The Optimas software was calibrated using a prefixed grid 2 of known dimensions (checkerboard pattern, square area of 2025 mm) using a 6.33 objective. Using a random number generator up to ten areas may be select for morphometry and quantitative analysis of the biliary epithelial cells or myocytes. Each freeze frame is saved on the hard disk (Tiff files, Tiff Image Archive System) or an external hard-drive permitting SATA files transfer. The outline shape may be drawn manually using the induction mouse on a drawing pad (WacomTM) or automatically using an open-source free-software Image J. Nuclear area and myofibril volume fraction are two morphometric variables, which have achieved an important status for their relationship to cardiac function and patients outcome. Both nuclear enlargement and reduced myofibril volume fraction (myofibril loss) are associated with a worse functional status and poorer prognosis. Electron Microscopy Even though the study of the ultrastructure is nonspecific, it may reveal characteristics associated with the myofibril loss. Electron microscopy studies show enlargement of nuclei with irregular shape and deep infoldings of the nuclear membrane, multiple nucleoli, nuclear inclusions of sarcoplasmic components, double nuclei, dilation and proliferation of T tubules, rough endoplasmic reticulum, and an increase of the number of sarcomeres and mitochondria (changes consistent with myocellular hypertrophy). Myofibril loss is characterized by a range from rarefaction to complete absence of sarcomeres. Conclusion Clinically, fatigue, exercise intolerance, and chest pain are the most frequent symptoms. Physical examination reveals a variable degree of cardiomegaly and aspects of congestive pump failure. Pulmonary vascular redistribution is detected by chest radiography and complex ventricular arrhythmias are common and have been linked to the degree of left ventricular functional impairment. In children, recurrent or incessant supraventricular tachycardias may be the cause, and not the effect of ventricular dysfunction, because arrhythmia therapy may induce improvement, or even regression of IDCM changes. In terms of risks and effectiveness the indications for endomyocardial biopsy are still controversial. However, it adds minimal risk to a cardiac catheterization in specialized centers and should be considered a useful complement in the diagnostic procedure in distinguishing IDCM
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from other common causes of cardiomyopathy, to exclude myocarditis (real-time PCR), to suspect a mitochondrial cardiomyopathy (electron microscopy), to identify abnormal deposition (myocardial toxicity), to address appropriate genetic counseling (clinical and molecular genetics), and to provide the basis for future research (autoimmunity?). Pathological anatomy of IDCM may show characteristics that taken singularly are not diagnostic, but together provide not only an important diagnostic help, but also the basis for the study of this fascinating chapter of cardiovascular pathology.

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Fig. 1. Cardiomyocytes of a patient with IDCM showing perinuclear halos, contraction bands, hyperchromatic enlarged nuclei and waving of the fibers shape (x 400, original magnification, hematoxylin and eosin staining).

Morphological features of the Idiopathic Dilated Cardiomyopathy

Figure 2. Myocardial tissue for morphometric analysis of cardiomyocytes in an endomyocardial biopsy from a patient affected with idiopathic dilated cardiomyopathy. We use a particular thin section of 1-2 micrometer to study cardiomyocyte morphometry (x 200, original magnification, alcian-hematoxylin staining).

Consolato Sergi, Francesco Cappello, Hans-Joerg Steiner, Valentina Di Felice, Giovanni Zummo and Gregor Mikuz

References [1] [2] [3] [4] [5] [6] Elliott P. Cardiomyopathy. Diagnosis and management of dilated cardiomyopathy. Heart 2000; 84:106112. Gallo P, dAmati G. Cardiomyopathies. In: Cardiovascular Pathology Silver, Gotlieb, Schoen (Eds). Third Edition, pp. 285-294. Henry WL, Gardin JM, and Ware JH. Echocardiographic measurements in normal subjects from infancy to old age. Circulation 1980; 62:10541061. Ardizzone N, Cappello F, Di Felice V, Rappa F, Minervini F, Maras S, Maras L, Rabl W, Zummo G and Sergi C. Atrial Natriuretic Peptide and CD34 Overexpression in Human Idiopathic Dilated Cardiomyopathies. AMPIS, in press 2007. Hamilton PW, Allen DC. Morphometry in histopathology. J Pathol 1995; 175:369379. Sergi C, Adam S, Kahl P, Otto HF. The remodeling of the primitive human biliary system. Early Hum Dev 2000; 58:167-178.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

HYDROGEN PEROXIDE IN ENDOTHELIUM: MULTIfACETED ROLES IN CELLULAR STRESS AND SIGNALLING


[Il perossido didrogeno nellendotelio: molteplici ruoli nello stress e nella segnalazione cellulare]

Rita Anzalone, Giampiero La Rocca*, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Francesca Pizzo, Giuseppina Maria Ciaccio, Antonino Di Stefano+, Pantaleo Giannuzzi, Felicia Farina and Giovanni Zummo
Sezione di Anatomia Umana, Dipartimento di Medicina Sperimentale, Universit degli Studi di Palermo, Palermo (I) - + Fondazione S. Maugeri IRCCS, Centro Medico di Veruno (NO) - The two authors contributed equally to this work - * Corresponding author

Key words: Hydrogen peroxide, Endothelial cells, Oxidative stress, Nitric oxide, Chronic heart failure Parole chiave: Perossido di idrogeno, Cellule endoteliali, Stress ossidativo, Ossido nitrico, Scompenso cardiaco cronico Abstract. The endothelial cells are a population of squamous cells that line the blood vessels throughout the body. These cells are being now viewed as key modulators of several physiological functions, and multiple cardiovascular diseases are due to impairment of their function. An increasing number of studies have shown the ability of a variety of vascular cells, not only endothelial cells, but also fibroblasts and smooth muscle cells, to produce reactive oxygen species (ROS). Moreover, as a distinctive feature, endothelial cells are able to manage with higher concentrations of ROS as well as reactive nitrogen species (RNS) with respect to the other cell types. The importance of hydrogen peroxide in endothelial biology is object of growing interest, and consequently the enzymes involved in both its generation and its clearance are now viewed as novel mediators of broad relevance. Historically, superoxide ion has been viewed as the most important ROS in vasculature. In fact, it can be produced by a number of intracellular enzymes, and it can directly influence nitric oxide bioavailability through its transformation in peroxynitrite. More recently, the dismutation product of superoxide, i.e. hydrogen peroxide, has been regarded as an important signaling agent; capable to transduce the initial stimulus longer and towards longer distances. Given the comprehension of the molecular mechanisms involving these molecules should be the key to better understanding the pathophysiology of cardiovascular diseases in which oxidative stress plays a key role. Riassunto. Le cellule endoteliali costituiscono una popolazione di elementi pavimentosi che rivestono I vasi ematici nel corpo. Queste cellule sono ormai considerate modulatrici di diverse funzioni fisiologiche, e diverse malattie cardiovascolari sono dovute ad uno sbilancio nella loro funzionalit. Un cresente numero di studi ha mostrato labilit delle cellule vascolari, non solo endoteliali, ma anche fibroblasti e cellule muscolari lisce, nella produzione di specie reattive dellossigeno (ROS). In particolare, le cellule endoteliali sono in grado di tollerare maggiori concentrazioni di ROS e di specie reattive dellazoto (RNS) rispetto ad altri citotipi. Limportanza del perossido di idrogeno nella biologia endoteliale oggetto di crescente interesse, e di conseguenza gli enzimi coinvolti nella generazione e nella eliminazione di questa molecola sono considerati mediatori di grande rilevanza. Storicamente, lo ione superossido stato considerato il pi importante ROS nella

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Rita Anzalone, Giampiero La Rocca, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Francesca Pizzo, Giuseppina Maria Ciaccio, Antonino Di Stefano, Pantaleo Giannuzzi, Felicia Farina, Giovanni Zummo

vascolatura. Infatti, esso pu essere prodotto da diversi enzimi cellulari, e pu rapidamente influenzare la biodisponibilit dellossido nitrico attraverso la sua trasformazione in perossinitrito. Pi recentemente, il prodotto di dismutazione del superossido, cio il perossido didrogeno, stato considerato come unimportante molecola di segnalazione, capace di trasdurre lo stimolo iniziale pi a lungo e a maggiore distanza. La comprensione dei meccanismi molecolari che coinvolgono tali molecole pu essere la chiave per meglio chiarire la fisiopatologia delle malattie cardiovascolari in cui lo stress ossidativo gioca un ruolo chiave.

1. Endothelium: origin, phenotype, in vitro models Endothelial cells (ECs) line the blood vessels in all our organs since the initial phases of development. They derive from mesoderm germ layer, and are characterized by a very early differentiation process, together with hematopoietic tissues. In fact, following angioblasts proliferation in the yolk sac, multiple foci of endothelial cells can be observed in the embryo. From the further proliferation of these cells, the heart as well as the embryonic vessels will be formed [1,2]. As a fundamental characteristic, these processes are finely regulated by a series of molecular switches between two endothelial phenotypes: migratory and non migratory, in order to extend the primitive vascular network following the developmental processes [3]. Multiple recent reports suggested that angioblasts (or cells classified as endothelial precursors) can be found also in adult tissues as well as in the bloodstream. The central importance of endothelial cells for the study of cardiovascular diseases has been highlighted in a growing number of experimental studies, both in vitro and in vivo. In particular, after the development of techniques to isolate and successfully propagate endothelial cells in vitro, with the use of appropriate substrates and supplements [4,5], in vitro studies increased by number giving a fundamental contribution to the definition of endothelial biology. In particular, HUVECs (human umbilical vein endothelial cells) have been widely used as a model system to investigate endothelial phenotype under physiologic and pathologic conditions (e.g. for experiments on angiogenesis, atherosclerosis, inflammation) [6]. The main counterpoint to the use of this model system was its derivation from an extraembryonic vascular structure, which lasts only some months and therefore, cannot develop significant vascular alterations in this period. Moreover, the need to obtain organ-specific endothelial cells has developed following the observations that vascular beds show a great heterogeneity between different organs and tissues and even inside the same organ [7]. To this regard, endothelial cells obtained from adult organs (also defined primary microvascular cells) can constitute a more valid model for the study of vascular diseases. Moreover, at the molecular level, the phenotypical characterization of endothelial cells should be based on more than one marker (the most common being CD31 and von Willebrand Factor, vWF) since not all endothelia in all types of vessels express uniformly
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Hydrogen peroxide in endothelium: multifaceted roles in cellular stress and signalling

these two markers, differences being present even inside the same organ [8]. More strikingly, other endothelial markers, as CD34, are used with caution due to their unconstant rate of distribution. In a classical work [9], it has been shown that while cells from the umbilical artery are highly positive to this molecule, HUVEC (which are extracted from the umbilical vein) present a lower number of CD34+ cells, therefore influencing experimental works based on the expression of this surface marker (i.e. homing of hematopoietic stem cells). In addition, the phenomenon of the lack of positivity to phenotypical as well as functional markers is accentuated in stable commercial endothelial cell lines, widely used in in vitro experiments. Most of the lines, in fact, even if capable to perform an higher number of population doublings with respect to primary cells, have been shown to lack one or more markers, adhesion molecules or receptors, therefore justifying the efforts of researchers in using primary cell lines as HUVEC or organ-specific microvascular cells as primary models for vascular pathophysiology [10]. Among the number of functions characterizing endothelial cells in vivo, they provide a structural barrier between the circulation and the surrounding tissues, with local variations in morphology and permeability, therefore regulating molecular trafficking via specific transport systems. In fact, ECs are able to transport specifically molecules from the luminal side to the basal one, by the process of transcytosis [11]. Moreover, the regulation of blood pressure and blood flow is influenced by endothelium-dependent release of molecular mediators: vasodilator molecules such as nitric oxide (NO) as well as vasoconstrictor ones. NO is receiving great attention for its multiple roles in vascular biology and cellular signaling processes. Endothelial cells elaborate NO constitutively, by the activity of one of the isoforms of NO synthase (NOS), eNOS or the NOS3 gene product, which is constitutively expressed in ECs. All isoforms of NOS require the formation of an active dimer to exert their enzymatic function, and the regulation of the enzyme dimeric state is an important regulatory process for the maintenance of its activity. Endothelial-cell derived NO has several important effects on the vasculature: first of all, NO maintains basal tone of vessels by relaxing vascular smooth muscle cells, therefore providing endothelium-dependent vasorelaxation. In addition, nitric oxide inhibits smooth muscle cell migration and proliferation, therefore providing signals which act limiting neointimal proliferation after vascular injury [12]. 2. Roles of ROS in endothelium The term endothelial dysfunction refers to a number of processes characteristic of several cardiovascular pathologies. While oxidative stress is a common denominator of endothelial dysfunction, the mechanisms underlying the impairment of endothelium-dependent responses can be pathology-specific and therefore markedly different [13]. Between the key factors that are associated to endothelial dysfunction, an important role is played by
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Rita Anzalone, Giampiero La Rocca, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Francesca Pizzo, Giuseppina Maria Ciaccio, Antonino Di Stefano, Pantaleo Giannuzzi, Felicia Farina, Giovanni Zummo

a disequilibrium in the cellular levels of ROS. Often this intracellular variation triggers a complex cascade of consequences, which may be accompanied by, or lead to, a reduced bioavailability of NO. As a final consequence, therefore, alterations in nitric oxide concentration should lead to the formation of other nitrogen-containing species, namely reactive nitrogen species (RNS), therefore linking together two of the fundamental outcomes of cellular stress events: oxidative stress and nitrosative stress [14]. A first way by which ROS can contribute to the development of cardiovascular pathologies should be by direct chemical interactions, e.g. by oxidation of the cellular constituents, or by influencing the bioavailability of NO, or even by influencing the signal transduction pathways [15]. Interestingly, the endothelium constitutes a particular example of tissue in which the basal levels of some ROS, as hydrogen peroxide, are more elevated than in other cell types. In addition, in vitro and in silico data have shown that the cellular permeability to molecules as hydrogen peroxide is enhanced in cultured endothelial cells. Moreover, several enzymes in these cells may constitute potential sources of ROS. One example may be the NADPH oxidase, together with uncoupled eNOS or xanthine oxidase (XO) [16]. These enzymes have been shown to be able to produce superoxide ion, as discussed further below. Finally, it is well established that the balance between ROS-producing and ROS-scavenging mechanisms is of key relevance for the maintenance of a proper endothelial function. Therefore, the reduction of ROS-scavenging processes, characteristic of chronic heart failure, may contribute to the development of oxidative stress conditions. More importantly, in heart failure additional processes influencing the delicate balance between ROS and RNS species should take place. In fact, xanthine oxidase, a superoxideproducing enzyme, is upregulated following heart failure [17]. Interestingly, it has been suggested that the unbalance between enzymes (NOS versus XO) in failing heart should reflect the disequilibrium of nitric oxide and superoxide ion levels. It has been hypothesized that, while in physiologic context the equilibrium is shifted towards an abundance of NO with respect to superoxide, favoring S-nitrosylation reactions, in heart failure superoxide is the prevalent molecular specie, and this should favor oxidative stress development due to oxidation reactions taking place [14]. 3. Hydrogen Peroxide in endothelium: sources and clearance Hydrogen peroxide is a reactive oxygen species which is capable to exert several physiological effects on endothelial cells. In vitro experiments have shown that endothelial cells isolated from different vascular beds increase their proliferative activity in presence of exogenous hydrogen peroxide in the low micromolar range (lower than 50 M, but higher in certain subtypes of endothelial cells) (reviewed in ref. 18). In fact, hydrogen peroxide action should lead to an increase of expression of VEGF, as well as other growth factors.
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On the other hand, excess levels of hydrogen peroxide should lead to an increase of cell death. Endogenous H2O2 is a ROS which is mainly generated by dismutation of superoxide ion. There are many intracellular sources of superoxide ion in endothelial cells, such as xanthine oxidase, uncoupled eNOS, or the mitochondrial respiratory chain. Therefore, even if this ion possesses a short half-life, it can trigger different responses in several pathways. For example, one of the most chemically-favored reactions involving superoxide is the production of peroxynitrite (ONOO-), thus lowering the intracellular levels of NO [15,18]. The second important reaction is the dismutation, which is carried out by SOD (superoxide dismutase) enzymes, in order to produce hydrogen peroxide, a molecule which is being viewed as the long-term effector of superoxide signaling. In fact, with respect to other ROS, it is not a free radical, since it does not possess an unpaired electron in its outer layer. The short half-life and potential of diffusion of superoxide constitute a severe limit to the role of this ROS as an important paracrine hormone in vascular biology. Therefore hydrogen peroxide has two advantages: it is more stable, and is also capable to diffuse into different cellular compartments. Regarding superoxide dismutation, several literature reports suggested a differential activity of the three SOD isoforms depending on the source of superoxide, i.e. the location of the enzymatic activity which is responsible for its production (reviewed in ref. 19). In fact, mitochondrial manganese SOD (Mn-SOD) is primarily involved in the processing of O2- derived from mitochondrial electron transport chain. Cytosolic copper/zinc (Cu/Zn)-SOD dismutes O2- derived from cytosolic oxidases. Moreover, extracellular superoxide produced by leukocytes (and probably other cell types) is determined by the extracellular form of Cu/Zn-SOD (ec-SOD) [20]. A so high number of cellular sources for hydrogen peroxide corresponds to several enzymatic system to properly manage the oxidant properties of this molecule. Therefore, hydrogen peroxide levels are maintained at a steady state by the actions of scavengers as catalase and glutathione peroxidase (Gpx1) [21]. Gpx1 is located in both cytoplasm and mitochondria, and is a key enzyme for the cellular defense against oxidative stress. It has been suggested that in cells exposed to doses of hydrogen peroxide lower than 100M, Gpx1 is the main enzyme involved in H2O2 reduction. Catalase is a peroxisomal enzyme and exclusively catalyzes the conversion of H2O2 to water, after the so-called catalase latency. More recently, a new class of antioxidants has been described. Peroxiredoxins are able to reduce hydrogen peroxide using thioredoxin as electron donor. Recent studies have characterized their involvement in the regulation of signaling processes by hydrogen peroxide [22]. As discussed earlier, the reduction of ROS-scavenging molecules, characteristic of chronic heart failure, may contribute to the development of oxidative stress conditions,
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therefore the proper maintenance of peroxide-producing as well as clearance mechanisms is essential for endothelial function. 4. The differential effects of hydrogen peroxide on endothelial biology In recent years, hydrogen peroxide emerged as a molecule possessing several effects on endothelium. First of all, in vitro experiments gave contrasting results on viability effects of exogenous hydrogen peroxide on ECs. In fact, while several reports indicated that doses of hydrogen peroxide over 50M in the culture medium should lead to an increase in apoptosis in HUVEC cells [23], which increased at higher doses, other studies have shown that artery-derived endothelial cells should overcome doses of H2O2 even four fold higher. Interestingly, clonogenic endothelial progenitor cells showed a high sensitivity to oxidative stress [24]. Moreover, similar doses of hydrogen peroxide may have differential effects on cellular viability with respect to the type of endothelial cells used for the experiments. In fact, in a very recent paper, La Rocca and collaborators [25] showed that exposure of HUVEC to 60M hydrogen peroxide for 3 and 6 hours resulted in a slight decrease of viability (4 to 7% less). When the same experiments were performed on endocardial endothelial cells, derived from the endocardium of CHF patients, these cells responded with a significant increase in proliferation. The authors therefore suggested that endothelial cells isolated from an organ primed for oxidative stress should be more resistant to the presence of stressor molecules [25]. Apart from these roles regarding endothelial proliferation, it has been shown a mutual interdependence between the concentrations of hydrogen peroxide and nitric oxide in endothelium: NO has been shown to possess a dual effect on H2O2 mediated toxicity, limiting or enhancing this process with respect to the concentrations of both species [26]. Endothelial hydrogen peroxide has been shown to exert endothelium-dependent vasorelaxation, a process due to the action of the endothelial isoform of NOS. It has been demonstrated that in vitro exogenous hydrogen peroxide upregulates eNOS transcription [18]. The increase of enzyme molecules leads to an increase in NO bioavailability, thereby causing vascular relaxation. Moreover, it has recently been suggested that hydrogen peroxide can function as a coupler of myocardial oxygen consumption to coronary blood flow increase. In fact, since superoxide ion is produced proportionally to the rate of cardiac metabolism, its dismutation leads to an increase in intracellular H2O2 concentration. Since one of the downstream effects is the upregulation of eNOS, the final point is a vasoactive effect [27]. On the contrary, in cardiovascular pathologies a number of factors should lead to the uncoupling of NOS. This process causes the arrest of NO production and the formation of superoxide ion. Self-maintaining circuits have been hypothesized for the generation of H2O2 in endothe74

Hydrogen peroxide in endothelium: multifaceted roles in cellular stress and signalling

lial cells. The ability of elevations in ROS to promote oxidase activation has been reported in some past experiments: Li et al. [28] showed that exogenous H2O2 was able to activate NAD(P)H oxidase to produce O2 independently of xanthine oxidase or eNOS. These findings were further confirmed by other groups, who proposed that even small concentrations of hydrogen peroxide derived from NAD(P)H oxidase can promote sustained activation of NAD(P)H oxidase itself [29]. Finally, as discussed earlier, H2O2 increase should lead to the reduced bioavailability of eNOS cofactors, therefore resulting in uncoupled eNOS and leading to further peroxide production. Enzymatic uncoupling should be caused by different processes: on one hand, it can be caused by the reduction of bioavailability of L-arginine, the NOS substrate, due to its chlorination by hypochlorous acid. On the other hand, the deficiency of tetrahydrobiopterine (BH4), an essential cofactor in NO synthesis, should be the result of the downregulation of dihydrofolate reductase (DHFR) by hydrogen peroxide. In fact, this enzyme is involved in recycling of oxidized BH4, a molecule which levels were linked to eNOS uncoupling by multiple reports [30,31]. Present data therefore reinforce the concept that the equilibrium of hydrogen peroxide levels, maintained by its intra- or extracellular sources on one hand, and by the clearance enzymes on the other hand, constitutes a key factor in endothelium physiology. Perturbations of this equilibrium, as seen in cardiovascular diseases, should have profound consequences, therefore misleading the cellular and paracrine signaling pathways regulating vascular homeostasis.

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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] Ferguson JE III, Kelley RW, PattersonC. Mechanisms of endothelial differentiation in embryonic vasculogenesis. Arterioscler Thromb Vasc Biol 2005; 25: 2246-2254. Ratajska A, Czarnowska E, Kolodzinska A, Kluzek W, Lesniak W. Vasulogenesis of the embryonic heart: origin of blood island-like structures. Anat Rec A Discov Mol Cell Evol Biol 2006; 288: 223-232. Schmidt A, Brixius K, Bloch W. Endothelial precursor cell migration during vasulogenesis. Circ Res 2007; 101: 125-136. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: Identification by morphologic criteria. J Clin Invest 1973; 52: 2745-2756. Marin V, Kaplanski G, Gres S, Farnarier C, Bongrand P. Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells. J Immunol Methods 2001; 254: 183-190. Jacobi J, Kristal B, Chezar J, Shaul SM, Sela S. Exogenous superoxide mediates pro-oxidative, proinflammatory, and procoagulatory changes in primary endothelial cell cultures. Free Radic Bol Med 2005; 39: 1238-1248. Aird WC. Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms. Circ Res 2007; 100: 158-173. Turner RR, Beckstead JH, Warnke RA, Woods GS. Endothelial cell phenotypic diversity. In situ demonstration of immunologic and enzymatic heterogeneity that correlates with specific morphologic subtypes. Am J Clin Pathol 1987; 87: 569-575. Fina L, Molgaard HV, Robertson D, Bradley NJ, Monaghan P, Delia D, Sutherland DR, Baker MA, Greaves MF. Expression of the CD34 gene in vascular endothelial cells. Blood 1990; 75: 2417-2426. Unger RE, Krump-Konvalinkova V, Peters K, Kirkpatrick CJ. In vitro expression of the endothelial phenotype: comparative study of primary isolated cells and cell lines, including the novel cell line HPMECST1.6R. Microvascular Res 2002; 64: 384-397. Simionescu M, Gafencu A, Antohe F. Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey. Microsc Res Tech 2002; 57: 269-288. Marks DS, Vita JA, Folts JD, Keaney JF, Welch GN, Loscalzo J. Inhibition of neointimal proliferation in rabbits following vascular injury by a single treatment with a protein adduct of nitric oxide. J Clin Invest 1995; 96: 2630-2638. Fltou M, Vanhoutte PM. Endothelial dysfunction: a multifaceted disorder. Am J Physiol Heart Circ Physiol 2006; 291: H985H1002. Hare JM, Stamler JS. NO/redox disequilibrium in the failing heart and cardiovascular system. J Clin Invest 2005; 115: 509-517. Linke A, Recchia F, Zhang X, Hintze TH. Acute and Chronic Endothelial Dysfunction: Implications for the Development of Heart Failure. Heart Failure Rev 2003; 8: 87-97. Bauersachs J, Bouloumie A, Fraccarollo D, Hu K, Busse R, Ertl G. Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: Role of enhanced vascular superoxide production. Circulation 1999; 100: 292298. Seddon M, Looi YH, Shah AM. Oxidative stress and redox signalling in cardiac hypertrophy and heart failure. Heart 2007; 93: 903-907. Cai H. Hydrogen peroxide regulation of endothelial function: Origin, mechanism and consequences. Cardiovasc Res 2005; 68: 26-36. Faraci FM, Didion SP. Vascular Protection: Superoxide Dismutase Isoforms in the Vessel Wall. Arterio-

[17] [18] [19]

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scler. Thromb. Vasc. Biol. 2004; 24: 1367-1373. [20] Stralin P, Karlsson K, Johansson BO, Marklund SL. The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase. Arterioscler Thromb Vasc Biol 1995; 15: 2032-2036. [21] Suttorp N, Toepfer W, Roka L. Antioxidant defense mechanisms of endothelial cells: glutathione redox cycle versus catalase. Am J Physiol 1986; 251: C671-C680. [22] Kang SW, Chae HZ, Seo MS, Kim K, Baines IC, Rhee SG. Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-alpha. J Biol Chem 1998; 273: 6297-6302. [23] Ramachandran A, Moellering D, Go YM, Shiva S, Levonen AL, Jo H, Patel RP, Parthasarathy S, DarleyUsmar VM. Activation of c-Jun N-terminal kinase and apoptosis in endothelial cells mediated by endogenous generation of hydrogen peroxide. Biol Chem 2002; 383: 693-701. [24] Ingram DA, Krier TR, Mead LE, McGuire C, Prater DN, Bhavsar J, Saadatzadeh MR, Bijangi-Vishehsaraei K, Li F, Yoder MC, Haneline LS. Clonogenic endothelial progenitor cells are sensitive to oxidative stress. Stem Cells 2007; 25: 297-304. [25] La Rocca G, Di Stefano A, Eleuteri E, Anzalone R, Cappello F, Magno F, Corrao S, Loria T, Farina F, Martorana A, Di Gangi C, Colombo M, Sansone F, Patan F, Rinaldi M, Giannuzzi P, Zummo G: Oxidative stress induces myeloperoxidase expression by HUVEC and Chronic Heart Failure endocardial endothelial cells. Lab Invest, 2007, submitted. [26] Rauen U, Li T, de Groot H. Inhibitory and enhancing effects of NO on H(2)O(2) toxicity: dependence on the concentrations of NO and H(2)O(2). Free Radic Res 2007; 41: 402-412. [27] Saitoh S, Zhang C, Tune JD, Potter B, Kiyooka T, Rogers PA, Knudson JD, Dick GM, Swafford A, Chilian WM. Hydrogen peroxide: A feed-forward dilator that couples myocardial metabolism to coronary blood flow. Arterioscler Thromb Vasc Biol 2006; 26: 2614-2621. [28] Li W-G, Miller FJ Jr, Zhang HJ, Spitz DR, Oberley LW, Weintraub NL. H2O2-induced O2 production by a non-phagocytic NAD(P)H oxidase causes oxidant injury. J Biol Chem 2001; 276: 29251-29256. [29] Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P) H oxidase activity. Upstream mediators. Circ Res 2002; 91: 406-413. [30] Chalupsky K, Cai H. Endothelial dihydrofolate reductase: Critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase. PNAS USA 2005; 102: 9056-9061. [31] Moens AL, Kass DA. Tetrahydrobiopterin and cardiovascular disease. Arterioscler Thromb Vasc Biol 2006; 26: 2439-2444.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

METODICHE MICROPERfUSIONALI E SEPSI


[Microcirculation and sepsis] Francesco Carini, Chiara Lo Piccolo, Giuseppe Alessandro Scardina*, Pietro Messina* e Vincenzo Valenza
Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana Normale - *Dipartimento di Scienze Stomatologiche, Universit degli Studi di Palermo (I)

Parole chiave: Microcircolo, Shock settico Key words: Microcircol, Septic shock Riassunto. La sepsi una complessa sindrome caratterizzata da infiammazione sistemica in risposta allinfezione. Lincidenza della sepsi aumentata negli ultimi decenni e si prospetta un ulteriore aumento. Lalta mortalit e il peso sul sistema sanitario nazionale ci convince che c urgenza necessit di migliorare la diagnosi e la gestione del paziente settico. La micro circolazione uno dei pi grandi organi del corpo e la funzione micro circolatoria il principale prerequisito per adeguare lossigenazione tissutale con la funzione dorgano. Il suo scopo trasportare lossigeno e nutrire le cellule, assicurarne adeguate funzioni immunologiche e nella patologia liberare sostanze terapeutiche. Immagini ottenute con OPS mostrano le prime osservazioni cliniche in organi umani interni e sono state messe in relazione le anomalie micro circolatorie ed i vari stadi della sepsi. Recentemente con il sistema SDF le osservazioni del micro circolo hanno offerto migliori dettagli. SDF utilizza una nuova metodica di immagine beneficiando del fatto che la uce che illumina e la luce riflessa hanno via riflessa. La micro circolazione visualizzata da una analisi del movimento cellulare la quale permette di misurare quantitativamente il flusso dei globuli rossi nei capillari; questa misura ritenuta una parametro sicuro indicativo di funzione o disfunzione cardiovascolare. Caratteristiche morfologiche della micro circolazione, densit capillare e morfologia dei micro vasi possono essere misurati usando SDF. In questa review discutiamo sul ruolo della disfunzione micro circolatoria nello sviluppo e nel trattamento dei difetti di distribuzione circolatori associati a sepsi. Abstract. Sepsis is a complex sindrome characterized by systemic inflammation in response to infection. The incidence of sepsis has increased in recent decades and is predicted to continue to rise. The high sepsis related mortalities and the burden on healthcare system means there is an urgent need to improve the diagnosis and management of sepsis patients. The microcirculation is one of the largest organs in the body and microcirculatory function is the main prerequisite for adequate tissue oxygenation and thus organ function. Its purpose is to transport oxygen and nutrients to tissue cells, ensure adequate immunological function and in disease to deliver therapeutic drugs to target cells. Orthogonal polarization spectral imaging allowed the first clinical observation in the microcirculation in human internal organ and has identified the pivotal role of microcirculatory abnormalities in defining the severity of sepsis. Recently sublingual sidestream dark field imaging (SDF) has been introducted allowing observations of the

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microcirculation in even greater detail. SDF imaging utilizes a novel method of reflectance avoidance in which the illuminated light and light travel independent pathways. The microcirculation is achieved by an analysis of the moving cells in the images, which permits the quantitative measurement of red blood cell flow in the capillaries; this measurement is belived to represent a truly sensitive measurement which s indicative of cardiovascular function or disfunction Morphological characteristics of the microcirculation such as functional capillary density and micro vessel morphology can be measured using SDF. In this review we discuss the role of microcirculatory dysfunction in the development and treatment of the circulatory distributive defect associated with sepsis.

I contributi pi recenti della letteratura suggeriscono un aumento dellincidenza della sepsi; tale aumento dovuto a vari fattori quali lavanzare dellet media della popolazione, il numero crescente di procedure invasive, limpiego di farmaci immunosoppressivi. Laumento dellincidenza della sepsi porta un danno endoteliale localizzato in vari distretti; stato questo aspetto che ci ha condotto a focalizzare i nostri studi sullendotelio e sulle indagini microperfusionali in vivo, al fine di tentare di portare un contributo scientifico in tale campo In questo lavoro viene analizzato lo stato dellarte su un tema attuale e complesso che impegna quotidianamente tutti coloro che sono coinvolti nellassistenza al paziente critico La perfusione tissutale dipende dal numero dei capillari che vengono irrorati dal sangue proveniente dalle arteriole progenitrici. In condizioni fisiologiche, lapporto ematico agli organi varia secondo le esigenze funzionali e metaboliche delle cellule. I meccanismi che consentono di aumentare il flusso ematico in un organo sono rappresentati dalle variazioni del diametro delle arteriole e dallaumento del flusso ematico attraverso i capillari. Le variazioni di diametro del vaso sono dovute allattivit del muscolo liscio vascolare che risponde a svariate stimolazioni quali: pressoria,. nervosa, ormonale, metabolica locale, endoteliale, dipendente dal flusso I batteri e le tossine possono determinare una profonda alterazione della circolazione periferica. La concentrazione di citochine infiammatorie contribuisce a determinare il danno a carico dellendotelio vascolare e degli organi bersaglio. I leucociti sono attivati e aderiscono alla parete dei vasi e la capacit di agglutinarsi causa di alterazioni della circolazione del sangue capace di determinare importanti lesioni ossidative del sistema vascolare. La pressione arteriosa si pu ridurre in modo marcato, in conseguenza di uno stato endotossico in grado di aumentare la permeabilit del sistema vascolare con conseguente edema. Nelle situazioni pi gravi pu determinarsi una condizione severa di coagulopatia da consumo. La maggior parte dei pazienti che muore a causa della sepsi presenta

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basse concentrazioni di anticoagulanti come la proteina C e lantitrombina e la terapia di rimpiazzo di queste proteine pu risultare efficace. Si sviluppa una complessa interazione tra endotelio, infiammazione e sistema della coagulazione. Inoltre, lattivazione dei fattori umorali e cellulari, con particolare riferimento allinterazione endotelio-neutrofili, altera la barriera endoteliale e la regolazione vasale causando disfunzioni nel trasporto di ossigeno e nel suo utilizzo da parte dei tessuti. La cascata della coagulazione attivata facilmente nei diversi modelli sperimentali; in clinica, la sindrome da coagulopatia da consumo e in particolare i fenomeni di trombosi rappresentano unevenienza abbastanza frequente e in grado di peggiorare il decorso della sepsi. La presenza di trombi intravascolari diffusi nel circolo periferico pu determinare una condizione di coagulazione intravasale disseminata. Le citochine proinfiammatorie interagiscono con il sistema della coagulazione mediante lattivazione del fattore tissutale, e sono in grado di alterare in modo profondo il profilo della cascata della coagulazione. La deplezione della proteina C, i ridotti livelli di ATIII e dellinibitore C1 delle esterasi, la conseguente inibizione della fibrinolisi sono tutti fattori in grado di determinare unimportante azione procoagulante. Lattivazione del sistema della coagulazione che alimenta la reazione infiammatoria in grado di automantenersi e amplificare gli effetti deleteri sulla perfusione degli organi. I fattori che interagiscono sono molteplici, tra laltro la trombina in grado di determinare una sovraregolazione della selectina P ed E; tale condizione attiva il fattore di contatto ed in grado di stimolare la produzione di bradichinina, con conseguente peggioramento del quadro dipotensione e dipoperfusione. In studi clinici stato dimostrato che i livelli di ATIII e di proteina C, in corso di sepsi, si riducono drasticamente; la mortalit dei pazienti settici inversamente correlata con i valori di questi due elementi. Lipotensione definita da valori della pressione sistolica arteriosa < 90 mmHg e della pressione arteriosa media < 60mmHg, o da una riduzione > 40 mmHg dal valore di partenza, malgrado adeguato riempimento e in assenza di altre cause; quando il sistema circolatorio non pi in grado di assicurare una perfusione tissutale adeguate si realizza lo shock. Nello shock settico si ha usualmente un profilo caratterizzato da una iniziale gittata cardiaca elevata e da basse resistenze vascolari sistemiche con ipotensione, maldistribuzione del flusso ematico nel microcircolo e compromissione della perfusione tissutale. Sul piano fisiopatologico compendia le caratteristiche dello shock ipovolemico, ostruttivo, cardiogeno, distributivo, citotossico. I pazienti settici che richiedono un supporto emodinamico sono per definizione instabili e hanno spesso anche una compromissione dorgano preesistente di grado diverso. La maldistribuzione di una normale gittata cardiaca pu compromettere la perfusione dorgano; allinterno dellorgano, la cattiva distribuzione per la compromis81

Francesco Carini, Chiara Lo Piccolo, Giuseppe Alessandro Scardina, Pietro Messina e Vincenzo Valenza

sione delle resistenze vascolari esacerba la disfunzione. Inoltre lazione sul metabolismo cellulare da parte dei mediatori della sepsi porta ad uninadeguata utilizzazione dellossigeno e degli altri nutrienti. Lendotelio polmonare svolge un ruolo fondamentale nella dinamica fisiopatologia della sepsi; sia perch il polmone pu ospitarne la causa o perch pu esserne il bersaglio Linsulto infiammatorio allendotelio vascolare ne provoca laumento di permeabilit alle proteine e danneggia il sistema di autoregolazione del tono arteriolare. Questi eventi contribuiscono allalterazione dellequilibrio tra pressioni oncotiche e idrostatiche, favorendo la fuoriuscita dei fluidi dai vasi. Il processo naturalmente coinvolge anche il circolo polmonare ed la causa delledema interstiziale, caratteristica anatomopatologica tipica dellALI/ ARDS. Il fattore fondamentale che spiega la patogenesi delledema interstiziale si riassume nellequilibrio tra i gradienti idrostatici e oncotici tra linterstizio e i vasi in rapporto al grado di permeabilit capillare. Quando la quantit di fluidi che si raccolgono nel tessuto polmonare non riesce ad essere drenata (attraverso il sistema linfatico), si accumula il liquido extravascolare. In corso di sepsi ci possono essere diversi gradi di permeabilit capillare, quindi il gradiente di pressione idrostatica rispetto a quello oncotico varia man mano che le molecole responsabili per il mantenimento della pressione oncotica attraversano le barriere endoteliali divenute molto permeabili. Laccumulo di liquido extravascolare e lessudato di proteine plasmatiche nello spazio alveolare crea ledema interstiziale che porta allARDS. Lipertensione polmonare nellARDS ha origine multifattoriale, ma indipendente dalla causa che la sottende; i pazienti che presentano un aumento significativo della resistenza vascolare polmonare hanno indiscutibilmente prognosi peggiore. Ledema perivascolare che predomina nelle fasi iniziali dellARDS ha un ruolo importante nella patogenesi dellipertensione vascolare; alleffetto delledema si aggiungono quello della vasocostrizione causata dallipossia alveolare e da altri mediatori vasoattivi come trombossani ed endoteline, oltre allostruzione causata da trombi piastrinici. Nellevoluzione della patologia il progredire dellipertensione riflette lo svilupparsi del processo di fibrosi, anchesso responsabile dellobliterazione de letto vascolare. Bench laumento della pressione dellarteria polmonare sia un evento caratteristico in corsi di ARDS, la resistenza vascolare polmonare in genere solo poco o moderatamente elevata e ci dovuto essenzialmente alla ridotta gittata cardiaca. La causa principale dipossiemia associata a sepsi legata allo shunt intrapolmonare. NellARDS lo shunt dovuto al persistere della perfusione di alveoli atelettasici e riempiti di liquido e dallabolizione del fisiologico riflesso di vasocostrizione ipossica. Dopo il danno iniziale al polmone si sviluppa un gradiente lungo lasse gravitazionale, le zone di polmone dipendenti (e quindi maggiormente perfuse) si consolidano diventando la fonte principale di shunt. Lo shunt del sangue attraverso le parti di polmone non ventilato spiega la natura refrattaria dellipossiemia nellARDS.
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Le cellule endoteliali liberano NO e PGI2 in rapporto a una serie di stimolazioni che si esercitano sulla superficie delle cellule. La PGI2 deriva dallacido arachidonico per azione dellenzima ciclossigenasi. Viene liberata dallendotelio e determina rilasciamento del muscolo liscio vascolare, aumentando i livelli intracellulari di cAMP. La PGI2 ha notevoli effetti antiaggreganti sulle piastrine, contribuendo alla perfusione dei tessuti periferici. Il NO liberato dalle cellule endoteliali induce lattivazione di una guanilatociclasi solubile, che attiva il meccanismo di formazione di cGMP nelle cellule muscolari lisce vascolari. Il risultato finale la riduzione dei flussi di calcio e il rilasciamento del muscolo liscio, che causa vasodilatazione. Laminoacido L-arginina funge da substrato per la formazione di NO, pertanto la sua somministrazione pu aumentare la formazione e la liberazione di NO. Il fattore iperpolarizzante di origine endoteliale il terzo fattore, che contribuisce alla dilatazione delle arteriole e allaumento del flusso ematico. La sua costituzione biochimica ancora da chiarire in molti distretti vascolari, ma in genere viene liberato dallendotelio e determina iperpolarizzazione delle cellule muscolari lisce, dando luogo a vasodilatazione. Tra i fattori che causano vasocostrizione vi sono le endoteline (ET1,ET2,ET3), i trombossani, che derivano dallacido arachidonico, lungo la catena di reazioni attivate da ciclossigenasi. Sono stati descritti anche fattori costrittori che sono indipendenti dalla ciclossigenasi. Dunque lendotelio pu modulare le risposte vasocostrittorie del muscolo liscio vascolare, agendo come una barriera tra il sangue e le cellule muscolari, limitando la quantit di sostanze attive sul muscolo liscio e liberando al contempo NO e fattori rilascianti, che provocano vasodilatazione Le variazioni di flusso ematico nelle arteriole sono in grado di indurre risposte endoteliali capaci di regolare il tono vascolare. Se aumenta il flusso ematico in unarteria, questa si dilata facilitando la riduzione della resistenza idraulica del sistema. Il meccanismo della vasodilatazione indotta da un aumento del flusso ematico dovuto alla liberazione di NO e PGI2 indotto dalle forze tangenziali che agiscono sulle cellule endoteliali (shear stress). Aumentando lo shear stress, si ha una dilatazione arteriolare, che contribuisce alla regolazione rapida della resistenza durante liperemia funzionale. Vi sono recettori endoteliali sensibili allo shear stress, che rispondono liberando NO e dando seguito allinterno delle cellule a messaggi trasferiti a livello nucleare. Sono, infatti, stimolati il fattore nucleare kappa B e il fattore proteico nucleare di attivazione I, che sono capaci di legarsi a elementi di risposta allo shear stress a livello di geni che rispondono alle stimolazioni meccaniche. Sepsi e disfunzioni dei sistemi di regolazione I predetti meccanismi autoregolatori e le loro funzioni microocircolatorie sono spesso
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alterati durante la sepsi e la loro disfunzione spesso da mettere in relazione a un definito fattore nella patofisiologia della sepsi. La disfunzione microcircolatoria caratterizzata da eterogenee anomalie nel flusso sanguigno con la presenza di alcuni capillari sottoperfusi, mentre altri hanno normale o alto flusso di sangue. La unit funzionali microcircolatorie divenute ipossiche, spiegano lestrazione deficitaria di ossigeno durante la sepsi. In queste condizioni, la pressione parziale di ossigeno scende sotto la pO2 venosa. La disparit stata chiamata lapertura pO2, ed una misura della severit dello shunt funzionale, avvenimento che pi severo nella sepsi che nella emorragia. Nella sepsi il microcircolo non adatto a compiere la funzione regolatoria perch disturbato dallinsieme di segnali di trasduzione e di comunicazioni elettrofisiologiche. Il sistema ossido nitrico, componente centrale nel controllo autoregolatorio microcircolatorio, severamente disturbato nella sepsi da espressione eterogenea delle iNOS in differenti aree di organo, risultando shunt patologico di flusso. Poich iNOS non espressa omogeneamente in sistemi dorgano, aree mancanti di iNOS hanno meno vasodilatazione NO-indotta e diventano ipoperfuse. Le cellule del muscolo liscio che circondano le arteriole e regolano la perfusione perdono la loro sensitivit adrenergica e il tono durante la sepsi. I globuli rossi diventano meno deformabili e si aggregano di pi. I globuli rossi giocano anche un importante ruolo nella regolazione del flusso sanguigno microcircolatorio determinato dalla loro capacit di rilasciare NO in presenza di ipossia e causare vasodilatazione. Questa propriet regolatoria dei globuli rossi pu anche essere assente durante la sepsi. Questi severi difetti, insieme con i disturbi della coagulazione durante la sepsi, impediscono ulteriormente la perfusione e la funzione microcircolatoria. In pi, lattivazione dei leucociti da infiammazione settica genera particolari reazione allossigeno che danneggia direttamente le strutture microcircolatorie, le interazioni cellulari e le funzioni coagulatorie. Questi e altri mediatori infiammatori alterano le funzioni di barriera nella microcircolazione, incluse le giunzioni tra le cellule dando edema tissutale e ulteriore deficitaria estrazione di ossigeno. La disfunzione microcircolatoria determina distress respiratorio delle cellule parenchimali risultandone fallimento dorgano. Sepsi, microcircolo, D.I.C. - Distress mitocondriale Se la causa primaria del deficit estrattivo di ossigeno nella sepsi causato da un debole shunt, lipossia microcircolatoria e lincapacit mitocondriale a processare ossigeno attualmente causa di intenso dibattito. Il mitocondrio funziona da centrale dintegrazione degli stimoli apoptoici. Questi possono essere di diversa natura (caspasi, ceramide, chinasi) e sono in grado di determinare lapertura di un complesso proteico chiamato poro di transizione mitocondriale (PTPC Permeability Transit Pore Complex) localizzato in alcuni punti di contatto tra le due membrane
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mitocondriali. Questevento fa cadere la differenza di potenziale, per uscita dei protoni, ed ingresso di molecole prima interdette allingresso. Come risultato finale, il mitocondrio si riempie di liquido e la membrana esterna scoppia liberando nel citoplasma fattori stimolanti lapoptosi come lAIF (Apoptosis Inducing Factor), che in grado di raggiungere il nucleo ed attiva una via indipendente delle caspasi in grado di degradare il DNA, ed il citocromo e che si lega alle proteine Apaf-1 (apoptotic protease activating factor) e caspasi 9 ed una molecola di ATP formando un complesso definito apoptosoma. La caspasi 9 presente diviene in grado di attivare altre caspasi che danno il via ad una cascata mitocondriale che si conclude con la degradazione del DNA ad opera di fattori nucleari. Ai processi di alterazione della permeabilit del mitocondrio prendono parte anche i membri della famiglia di Bcl-2, composta da almeno 16 proteine, le quali sono in grado di interagire con le membrane mitocondriali. Tale famiglia proteica contiene elementi sia antiapoptotici, come Bcl-2 e Bcl-XL, sia proapoptotici, come Bax, Bid, Bcl-XS. Tali membri possono unirsi formando omodimeri ed eterodimeri che hanno attivit sia proapoptotica sia antiapoptotica. Levento chiave consiste nellabbondanza dei fattori proaptotici rispetto a quelli protettivi. Se questo evento avviene potranno allora formarsi dimeri in grado di alterare la permeabilit del mitocondrio. In alcuni studi effettuati su cuore di topo con sepsi fu osservata endotossemia nellarea ipossica; tuttavia in questo modello non fu trovata disfunzione mitocondriale, come evidenziato dalla normale risposta dello stato energetico mitocondriale allipossia in situ. Probabilmente con il progredire a sepsi severa si potrebbe presentare la disfunzione mitocondriale, accompagnata o anche possibilmente causata da pi gravi disfunzioni microcircolatorie. Brealey et al. [33] mostrano che la disfunzione mitocondriale effettivamente gioca un importante ruolo nella sepsi dove i livelli di disfunzione respiratoria dei mitocondri sono correlati con loutcome del paziente. Fallimento mitocondriale associato con sepsi contribuisce al distress respiratorio, specialmente in aree ipossiche e pu alimentare il distress tissutale conducendo a disfunzione dorgano. Microcircolazione e sindrome di distress mitocondriale Leventuale recupero di un fallimento circolatorio associato con sepsi consiste nella correzione del sistema emodinamico e dalle variabili ossigeno-derivate, ma quando il distress respiratorio persiste si parla di sindrome da distress mitocondriale e microcircolatorio (MMDS). Questo concetto stato formulato per identificare il compartimento fisiologico vulnerabile mascherato dal sistema circolatorio e responsabile del trasporto di ossigeno e della respirazione cellulare che diventa da disfunzione a sepsi, e pu condurre a disfunzione dorgano. Gli elementi definiti della natura e della severit della sepsi includono la natura delliniziale colpo conducente alla sepsi, comorbidit, mappaggio
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genetico individuale, precedenti terapie e tempo di trattamento. Il tempo che la sindrome durata e la precedente terapia ricevuta hanno una responsabilit ed effetti modulatori sulla patofisiologia e definiscono le subclassi della sindrome. La natura patogenetica del tempo fu dimostrata dagli studi di Rivers dove un trattamento precoce mostrava essere associato con improvviso outcome. La MMDS quando terapia e tempo sono inclusi nella sua definizione indica che le valutazioni integrative di questi determinati fattori microcircolatori e funzionali mitocondriali sono necessario per la valutazione della severit della sindrome. Strategie di salvezza della microcircolazione La presenza di distress respiratorio, nonostante la rianimazione basata sullemodinamica e su derivati dellossigeno, suggerisce fortemente che il fallimento microcircolatorio un fattore chiave da mettere in relazione anche nellelevato livello di lattato, ai disturbi del bilancio acido base ed alti livelli di CO2 evidenziati talvolta in queste condizioni. Il fallimento microcircolatorio pu avvenire con la presenza di normali o sopranormali variabili emodinamiche e ossigeno derivate, con distress microcircolatorio essendo mascherato dalla circolazione sistemica dallinsieme di shunts. Cos grande importanza assumono le tecniche di monitoraggio per Immagine statica di videocapillaroscopia verificare le strategie da assumere e per valutare la microcircolazione.

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Immagine ottenuta con SDF. possibile visualizzare e quantizzare il flusso del sangue ed evidenziare il movimento degli elementi corpuscolati

Monitorare la microcircolazione per ottimizzare il trattamento. Svariati metodi sono stati usati per monitorare la microcircolazione durante fallimento circolatorio in chirurgia e in terapia intensiva. Questi includono le misure di CO2 sublinguale, buccale e i livelli di CO2 sottocutanea, cos come lassorbimento, la riflettanza e la spettroscopia a infrarosso, per misurare la saturazione di emoglobina microcircolatoria. La spettroscopia ortogonale polarizzante (OPS) fu introdotta in chirurgia e mostrava la prima diretta osservazione della microcircolazione di organi interni umani. La tecnica mostra visualizzazione microscopica del microcircolo profondo e il flusso dei globuli rossi nella variabilit del microcircolo. Quando applicata sotto la lingua OPS provvede a una buona specificit per misurare la severit del difetto distributivo nella sepsi non realizzabile col monitoraggio convenzionale dellemodinamica sistemica e dellossigeno-derivati. La capnografia sublinguale combinata con lOPS immaging stata usata per investigare le relazioni tra la microcircolazione e lo stato metabolico durante la rianimazione. In cardiochirurgia, misure simultanee di spettroscopia a infrarosso sublinguale di diverse regioni misurate profondamente e spettrofotometria di regioni microcircolatorie superficiali, davano informazioni integrative circa la redistribuzione di ossigenazione microcircolatoria occorrente tra questi comparti durante cardiochirurgia. Tale combinazione, guardando ai differenti comparti funzionali della microcircolazione, pu accertare la distribuzione del trasporto di ossigeno durante sepsi, shock settico, e terapie che non sono previste dal monitoraggio convenzionale dellemodinamica sistemica e ossigeno-derivati. Studi di De Backer [7,10,2] sulla microcircolazione sublinguale usando OPS in pazienti settici hanno direttamente associato il grado di distress respiratorio con la severit della malattia e la risposta alla terapia. Questi studi con OPS mostrano che il difetto distributivo associato con sepsi caratterizzato da stasi del flusso nei piccoli capillari e normale flusso nei pi grandi vasi vicini. Questo sottolinea la necessit del monitoraggio clinico del flusso sanguigno in questi piccoli capillari.
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Limmagine OPS limitata, le immagine dei capillari sono talvolta macchiate e non possono essere sempre determinate. A quanto detto gli studi di Ince sviluppano una nuova modalit di immagini per osservare la microcircolazione chiamato sidestream dark field imaging (SDF). SDF consiste di una leggera guida circondata da 530 nm diodi a emissione di luce (LEDs), una lunghezza donda di luce che assorbita dallemoglobina dei globuli rossi, permettendo la loro osservazione come cellule scure che scorrono nella microcircolazione. I LEDs alla punta della guida sono otticamente isolati dallinterno dellimmagine conducente e danno luce profonda nei tessuti illuminando la microcircolazione. Questa illuminazione in campo oscuro applicata evita riflessi di superficie, dando chiare immagini delle strutture microcircolatorie e del flusso del sangue. Ci si aspetta che le immagini con SDF miglioreranno le immagini della microcircolazione specialmente per i capillari. Scelte terapeutiche Numerose scelte terapeutiche sono disponibili per migliorare la microcircolazione nel paziente settico. Volume rianimatorio. Se i meccanismi di autoregolazione sono intatti questi assicurano la rianimazione dallo shock ipovolemico attraverso volume, che effettivamente recupera letto microcircolatorio. Il volume fornito ripristina anche la funzione di barriera microcircolatoria e promuove il trasporto di ossigeno microcircolatorio [23,50,52]. Tuttavia induce emodiluizione, effetto che pu causare una redistribuzione della distribuzione di ossigeno. Il significato di una redistribuzione di approvvigionamento di ossigeno e il suo ruolo nella patofisiologia della sepsi e nella rianimazione, tuttavia, ancora da essere stabilito. Il sangue il miglior trasportatore di ossigeno, migliore dei cristalloidi e dei colloidi. Let della riserva dei globuli rossi tuttavia, pu modificare le propriet del sangue e ci deve essere preso in considerazione [53]. Il trasporto di ossigeno con lemoglobina, anche se valido non pu essere adoperato come routine clinica. Inibitori di iNOS e steroidi. Lossido di azoto, impropriamente chiamato ossido nitrico, uno dei pi potenti mediatori biochimici che gli organismi viventi producono; sicuramente degno di nota il fatto che a questa sostanza sia legato il premio Nobel 1998 per la Medicina/Fisiologia, attribuito al ricercatore americano Louis Ignaro per le sue scoperte riguardanti lossido nitrico come molecola segnale nel sistema cardiovascolare. Sei anni prima la rivista Science aveva eletto lNO come molecola dellanno. LNO una sostanza prodotta a partire dallamminoacido L-arginina in una reazione multi-step catalizzata dallenzima ossido nitrico sintetasi. Questultimo esiste in numerose isoforme, alcune costitutive (cellule endoteliali, piastrine, sistema nervoso) ed altre inducibili (macrofagi,
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leucociti, cellule muscolari lisce, epatociti). LNO agisce come importante messaggero intra ed intercellulare regolando numerose funzioni. Le cellule endoteliali producono NO che diffonde nel circolo, riducendo laggregabilit delle piastrine e ladesivit dei leucociti alle pareti dei vasi sanguigni e, in parte raggiunge la sottostante muscolatura liscia vascolare inducendone il rilasciamento. I conseguenti effetti antiaggreganti, antinfiammatori ed antiipertensivi sono di grande importanza. Oltre alleffetto sullendotelio il NO ha altre funzioni: a livello cerebrale (controllo dellapprendimento e della memoria), gastrointestinali (modulazione dele secrezioni e della motilit), respiratorio (modulazione del tono della muscolatura liscia bronchiale). Sono in corso studi sulle sue possibili azioni nei confronti delle infezioni batteriche e nel controllo della crescita dei tumori. Spesso nella sepsi, i meccanismi di autoregolazione sono danneggiati [55]. Semplici fluidi, possono correggere lemodinamica sistemica e recuperare deboli aree della microcircolazione ipossica [11,12]. Questa distribuzione di flusso in relazione, fra altri meccanismi, alla eterogenea espressione delle iNOS in parti differenti di letti dorgano risultando shunt patologici di flusso [15,16]. Di nota che deficienze di iNOS nel topo non espongono le disfunzioni circolatorie associate con le endotossine che sopravvengono in topi di tipo selvatico, sottolineando limportanza del controllo di iNOS nella sepsi [57]. In studi recenti di maiali con sepsi, liquidi combinati con inibitori di iNOS, reclutavano letto circolatorio nellintestino [50, 58]. Linibizione di iNOS protegge anche le funzioni di barriera della microcircolazione e pu essere guardata come una misura per il reclutamento microcircolatorio [59]. Agenti antinfiammatori, cos come gli steroidi, sono estremamente validi allinibizione di iNOS e possono prevenire lipotensione indotta dalle endotossine. Somministrandoli in ritardo, tuttavia, non danno inbibizione di iNOS dovuto dagli inibitori di NO sepsi evocata dei recettori dei glucocorticoidi [60]. Tali studi mostrano la razionalit di una terapia precoce. Gli steroidi migliorano anche la funzione autoregolatoria come osservato in studi su ratto delle propriet autoregolatorie di un cuore isolato in sepsi [55]. La maggior parte degli studi sperimentali tuttavia usa quantit elevate di steroidi e lorientamento clinico mette al corrente contro luso di alti livelli di steroidi nel trattamento della sepsi [61]. Ci nonostante questi studi indicano che riducendo lespressione di iNOS si pu avere un controllo importante nella distribuzione emodinamica deficitaria nella sepsi. Vasodilatatori e vasopressori Reclutare la perfusione microcircolatoria sotto normovolemia pu essere realizzato da terapia vasodilatatoria perch ci aumenta la pressione giuda del flusso sanguigno allingresso della microcircolazione [62]. In un modello di maiale con sepsi, NO somministrato in combinazione con liquidi migliora lossigenazione microcircolatoria intestinale e corregge
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la pressione parziale gastrica di CO2 mentre ci non avviene somministrando solo fluidi [63]. In uno studio clinico di pazienti in shock settico, dove la microcircolazione sublinguale fu osservata con OPS, la pressione-guida risultava in conseguenza del flusso nei larghi vasi ma non nei capillari, dove rimaneva lenta o stagnante. Questo scenario visualizza direttamente lazione dello shunt e identifica la microcircolazione come il luogo dei difetti di distribuzione durante sepsi. Terapia vasodilatatoria da nitroglicerina con adeguati supporti di volume, tuttavia, fu capace di reclutare questo flusso stazionario di capillari e ripristinare la microcircolazione sublinguale [10]. De Backer et al. riportarono simili anomalie microcircolatorie in pazienti settici [7]. Essi mostrarono ulteriormente che la risposta vasodilatatoria endoteliale fu intatta dimostrando che lapplicazione topica di acetilcolina era effettiva nel reclutamento dellapertura/chiusura dei capillari. Studi di immagini di OPS sublinguale in pazienti con sepsi trovarono che mentre la pressione guida rianimatoria era effettivamente in ripristino, la pressione sanguigna sistemica non ha un effetto correttivo sulla perfusione microcircolatoria [2]. Da una prospettiva microcircolatoria, i vasopressori dovrebbero essere applicati con attenzione e sotto condizioni di monitoraggio microcircolatorio. Uno studio di Dubois et al. [84] dimostra che la pressione sistemica sanguigna fu ripristinata da vasopressina in pazienti con shock. Qui dirette osservazioni della circolazione sublinguale con immagini OPS non mostravano effetti dannosi sulla perfusione microcircolatoria. Tuttavia, in altri casi di pazienti studiati con shock settico, la vasopressina anche se aumentava la pressione del sangue e le urine eliminate, causava una completa cessazione del flusso microcircolatorio sublinguale, comprimendo la circolazione regionale e portando a morte. Esperimenti su animali hanno anche mostrato risultati conflittuali: alcuni studi hanno mostrato che la vasopressina ha effetti benefici sulla microcircolazione renale [65], mentre altri hanno mostrato che la vasopressina causa sospensione dellattivit microcircolatoria intestinale [66]. Terapia con multiple azioni I fluidi in combinazione con vasoattivi e supporto inotropo reclutano effettivamente la microcircolazione, anche se i loro effetti sulla microcircolazione non si possono dedurre solo con le variazioni sistemiche [2, 38]. I pazienti dove la microcircolazione non era responsabile di rianimazione, tuttavia, hanno una cattiva prognosi [2]. Il reclutamento della microcircolazione pu essere portato a termine con differenti modi e combinazioni di terapie. In questo modo, un agente donatore di NO pu aprire la microcircolazione e la terapia perfusionale lentamente recuperare le unit microperfusionali, mentre un agente antinfiammatorio o specifici inibitori di iNOS possono ridurre lo shunt patologico e reinstradare il flusso sanguigno a reclutare lentamente le unit microcircolatorie. Questo pu apparire paradossale da una certa posizione, ma entrambi le terapie reclutano effettivamente la
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microcircolazione e potrebbero essere teoricamente combinate. chiaro che quando applicate come combinazione di terapia la loro efficacia sul reclutamento microcircolatorio necessita di essere verificato per differenti sistemi dorgano. Prendendo in considerazione che la rianimazione dalle forme di sepsi multifattoriale, medicine con multipli siti di azione possono provvedere a uneffettiva strategia di trattamento per reclutare le funzioni microcircolatorie durante sepsi. Attivata la proteina C (APC) [67] provvede solo cos a un approccio integrato agendo su diversi meccanismi coinvolti in distress respiratorio. stato mostrato per esempio, che APC inibisce lespressione di iNOS e protegge contro lipotensione endotossina indotta [68]. Inoltre, attraverso la sua azione sul fattore nucleare kB, APC riduce anche il livello del fattore di necrosi tumorale, un effetto non visto quando gli inibitori di iNOS sono somministrati da soli [69]. In pi APC riduce lattivazione leucocitaria e la liberazione di specie reattive di ossigeno, cos come intervenendo sulla via coagulatoria [70]. Ulteriori studi hanno mostrato che queste azioni multifattoriali migliorano la microcircolazione in animali con sepsi [71,72]. La proteina C scatena un numero di effetti che possono essere visti come una liberazione strategica per la disfunzione microcircolatoria nella sepsi. Tuttavia, parecchie questioni rimangono nelle modalit di azione della proteina C. In questa review si discusso sul ruolo delle disfunzioni microcircolatorie nello sviluppo e sul monitoraggio e trattamento della distribuzione circolatoria deficitaria associata con sepsi. Lemodinamica sistemica tradizionale e le variabili derivate dovrebbero scoprire le disfunzioni microcircolatorie e le risposte alla terapia. Disfunzioni microcircolatorie possono dare distress cellulare e portare a disfunzione dorgano. Da queste prospettive la microcircolazione si pu guardare come motore della sepsi. Ulteriori monitoraggi delle funzioni microcircolatorie contribuiranno alla diagnosi e al trattamento della sepsi.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

IMMUNOHISTOCHEMICAL EXPRESSION Of INOS AND MATRIX METALLOPROTEINASES MMP-2 AND MMP-9 IN AORTIC ANEURYSMS
[Espressione immunoistochimica della iNOS e delle metalloproteinasi MMP-2 e MMP-9 negli aneurismi aortici] Giovanni Francesco Spatola
DI.ME.S., Sezione di Istologia ed Embriologia, Facolt di Medicina, Universit degli Studi di Palermo (I)

Key words: Aortic aneurysm, iNOS, Metalloproteinases, MMP-2, MMP-9 Parole chiave: Aneurisma aortico, iNOS, Metalloproteinasi, MMP-2, MMP-9 Abstract. Aortic aneurysms (AA) is a degenerative vascular disease characterized by localized dilatation of the aortic wall as a result of altered matrix composition (elastin and collagen degradation). Howewer the pathogenesis of the changes is elusive and unclear. Some experimental evidences suggest that iNOS (who synthesize a large amount of NO in inflammatory processes) and the metalloproteinases (MMP) are implicated in the pathogenesis of AA but the relationship between NO and MMP to aneurismal disease is currently unknown. The aim of this study is to investigate the immunohistochemical expression of iNOS and MMP-2 and MMP-9 in human aneurysmal tissues. Riassunto. Gli aneurismi aortici (AA) sono una patologia vascolare degenerativa caratterizzata da una locale dilatazione della parete aortica derivante da una alterazione della matrice extracellulare (in particolare deriva da una degradazione delle fibre collagene ed elastiche). Tuttavia, a tuttoggi non si sono del tutto chiariti i meccanismi patogenetici. Diverse evidenze sperimentali suggeriscono che liNOS (che sintetizza grandi quantit di NO durnate i processi infiammatori) e le metalloproteinasi (MMP) sono implicate nella patogenesi degli AA anche se la relazione tra NO e MMP nella patologia aneurismatica attualmente poco chiara. Lo scopo di questo studio quello di investigare lespressione immunoistochimica della iNOS e delle MMP-2 e MMP-9 in tessuti aneurismatici umani.

Introduction The cellular components of blood vessels wall are supported and organized by a complex structure of collagens, elastins, laminins, fibronectins, and proteoglycans known as the extracellular matrix (ECM) [1]. Researchers in nearly every medical discipline are examining the ECM in their quest to arrest disease, with many of the most promising advances taking place in cardiovascular research. Much of this cardiovascular research is focused on the family of ECM-remodeling enzymes collectively termed the matrix metalloproteinases (MMPs). Twenty-three MMPs have been described in humans, although they share a high degree of homology in their structure. Most MMPs are dismissed freely into the extracellular space immediately after synthesis as proenzymes, but some are stored
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within cells (eg, MMP-9 in neutrophil granules), and others are bound to cell surface membranes (eg, MT1-MMP) [2,3]. Rigorous regulation of MMP production and activity is a crucial part of ECM homeostasis. MMPs are formed as inactive proenzymes and are activated by proteolysis in the extracellular fluid, a process that is tightly regulated by other proteases and by endogenous MMP inhibitors. Plasma proteins and tissue inhibitors of metalloproteinases (TIMPs) are the primary endogenous inhibitors of MMPs, although they also serve other physiologic functions [2,3]. For example, TIMP-2 inhibits MMP-2, but is also required for MT1-MMPmediated activation of proMMP-2 [4]. The vascular microenvironment provides several specific modes of MMP regulation. For example, the cyclic strain on endothelial cells created by arterial pulsation has been shown to increase expression and activity of MMP-2 and its activator, MT1-MMP [5,6,7]. Other recent studies clarified a leading role of MMP2 in the angiogenesis and in the hypoxia. The endothelial cells overexpression MMP2 not induced by MT1-MMP reduced during hypoxia on the membrane cell, caused a migration of endothelial cells in accordance with the proangiogenetic role ascribed to MMP2. The involvement of this protease in the hypoxia-related death of endothelial cells support an additional apoptotic role of this protease [8,9,10,11,12]. Furthermore, emerging evidence suggests that nitric oxide inhibits gene expression of MMP-2 from endothelial cells, and increase a production of MMP-9 from endothelial cells and vascular smooth muscle cells. MMPs contribute to many normal and necessary physiologic processes through their modification of the ECM. Inflammation accompanied by increased MMP activity also contributes to many disease processes several are sequestered in inflammatory cells. In fact, increased inflammation and loss of MMP regulation is the hallmark of many pathologic states, including many of the disease processes treated by vascular surgeons [13,14]. The permanent and irreversible expansion of a tract of artery is defined aneurysm. Related of the seat distinguish, thoracic (TAA), abdominal thoracic (TAAA) and abdominal (AAA) aneurysms. The TAA can be localized to level of the ascending aorta, with eventual interest of the aortic valve, arc or the descending aorta. The TAAA and the AAA imply the involvement, with variable extension, of the descendant thoracic aorta and the abdominal aorta [15,16]. The pathogenesis of several types of aneurysms often differ based of their localization: in particular the TAA are in kind to degenerate, dilatative character or dissecting [17,18] while the AAA recognize a multifactorial genesis and an inflammatory origin (infective processes, aortities type Takayasu etc.) [19,20]. According the anatomo-pathologic point of view, the aneurysms have a degenerative
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vascular basis that are caused by altering of the cellular components of ECM with degradation of elastic and collagen fibers. Recent experimental studies have clarified that MMP are responsible of these alteration particularly the MMP-2 and the MMP-9. The MMP-2 is physiologic formed by tiny quantity in the muscle cells and in the fibroblast [21,22] and only a little quantity is formed by macrophages. Furthermore, the MMP-2 as we have seen before, it is perceptible in the hipoxia and it makes up one case of the apoptotic endothelial processes because it is produced also by these cells in particulary pathologic situation [8,9,10,11,12]. This production, localized, in the first time, in the aneurysm wall it will be exalted because it is the primum movens of degradative processes of the collagens fibres and causes the display of elastic fibres. The MMP-9 are formed by a huge quantity by macrophages [21,22] and only in one little side by the fibroblasts, act, on the contrary, degrading mostly elastic fibres. In addition, the inflammatory processes, that are involved in the first step of the pathological aneurysm process, provoke in the vessel wall forms an infiltrate consisting of, in the main way, macrophages that are producing a huge quantity of MMP-9. MMP-9, working at the same time with MMP-2, reduces in a full way the extracellular matrix by causing the dilatation by deterioration of the vessel [22]. The aim of our study is to investigate and compare the immunohistochemical expression of MMP-2 and MMP-9 and iNOS in human probable inflammatory abdominal aneurysmal tissues and in the dilatative and dissecting thoracic aneurysm. Materials and Methods Fragments of human 10 AAA, 10 dilatative TAA and 10 dissecting TAA were obtained during surgical procedure. However, during surgical aorto-coronaric bypass were obtained 10 punches of normal aorta in the side of joint of bypass, to use like normal control (the sample came from GENURTO O.U. of Cardiac surgery and O.U. of Vascular surgery, University of Palermo). All the specimens were fixed in Bouins mixture and embedded in paraffin; obtained sections were processed with anti MMP-9 (Chemicon International), monoclonal anti MMP-2 (Chemicon International), anti iNOS (Transduction laboratories) by EnVision+System HRP (AEC) (Dako Cytomation). In the same time, the negative controls have been realized on sections that are near by and processed on the base of the same protocol but without the passage of the primary antibody. Other sections near by those processed have been submitted at the histological stain using the method of Mallory Azan, and this to look forward to seeing the fibrous component and the miocytes. All the samples have been studied with microscope Leica DM1000 and Nikon OPTIPHOT 2.
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Results Morphological results The microscopic observation of the normal and pathologic fragments that are coloured with the Mallory-Azans method, who has pointed out the total subversion of the structure of arterial media and intima layers in aneurysmatic fragments compared to the controls. Furthermore, in the specimens of AAA aneurysm, was emphasized a plentyfull inflammation infiltrate in the media and adventitial tonaca. This infiltrate shows to be less evident in the dilatative aneurysm TAA and totally absent in those dissecant. However, the structure of the layers shows to be adulteraded in the aneurysm inflammatory, in those dilatative and in some points it becomes really difficult to distinguish it (Fig. 1,2,3,4). However, in the dissecant aneurysm, we emphasize the presence of the lesion at the level of the muscular layer that seem to be fractured in different points (Fig.5). Immunohistochemical results iNOS: In the fragments of the normal aorta hasnt been emphasized any activity of iNOS (fig. 6). In the fragments of the AAA has been emphasized a widespread immunoreactivity more intense at the level of the inflammatory cells that are in the context of the vascular wall (fig. 7). There is a weak reactivity in the dilatative TAA aneurysm endothelial cells (Fig.8) and not have reactivity in the specimens of the aortic dissection (Fig.9). MMP-2: There is a discrete reactivity of MMP-2 in the normal aortic wall localized in the tonaca media, where there are positive some miocytes, as in the tonaca adventitia, where some fibroblasts are immunopositive (Fig.10). In the aneurysm samples of inflammatory nature (AAA), we emphasize an intense MMP-2 immunoreactivity, that is located both in musculary cells and in fibroblasts. (Fig. 11). In the TAA of dilatative nature, we emphasize a strong granular reactivity in the cytoplasm of the endothelial cells (Fig.12,13). However, this reactivity shows to be absent in the dissecant TAA, while we emphasize a reactivity in the capillars and in a few fibroblasts (Fig.14). MMP-9: The fragments of the normal aorta show a weak immunoreactivity located in the tonaca media (Fig. 15). In the AAA there is an intense reactivity widespread in all components of the wall, more evident in the areas where there is much more presence of the inflammatory infiltrate (Fig. 16). In the dilatative TAA there is the presence of an intense immunoreactivity at the level of the endothelial cells (Fig.17) and moreover there is a modest immunoreactivity in some cell members of probable fibroblastic nature. This distribution is the same also in the dissecant aneurysm.

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Conclusions Our results document that the iNOS is present only in the aneurysm wall of AAA, in fact according the recent literature, they are considered as a inflammatory genesis, while it proves to be absent or present in small amount, in the TAA dilatative or dissecant and totaly absent in the normal vessels used as control. The reactivity of MMP are significantly increased in the pathologic specimens compared to those of the normal aorta. MMP-2, in AAA is more present at the level of the muscle cells and in the fibroblast, while in TAA of dilatative nature, that are present at in the endothelial cells according with the recent letterature that invest those cytotipes of angiogenetic role (the intima to be absolutely disarranged with an obvious and followed tissutal hypoxia), and, in second time, of apoptotic functions. MMP-9 in the AAA is produced in a big quantity in the inflammatory cells, while in the TAA it presents a reactivity like to MMP-2. This research gives the immunohistochemical basis to support the role of the NO and the metalloproteinases in the AAAs pathogenesis and suggests the hypothesis that the iNOS is produced in huge quantity below the inflammatory cytochines induction and it stimulates the activity of the MMP during the formation of the aneurysm. Moreover it confirm other biochemical data that give to the MMP-2 a fundamental role in the angiogenetic and apoptotic processes in the TAA.

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[1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

Figures 1-8: [1] Normal Aorta Mallory-Azan 10x aortic media and adventitia with the normal parallel distribution of myocites and the presence of collagen and elastic fibers, fibroblast and vasa vasorum - [2] AAA Mallory-Azan 10x inflammatory infiltrate in the disarranged media and adventitia - [3] Dilatative TAA Mallory-Azan 20X endothelial cells and the totally disrupted intima layer - [4] Dilatative TAA MalloryAzan 20X alteration of aortic wall - [5] Dissecant TAA Mallory Azan 20X the muscular layer of aorta that seem to be fractured - [6] Aorta iNOS 10x iNOS reactivity is totally absent - [7] AAA iNOS 40x immunoreactivity iNOS more intense in the inflammatory cells - [8] Dilatative TAA iNOS 63X iNOS immunopositive endothelial cells.

Espressione Immunoistochimica della inos e delle metalloproteinasi MMP-2 e MMP-9 negli aneurismi aortici

[9]

[10]

[11]

[12]

[15]

[13]

[14]

[16]

[17]

Figures 9-17: [9] Dissecant TAA iNOS 20X poor and aspecific immunoreactivity - [10] Aorta MMP-2 40x presence of MMP-2 reactivity in the myocites and in adventitia layer - [11] AAA MMP-2 20x strong immunoreactivity in the muscular layer - [12] Dilatative TAA MMP-2 20x positive endothelial cells - [13] Dilatative TAA MMP-2 100x oil positive endothelial cells - [14] Dissecant TAA MMP-2 40x reactivity in the capillars and in a few fibroblasts - [15] Aorta MMP-9 20x immunoreactivity located in the tonaca media - [16] AAA MMP-9 20x strongly immunoreactivity in the media and the adventitia (macrophages positive) - [17] Dilatative TAA MMP-9 20x positive endothelial cells.

Giovanni Francesco Spatola

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] Kelleher CM, Mc Lean SE, Mecham RP. Vascular extracellular matrix and aortic development. Curr Top Dev Biol 2004; 62:153-88. Vise R, Nagase H. Matrix metaloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003; 92:827-39. Nagase H. Woessner JF. Matrix Metalloproteinases. J Biol Chem 1999; 274:21491-4. Wang Z, Juttermann R, Soloway PD. TIMP-2 is required for efficient activation of proMMP-2 in vivo. J Biol Chem 2000; 275:26411-5. Von Offenberg Sweeney N, Cummins PM, Birney YA, Cullen JP, Redmond EM, Cahill PA. Cyclic strainmediated regulation of endothelial matrix metalloproteinase-2 expression and activity. Cardiovasc Res 2004; 63:625-34. Wang BW, Chang H, Lin S, Kuan P, Shyu KG. Induction of matrix metalloproteinases-14 and 2 by cyclical mechanical stretch is mediated by tumor necrosis factor-alpha in cultured human umbilical vein endothelial cells. Cardiovasc Res 2003;59:460-9. Hobeika MJ, Thompson MD, Muhs BE, Brooks PC, Gagne PJ. Matrix metalloproteinases in pheripheral vascular disease. Journ Vasc Surg 2007; 45: 849-57. Boyd PJ, Doyle J, Gee E, Pallan S, Haas TL. MAPK signalling regulates endothelial cell assembly into network and expression of MT1-MMP and MMP2. Am Journ Physiol 2005; 57:3 C659-C668. Puyraimond A, Fridman R, Lemesle M, Arbeille B, Menashi S. MMP2 Colocalizes with Caveolae on the surface of endothelial cell. Exp Cell Res 2001; 262: 28-36. Ben-Yosef Y, Miller A, Shapiro S, Lahat N. Hypoxia of endothelial cells leads to MMP2-dependent survival and death. Am Journ Physiol Cell Physiol 2005; 289:1321-1331. Wang L, Zhang ZG, Zhang RL, Gregg SR, Hozeska-Solgot A, LeTourneau Y, Wang Y, Chopp M. Matrix metalloproteinase 2 (MMP-2) and MMP-9 secreted by erythropoietin-activated endothelial cells promote neural progenitor cell migration. Journ Nurosci 2006; 26(22): 5996-6003. Finetti F, Solito R, Morbidelli L, Giachetti A, Ziche M, Donnini S. Prostaglandin E2 regulates angiogenesis via activation of fibroblast growth factor receptor-1. Journ Biol Chem November 2007 (paper in press). Thompson RW et al. Pathophysiology of abdominal aortic aneurysma: insights from a the elastase-induced model in mice different genetic backgrounds. Ann NY Acad Sci 2006 Nov.;1085:59-73. Sun MH et al. Expression of inducible nitric oxide synthase and matrix metalloproteinase-9 and their effects on angiogenesis and progression of hepatocellular carcinoma. World J Gastroenterol 2005 Oct 14; 11(38): 5931-7. Svensson LG, Crawford ES, Hess KR, et al. Composite valve graft replacement of the proximal aorta: comparison of techniques in 348 patients. Ann Thorac Surg 1992;54:427-39. Crawford ES, Crawford JL, Safi HJ et al. Thoracoabdominal aortic aneurysms: preoperative and intraoperative factors determining immediate and long-term results of operations in 605 patients. J Vasc Surg 1986;3:389-404. De Bakey ME, Henley WS, Cooley DA et al. Surgical management of dissecting aneurysm of the aorta. J Thorac Cardiovasc Surg 1965; 49: 130-149. Daily PO, Trueblood HW, Stinson EB, Werflein RD, Shumway NE. Management of acute aortic dissections. Ann Thorac Surg 1970; 10: 237-247. Maffei S, Di Renzo M, Bova G, Auteri A, Pasqui AL. Takayasus arteritis: a review of the literature. Intern Emerg Med 2006;1(2):105-12.

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[20] Pearce WH, Shively VP. Abdominal aortic aneurysm as a complex multifactorial disease: interactions of polymorphisms of inflammatory genes, features of autoimmunity, and current status of MMPs. Ann N Y Acad Sci 2006 Nov;1085:117-32. [21] Davis VPR et al. Matrix metalloproteinase-2 production abd its binding to the matrix are increased in abdominal aortic aneurysm. Arter Thromb Vasc Biol 18: 1625-1633, 1998. [22] Longo GM et al. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. Journ Clin Invest 110, 5, 2002.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

CHAPERONOLOGY: A NOvEL RESEARCH fIELD fOR EXPERIMENTAL MEDICINE IN THE XXI CENTURY
[Chaperonologia: un moderno campo di ricerca per la Medicina Sperimentale del XXI secolo] Francesco Cappello, Fabio Bucchieri, Sabrina David, Claudia Campanella, Anna Ribbene, Antonella Marino-Gammazza, Nella Ardizzone, Anna Merendino, Vito Marcian, Giovanni Peri, Everly Conway de Macario*, Alberto J.L. Macario* and Giovanni Zummo
Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana, Universit degli Studi di Palermo, via del Vespro 129, 90127 Palermo, Italy. - * University of Maryland Biotechnology Institute (UMBI), Columbus Center, 701 E. Pratt Street, Baltimore, MD 21202, USA

Key words: Chaperones, Chaperonins, Hsp60, Hsp10, Chaperonopathies, Chaperonotherapy Parole chiave: Chaperoni, Chaperonine, Hsp60. Hsp10, Chaperonopatie, Chaperonoterapia Abstract. We have been studying for some years two mitochondrial heat shock proteins (Hsps), the chaperonins Hsp60 and Hsp10, that are necessary for folding of mitochondrial proteins. In recent times, the interest in these Hsps has been growing since it has been shown that they can also be present in the cytoplasm and secreted outside cells. We still do not know all their functions, but we are aware that they could represent important biomarkers for some tumours and inflammatory diseases. Riassunto. Da alcuni anni ci occupiamo di studiare due proteine da shock termico mitocondriali, la chaperonina Hsp60 e la sua co-chaperonina Hsp10, necessarie per il folding delle proteine mitocondriali. Linteresse nei loro confronti negli ultimi tempi aumentato perch si scoperto che queste molecole possono anche essere presenti nel citoplasma ed essere secrete allesterno della cellula. Non sono ancora note tutte le loro funzioni, che possono anche essere degli importanti biomarcatori in alcuni tumori e in alcune patologie infiammatorie.

Introduction For some years, our group has been studying morphology and function of mitochondria both in normal cells and pathological models, in order to achieve a better understanding of the mitochondrial involvement in the pathogenesis of some disease, like cancer. More recently, we have focused our attention on two mitochondrial proteins, namely Hsp60 and Hsp10, that are important for the survival of such organelles and thus of the whole cell. The Hsps constitute a heterogeneous group of molecules highly preserved during evolution as they are involved in many crucial cellular functions [1-3]. One of the most relevant is the chaperoning role, which is responsible not only for the acquisition of functional conformation of other proteins, but also of their preservation after stress caused by a variety of stressors affecting diverse tissues (Table 1). Chaperones are also involved in the

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degradation of damaged proteins [4-6]. So, chaperones are proteins whose function is to promote correct folding of nascent polypeptides, refolding of partially denatured proteins, and degradation of irreversibly damaged molecules. Not all Hsps are chaperones, since other molecules, different from Hsps, have such a function, too. Table 1: Chaperone inducers (Cell stressors)a Type Description

Physical Heat, irradiation, UV light, etc Chemical Oxygen derived free radicals, hypoxia-anoxia-reperfusion Biological Infection, Inflammation Psychological Emotions, hormonal imbalance Mechanical Compression, shearing stretching Others: ethanol, methanol, tetracycline, teratogens, mutagens, carcinogens
a

Reproduced with permission from reference 9.

Chaperonin and co-chaperonins in normal and pathologic tissues Hsp60 and Hsp10 are mitochondrial chaperones, commonly named chaperonin (Hsp60) and co-chaperonin (Hsp10). It is known from studies in prokaryotes that these two chaperonins participate in the folding of nascent polypeptides (Fig.1) [7-8]. Since the mitochondrial Hsp60 and Hsp10 are phylogenetically close to those from bacteria it is generally assumed that the mitochondrial molecules have similar functions than those of the bacterial counterparts and act through similar mechanisms. A number of studies carried out by different groups have shown that Hsp60 and Hsp10, as well as other chaperones, are associated with several diseases, now referred to as

Fig. 1: Schematic representation of Hsp60-Hsp10 performing their protein-folding function as envisioned from data from the prokaryotic system. 110

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chaperonopathies [9-11]. A first classification included two main classes of chaperonopathies, genetic and acquired. Some of the genetic chaperonopathies are, CharcotMarie Tooth disease, distal hereditary motor neuropathy, childhood cataracts, distal motor neuropathy, hereditary spastic paraplegia, X-linked retinitis pigmentosa, and Leber congenital amaurosis. Examples of acquired chaperonopathies are Alzheimer disease, amyotrophic lateral sclerosis, Huntingtons disease, and other pathologic conditions affecting the vascular, respiratory tract, intestinal tract, and hematopoietic tissues. More recently, a third class has been proposed, the chaperonopathies by collaborationism (or by mistake) [11], consisting of a number of tumors in which Hsps have been found overexpressed or downregulated and in which Hsps seem to play a role in tumor growth. Our recent studies have contributed to the understanding of the involvement of both Hsp60 and Hsp10 in the pathogenesis of some solid tumors. In particular, we found an overexpression of such molecules during carcinogenesis of large bowel (Fig. 2), uterine exocervix, and prostate [12-18]. By contrast, we found downregulation during bronchial carcinogenesis and vesical cancer progression [19-20]. Our data have been confirmed by other studies [21-24]. Moreover, our work has highlighted that Hsp60 and Hsp10 may also have a cytoplasmic localisation, even if not always together. We have postulated that these proteins, when present in the cytoplasm, may perform functions that are different from their canonical role in protein folding, For example, cytosolic Hsp60 and Hsp10 could participate in the mechanisms of cell proliferation and differentiation. In addition, we have shown that these molecules occur also in the peritumoral stroma [12-15]; their presence there due, perhaps, to a secretory mechanism.

Fig. 2: Hsp60 is overexpressed in colon adenocarcinoma (a), compared to normal colonic mucosa (b). 111

Francesco Cappello, Fabio Bucchieri, Sabrina David, Claudia Campanella, Anna Ribbene, Antonella Marino-Gammazza, Nella Ardizzone, Anna Merendino, Vito Marcian, Giovanni Peri, Everly Conway de Macario, Alberto J.L. Macario and Giovanni Zummo

Perspectives Our attention is now directed to elucidating the molecular mechanisms by which increased or reduced expression of Hsp60 and Hsp10 determine the development of certain neoplasms [25-27]. Preliminary data suggest that Hsp60 plays both pro- and anti- apoptotic roles, depending on the expression of other molecules, like Hsp70, and p53; therefore, Hsp60 is probably connected to other pro- and anti-proliferative molecules in a complex network with a fine regulation. Our studies are also focused on the mechanisms underlying the presence of Hsp60 and Hsp10 outside cells, since we assume that such molecules, when released in the interstitium, constitute a powerful pro-inflammatory stimulus [28]. In parallel, we are determining the levels of Hsp60 and Hsp10 in sera of patients with certain types of cancer in the hope that they could become biomarkers useful in clinical oncology. Conclusions In summary, our research pertains to the field of Chaperonology, encompassing Chaperonomics and Chaperonotherapy. Chaperonology is the study of intracellular and extracellular chaperones in all their aspects (structure, function, genetics, evolution, pathology) aiming to expand our knowledge on disease pathogenesis, and to use chaperones as diagnostic markers and prognostic indicators. Chaperonomics refers to the analysis of chaperone genes in genomes and at their products, including identification and classification of genes and pseudogenes, transcripts, polymorphisms, mutations, and inheritance. These studies should provide a solid basis for further bioinformatics analyses and experimental studies on the role of chaperone genes-proteins in ageing and associated diseases. Chaperonotherapy defines the use of chaperones in prevention and treatment of pathological conditions in which malfunctioning or absence of chaperones play a pathogenetic role and that may, therefore, benefit from the use of chaperones as therapeutic agents. Chaperonotherapy also includes the development and use of anti-chaperone agents to control conditions in which chaperones play a pro-disease role, like those types of malignant tumors mentioned above that need chaperones to grow and metastasize. We hope that research and clinico-pathological activities in these fields will be useful and will contribute to improve human health in the XXI century.

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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] Ohtsuka K, Kawashima D, Gu Y, Saito K. Inducers and co-inducers of molecular chaperones. Int J Hyperthermia 2005 Dec;21(8):703-11. Walsh D, Grantham J, Zhu XO, Wei Lin J, van Oosterum M, Taylor R, Edwards M. The role of heat shock proteins in mammalian differentiation and development. Environ Med 1999 Dec;43(2):79-87. Bensaude O, Bellier S, Dubois MF, Giannoni F, Nguyen VT. Heat-shock induced protein modifications and modulation of enzyme activities. EXS 1996;77:199-219. Latchman DS. Heat shock proteins and cardiac protection. Cardiovasc Res. 2001 Sep;51(4):637-46. Latchman DS. Protective effect of heat shock proteins in the nervous system. Curr Neurovasc Res 2004 Jan;1(1):21-7. Alsbury S, Papageorgiou K, Latchman DS. Heat shock proteins can protect aged human and rodent cells from different stressful stimuli. Mech Ageing Dev 2004 Mar;125(3):201-9. Richardson A, Schwager F, Landry SJ, Georgopoulos C. The importance of a mobile loop in regulating chaperonin/ co-chaperonin interaction humans versus Escherichia coli. J Biol Chem 2001 Feb 16;276(7):4981-7. Dubaquie Y, Looser R, Rospert S. Significance of chaperonin 10-mediated inhibition of ATP hydrolysis by chaperonin 60. Proc Natl Acad Sci (USA) 1997 Aug 19;94(17):9011-6. Macario AJL, Conway de Macario E. Sick chaperones, cellular stress, and disease. N Engl J Med 2005 Oct 6;353(14):1489-501. Macario AJL, Conway de Macario E. Chaperonopathies and chaperonotherapy. FEBS Lett 2007 Jul 31;581(19):3681-8. Macario AJL, Conway de Macario E. Chaperonopathies by defect, excess, or mistake. Ann N Y Acad Sci 2007 May 4; [Epub ahead of print] Cappello F, Bellafiore M, Palma A, Marciano V, Martorana G, Belfiore P, Martorana A, Farina F, Zummo G, Bucchieri F. Expression of 60-kD heat shock protein increases during carcinogenesis in the uterine exocervix. Pathobiology 2002-2003;70(2):83-8. Cappello F, Bellafiore M, Palma A, David S, Marciano V, Bartolotta T, Sciume C, Modica G, Farina F, Zummo G, Bucchieri F. 60KDa chaperonin (HSP60) is over-expressed during colorectal carcinogenesis. Eur J Histochem 2003;47(2):105-10. Cappello F, Rappa F, David S, Anzalone R, Zummo G. Immunohistochemical evaluation of PCNA, p53, HSP60, HSP10 and MUC-2 presence and expression in prostate carcinogenesis. Anticancer Res 2003 Mar-Apr;23(2B):1325-31. Cappello F, Bellafiore M, David S, Anzalone R, Zummo G. Ten kilodalton heat shock protein (HSP10) is overexpressed during carcinogenesis of large bowel and uterine exocervix. Cancer Lett 2003 Jun 30;196(1):35-41. Cappello F. HSP60 and HSP10 as diagnostic and prognostic tools in the management of exocervical carcinoma. Gynecol Oncol 2003 Dec;91(3):661. Cappello F, David S, Rappa F, Bucchieri F, Marasa L, Bartolotta TE, Farina F, Zummo G. The expression of HSP60 and HSP10 in large bowel carcinomas with lymph node metastase. BMC Cancer 2005 Oct 28;5:139. Cappello F, Di Stefano A, DAnna SE, Donner CF, Zummo G. Immunopositivity of heat shock protein 60 as a biomarker of bronchial carcinogenesis. Lancet Oncol 2005 Oct;6(10):816. Czarnecka AM, Campanella C, Zummo G, Cappello F. Mitochondrial chaperones in cancer. From molecular biology to clinical diagnostics. Cancer Biol Ther 2006 Jul;5(7):714-20.

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[20] Cappello F, Di Stefano A, David S, Rappa F, Anzalone R, La Rocca G, DAnna SE, Magno F, Donner CF, Balbi B, Zummo G. Hsp60 and Hsp10 down-regulation predicts bronchial epithelial carcinogenesis in smokers with chronic obstructive pulmonary disease. Cancer 2006 Nov 15;107(10):2417-24. [21] Urushibara M, Kageyama Y, Akashi T, Otsuka Y, Takizawa T, Koike M, Kihara K. HSP60 may Predict Good Pathological Response to Neoadjuvant Chemoradiotherapy in Bladder Cancer. Jpn J Clin Oncol 2007 Jan;37(1):56-61. [22] Johansson B, Pourian MR, Chuan YC, Byman I, Bergh A, Pang ST, Norstedt G, Bergman T, Pousette A. Proteomic comparison of prostate cancer cell lines LNCaP-FGC and LNCaP-r reveals heatshock protein 60 as a marker for prostate malignancy. Prostate 2006 Sep 1;66(12):1235-44. [23] Mori D, Nakafusa Y, Miyazaki K, Tokunaga O. Differential expression of Janus kinase 3 (JAK3), matrix metalloproteinase 13 (MMP13), heat shock protein 60 (HSP60), and mouse double minute 2 (MDM2) in human colorectal cancer progression using human cancer cDNA microarrays. Pathol Res Pract 2005;201(12):777-89. [24] Castle PE, Ashfaq R, Ansari F, Muller CY. Immunohistochemical evaluation of heat shock proteins in normal and preinvasive lesions of the cervix. Cancer Lett 2005 Nov 18;229(2):245-52. [25] Cappello F, Zummo G. HSP60 expression during carcinogenesis. A molecular proteus of carcinogenesis? Cell Stress Chaperones 2005 Winter;10(4):263-4. [26] Cappello F, Zummo G. HSP60 expression during carcinogenesis. Where is the pilot? Pathol Res Pract 2006;202(5):401-2. [27] Czarnecka AM, Campanella C, Zummo G, Cappello F. Heat shock protein 10 and signal transduction. a capsula eburnea of carcinogenesis? Cell Stress Chaperones 2006 Winter;11(4):287-94. [28] Cappello F, Czarnecka AM, Rocca GL, Stefano AD, Zummo G, Macario AJ. Hsp60 and Hsp10 as Antitumor Molecular Agents. Cancer Biol Ther 2007 Apr 1;6(4).

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

THE SUBSTANTIA NIGRA DOPAMINERGIC NEURONS


[I Neuroni Dopaminergici della Substantia Nigra] Arcangelo Benigno, Attilio Licciardi and Giuseppe Di Giovanni
Dipartimento di Medicina Sperimentale, Sezione di Fisiologia Umana, G. Pagano, Universit degli Studi di Palermo (I)

Key words: Parkinsons Disease, Neurogenesis, Apoptosis, Neuroprotection Parole chiave: Malattia di Parkinson, Neurogenesi, Apoptosi, Neuroprotrezione Abstract. Since the 1950s, when dopamine (DA) was discovered in the mammalian central nervous system (CNS), an enormous amount of experimental evidence has revealed the pivotal role of this biogenic amine in a number of cognitive and behavioural functions. Moreover, DAergic neurons, although their numbers are few, are of clinical importance because it is implicated in several psychiatric disorders. The lost of DAergic neurons of the substantia nigra compacta (SNc) is associated with one of the most prominent human neurological disorders, Parkinsons disease (PD). The mechanisms whereby nigral dopaminergic neurons may degenerate still remain controversial. Hitherto, several data have shown that the earlier cellular disturbances occurring in dopaminergic neurons include oxidative stress, excitotoxicity, inflammation, mitochondrial dysfunction, and altered proteolysis. These alterations, rather than killing neurons, trigger subsequent deathrelated molecular pathways, including elements of apoptosis. In this review, the characteristics of the SNc dopaminergic neurons and their life cycle from birth to death are discussed. Furthermore, the new evidence of a possible de novo neurogenesis in the SNc of adult animals and in PD patients will also be examined. Riassunto. Dal 1950, quando la dopamina (DA) stata scoperta nel sistema nervoso centrale (SNC) dei mammiferi, una enorme quantita` di evidenze sperimentali ha rilevato il ruolo fondamentale di questa amina biologica in numerose funzioni cognitive e comportamentali. Inoltre, i neuroni dopaminergici, sebbene pochi di numero, sono di importanza clinica perch implicati in numerose malattie psichiatriche. La perdita dei neuroni dopaminergici della substantia nigra pars compacta (SNc) associata con una delle principali malattie neurologiche, il morbo di Parkinson (PD). Il meccanismo con cui i neuroni dopaminergici degenerano rimane ancora controverso. Fino ad ora numerosi dati sperimentali hanno mostrato che anomalie cellulari avvengono in stadi precoci come stress ossidativo, citossicita`, infiammazione e disfunzione dei mitocondri e alterata proteolisi. Queste alterazioni, piuttosto che uccidere i neuroni, scatenano un meccanismo molecolare di morte cellulare, probabilmente lapoptosi. In questa review verranno trattate le caratteristiche dei neuroni dopaminergici e il loro ciclo vitale dalla nascita alla morte. Inoltre, verranno esaminate le nuove evidenze di una possible neurogenesi nella SNc in animali adulti e nei parkisoniani.

Parkinsons disease (PD) is the second most common neurodegenerative disorder in the elderly population for which, unfortunately, there is no cure as yet [1-3]. The cardinal motor symptoms are the result of the loss of dopaminergic (DAergic) neurons in the substantia

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nigra pars compacta (SNc), which causes a consequent reduction of dopamine (DA) levels in the striatum [4,5]. Since the underlying mechanisms of neuronal loss in patients are not known, current therapies are mainly symptomatic and do not halt the progression of the disease [1,3,6]. It appears clear that understanding the etiopathogenesis of PD, the modalities whereby the neurodegenerative process begins and progresses, is fundamental for the development of drugs to slow or prevent the progression of PD. There have certainly been major advances in these areas over the past few years, but, the modalities whereby the neurodegenerative process begins and progresses remain unclear. The situation is complicated further by the large number of factors that seem to be involved in the onset of this disease, such as aging, genetic vulnerability, exogenous or endogenous toxins, hydroxyl radicals (.OH) production, neuronal metabolic disturbances and inflammation (Fig. 1) [1,3,7-9]. Thus, the cumulative neuronal insults attributable to these metabolic stress factors may promote premature SNc DAergic degeneration through the activation of apoptotic programs [10] (Table). The SNc DAergic neurons Dopamine is one of the most intensively studied neurotransmitters in the brain due to its involvement in several mental and neurological disorders, such as schizophrenia, depression and PD. The most prominent DAergic cell group resides in the ventral part of mesencephalon, which contains approximately 90% of the total number of brain DAergic cells. Essentially, they are restricted to two nuclei, the ventral tegmental area of Tsai (VTA; A10) and the lateral SNc (A9). Nevertheless, cells expressing tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, have also been described in the striatum of rodents, monkeys and even humans [11-13]. The DAergic neurons localised in the SNc preferably project to the caudate nucleus and the putamen, i.e. the dorsal part of the striatum, and therefore this pathway is often called nigrostriatal DAergic system. The substantia nigra (SN) has been cytoarchitecturally divided into three different parts: the SNc, a horizontal sheet of densely packed medium and large cells that occupies its dorsal one-third; the SN pars reticulata (SNr), a more diffuse and cell-poor division, containing small and medium neurons lying between the SNc and the cerebral peduncles, and the SN pars lateralis (SNL), a small cluster of medium cells that extends rostrocaudally along the lateral border of SNc and SNr [14,15]. According to their neurotransmitter, nigral neurons were classified into DAergic and -aminobutyric acid (GABA)-ergic neurons [16]. Most DAergic neurons are localized in the SNc, some of them in the SNr, and to a lesser extent, in the SNL. The majority (>90%) of cells in the SNc are medium sized aspiny DAergic neurons with sparsely branching dendritic trees. Most pars compacta DAergic neurons also send one or two very long dendrites ventrally into pars reticulata that may be over 1
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mm in length. The axon is thin and unmyelinated, and does not give off local collaterals. Electrophysiologically, DAergic neurons in the SNc display the typical firing properties of DAergic neurons i.e. broad action potential (mean biphasic = 2.17 ms), slow firing rate (mean = 4.15 Hz) and regular firing pattern [17] (Fig. 2). They are composed of subsets of neurochemically different neurons, and these chemical differences may be involved in their physiological properties and vulnerability to aggression. DA-nigral cells are far from being a homogeneous group, considering the distribution of imureactivity for calbindin-D28k (CB) the SNc is compartmentally organized along the lines of a nigrosome-matrix [18]. According to Damier and colleagues [19,20], 60% of DA neurons in the human SNc are sparsely distribuited whithin the large region of intense CB staining, which they named nigral matrix; the other 40% of DA-containing neurons are included within 5 different nigrosomes, numered from 1 to 5. The birth of SNc DAergic neurons The number of nigral neurons declines during normal aging, and it is possible that many normal elderly subjects, as well as would-be PD cases, do not develop signs of the condition because DAergic cell loss has not reached the threshold level. Hence, it is theoretically feasible that individuals born with smaller numbers of nigral neurons might be more susceptible to reaching the critical level of neuronal loss, for example by exposure to environmental toxins or even through aging. The quality of the contact and/or the degree of trophic support in early life might be important in determining the number, health and length of survival of cells such as DAergic neurons. Moreover, the transcription factors involved are expressed throughout life in the basal ganglia, suggesting that they have a role in maintaining the health of specific neurons. Recent advances in molecular biology and mouse genetics have helped to unravel the mechanisms involved in the development of mesodiencephalic DAergic (mdDA) neurons, including their specification, migration and differentiation, as well as the processes that govern axonal pathfinding and their specific patterns of connectivity and maintenance [21]. The precise time point of origin of the first postmitotic mdDA neurons is still a matter of debate. The problem has been complicated by the fact that the mdDA system is not a homogenous group of neurons. The development of mdDA neurons follows a number of stages marked by distinct events. The first mDA neurons are born around embryonic day (E)12 in Sprague-Dawley rats, and develop from a single embryological cell group that originates at the mesencephalic-diencephalic junction, known as the isthmus. Developmental studies of the pathways involved, have led to the identification of several factors that influence the final formation of midbrain DA-neurons in the adult animal. The specification of the permissive region for DA neuron generation occurs through the secretion by the isthmus of two secreted signalling proteins;
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the sonic hedgehog (SHH) and the fibroblast growth factor 8 (FGF8). This permissive region is also defined by a specific pattern of gene expression in the mesodiencephalon ventricular zone; i.e. orthodenticle homologue 2 (OTX2), gastrulation brain homeobox 2 (GBX2) and transforming growth factor-b (TGF b). Studies analyzing the functions of these factors have not only increased the understanding of how DAergic neurons are generated in vivo, but also allowed for the development of new strategies in stem cells for engineering DAergic neurons in vitro. These results may be significant in terms of the development of future therapies for PD patients [21]. The death of the SNc DAergic neurons Considerable differences exist in the numbers of midbrain DAergic cell bodies in various mammals ranging from about 45,000 in the rat, 165,000 in the macaca monkey, to 590,000 in human beings [1]. This latter number applies to humans in their fourth decade of life but drops to an average of about 350,000 during the sixth decade of life [1]. Such an age-dependent decrease in the numbers of SNc DA-cells has also been reported for nonhuman primates. It is intriguing to note that parkinsonian neurodegeneretion it is not simply an accelerated form of cell loss seen during the normal aging, even though they share some pathological characteristics. For example, the pattern of loss is opposite to ageing, with greatest in the ventral part of SNc [22]. DAergic neurons are peculiarly prone to oxidative stress due to their high rate of oxygen metabolism, low levels of antioxidants, and high contents of iron and neuromelanin pigment (NM). Moreover, DA is thought to be capable of generating toxic reactive oxygen species (ROS) via both its enzymatic and non-enzymatic catabolism [2,7]. Furthermore, the midbrain region that encompasses the SN is particularly rich in microglia [2,7], therefore, activation of nigral microglia and the release of these pro-inflammatory neurotoxic factors may be a crucial component of the degenerative process of DAergic neurons in PD. We are far from seeing the whole picture; the mechanisms responsible for nigral DA cell death are only beginning to be understood. It is well known that DAergic neurons degenerate in PD in a disease-duration pattern [19]. Human DA nigral neurons in the calbindin-CB-poor nigrosomes in contrast to those in the CB-rich matrix are more likely to degenerate in PD. Within the nigrosomes, cell loss follows a strict order, depletion being maximal, with a maximum cell loss of 98%, in nigrosome 1 located in the caudal and mediolateral part of the SNc and then to other nigrosomes and finally to the matrix. The reason for such differential vulnerability of DAergic neurons is not known. Yet, the different nigral compartments may differ in terms of their content of growth factor, receptors, compounds related to exocitoxicity, agents involved in oxidative metabolism, and activity of potentially predisposing genes such as those for a-synuclein and parkin. Much attention has been
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given to the role of neuromelanin (NM), which has long been recognized as a marker of increased oxidative stress. Interestingly, the distribution pattern of NM within the SNc is inverse to the density of CB-immunostaining intensity and clearly overlaps the degeneration disease-duration pattern showed by Damier et al. [19,20]. In so far as ventral SNc DAergic neurons of nigrosome 1, the first to degenerate in PD, contain a high degree of pigmentation. NM pigment could be toxic to aminergic neurons as it physically interferes with intracellular communication, causing a macromolecular crowding effect, thereby interfering with the synthesis and degradation of cellular proteins. How the DAergic neurons die Two major mechanisms of neuronal demise have been discussed in neurodegeneration: apoptosis and necrosis. These cell death types are different, frequently divergent, but sometimes overlapping cascades of cellular breakdown. Apoptosis, a specific form of gene-directed programmed cell death (pcd) brings about the removal of unnecessary, aged or damaged cells and is distinguished by distinct morphological and biochemical features. It is performed by an intrinsic suicidal machinery of the cell and can be set off by environmental stimuli including irradiation leading to DNA damage, oxidative stress, toxins, viruses, withdrawal of neurotrophic support, etc. It is well known that adult mature neurons die at a low rate during the normal aging process but at an accelerated pace in cases of neurodegenerative disorders, like PD. Some groups have reported that dying neurons displaying the morphological features of apoptosis are present in the post mortem human brain of patients with neurodegenerative disorders. These features include cell shrinkage, chromatic condensation, DNA fragmentation, and increased expression of both proapototic, and antiapoptotic proteins, or DNA repair enzymes [23]. However, other groups have observed little or no evidence of apoptotic neuronal cell death associated with neurodegenerative diseases. The current view about the apoptotic mechanism underlying nigrostriatal DA neuron degeneration in PD is quite mixed. Recent evidence in experimental models of PD, point to the fact that neuroapoptosis might quite possibly be an early pathological event, and may or may not be present at the end of disease stage, when postmortem samples are collected and analyzed. The SNc DAergic neurons resurrection Recently, another dogma of science has been disproved; the adult mammalian brain does have the potential to generate new neurons and to integrate them into existing circuits. Neurogenesis has been shown at least in two rather discrete areas of the brain, the dentate gyrus of the hippocampal formation and the subventricular zone (SVZ) [24,25].
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Low levels of neurogenesis also occur in various other regions of the adult CNS including the SNc [26,27], a finding, which may have profound implications for the treatment of PD. Whether DAergic neurogenesis occurs in the adult SNc in normal brain or in PD animal models is still a matter of debate [28]. The existence of endogenous neurogenesis in the striatum and the subventricular zone in PD would open possibilities for a new cell-based approach to the treatment of neurodegeneration in PD patients, bypassing the need for transplantation. However, there is some evidence supporting neurogenesis or a more general degree of plasticity in the brains of PD patients and of PD animal models [29]. Conclusions From the literature here reviewed it appears evident that the progress in understanding the neuropathological process that induces DAergic cell demise in PD has been impressive. However, despite these advances, the processes that initiate cell death remain unclear. Whether they involve energy metabolism deficiencies, inadequate control of the redox state, low amounts of neurotrophic support and/or the action of environmental and endogenous toxins, remains to be elucidated. Clearly, a better understanding of the DAergic cell biology, the mode of cell death in PD, and the molecular mechanisms that controls it, is required. Indeed, DAergic neurons are sui generis cells, involved in a large number of physiological and pathological conditions and are also very delicate. SNc DAergic cells for some unknown reason are prone to degenerate and they are very sensitive to oxidative stress and inflammation. A better comprehension of the difference in resistance among DAergic cells of the mesencenphalic region will bring further insight into their peculiar characteristics.

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Table SNc dopaminergic neurons characteristic The first DAergic neurones are born around day E12 of rat development from a single embryological cell group that originates at the the isthmus. 590,000 DAergic cells in human beings, that drops to an average of about 350,000 during the sixth decade of life. The majority of brain DAergic cells are restricted in the VTA and SNc, from which originate the mesocorticolimbic and nigrostriatal DAergic system, respectively. SNc DAergic cells are medium sized aspiny neurones, coexpresing also CCK, CR, and CB. According to CB staining the SNc is compartmentally organized along the lines of nigrosome-matrix. SNc DAergic neurones degenerate in PD in a disease-duration pattern: from nigrosome 1 to nigrosoma 5 and then to the matrix. SNc DAergic neurons are likely to die for apoptosis in PD degeneration. A de novo neurogenesis is possible that occurs in the SNc of adult brain and in the SNc and striatum of PD patients, suggesting a new interventional approach.

Arcangelo Benigno, Attilio Licciardi and Giuseppe Di Giovanni

Fig 1. A simplified schematic of the vicious cycle that lead to the final demise of nigral DA cells. Activated microglia (subsequent to immune activation or neuronal lesion caused by exposure to toxins such as MPTP or 6-OHDA) can contribute to the degeneration of DA neurones by releasing neurotoxic factors. Cytokines can then activate receptor-mediated proapoptotic pathways within the DA neuron as well as further stimulation of the microglia in the form of iNOS and COX2 induction. The former will lead to greatly increased NO generation and resulting elevation of ROS, which damage the cell as a result of DNA damage, protein disruption and lipid peroxidation. The latter causes increased PGE2 production leading to direct toxicity to the DA neuron. Superoxide further stimulates microglial cytokine production as well as increases the quantities of ROS in the dopaminergic neuron. As signals from damaged DA cells further recruit and also stimulate microglia, the process can readily spiral out of control into full-blown neurodegeneration.

The Substantia Nigra Dopaminergic Neurons

Fig. 2. Anatomical and electrophysiological characteristics of a SNc DAergic neurone. Left panel: Reconstruction of an electrophysiologically identified nigrostriatal DAergic neurone intracellularly labeled with HRP following in vivo intracellular recording. The inset at lower right shows the position of the neurone with respect to the boundaries of substantia nigra. Right panel: Identification of SNc neurones in extracellular (A) and whole-cell patch-clamp (B) recordings. (A) Example of extracellular recording from a DAergic neurone. A(1): spontaneous firing. A(2): interval histogram demonstrating regularity of spontaneous firing. A(3): shape of individual action potentials (average of 16 consecutive potentials). Note inflection on the rising phase of the action potential (arrow). A(4): inhibition of firing after bath application of dopamine (30 M). (B) Example of whole-cell patch-clamp recording. B(1): DAergic neuron. From top: Ih current induced by hyperpolarizing voltage commands (voltage-clamp), and voltage responses to step current pulses recorded in current-clamp. Note depolarizing sag of the membrane potential (arrow) evoked by hyperpolarizing current pulses. B(2): non-DAergic neurone. From above: currents induced by voltage steps (voltage-clamp), and responses to step current pulses (current-clamp).

Arcangelo Benigno, Attilio Licciardi and Giuseppe Di Giovanni

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] Esposito E, Di Matteo V, Di Giovanni G. Death in the substantia nigra: a motor tragedy. Expert Rev Neurother 2007;7(6):677-97. Sian J, Gerlach M, Youdim MB, Riederer P. Parkinsons disease: a major hypokinetic basal ganglia disorder. J Neural Transm 1999;106(5-6):443-76. Esposito E, Di Matteo V, Benigno A, Pierucci M, Crescimanno G, Di Giovanni G. Non-steroidal anti-inflammatory drugs in Parkinsons disease. Exp Neurol 2007;205(2):295-312. Bernheimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F. Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J Neurol Sci 1973;20(4):415-55. Hornykiewics O. Neurochemical pathology and etiology of Parkinsons disease: basic facts and hypothetical possibilities Mt Sinai J Med 1988;55(10):11-20. Schapira AH. Present and future drug treatment for Parkinsons disease. J Neurol Neurosurg Psychiatry 2005;76(11):1472-78. Jenner P, Olanow CW. The pathogenesis of cell death in Parkinsons disease. Neurology 2006;66(10 Suppl 4):S24-36. Di Matteo V, Pierucci M, Di Giovanni G, Di Santo A, Poggi A, Benigno A, Esposito E. Aspirin protects striatal dopaminergic neurons from neurotoxin-induced degeneration: an in vivo microdialysis study. Brain Res 2006;1095(1):167-77. Di Matteo V, Benigno A, Pierucci M, Giuliano DA, Crescimanno G, Esposito E, Di Giovanni G. 7-nitroindazole protects striatal dopaminergic neurons against MPP+-induced degeneration: an in vivo microdialysis study. Ann NY Acad Sci 2006;1089:462-71. Novikova L, Garris BL, Garris DR, Lau YS. Early signs of neuronal apoptosis in the substantia nigra pars compacta of the progressive neurodegenerative mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/ probenecid model of Parkinsons disease. Neuroscience 2006;140(1):67-76. Cossette M, Lecomte F, Parent A. Morphology and distribution of dopaminergic neurons intrinsic to the human striatum. J Chem Neuroanat 2005;29(1): 1-11. Baker H, Kobayashi K, Okano H, Saino-Saito S. Cortical and striatal expression of tyrosine hydroxylase mRNA in neonatal and adult mice. Cell Mol Neurobiol 2003;23(4-5):507-18. Andn NE, Carlsson A, Dahlstroem A, Fuxe K, Hillarp NA, Larsson K. Demonstration and mapping out of nigro-neostriatal dopamine neurons. Life Sci 1964;3: 523-30. McRitchie DA, Halliday GM. Calbindin D28k-containing neurons are restricted to the medial substantia nigra in humans. Neuroscience 1995;65(1):87-91. Dahlstrom A, Fuxe K. Localization of monoamines in the lower brain stem. Experientia 1964; 20(7): 398-99. Oertel WH, Mugnaini E. Immunocytochemical studies of GABAergic neurons in rat basal ganglia and their relations to other neuronal systems. Neurosci Lett 1984; 47(3): 233-38. Di Giovanni G, De Deurwaerdere P, Di Mascio M, Di Matteo V, Esposito E, Spampinato U. Selective blockade of serotonin-2C/2B receptors enhances mesolimbic and mesostriatal dopaminergic function: a combined in vivo electrophysiological and microdialysis study. Neuroscience 1999;91(2):587-97. McRitchie DA, Hardman CD, Halliday GM. Cytoarchitectural distribution of calcium binding proteins in midbrain dopaminergic regions of rats and humans. J Comp Neurol 1996;364(1):121-50. Damier P, Hirsch EC, Agid Y, Graybiel AM. The substantia nigra of the human brain. II. Patterns of loss of dopamine-containing neurons in Parkinsons disease. Brain 1999;122(Pt 8):1437-48.

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[20] Damier P, Hirsch EC, Agid Y, Graybiel AM. The substantia nigra of the human brain. I. Nigrosomes and the nigral matrix, a compartmental organization based on calbindin D (28K) immunohistochemistry. Brain 1999;122(Pt 8):1421-36. [21] Smidt MP, Burbach JP. How to make a mesodiencephalic dopaminergic neuron. Nat Rev Neurosci 2007;8(1):21-32. [22] Fearnley JM, Lees AJ. Ageing and Parkinsons disease: substantia nigra regional selectivity. Brain 1991;114(Pt 5):2283-301. [23] Graeber MB, Grasbon-Frodl E, Abell-Aleff P, Ksel S. Nigral neurons are likely to die of a mechanism other than classical apoptosis in Parkinsons disease. Parkinsonism. Relat Disord 1999;5(4):187-92. [24] Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, Weisstaub N, Lee J, Duman R, Arancio O, Belzung C, Hen R. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 2003;301(5634):805-9. [25] Kempermann G, Jessberger S, Steiner B, Kronenberg G. Milestones of neuronal development in the adult hippocampus. Trends Neurosci 2004;27(8):447-52. [26] Lie DC, Dziewczapolski G, Willhoite AR, Kaspar BK, Shults CW, Gage FH. The adult substantia nigra contains progenitor cells with neurogenic potential. J Neurosci 2002;22(15):6639-49. [27] Zhao M, Momma S, Delfani K, Carlen M, Cassidy RM, Johansson CB, Brismar H, Shupliakov O, Frisen J, Janson AM. Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci (USA) 2003;100(13):7925-30. [28] Frielingsdorf H, Schwarz K, Brundin P, Mohapel P. No evidence for new dopaminergic neurons in the adult mammalian substantia nigra. Proc Natl Acad Sci (USA). 2004;101(27):10177-82. [29] Borta A. Hoglinger, G.U. Dopamine and adult neurogenesis. J Neurochem 2007;100(3):587-95.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

MULTIvARIATE ANALYSIS AS TOOL fOR THE STUDY Of BEHAvIOR


[Analisi multivariata come strumento di studio del comportamento] Maurizio Casarrubea
University of Palermo, School of Medicine, Dept. of Experimental Medicine, Human Physiology Section Palermo (I)

Key words: Multivariate analysis, Stochastic analysis, Cluster analysis, T-Pattern analysis Parole chiave: Analisi multivariata, Analisi stocastica, Cluster-analisi, T-Pattern analisi Abstract. Crucial feature of emergent phenomena is that, even if basic processes are fully elucidated and/or described, they may be impossible to predict. This idea perfectly fits with the evidence that, concerning behavioral studies, reductionistic descriptions (durations, per cent distributions and so on) of isolate events are far to be representative of the whole behavior. This last can be exhaustively understood only if inter-relations among events are taken into account. It is exactly here that traditional descriptive approaches to behavioral study give way to multivariate analysis. In fact, an important characteristic of multivariate techniques dwells in the possibility to analyze relationships between the elements of a behavioral sequence. Riassunto. Laspetto principale di un qualsiasi fenomeno emergente risiede nella sua assoluta imprevedibilit, anche se i processi di base che lo determinano sono noti e/o descritti. Questa idea si adatta perfettamente con levidenza che, per quanto concerne gli studi comportamentali, descrizioni puramente riduzionistiche (durate, frequenze, distribuzioni percentuali ecc.) di isolati eventi non sono affatto rappresentative dellintero comportamento che si prende in esame. Questultimo pu essere esaustivamente compreso solo se le inter-relazioni tra i diversi elementi che lo compongono sono prese in considerazione. esattamente qui che gli approcci puramente descrittivi allo studio del comportamento cedono il passo allanalisi multivariata. Infatti, una delle caratteristiche pi importanti delle tecniche di analisi multivariata risiede proprio nella possibilit di analizzare le relazioni esistenti tra gli elementi di una data sequenza comportamentale.

1. Introduction Multivariate analysis (MVA) encompasses techniques dedicated to the study of data sets comprising more than one variable. Along the last two decades, MVA has been considered as an useful tool to study the structure of a behavior in different experimental approaches. For instance, previous papers have illustrated different characteristics of rats social behavior [1], rodents responses in the hot plate [2-4], in the elevated plus maze test [5, 6] and in the swimming test [7]. MVA has also been used to describe effects of isolation on behavior [8], relationships between rat behavioral structure and EEG activity [9] and rat responses in specific conditions of fear or aggressiveness [10-12]. Moreover, in a recent paper, through a MVA approach, we have demonstrated specific behavioral modifications

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following administration of dopaminergic drugs, in rats tested on hot plate [13]. Aim of the present review is to present an outline of different multivariate techniques useful for the behavioral analysis. 2. Transition matrix The first step is the construction of an ethogram that is, in its simplest form, a formal list containing descriptions of animal behaviors. After that, behavioral transitions from a behavioral element to another can be noted down in a square transition matrix. Basically, a transition matrix is a table representing shifts among the behavioral elements, according to the selected ethogram. Fig. 1 summarizes the pivotal structure of a transition matrix:

Fig. 1 - Transition matrix. See text for details.

A = behavioral element A B = behavioral element B a = transitions from A to B b = transitions from B to A a = Total transitions of A b = Total transitions of B The transition matrix (fig. 1) represents the starting point of all MVA, except T-Pattern analysis (see below).

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2.1. Stochastic analysis A relatively simple approach to the analysis of transition matrices is represented by the stochastic analysis. The stochastic analysis requires transition matrices to be transformed into probability matrices which represent relative frequencies of transition from a behavioral element to another one. Thus, within a probability matrix, the following qualifications need to be taken into account: (a) switching probability from an element to all others must be 1; (b) all elements must be between 0 and 1; (c) each row must sum 1. Matrices containing relative frequencies can be graphically expressed through corresponding stochastic pathway diagrams. Fig. 2 illustrates a pathway diagram representing probabilistic relations among five different behavioral elements in a group of 42 rats in open field. Three probability ranges were selected: between 0.10 and 0.24 (thin dotted arrows), between 0.25 and 0.49 (medium arrows), and between 0.50 and 1 (large arrows). All behavioral elements show convergences on immobile-sniffing. Also, high probabilities of transitions are shared between walking and climbing and between immobility and immobile sniffing. 2.2. Cluster analysis In the cluster analysis transition matrices are transformed into similarity matrices through an aggregative procedure which allows to identify clusters on the basis of the overall reciprocal number of transitions. It is important to mention that the similarity table (containing only absolute values concerning the affinity between two behavioral elements) is not a square from-to matrix but an half matrix where each cell indicates the vicinity between two given elements. Cluster analysis is to some extent less intuitive than stochastic analysis probably because of the underlying aggregative algorithm that converts transitions into similarity values (i.e. not expressing directions of transitions). Also, different aggregative procedures can be used to obtain an half similarity matrix. For a detailed description of specific aggregative procedures see E.F. Espejo et al. [2] or M. Casarrubea et al. [13]. The main outcome of the cluster analysis is a dendrogram, that is a tree diagram. Dendrogram in fig. 3 represents vicinity relations among the same five behavioral elements presented in fig. 2. Four elements group in two different behavioral clusters including respectively immobility - immobile sniffing (S = 100) and walking - climbing (S = 110). 2.3. Transition matrix: elaborations Statistics between groups (for instance a control and an administered group) is both an important and neuralgic part of an experiment. Concerning MVA, it goes without saying that a comparison between groups could involve matrices. However, procedures concerning
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Fig. 2 - Pathway diagram representing probabilistic relations among five different behavioral elements in a group of 42 rats in open field. Three different probabilities of transitions are indicated on the right side. Im = immobility; Cl = climbing; Wa; walking; Re = rearing; IS = immobile sniffing.

Fig. 3 - Dendrogram representing similarities between five different behavioral elements in a group of 42 rats in open field. Similarity values (y-axis) mould two different clusters (x-axis): Im-IS and Wa-Cl. See fig. 2 for abbreviations.

comparison of matrices represent a challenging issue. For example, it has been pointed out that the classical methods such as chi-square test should not be applied to transitional tables because behavioral transitions are dependent each other. So, for instance, Tavar and Colleagues proposed the so called gamma correction factor to multiply by the chi-square [14]. However, an elegant, albeit not simple, method to assess significance of individual cells within and between transition matrices is probably the one successfully used by B.S. Everitt in 1977 [15] and B.M. Spruijt in 1984 [1] and, after that, in different recent papers. In this method a transition matrix is transformed into a matrix containing adjusted residuals. In brief, for each given transition, the adjusted residual represents the difference between the observed cell value and an expected one, calculated by a specific software engine on the basis of a random distribution of transitions. Positive residuals indicate transitions occurring more often than expected, negative residuals represent transitions occurring less often than expected. The great advantage of adjusted residuals is that they can be expressed according to a Z-distribution so that P-values can be found in a Z-table.
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2.4. T-Pattern analysis Stochastic and cluster analyses have both benefits and negative aspects. Noticeable advantages provided by dendrograms and stochastic pathway diagrams is that they represent patterning among behavioral elements in a very original, simple and intuitive way. On the other hand it should be taken into account that stochastic and/or cluster analyses: need heavy additional elaborations (see par. 2.3.); represent a whole observational period, similar to snapshots of everything; are extremely sensitive to noise (i.e. disturbing behavioral elements need to be removed before running the analysis). Fig. 4 represents an observational period (T0 Tx) with various behavioral elements and few uncommon ones (for instance: Y, T, Z): they are the noise that must be removed before running a cluster or a stochastic analysis. These drawbacks are not present in a T-Pattern analysis in fact, not only the a-priori noise reduction is not necessary but, importantly, T-Patterns represent events along the session time. T-Pattern analysis is carried out through a specific software known as Theme. This program repeats a recursively test, checking the distribution of every combination of events along a specific time window [16]. If the time lag of a sequence of events is not randomly distributed a T-Pattern is defined (elements l and f in fig. 4). In a second step, detected patterns are processed again and if there is a temporal relation with other events, they are combined (l-f-b, see fig. 4) into higher order patterns, and so on, over and over again, following a bottom-up process. Since the graphical representation of T-patterns and the results of cluster analysis are both visualized through tree diagrams (fig. 4 and fig. 5), it is important to remember that cluster analysis is based on the similarities between events (fig. 3). On the other hand, the tree structure provided by Theme does not represent such similarities, but the existence of significant relationships along time. Fig. 5 shows a T-Pattern encompassing Immobility, Immobile-Sniffing and Walking in ten subjects randomly taken from the main group of animals represented in fig. 2 and fig. 3. The T-pattern structure is shown in the upper left panel. All occurrences of the responses are shown in the upper right panel. The bottom panel shows nine occurrences of one significant (p < 0.0001) T-pattern.

Fig. 4 - Observational period (T0 Tx) with various behavioral elements (letters). T-Pattern analysis is carried out through a specific software that repeats a test over and over again, checking the distribution of every combination of events along the specified time window. If a sequence of events is found and their time lag is not randomly distributed, then a simple T-Pattern is defined (l-f). In a second step, detected patterns are processed again and if there is a temporal relation with other events, they are combined (l-f-b) into higher order patterns and so on, recursively, following a bottom-up process. The final result is a tree representation. 133

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Fig. 5 - T-pattern structure is shown in the upper left panel. All occurrences of the responses are shown in the upper right panel. The bottom panel shows nine occurrences of one significant (p < 0.0001) T-pattern between immobility, immobile-sniffing and walking. Data concerning ten subjects randomly taken from the main group of 42 rats in open field. b = beginning point of the event; e = ending point. See fig. 2 for abbreviations.

3. Discussion An even swift comparison among fig. 2, fig. 3 and fig. 5 makes clear how three rather different MV approaches strengthen each other in representing animals behavior. Moreover, each representation perfectly fits with the remaining ones. In other terms, these MV techniques can be successfully used together to depict behavioral patterning from different point of views. Nonetheless, the pivotal question is why do we need to represent behavior through these unconventional approaches? Behavior is much more than simple durations or frequencies of isolate behavioral elements: the meaning of behavior largely resides in its sequential organization. It is my contention that even hundreds of purely descriptive parameters make available only a short-sighted, reductionistic, view of the behavior and, whats more, the possible illusion of deepness and exhaustivity concerning presented data. To tell it like it is, descriptive analyses reduce behavior to simple numbers. This is a reductionistic approach, extremely similar to the so called Cartesian Reductionism. The Cartesian reductionism can be easily summarized through the so called machine metaphor. The meaning of the
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machine metaphor lies in the intuitive idea of machine that we have: a machine is built up from separate pieces, nonetheless it can be reduced to those pieces without losing its machine-like qualities. This approach does not work for behavioral analysis because behavior is complex, characterized by emergent phenomena arising from the inter-relations among events. Thus, durations, per cent distributions and/or frequencies of disjointed behavioral elements cant offer answers to the crucial question in all behavioral studies: what about the relationships between the observed elements? It is exactly here that traditional descriptive approaches to behavioral study give way to MVA: the pivotal aspect of MVA techniques dwells in the possibility to analyze the existing relationships between the elements of a behavioral sequence. On the basis of these inter-relations it becomes possible to explore behavior from very different points of view, greatly beyond what the human eye can intuitively interpret.

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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] Spruijt BM, Gispen WH. Behavioral sequences as an easily quantifiable parameter in experimental studies. Physiol Behav 1984; 32: 707-10. Espejo EF, Mir D. Structure of the rats behaviour in the hot plate test. Behav Brain Res 1993; 56: 171-6. Espejo EF, Mir D. Differential effects of weekly and daily exposure to the hot plate on the rats behavior. Physiol Behav 1994; 55: 1157-62. Espejo EF, Stinus L, Cador M, Mir D. Effects of morphine and naloxone on behaviour in the hot plate test: an ethopharmacological study in the rat. Psychopharmacology 1994; 113: 500-10. Cruz AP, Frei F, Graeff FG. Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol Biochem Behav 1994; 49: 171-6 Espejo EF. Structure of the mouse behaviour on the elevated plus-maze test of anxiety. Behav Brain Res 1997; 86: 105-12. Lino-de-Oliveira C, De Lima TCM, Carobrez AP. Structure of the rat behaviour in the forced swimming test. Behav Brain Res 2005; 158: 243-50. Van Den Berg CL, Van Ree JM, Spruijt BM. Sequential analysis of juvenile isolation-induced decreased social behavior in the adult rat. Physiol Behav 1999; 67: 483-8. Van Lier H, Drinkenburg WH, Coenen AM. Strain differences in hippocampal EEG are related to strain differences in behaviour in rats. Physiol Behav 2003; 78: 91-7. Mos J, Olivier B, Van Der Poel M. Modulatory actions of benzodiazepine receptor ligands on agonistic behaviour. Physiol Behav 1987; 41: 265-78. Aguilar R, Gil L, Gray JA, Driscoll P, Flint J, Dawson GR, Gimenez-Llort L, Escorihuela RM, FernandezTeruel A, Tobena A. Fearfulness and sex in F2 Roman rats: males display more fear though both sexes share the same fearfulness traits. Physiol Behav 2003; 78: 723-32. Aguilar R, Gil L, Flint J, Gray JA, Dawson GR, Driscoll P, Gimenez-Llort L, Escorihuela RM, FernandezTeruel A, Tobena A. Learned fear, emotional reactivity and fear of heights: a factor analytic map from a large F(2) intercross of Roman rat strains. Brain Res Bull 2002; 57: 17-26. Casarrubea M, Sorbera F, Crescimanno G. Effects of 7-OH-DPAT and U 99194 on the behavioral response to hot plate test, in rats. Physiol Behav 2006; 89: 552-62. Tavar S, Altham PME. Serial dependence of observations leading to contingency tables, with corrections to c2 statistics. Biometrika 1983, 70: 139-144. Everitt BS. The analysis of contingency tables. New York: John Wiley and Sons, 1977. Magnusson MS. Repeated Patterns in Behavior and Other Biological Phenomena. Eds: Oller DK, Griebel U. Evolution of Communication Systems: A Comparative Approach, Cambridge: The MIT Press, 2004: 111-128.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

MODIfICAZIONI INDOTTE DALLALOPERIDOLO SUL COMPORTAMENTO ATTENTIvO DEL RATTO


[Modifications induced by haloperidol on rat attentive behavior] Giuseppe Crescimanno, Maurizio Casarrubea e Filippina Sorbera
Dipartimento di Medicina Sperimentale, Sezione di Fisiologia Umana, Universit di Palermo (I)

Parole chiave: Comportamento attentivo, Dopamina, Aloperidolo, Ratto Key words: Attentive behavior, Dopamine, Haloperidol, Rat Riassunto. Scopo della presente ricerca stata la individuazione di possibili effetti dellaloperidolo, antagonista della famiglia recettoriale dopaminergica D2, su alcune manifestazioni comportamentali caratterizzanti lattenzione focalizzata nel ratto. Sono stati adoperati 5 gruppi di ratti Wistar maschi analizzati nel corso di unattivit esploratoria durante la quale stimoli acustici erano erogati secondo sequenze casuali per mantenere uno stato di continua vigilanza. Un gruppo riceveva solo stimolazione acustica, uno stimolazione acustica e salina IP, e tre gruppi erano stimolati dopo la somministrazione di aloperidolo a differenti dosi (0.05, 0.1, 0.3 mg/kg). Due parametri sono stati scelti per analizzare gli episodi di attenzione focalizzata: il numero di episodi e la loro durata. Laloperidolo induceva una diminuzione del numero di episodi e un aumento della loro durata, eccetto che alla dose minore che, agendo a livello autorecettoriale, provocava effetti opposti. I risultati suggeriscono che laloperidolo potrebbe influenzare specifici meccanismi neurali coinvolti nella regolazione del comportamento attentivo, probabilmente influenzando i processi di gating sensoriale dopamino-dipendenti. Abstract. Aim of the present research was to analyze effects of haloperidol (HAL), a dopamine D2 receptor antagonist, on focused attention, in the rat. Five groups of male Wistar rats, observed during exploration of a novel environment and subjected to randomly delivered acoustic stimuli to induce a condition of attentional focusing, were used. One group received acoustic stimulation, one was stimulated and received saline i.p., and three groups were stimulated and administered different doses of HAL (0.05, 0.1, 0.3 mg/kg. i.p.). Episodes of attentional focusing were characterized by immobility, auricle erection and orientation, eyes open and head oriented toward the stimulus source. With the aid of a videoanalysis apparatus, number and duration of focusing episodes were recorded during experimental sessions lasting 20 min. HAL provoked a decrease of the episodes of focusing and an increase of their duration except for the lower dose which likely affected presynaptic receptors. Results suggest that HAL might influence specific neural mechanisms involved in the regulation of attentive behavior likely by affecting sensory-gating dopamine-dependent processes.

Introduzione stato ipotizzato che alterazioni della trasmissione dopaminergica, spesso presenti in alcune forme di psicosi [1, 6, 13], possano causare delle modificazioni dei processi di sensory gating conducendo a disordini della sfera attentiva. Laloperidolo, antagonista
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dei recettori dopaminergici appartenenti alla famiglia D2, stato dimostrato possedere unefficace attivit antipsicotica assieme a quella di controllo sui sintomi ipercinetici di alcune sindromi neurologiche [8]. stato inoltre dimostrato che laloperidolo provoca rapidi cambiamenti nei profili di attivit dei neuroni dopaminergici della pars compacta della substantia nigra (neuroni A9) e dellarea ventrale tegmentale (neuroni A10) [7, 11]. Daltra parte, lesioni di queste popolazioni neuronali provocano nel ratto deficits sensorimotori e disordini attentivi caratterizzati dalla impossibilit di orientarsi verso nuovi stimoli provenienti dallambiente [3, 10]. stato quindi possibile ipotizzare una relazione tra neuroni dopaminergici troncoencefalici e condizione attentiva ponendo in particolare i neuroni A9 in relazione ai fenomeni di shift attentivo e i neuroni A10 ai fenomeni di focalizzazione dellattenzione. La presente ricerca si prefigge di studiare se esiste una relazione tra i rapidi cambiamenti osservati nel firing dei neuroni dopaminergici A9 e A10 in seguito alla somministrazione di aloperidolo e modificazioni di alcuni parametri comportamentali riconducibili allattenzione focalizzata. Tale condizione riconoscibile per la presenza di immobilit, occhi aperti, padiglioni auricolari eretti e orientati assieme al capo verso la sorgente dello stimolo. Per mezzo di un sistema di videoregistrazione sono stati analizzati in differenti gruppi di ratti, gli effetti della somministrazione per via generale di aloperidolo sul numero e la durata degli episodi di attenzione focalizzata (focusing attentivo). Tali episodi erano riscontrabili in ratti esposti a brevi stimoli acustici erogati secondo sequenze casuali per intervallo e localizzazione. Materiali e metodi La ricerca stata condotta su ratti wistar maschi del peso di 250-300 gr stabulati in gabbie singole, in un ambiente a temperatura costante (21 + 1 C) e con libero accesso al cibo ed allacqua. Gli esperimenti si svolgevano tra le 07.00 e le 19.00 in una stanza insonorizzata e mantenuta a temperatura e illuminazione costante. Per le osservazioni comportamentali, gli animali erano posti in box di perspex (30 x 30 x 30 cm) illuminati e ventilati. Sia nella parete di destra che in quella di sinistra del box, era inserito un altoparlante controllato da uno stimolatore acustico Coulbourn il cui output accendeva dei LEDs indicanti quale fosse laltoparlante attivo e inizio e durata dello stimolo. Gli stimoli acustici, i cui parametri (300 Hz, 2 sec, 72 dBA ) erano selezionati per non indurre risposte di evitamento, erano anche erogati secondo sequenze casuali per intervallo interstimolo (da 1 a 5 minuti) e localizzazione dellaltoparlante attivo (parete di destra o di sinistra) per evitare labitudine. Sono stati adoperati 5 gruppi di animali, ciascuno composto da 10 ratti. Ogni animale era testato una sola volta. Un gruppo riceveva soltanto stimolazione acustica ed un se138

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condo gruppo riceveva stimolazione acustica e soluzione salina IP. I rimanenti 3 gruppi ricevevano stimolazione acustica e 3 differenti dosaggi di aloperidolo IP (0.05, 0.1, e 0.3 mg/kg). Gli esperimenti duravano 20 minuti, erano registrati su videocassette per mezzo di una videocamera (Bauer VCC 550 AF) e successivamente analizzati per mezzo di un videoregistratore (JVC Super VHS HR-S6800E) ed una TV ad alta definizione (Sony Trinitron KV-X2151A). La sequenza comportamentale era studiata in playback, fotogramma per fotogramma, con una risoluzione temporale di 40 msec. Degli episodi di focusing attentivo era valutato il numero e la durata (solo in quelli non interrotti da stimoli acustici sopraggiungenti). I risultati sono espressi come media + D.S. Lanalisi statistica sta condotta adoperando lanalisi della varianza ad una via (ANOVA) seguita dal Newman-Keuls post-hoc test per comparazioni multiple. Il t-test di Student era adoperato per comparare il gruppo acusticamente stimolato e trattato con soluzione salina con quello soltanto acusticamente stimolato. Risultati La stimolazione acustica provocava negli animali un movimento di orientamento della testa verso la sorgente dello stimolo. Terminata la fase di orientamento, gli animali mostravano episodi di focus attentivo caratterizzati da immobilit, occhi aperti, padiglioni auricolari eretti e orientati, assieme alla testa, verso la sorgente dello stimolo. Degli episodi di focusing attentivo furono analizzati, per i 20 minuti di osservazione: il numero (media + S.D. 7.86 + 1.26 nel gruppo controllo) e la durata (media + S.D. 16.72 + 3.53 sec). La somministrazione di soluzione fisiologica non apportava variazioni significative ai parametri osservati: media + S.D. 8.41 + 1.57 per il numero di episodi di focusing e 15.53 + 4.04 sec per la durata degli episodi. La somministrazione di aloperidolo (0.05, 0.1, 0.3, mg/kg IP) induceva una diminuzione statisticamente significativa e dose dipendente del numero di episodi (7.23 + 1.2 per 0.1 mg/kg e 6.54 + 0.71 per 0.3 mg/kg) eccetto che per la dose di 0.05 mg/kg (12.37 + 1.74) (F 3,39 = 36.51, p < 0.0001). Il N-K post-hoc test mostrava effetti significativi per i dosaggi 0.05 e 0.03 mg/kg. Per quanto riguarda la durata degli episodi, la somministrazione di aloperidolo ne provocava un incremento dose dipendente e statisticamente significativo (21.45 + 4.91 sec per 0.1 mg/kg e 40.22 + 4.47 sec per 0.3 mg/kg) eccetto che per la dose di 0.05 mg/kg (13.06 + 4.33 sec) (F 3,39 = 76.26, p < 0.0001). Il N-K post-hoc test mostrava effetti altamente significativi per i dosaggi di 0.1 e 0.3 mg/kg. Discussione I risultati della presente ricerca mostrano che il numero degli episodi di attenzione focalizzata (focusing), individuati in ratti sottoposti a stimuli acustici erogati secondo sequenze
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casuali, decrescono in maniera statisticamente significativa dopo la somministrazione di aloperidolo, antagonista della famiglia recettoriale dopaminergica D2. Un effetto opposto stato osservato per la durata degli episodi di focusing. Precedenti ricerche hanno mostrato un coinvolgimento della dopamina nella capacit dellanimale di adattare i propri comportamenti a contesti ambientali differenti [2, 4, 5, 15]. Inoltre, stato dimostrato che i neuroni dopaminegici centrali modificano il proprio profilo di attivit in risposta alla presentazione di stimoli di differente natura [9, 11, 14], ipotizzabile, di consequenza, un ruolo del mediatore dopaminergico nella regolazione del traffico degli inputs sensoriali. Laloperidolo, antagonizzando gli effetti della dopamina potrebbe porre lanimale in una condizione di minore reattivit agli stimoli sensoriali facendogli trascorrere un tempo maggiore in atteggiamenti comportamentali tipici dellattenzione focalizzata (aumento della durata del focusing attentivo). Laumento della durata degli episodi di focusing attentivo ben si correla con la diminuzione del loro numero. Infatti, una maggiore quantit di tempo trascorsa in atteggiamenti comportamentali caratteristici dellattenzione focalizzata provoca una diminuzione del numero totale degli episodi. Leffetto opposto osservato negli animali trattati con aloperidolo al dosaggio di 0.05 mg/kg potrebbe trovare spiegazione in una prevalente azione di blocco esercitata dalla sostanza a livello autorecettoriale. I risultati mostrano che modificazioni della trasmissione dopaminergica, provocando alterazioni dei processi di sensory gating, possono ripercuotersi sulle manifestazioni comportamentali caratteristiche dellattenzione focalizzata.

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Bibliografia [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] Andreasen NC, Swayze V, Oleary DS, Nopoulos P, Cizadlo T, Harris G, Arndt S, Flaum M. Abnormalities in midline attentional circuitry in schizophrenia: evidence from magnetic resonance and positron emission tomography. Eur Neuropsychopharmacol 1995; 5:37- 41. Bos van den R, Charria-Ortiz GA, Bergmans AC, Cools AR. Evidence that dopamine in the nucleus accumbens is involved in the ability of rats to switch to cue-directed behaviours. Behav Brain Res 1991; 42:107-14. Brown VJ, Robbins TW. Simple and choice reaction time performance following unilateral striatal dopamine depletion in the rat. Impaired motor readiness but preserved response preparation. Brain 1991; 114:513-25. Crescimanno G, Sorbera F, Emmi A, Amato G. Inhibitory effect of A10 dopaminergic neurons of the ventral tegmental area on the orienting response evoked by acoustic stimulation in the cat. Brain Res Bull 1998; 45: 61-5. Crescimanno G, Mannino M, Casarrubea M, Amato G. Effects of sulpiride on the orienting movement evoked by acoustic stimulation in the rat. Pharmacol Biochem Behav 2000; 66: 747-50. Ellembroek BA. Treatment of schizofrenia: A clinical and preclinical evaluation of neuroleptic drugs. Pharmacol Ther 1993; 57: 1-78. Freeman AS, Bunney BS. Activity of A9 and A10 dopaminergic neurons in unrestrained rats: further characterization and effects of apomorphine and cholecystokinin. Brain Res 1987; 405: 46-55. Green MF, Marder SR, Glynn SM, McGurk SR, Wirshing WC, Wirshing DA, Liberman RP, Mintz J. The neurocognitive effects of low-dose haloperidol: a two-year comparisons with risperidone. Biol Psychiatry 2002; 51: 972-78. Horvitz JC, Stewart T, Jacobs BL. Burst activity of ventral tegmental dopamine neurons is elicited by sensory stimuli in the awake cat. Brain Res 1997; 759: 251-58. Le Moal M, Simon H. Mesocorticolimbic dopaminergic network: functional and regulatory roles. Physiol Rev 1991; 71:155-233. Overton PG, Clark D. Burst firing in midbrain dopaminergic neurons. Brain Res Rev 1998; 25: 312-34. Robbins TW, Everitt BJ. Functions of dopamine in the dorsal and ventral striatum. Sem Neurosci 1992; 4: 119-27. Stratta F, Bonci A, Calabresi P, Stefani A, Pisani A, Bernardi G, Mercuri NB. Basic research. in substantia nigra and ventral tegmental area: Clinical implications. J Neural Transm 1995; 45: 47-55. West CHK, Michael RP. Responses of units in the mesolimbic system to olfactory and somatosensory stimuli: modulation of sensory input by ventral tegmental stimulation. Brain Res 1990; 532: 307-16. Zhang H, Kiyatkin EA, Stein EA. Behavioral and pharmacological modulation of ventral tegmental dendritic dopamine release. Brain Res 1994; 656: 59-70.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

HUMAN UMBILICAL CORD AT TERM. IMMUNOHISTOCHEMICAL EXPRESSION Of MMP-2, MMP-9, ORPHANIN fQ AND OTHERS PEPTIDES INvOLvED IN THE REGULATION Of vASCULAR TONE

[Cordone ombelicale umano a termine. Espressione immunoistochimica di MMP-2, MMP-9, Orfanina FQ ed altri peptidi implicati nella regolazione del tono vascolare] Annamaria Mauro, Maria Buscemi, Angelo Leone and Aldo Gerbino
Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia Arcangelo Pasqualino di Marineo, Facolt di Medicina e Chirurgia, Universit di Palermo, (I)

Key words: Human umbilical cord, Orphanin, Oxytocin, ANF, eNOS, iNOS, MMP-2, MMP-9 Parole chiave: Cordone ombelicale umano, Orfanina, Ossitocina, ANF, eNOS, iNOS, MMP-2, MMP-9 Abstract. Umbilical cord (UC) is the structure that connects the fetus to the placenta. Umbilical cord at term is made of three vessels surrounded by the Whartons jelly, a mucous connective tissue rich in glycosaminoglycans, proteoglycans and different types of collagens and other microfibrils, which are responsible for its elasticity and its mechanical properties. Umbilical cord seems to lack any capillaries or lymphatics and umbilical vessels are unique in lacking any innervation; thus endothelial cells may play the major role in the local control of the blood flow. In this paper we investigated in the human umbilical cord at term the expression of some peptides involved in the regulation of the vascular tone, and the expression of some enzymes responsible for the remodelling of the extracellular matrix. Through immunohistochemistry we identified the presence of orphanin FQ, oxytocin, ANF, eNOS, iNOS, MMP-2 and MMP-9 in different territories of the human umbilical cord. Riassunto. Il cordone ombelicale (UC) la struttura che connette il feto alla placenta. Il cordone ombelicale a termine costituito da tre vasi circondati dalla gelatina di Wharton, un tessuto connettivo mucoso ricco di glicosamminoglicani, proteoglicani e diversi tipi di collagene ed altre microfibrille, che sono responsabili della sua elasticit e delle sue propriet meccaniche. Il cordone ombelicale sembra essere privo di capillari e vasi linfatici e i vasi ombelicali sono unici in quanto privi di innervazione; per questo motivo le cellule endoteliali possono giocare il ruolo principale nel controllo locale del flusso sanguigno. In questo lavoro abbiamo studiato nel cordone ombelicale umano a termine lespressione di alcuni peptidi coinvolti nella regolazione del tono vascolare e lespressione di alcuni enzimi responsabili del rimodellamento della matrice extracellulare. Per immunoistochimica abbiamo identificato la presenza di orfanina FQ, ossitocina, ANF, eNOS, iNOS, MMP-2 e MMP-9 in diversi territori del cordone ombelicale umano.

Introduction Histo-physiology Umbilical cord (UC) is the structure that connects the fetus to the placenta, the organ providing blood oxygenation and fetal nourishment. Umbilical cord at term encases three
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vessels (two arteries arranged in Umbilical c coils around a vein) that are imb Vein mersed within the Whartons jelly, a a mucous connective tissue very rich d e of water (about 90%), described as f a three-dimensional spongy network of interlacing collagen fibers, small woven bundles of glycoprotein microfibrils and an interfibrillar soluble phase (hyaluronans, proteoglycans) [1]. UC is covered by the umbilical epithelium, that is originated from the amniotic epithelium. Umbilical cord seems to lack any capillaries Umbilical or lymphatics. Artery The human UC at term weighs around 40 g, its length reaches ap- Fig. A - Azan staining for human Umbilical Cord. A shows the proximately 60-65 cm with a mean 6 representative regions we have considered in our experiments: [a] amniotic epithelium [b] subamniotic Whartons jelly diameter of 1.5 cm. [c] inner Whartons jelly [d/e] circular and longitudinal vascular Today six distinctive zones are muscolature [f] vascular endothelium. recognized based on structural and functional studies; from outer to inner; 1) surface epithelium; 2) subamniotic stroma; 3) clefts area; 4) intervascular stroma (Whartons jelly); 5) perivascular stroma; and 6) vessels [2]. Whartons jelly is a connective tissue rich in glycosaminoglycans, proteoglycans, and different types of collagens and other microfilaments, which are responsible for the mechanical properties of the umbilical cord [3]. Proteoglycans (PGs) are macromolecules built of core proteins covalently attached to sulfated glycosaminoglycans (GAGs). PGs perform numerous functions: they affect the mechanical properties of tissues, regulate collagen matrix organization, participate in cellcell and cellextracellular matrix interactions, bind growth factors, enzymes, viruses, etc. [4]. Proteoglycans of Whartons jelly contain mainly chondroitin/dermatan sulphate chains. The predominant proteoglycan is decorin (core proteins of 45 and 47 kDa), although the core proteins of biglycan (45 kDa), versican (260 kDa) and of other proteoglycans (90, 110, 220 kDa) were also detected [5]. Sulfated glycosaminoglycans (GAGs) are long unbranched polysaccharides containing
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repeated disaccharide unit. The disaccharide units contain either of two modified sugars N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc) and a uronic acid such as glucuronate or iduronate. Hyaluronic acid is the most abundant (70%) component of GAGs contained in Whartons jelly while the amounts of keratan sulphate, heparan sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate, dermatan-sulphate and heparin is very low [3]. Hyaluronan is known to influence cell behaviour and to play a crucial role in angiogenesis, morphogenesis and tissue remodelling especially during embryogenesis [6]. Remodelling tissues, in both normal and pathological situations, involves a greatly increased synthesis and turnover of hyaluronan and new blood vessel formation. Whereas native hyaluronan has been reported to inhibit angiogenesis in vivo, partial degradation products (4-25 disaccharide units) have been found to stimulate angiogenesis in several in vivo systems. Thus the metabolic state of hyaluronan could have profound effects on tissue neovascularization [6]. The amount and distribution of hyaluronan can be related to some pathological state; staining for hyaluronan using bHABP revealed hyaluronan to be present mostly in the subamniotic zone and around the vessels of the normal umbilical cord. In Down syndrome cords, a high concentration of hyaluronan was detected. Moreover, compared to the normal umbilical cord, hyaluronan was homogeneously distributed from the subamniotic zone to the umbilical cord vessels [7]. The mechanical properties of the umbilical cord depend on the nature of its extracellular matrix with its connective tissue fibers and related soluble proteins. The most abundant fibrillar component is definitely collagen fibers while elastic fibers are absent [8]. Collagen fibers build strong ionic bonds with glycosaminoglycans and proteoglycans, therefore providing the stroma with an extraordinary strength [3]. A regional distribution of collagen fibers was noted: types I, III, VI collagen is present in the subamniotic and intervascular regions as well as in the vessel walls. Type IV is found in both the umbilical epithelium basement membrane and external lamina of subamniotic stromal cells, whereas type VII was restricted to the epithelial basement membrane in term cord. Type I and III collagens were found to be the most abundant forms in the intervascular region and arterial wall [9]. The epithelium of the human umbilical cord from pregnancy to full term was studied by Hoyes [10]. At 8-10 weeks the epithelium consists of a single layer of cuboidal cells which show little evidence of differentiation. The epithelium is separated from the underlying mesenchyme by a basement membrane. The cells contain a rather well developed Golgi apparatus, numerous mitochondria, sacs of endoplasmic reticulum and many free ribosomes. In the latter part of the 3rd month the epithelium becomes bilaminar and the
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superficial cells show a collection of organelles above the nucleus, which suggests that they may have already begun to undergo differentiation. The intermediate layers of cells are formed during the 5th and 6th month of pregnancy. Between 22-26 weeks the zonulae occludentes disappear and at least partial separation of the cells from the surface can be seen. At term the epithelium 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 of these cells is thickened and zonulae occludentes can no longer be identified between them and the adjacent cells. The endothelium of the umbilical vessels was studied by Parry [11]. In full-term umbilical cord both veins and arteries endothelium is made of a single layer of endothelial cells. Endothelium in arteries is made of cells closely apposed laterally; mitochondria and Golgi apparatus can be seen. Arterial endothelial cells are packed with cisternae of endoplasmic reticulum. Large accumulations of glycogen occur, particularly in the supranuclear part of the cytoplasm. Weibel & Palade bodies are present as well as irregular lipid droplets. Interesting is the presence of an organelle-free zone at the base of the endothelial cells.These zones show morphological similarity to the myofilament zones of underlying smooth muscle cells. The most obvious difference between arteries and the vein endothelium is the paucity of glycogen in venous endothelial cells while lipid droplets are not encountered. Mitochondria are large and plentiful and Weibel-Palade bodies are more frequently seen.Venous endothelial cells contain very few recognizable dilated cisternae of rough endoplasmic reticulum.The Golgi apparatus is well developed [11]. Weibel-Palade bodies of human umbilical vein endothelial cells are storage sites of both histamine and Von Willebrand factor [12]. Although these cells constitutively release VWF, they also contain a storage pool of this protein that can be rapidly mobilized [13]. Human umbilical vessels are unique in lacking any innervation; thus endothelial cells may play the major role in local control of the blood flow; Cai et al. observed colocalization of atrial natriuretic peptide (ANP) and neuropeptide Y (NPY) with other vasoactive substances such as vasoactive intestinal peptide (VIP), substance P, calcitonin gene-related peptide (CGRP) and vasopressin [14]. Parry was among the firsts who studied the ultrastructure of human umbilical cord at term by electronic microscopy [15]. The cells of Whartons jelly are thin and elongated. The organelles are aggregated at the nuclear poles, the well developed rough endoplasmic reticulum and Golgi apparatus indicate the capacity of protein synthesis and secretion, so its likely to tought that they are the source of cord collagen and other extracellular matrix components. Moderate amount of glycogen and some lipid droplets are present in the
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cells. The cells contain numerous mitochondria and long, thin processes extending in both directions away from the nucleus. Bundles of fine filaments are arranged along the long axis of the cells, they continue into the elongated processes and are often concentrated at the periphery of the cell under the plasma membrane. Junctional complexes are occasionally seen between neighbouring elongated processes. Collagen fibres, recognizable by their size and characteristic cross-banding after staining, are present in the extracellular matrix. The main role of the embryonic mucous connective tissue called Whartons jelly is to prevent the compression, torsion and bending of the enclosed vessels, which provide bidirectional blood flow between fetal and maternal circulation [2]. UC stromal cells basically resemble mesenchymal fibroblasts. Ultrastructural studies indicated that their intrinsic properties were also similar to smooth muscle cells [16] and they are therefore considered as myofibroblasts. Several studies demonstrated the expression of some muscle-specific cytoskeletal filaments in stromal cells, a finding which supports the notion that rather than fibroblasts or smooth muscle cells, they are true myofibroblasts. Specifically, contractile proteins such as actin, non-muscle myosin, desmin, and a-smooth muscle actin (a marker for myofibroblasts) are expressed in stromal cells while muscle-myosin is lacking [17,16]. Those cells also express vimentin [17,18], an intermediate filament protein specifically expressed in mesenchyme-derived cells such as fibroblasts while not expressed in smooth muscle cells, and cytokeratin [18], which is basically expressed in endoderm and ectoderm originated epithelial cells. The density of cells in the different area of the Whartons jelly is variable; cell frequency is considerably low in subamniotic regions whereas perivascular areas possess the highest cell density [17]. Further analyses revealed an increasing cytoskeletal complexity in human UC stromal cells from the superficial layers towards the blood vessels [16]. In parallel to this increasing differentiation status, a gradual decrease in the proliferation rate of stromal cells was noted from outer to inner cells [9]. Conclusively, the immature cells retaining the ability to proliferate are located close to the amniotic surface, whereas highly differentiated perivascular cells are found in closer proximity to the umbilical vessels. UC stromal cells express CD105, CD73 and CD90 (known to characterize MSCs) [19] while they do not express hematopoietic stem cell markers. Stromal cells were also found to express low levels of some transcriptional factors which are mainly expressed in embryonic stem cells such as Oct- 4 and Nanog, and express low amount of Wnt signalling pathway molecules [2]. It is realistic to suppose that stromal cells contain more than one set of stem cell population. Karahuseyinoglu [20] demonstrated a higher than normal telomerase activity in UC
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stromal cells during early passages, which then decreased in later passages. This provide cells with the ability to proliferate up to 30-60 divisions but never to a level of tumorigenic state. The two most basic properties of mesenchymal stem cells (MSCs) are the capacities to self-renew indefinitely and differentiate into multiple cells and tissue types. The cells from human umbilical cord Whartons jelly have properties of MSCs and represent a source of primitive cells. In vitro differentiation potential of UC stromal cells has been extensively investigated: adipogenic, chondrogenic, osteogenic, cardiomyogenic and skeletal myogenic inductions have been the most studied cell lineages. UC stromal cells are capable of forming premature adipocytes [20]. Chondrogenic induction was investigated by Karahuseyinoglu [20] and Wang [21] who also demonstrated the osteogenic potency of UC stromal cells. Differentiation into cardiomyocytes or skeletal myocytes was also investigated by Conconi [22]. UC stromal cells were partially differentiated in vitro into neurons and glia cells [23]. Human UC stromal cells were found to secrete several neurotrophic factors, like FGF-2 and BDNF [24]. In a recent study by Wu et al. [25] human UC stromal cells were induced to differentiate into endothelial cells both in vitro and in vivo by basic FGF and VEGF. In vivo studies concerning UC matrix cells are very few in number but quite promising: xenotransplantation of porcine UC stroma cells into rat brain gave neither any immune rejection nor any teratoma formation. Secondly, transplanted undifferentiated stromal cells were stained positively with a neurofilament antibody and showed TH-positivity, which together indicated that donor cells could transform into neurons or neuron precursors in vivo [26,27]. UC-derived stromal cells should be considered as a valuable source of stem cells since they are more efficient in terms of stem cell potency as compared to bone-marrow derived MSCs. The ability to introduce exogenous DNA into the perivascular cells of UC stroma makes them potent stem cells for gene therapies similar to bone marrow derived MSCs. Human umbilical cord blood (HUCB) is now considered a valuable source for stem cellbased therapies. HUCB has abundant stem cells with impressive proliferative capacity and tolerant immunological features; furthermore HUCB cells are easily available and less immunogenic compared to other sources for stem cell therapy such as bone marrow. There are few advantages of CB over bone marrow, especially the lower rates of acute and chronic graft-versus-host disease (GVHD) after transplantation. Moreover no ethical problems are related with the use of cord blood stem cells. The clinical experience in CBT, especially in children, is encouraging; when using adequate number of NC/kg, results in CBT for malignant and non-malignant diseases are
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comparable to bone marrow transplantation (BMT). Future uses of CB might also be in the field of gene transfer and non hematopoietic injured tissues repair [28]. Umbilical cord blood (UCB) is a rich source of haematopoietic stem cells that can be used to reconstitute the blood system, but recently, it has been demonstrated that umbilical cord blood stem cells have the potential to give rise to non-haematopoietic cells, such as bone, neural and endothelial cells [29]. Korbling [30] showed in xenotransplantation experiments the ability of UCB cells to generate tissue-specific cells in liver, pancreas, CNS and endothelium. Teramoto [31] studied in vitro the differentiation of umbilical cord blood cells into hepatocytes. Neural stem/progenitor cells can be selected from human cord blood nonhematopoietic (CD34-negative) mononuclear fraction, and these progenitors could be effectively differentiated into functional neuron-like cells in vitro [32]. Differentiation of umbilical cord blood-derived multilineage progenitor cells into respiratory epithelial cells is reported by Berger [33]. Gene expression eNOS (endothelial Nitric Oxide Synthase or Type III NOS or Constitutive/cNOS, 140 kDa) is an enzyme that catalizes the synthesis of nitric oxide, a short life radical responsible for signals correlated with vasorelaxation, neurotransmission and citotoxicity. eNOS generates NO in blood vessels and is involved in the mechanisms that regulate vascular function. Its a constitutive form of NOS activated by agonists that increase the intracellular levels of Ca++. The expression of endothelial nitric oxide synthase (eNOS) in human umbilical vascular endothelial cells (HUVEC) has been extensively reported by many autors. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor (VEGF) in HUVEC [34]. Permeability of HUVEC can be modulated by eNOS- and iNOS-related mechanisms [35]. Statins affect the expression levels of genes involved in inflammation, coagulation, and vascular constriction; they increase the mRNA levels of nitric oxide synthase-3 (eNOS) in HUVEC [36]. iNOS (macNOS, Type II NOS) is the inducible form of Nitric Oxide Synthase and is expressed by macrophages. It is activated by inflammatory signals such us cytokines and interferon- or oxidative stresses such exposure to H2O2. The expression of iNOS is reported in human umbilical cord endothelial cells. Incubation of HUVEC with combinations of interleukin-1b, tumor necrosis factor a, and interferon- stimulates the synthesis of iNOS mRNA and also hydrogen peroxide is able to stimulate iNOS directly [37]. MMP-2 (Matrix metallopeptidase 2, gelatinase A, 72 kDa) is a matrix degradative enzyme which degrades type IV and V collagen, denatured collagen and elastin. It is costitutive151

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ly produced by several types of cell and its activity is Ca++ and Zn++-dependent. MMP-9 (Matrix metallopeptidase 9, gelatinase B, 92kDa) is a component of the matrix metalloproteinase (MMP) family that are involved in the breakdown of extracellular matrix both in physiological and pathological processes. It degrades type IV and V collagens. HUVEC express both MMP-2 and MMP-9. Some authors have reported a relationship between MMP-2 and MMP-9 production by endothelial cells and the onset of angiogenic events [38]. Alterations in both cell-cell and cell-matrix interactions are associated with the activation of endothelial cells that initiate angiogenesis. One of the molecular mechanisms involved in changes of cell-matrix interactions is the action of neutral proteinases, particularly matrix metalloproteinases. MMP-9 and MMP-2 were also detected in CD34+ cells isolated from umbilical cord blood [39] and in the cells of Whartons jelly [40]. Human mesenchymal stem cells (MSCs) derived from bone marrow (BM) and umbilical cord blood (CB) express MMP-2 [41]. Orphanin FQ/ Nociceptin (OFQ/N) is a peptide structurally similar to the oppioid dinorphin. It is a heptadecapeptide that bind to the NOP1 receptor (ORL1) which is homolog to the classical oppioid receptors coupled to G protein. Orphanin is mainly involved in the mediation and modulation of pain, but its also involved in anxiety, stress, memory, learning, locomotor activity, tolerance, dependence and withdrawal phenomena. N/OFQ is also involved in vasorelaxation phenomena; it causes hypotension and vasodilation. Proinflammatory and vasodilator effects of orphanin is reported in the rat mesenteric microcirculation [42]. Xu [43] showed that orphanin relaxed the porcine coronary arterial rings and inhibited the vasocontractivity of PGF2a. Orphanin induces impairment of NMDA dilation after cerebral hypoxia/ischemia [44] and have vasodilator activity in the vascular bed of the hindquarters of the rat [45]. No information was found in litterature about the expression of Orphanin FQ/Nociceptin in human umbilical cord. Atrial natriuretic peptide (ANP), atrial natriuretic factor (ANF), or atriopeptin is a 28 amino acid peptide with a 17 amino acid ring in the middle of the molecule; it is closely related to BNP (brain natriuretic peptide) and CNP (C-type natriuretic peptide) which all share the same amino acid ring. ANP is produced, stored and released by atrial myocytes and is secreted in response to: high blood pressure; atrial distention; sympathetic stimulation of b-adrenoreceptors; raised sodium concentration; angiotensin-II and endothelin. It acts to reduce water, sodium and adipose loads on the circulatory system, thereby reducing blood pressure. ANP also has vascular effects; it relaxes vascular smooth muscle in arterioles and venules. Several studies demonstrated the expression of ANP in endothelial cells of human um152

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bilical vessels confirming that these endothelial cells have the ability to synthesize this peptide [14,46]. ANP exerts anti-inflammatory effects on endothelial cells; it has been shown to reduce tumor necrosis factor-a (TNF-a)-induced activation of HUVEC via inhibition of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB pathways [47,48]. Physiological concentration of ANP induces HUVEC survival in vitro [49]. Templeton investigated a role for atrial natriuretic peptide in maintaining low vascular resistance within the fetoplacental circulation. He showed that physiological levels of ANP significantly reduced the vasoconstrictor effects of 5-hydroxytryptamine, and endothelin-1 in the human umbilical artery. These data suggest that ANP may modify vascular tone in vivo thereby counterbalancing several humoral factors which act to increase vascular resistance within the fetoplacental circulation [50]. Oxytocin is a mammalian hormone that also acts as a neurotransmitter in the brain. It is made in magnocellular neurosecretory cells in the supraoptic nucleus and paraventricular nucleus of the hypothalamus and is released into the blood from the posterior lobe of the pituitary gland. Oxytocin is a nonapeptide (CYIQNCPLG) with a sulfur bridge between the two cisteines; its structure is very similar to that of vasopressin. Its effects are mediated by specific, high affinity receptors which are G-protein-coupled receptor which requires Mg2+ and cholesterol. Its peripheral actions are: 1) letdown reflex in lactating (breastfeeding) mothers, 2) uterine contractions during labor 3) due to its similarity to vasopressin, it can reduce the excretion of urine slightly and can stimulate sodium excretion from the kidneys. Oxytocin has a role in social behaviors in many species: it is involved in bonding, maternal behavior, sexual arousal, increasing trust and reducing fear [51, 52], it also has various anti-stress functions: it reduces blood pressure and cortisol levels, increasing tolerance to pain and reducing anxiety. Oxytocin also has effect on vasorelaxation; it causes endothelium-dependent relaxation of the basilar artery by activating V1-vasopressinergic receptors [53]. Oxytocin also caused vasodilation in the preconstricted pulmonary vasculature and the vasodilation produced by oxytocin was blocked by prior administration of a selective V1receptor antagonist, suggesting that oxytocin acts via V1-vasopressinergic receptor stimulation [54]. Infusion of oxytocin into the vertebral artery dilated major vessels including the vertebral, anterior spinal, and basilar arteries [55] while high concentrations of oxytocin induce renal vasoconstriction in the rat by activating vasopressin V1A receptors [56]. No information about the expression of oxytocin in human umbilical cord is given in literature.
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Materials and Methods Samples: The analysis was performed on human umbilical cords obtained from caesarian delivery. The umbilical cords were provided by the gynecologic and ostetric clinic of the hospital Paolo Giaccone in Palermo. Immunohistochemistry: Human umbilical cords were collected in physiological solution. One section (1 cm thick) were dissected from each umbilical cord and fixed in Bouins solution. After fixation the tissue was dehydrated in a graded series of alcohols, cleared in xylene and paraffin embedded. Sections of 7 m were cut on to Leica microtome RM2145, dryed overnight at 37C 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. The immunohistochemistry was performed using the DakoCytomation EnVision + System-HRP (AEC) kit from Dako, following the manufacturers instructions. Briefly: sections were covered with the Peroxidase block reagent and incubated 5 min R.T. The samples were rinsed once in PBS buffer pH 7.2. The sections were covered with the antibody solution and incubated at 4C O/N. Rabbit Anti-Orphanin FQ polyclonal antibody (Chemicon, cat n AB5515) (1:500 dilution), Mouse Anti-eNOS/NOS type III monoclonal antibody (BD Bioscience, cat n 610297) (1:25 dilution), Rabbit Anti-oxytocin polyclonal antibody (Chemicon, cat n AB911) (1:800 dilution), Rabbit Anti-ANP polyclonal antibody (Chemicon, cat n AB1970) (1:800 dilution), Mouse anti human MMP-2 monoclonal antibody (Chemicon, cat n. MAB3308) (1:800 dilution), Rabbit anti mouse MMP-9 full lenght polyclonal antibody (Chemicon, cat n. AB19047) (1:100 dilution), Rabbit anti iNOS/ NOS type II polyclonal antibody (BD biosciences, cat n. 610332) (1:25 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 Peroxidase Labelled Polymer reagent. Samples were rinsed twice in PBS pH 7.2, then incubated with the Substrate-Chromogen 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 an identical manner, omitting primary antibody. Slides were coverslipped using the DakoCytomation Faramount Aqueous Mounting Medium. The specimens were observed under a Leica DM1000 light microscope. Results We identifyed the expression of eNOS in human umbilical cord. Endothelial cells show a moderate cytoplasmic reactivity for eNOS (+) (fig. 1). The muscular cell layers of the umbilical vessels are negative (-).
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The cells of the Whartons jelly express eNOS (++) mainly in the region close to the ectoblast, while the number of positive cells decreases towards the inner part of the jelly. The amniotic epithelium shows positive cells (++) difficult to distinguish due to the folding of the epithelium (fig. 2). We identifyed the presence of iNOS enzyme in the umbilical cord through immunohistochemistry. The endothelium was positive and showed a clear and strong cytoplasmic reactivity (+++) (fig. 3). The perivascular muscle cells of the circular musculature showed in their sarcoplasm positive granules (+) (fig. 4), while longitudinal musculature was negative (-) for iNOS as well as the Whartons jelly (-). We identifyed a faint expression (+) of iNOS in isolated cells of the Whartons jelly immediately below the ectoblast. The amniotic epithelium shows a faint diffused reactivity that probably its only background (-). Our immunohistochemistry experiments gave evidence that in human umbilical cord also MMP-2 is expressed. The endothelium shows a strong cytoplasmic reactivity (+++) (fig. 5) and also the longitudinal and circular musculature of the vessels seems to express

[1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

Figures 1-8: [1] Immunohistochemistry for eNOS in human umbilical cord vascular endothelium. Magnification 100X - [2] Immunohistochemistry showing eNOS expression in the amniotic epithelium of the umbilical cord, and in some cells of the subamniotic zone. Magnification 100X - [3] Immunohistochemistry for iNOS in umbilical cord vascular endothelium. Magnification 100X - [4] Immunohistochemistry for iNOS in the perivascular musculature of human umbilical cord. Magnification 20X - [5] Immunohistochemistry for MMP-2 in the vascular endothelium of umbilical cord. Magnification 100X - [6] Immunohistochemistry for MMP-2 in the cells of the Whartons jelly. Magnification 100X - [7] Immunohistochemistry for MMP-2 in the perivascular musculature of human umbilical cord. Magnification 100X - [8] Immunohistochemistry for MMP-9 in the vascular endothelium of umbilical cord. Magnification 100X. 155

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MMP-2 (++) (fig. 7); in some cells single granules can be seen while in other cells the reactivity is diffused. The Whartons jelly is disseminated with mesenchimal cells positive for MMP-2 (+++) (fig. 6), these positive cells are homogenously distributed in the jelly. The amniotic epithelium is negative (-). We identified the expression of MMP-9 in human umbilical cord. The endothelial cells were positive for MMP-9 (+++) and they showed a cytoplasmic reactivity (fig. 8). We identified a moderate reactivity for MMP-9 in the sarcoplasm of the muscle cells of both the circular (++) and longitudinal (++) perivascular musculature; the reactivity was diffused and no granules were seen. Some fibroblasts and mesenchimal cells of the Whartons jelly express MMP-9 (+++), but the distribution is not homogeneous; the positive cells are more concentrated in the perivascular region (+++) and in the external region of the jelly below the ectoblast (+++). The amniotic epithelium seems to be positive for MMP-9 (+++), even if its not possible to distinguish the single cells but a diffuse labelling, we hypothesized this diffuse labelling is not background reactivity but corresponds to the cells cytoplasm. Our immunohistochemical localization of orphanin FQ in human umbilical cord sections gave the following results. Endothelial cells were positive for orphanin (++) (fig. 9). Some cells of the longitudinal musculature of the vessels contained positive granules (++); the reactivity was more evident in the cells close to the vessel and gradually disappeared in the cells far from the vessel. Circular musculature was negative (-). Isolated storage sites for orphanin were seen along the ectoblast, and positive cells were also seen in the Whartons jelly immediately under the ectoblast (++). The ectoblast was negative (-). ANP immunostaining gave the following results. The endothelium of both the vein and the arteries shows a quite strong cytoplasmic reactivity for ANP (++) (fig. 10). Both the longitudinal and the circular musculature of the vessels is negative for ANP. Fibroblasts and mesenchimal cells positive for ANP (+++) are scattered in Whartons jelly (fig. 11). Positive cells are more concentrated in the jelly close to the ectoblast and their number decreases towards the vessels region. The immunostaining revealed a strong reactivity (++) in the ectoblast and in the region immediately below; in some region the single cells could be seen, while in other region just a diffused staining was visible. Our immunohistochemical localization of oxytocin in human umbilical cord sections gave the following results. Endothelial cells show a strong citoplasmic reactivity (+++) (fig. 12). The longitudinal musculature has a faint (+) diffused staining, while the circular is negative. In the Whartons jelly mesenchimal cells show a very strong (+++) and clear reactivity (fig. 13); these positive cells are uniformly scattered from the subamniotic region to the vessels musculature. The amniotic epithelium has a weak staining (+); the reactivity
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[9]

[10]

[11]

[12]

[13]

Figures 9-13: [9] Immunohistochemistry for Orphanin FQ in the vascular endothelium of umbilical cord. Magnification 100X - [10] Immunohistochemistry for ANF in the vascular endothelium of umbilical cord. Magnification 20X - [11] Immunohistochemistry for ANF in the cells of the Whartons jelly. Magnification 100X - [12] Immunohistochemistry for Oxytocin in the vascular endothelium of umbilical cord. Magnification 100X - [13] Immunohistochemistry for Oxytocin in the cells of the Whartons jelly. Magnification 20X.

Annamaria Mauro, Maria Buscemi, Angelo Leone and Aldo Gerbino

is mainly diffused with few single cell showing granules. Table 1 summarizes immunohistochemical results.

Discussion Umbilical cord seems to lack any capillaries or lymphatics and umbilical vessels are unique in lacking any innervation; thus endothelial cells may play the major role in the local control of the blood flow. In this paper we investigated in the human umbilical cord at term the expression of some peptides involved in the regulation of the vascular tone, and the expression of some enzymes responsible for the remodelling of the extracellular matrix. Through immunohistochemistry we identified the presence of orphanin FQ, oxytocin, ANF, eNOS, iNOS, MMP-2 and MMP-9 in different territories of the human umbilical cord. The expression of the enzyme catalyzing the synthesis of nitric oxide (eNOS), involved in the endothelial cytogenesis and contributing to the angiogenetic properties of vascular endothelial growth factor (VEGF) in HUVEC [34], seems not to be of great relief. Interesting is the expression of the inducible isoform of nitric oxide synthase (iNOS) mainly localized to the endothelium; iNOS could be probably involved in the mechanism of modulation of HUVEC permeability [35]. The immunoreactivity of the matrix metalloproteinases we investigated (MMP-2 and MMP-9) is evident in almost all the regions of the human umbilical cord at term. Probably the meaning of the wide distribution of these enzymes has to be linked to their functions in the remodelling of the extracellular matrix, since the umbilical cord, as source of stem cells, is a site of intense remodelling phenomena. Of great interest is the presence of gelatinase A and B in the cells of the endothelium, where these enzymes could contribute to the onset of angiogenic events [38], and in the cells of the Whartons jelly [40, 59, 60, 61, 62]. Fig. 14 shows the expression of MMP-2 and MMP-9 in the plasma membrane and in the
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Fig. 14. The sampling was performed on a 100X magnification field using 100 different detection points in the endothelial cells, both on the plasma membrane and in the cytoplasm around the nucleus. The diagram shows the results of immunohistochemical reactions for MMP-2 and MMP-9. The antibodies reaction time was the same.

cytoplasm of endothelial cells. The diagram shows that MMP-2 expression is more evident than MMP-9, and this expression is mainly concentrated on the plasma membranes. This result is in agrement with biochemical data from literature reporting an high expression of a MMP-2 activator (MT1-MMP) on the plasma membrane of andothelial cells. The expression of MMP-9 was more uniformely distributed between the two different regions. Atrial natriuretic factor (ANF) is mainly expressed in the cells of the Whartons jelly, whose cellular environment has to be considered a regulator of the concentration of different substances affecting circulation. In spite of the well known role of ANF on the regulation of muscle contractility, in our samples we didnt find vascular musculature express ANF. The expression of oxytocin is mainly localized to the mesenchimal cells of the Whartons jelly and to the vascular endothelium. This nonapeptide, with functions and molecular structure very similar to vasopressin, plays an important role in the regulation of the vascular compartment (vasodilation) through the interaction with vasopressinergic receptors on the plasma membrane [54]. Even if oxytocin is a peptide typically expressed by the hypothalamus, many authors demonstrated that it can be synthesized also by other tissues, for example the prostatic gland [57]. The strong immunoreactivity of oxytocin we detected in the endothelium suggests that this peptide could be synthesized also by endothelial cells.
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Pharmacological studies show the existence of oxytocin receptors in HUVEC cells. The interaction between oxytocin and its receptor causes an increase of the intracellular levels of calcium that activates the pathway responsible for the production of nitric oxide, thus leading to vasodilatation [58]. Interesting is the expression of orphanin FQ in the endothelium and in the longitudinal musculature of the vessels, probably connected with the role of this peptide both in the regulation of contraction and vascular tone.

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[38] Kiran MS, Sameer Kumar VB, Viji RI, Sudhakaran PR. Temporal relationship between MMP production and angiogenic process in HUVECs. Cell Biol Int 2006 Sep;30(9):704-13. [39] Rao Q, Geng YQ, An LL, Wu KF. Expression of gelatinase A and B in cord blood CD34+ cells and leukemic cell lines. Zhonghua Xue Ye Xue Za Zhi 2003 Feb;24(2):78-81. [40] Sobolewski K, Galewska Z, Wolaska M, Jaworski S. The activity of collagen-degrading enzymes of Whartons jelly in EPH gestosis (pre-eclampsia). Biol Neonate 2001;80(3):202-9. [41] Son BR, Marquez-Curtis LA, Kucia M, Wysoczynski M, Turner AR, Ratajczak J, Ratajczak MZ, JanowskaWieczorek A. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases. Stem Cells 2006 May;24(5):1254-64. [42] Brookes ZL, Stedman EN, Guerrini R, Lawton BK, Calo G, Lambert DG. Proinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine. Am J Physiol Heart Circ Physiol 2007 Nov; 293(5):H2977-85. [43] Xu PH, Chang M, Cheng LX, Cheng Q, Yan X, Wang R. The relaxant effect of nociceptin on porcine coronary arterial ring segments. Can J Physiol Pharmacol 2004 Nov;82(11):993-9. [44] Philip S, Armstead WM. Newborn pig nociceptin/orphanin FQ activates protein tyrosine kinase and mitogen activated protein kinase to impair NMDA cerebrovasodilation after ischemia. Neuroreport 2003 Feb 10;14(2):201-3. [45] Champion HC, Bivalacqua TJ, Zadina JE, Kastin AJ, Hyman AL, Kadowitz PJ. Role of nitric oxide in mediating vasodilator responses to opioid peptides in the rat. Clin Exp Pharmacol Physiol 2002 Mar; 29(3):229-32. [46] Cai WQ, Terenghi G, Bodin P, Burnstock G, Polak JM. In situ hybridization of atrial natriuretic peptide mRNA in the endothelial cells of human umbilical vessels. Histochemistry 1993 Oct;100(4):277-83. [47] Weber NC, Blumenthal SB, Hartung T, Vollmar AM, Kiemer AK. ANP inhibits TNF-alpha-induced endothelial MCP-1 expression--involvement of p38 MAPK and MKP-1. J Leukoc Biol 2003 Nov;74(5):932-41. [48] Frst R, Brueckl C, Kuebler WM, Zahler S, Krtz F, Grlach A, Vollmar AM, Kiemer AK. Atrial natriuretic peptide induces mitogen-activated protein kinase phosphatase-1 in human endothelial cells via Rac1 and NAD(P)H oxidase/Nox2-activation. Circ Res 2005 Jan 7;96(1):43-53. [49] Kook H, Itoh H, Choi BS, Sawada N, Doi K, Hwang TJ, Kim KK, Arai H, Baik YH, Nakao K. Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro. Am J Physiol Heart Circ Physiol 2003 Apr;284(4):H1388-97. [50] Templeton AG, Kingdom JC, Macmillan JB, McGrath JC, Whittle MJ. Atrial natriuretic peptide counteracts the vasoconstrictor effects of 5-hydroxytryptamine, U46619 and endothelin-1 in the human umbilical artery. Placenta 1994 Oct;15(7):715-20. [51] Gimpl G, Fahrenholz F. The oxytocin receptor system: structure, function, and regulation. Physiol Rev 2001 Apr; 81(2):629-83. Review. [52] Kosfeld M, Heinrichs M, Zak PJ, Fischbacher U, Fehr E. Oxytocin increases trust in humans. Nature 2005 Jun 2; 435(7042):673-6. [53] Katusic ZS, Shepherd JT, Vanhoutte PM. Oxytocin causes endothelium-dependent relaxations of canine basilar arteries by activating V1-vasopressinergic receptors. J Pharmacol Exp Ther 1986 Jan; 236(1):166-70. [54] Russ RD, Resta TC, Walker BR. Pulmonary vasodilatory response to neurohypophyseal peptides in the rat. J Appl Physiol 1992 Aug; 73(2):473-8. [55] Suzuki Y, Satoh S, Kimura M, Oyama H, Asano T, Shibuya M, Sugita K. Effects of vasopressin and oxytocin on canine cerebral circulation in vivo. J Neurosurg 1992 Sep;77(3):424-31.

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[56] Loichot C, Krieger JP, De Jong W, Nisato D, Imbs JL, Barthelmebs M. High concentrations of oxytocin cause vasoconstriction by activating vasopressin V1A receptors in the isolated perfused rat kidney. Naunyn Schmiedebergs Arch Pharmacol 2001 Apr; 363(4):369-75. [57] Farina-Lipari E, Lipari D, Bellafiore M, Anzalone R, Cappello F, Valentino B. Presence of atrial natriuretic factor in normal and hyperplastic human prostate and its relationship with oxytocin localization. European Journal of Histochemistry 2003; vol. 47 issue 2 (Apr-Jun): 133-138. [58] Thibonnier M, Conarty DM, Preston JA, Plesnicher CL, Dweik RA, Erzurum SC. Human vascular endothelial cells express oxytocin receptors. Endocrinology 1999 Mar;140(3):1301-9. [59] Von Offenberg Sweeney N, Cummins PM, Birney YA, Cullen JP, Redmond EM, Cahill PA. Cyclic strainmediated regulation of endothelial matrix metalloproteinase-2 expression and activity. Cardiovasc Res 2004; 63:625-34. [60] Wang BW, Chang H, Lin S, Kuan P, Shyu KG. Induction of matrix metalloproteinases-14 and 2 by cyclical mechanical stretch is mediated by tumor necrosis factor-alpha in cultured human umbilical vein endothelial cells. Cardiovasc Res 2003; 59:460-9. [61] Hobeika MJ, Thompson MD, Muhs BE, Brooks PC, Gagne PJ. Matrix metalloproteinases in pheripheral vascular disease. Journ Vasc Surg 2007; 45: 849-57. [62] Mauro A, Buscemi M, Leone A and Gerbino A. Role of MMP-2, MMP-9, Orphanin FQ and others peptides in vascular tone of human umbilical cord at term. Dent Med Probl, submitted.

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ONTOGENESI DEI PEPTIDI vASOPRESSINA, OSSITOCINA, ANP (PEPTIDE ATRIALE NATRIURETICO) IN ALCUNI NUCLEI IPOTALAMICI IN EMBRIONI DI RATTO
[Ontogeny of peptides vasopressin, oxytocin, ANP (Atrial Natriuretic Peptide) in some hypothalamic nuclei in rat embryos] Elvira Farina Lipari, Alessio Lipari e Biagio Valentino
Dip. Medicina Sperimentale, Sez. Anatomia umana, Universit degli Studi di Palermo (I)

Parole chiave: Ontogenesi, Ipotalamo, Neuropeptidi Key words: Ontogeny, Hypothalamus, Neuropeptides Riassunto. Uno studio immunoistochimico condotto sui alcuni nuclei ipotalamici mette in evidenza che ANP e vasopressina compaiono al 16 giorno di vita fetale nel nucleo sopraottico e che vasopressina al 18 giorno di vita fetale nel nucleo paraventricolare, mentre lossitocina compare 2 giorni dopo la nascita. Pertanto si evidenzia che ANP e vasopressina compaiono al medesimo stadio di sviluppo svolgendo unazione antagonista, mentre lossitocina ipotalamica non sembra non avere un ruolo durante la vita fetale probabilmente poich essa viene sostituita dallossitocina materna. Abstract. In rat developing brain, by immunohistochemical tecniques, the ontogeny of the vasopressin and oxytocin peptides in the hypothalamic supraopticus and paraventricular nuclei and the ontogeny of ANP in the hypothalamic supraopticus and suprachiasmatic nuclei and in the choroid plexus was studied. The results showed that in the supraoptic nucleus and choroid plexus ANP and vasopressin appear at 16 day of foetal life, in the paraventricular nucleus the vasopressin appears at 18 day of foetal life and that in the supraoptic and paraventricular nuclei the oxytocin appears after the birth. The results showed that ANP and vasopressin appear at same developmental stages with a antagonist role, while the oxytocin has no effect on the cardiac release in foetal life, differently than in the adult life.

Introduzione Il sistema magnocellulare neurosecernente ipotalamico un complesso di nuclei situati nellarea preottica anteriore e laterale ed costituito dai nuclei sopraottico, paraventricolare e soprachiasmatico; inoltre, tra il nucleo sopraottico ed il nucleo paraventricolare vi sono raggruppamenti neuronali magnocellulari che costituiscono i nuclei accessori [1]. La presenza e la sintesi di neurosecreto nei neuroni magnocellulari dei nuclei ipotalamici, da molto tempo, stata determinata con tecniche istologiche quali il metodo di Gomori [9], successivamente con tecniche immunoistochimiche stato stabilito che il neurosecreto fosse costituito da granuli dei peptidi vasopressina e ossitocina ed in particolare stato

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determinato che i nuclei sopraottico e paraventricolare sintetizzano i neuropeptidi vasopressina e ossitocina ed il nucleo soprachiasmatico soltanto vasopressina. Per quanto concerne i nuclei accessori, situati nellarea compresa tra il nucleo sopraottico e paraventricolare, lo studio istologico evidenzia che essi sono simili per gli aspetti istomorfolofgici ai neuroni neurosecernenti dei nuclei sopraottico e paraventricolare e presentano rapporti di contiguit con piccoli casi [10]; inoltre con ulteriori indagini abbiamo stabilito che i nuclei accessori effettivamente sintetizzano vasopressina (Fig.2) [2] ed ossitocina (Fig. 1). Pi recentemente nellencefalo di ratto sono stati riscontrati altri peptidi ANP, CNP, salusina, orexina etc. e negli ultimi decenni la letteratura si arricchita di numerosi dati che mettono in evidenza una interrelazione tra i peptidi ANP ed ossitocina in diversi organi, quali cuore [8] e prostata normale ed ipertrofica [5]. Prendendo in considerazione i dati bibliografici riportati noi abbiamo ritenuto importante indagare sullontogenesi di alcuni peptidi, pertanto intraprendendo uno studio immunoistochimico sullontogenesi di vasopressina, ossitocina e ANP allo scopo di stabilire lo stadio di sviluppo in cui ciascuno di questi peptidi compare, perch la conoscenza della sequenza temporale della comparsa dei peptidi nei diversi nuclei ipotalamici ci permette di conoscere meglio la interrelazione tra i peptidi stessi. In particolare abbiamo intrapreso lo studio immunoistochimico concernente lontogenesi dei peptidi vasopressina, ossitocina ed ANP (atrial natriuretic peptide) nellencefalo di ratto dal 15 giorno di vita prenatale al 2 giorno di vita postnatale. Lo studio immunoistochimico condotto in embrioni di ratto a partire dal 15 giorno di vita fetale dimostra che la vasopressina compare al 16 giorno di vita intrauterina nel nucleo sopraottico (Fig.3) ed al 18 giorno di vita intrauterina nel nucleo paraventricolare (Fig.4) [4] la ossitocina riscontrabile dal 2 giorno di vita postatale sia nel nucleo sopraottico (Fig.5) sia nel nucleo paraventricolare (Fig.6); la PCR evidenzia che lmRNA vasopressina compare contemporaneamente al peptide, mentre tale sincronismo tra peptide e mRNA non stato riscontrato per lossitocina; infatti la PCR mette in evidenza che lossitocina mRNA compare molto pi precocemente del peptide, precisamente al 18 girono di vita fetale. Questo risultato ci ha indotto ad ipotizzare che probabilmente lossitocina materna attraversa la barriera placentare inibendo la sintesi del peptide pur essendo presente il suo mRNA. Considerazioni recenti Lo studio seguente sullontogenesi di ANP stato condotto nei nuclei sopraottico e soprachiasmatico; i risultati hanno messo in evidenza che ANP nel nucleo sopraottico [6] compare in entrambi i nuclei 16 giorno di vita fetale (Fig.7) e tali i risultati sono confermati dalla PCR, anche nel nucleo soprachiasmatico ANP compare al 16 giorno di vita fetale
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Ontogenesi dei peptidi vasopressina, ossitocina, ANP (Peptide Atriale Natriuretico) in alcuni nuclei ipotalamici in embrioni di ratto

(Fig.8) ed i neuroni sintetizzanti ANP sono dapprima uniformemente diffusi, mentre si spostano in sede ventro-mediale del nucleo [7] nella vita postatale. Lo studio per la ricerca di ANP stato esteso anche ai plessi corioidei in encefalo di ratto adulto sia di embrioni); nei plessi di ratti adulti ANP stato riscontrato ratto sia nelle cellule ependimali della parete del ventricolo laterale sia nelle cellule ependimali del plesso corioideo in cui numerosi granuli intensamente immunopositivi sono stati riscontrati diffusi nel citoplasma; in embrioni in embrioni al 16 e al 18 giorno di vita intrauterina una intensa immunopositivit per ANP stata riscontrata nelle cellule ependimali del plesso corioideo, immunopositivit era localizzata in alcune cellule nella loro area apicale, in altre nella loro area basale [11]. Conclusioni Nel complesso dei risultati ottenuti nei sopracitati lavori noi possiamo dedurre che la vasopressina ed ANP compaiono in un medesimo stadio di sviluppo e ci comprensibile in considerazione del ruolo che entrambi svolgono riguardo lomeostasi dei fluidi corporei esplicando una azione antagonista, mentre lossitocina compare in uno stadio di sviluppo nettamente differente, nella vita postnatale. Nonostante sia stato riportato in letteratura che lossitocina ipotalamica sarebbe responsabile del rilascio di ANP nel cuore [8], ANP cardiaco presente in vita fetale, quindi la sua sintesi inizia in uno stadio di sviluppo molto pi precoce rispetto a quello della sintesi di ossitocina; questi differenti dati potrebbero indicare che il rilascio di ANP cardiaco non sia necessariamente o unicamente stimolato dalla ossitocina ipotalamica.

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Fig.1 Nucleo accessorio. Neuroni immunopositivi per ossitocina.

Fig.2 Nucleo accessorio. Neuroni immunopositivi per vasopressina.

Fig.3 Embrione al 16 giorno di vita fetale. Nucleo sopraottico: neuroni immunopositivi per vasopressina. Fig.4 Embrioni al 18 giorno di vita fetale. Nucleo paraventricolare: neuroni immunopositivi per vasopressina.

Ontogenesi dei peptidi vasopressina, ossitocina, ANP (Peptide Atriale Natriuretico) in alcuni nuclei ipotalamici in embrioni di ratto

Fig.5 Neonati a 2 giorni di vita. Nucleo sopraottico: neuroni immunopositivi per vasopressina.

Fig.6 Neonati a 2 giorni di vita. Nucleo paraventricolare: neuroni immunopositvi per ossitocina.

Fig.7 Embrioni al 16 giorno di vita fetale. Nucleo sopraottico: neuroni immunopositivi per ANP.

Fig.8 Embrioni a 18 giorni di vita fetale. Nucleo soprachiasmatico: neuroni immunopositivi per ANP.

Elvira Farina Lipari, Alessio Lipari, Biagio Valentino

Bibliografia Armstrong WE. Hypothalamic supraoptic and paraventricular nuclei. In The Nervous System, vol 1, Ed G Phaxinos, Academic Press,125, 1985. [2] Farina Lipari E and Valentino B. Immunohistochemicaln research on vasopressin in the hypothalamic accessory nuclei. It J Anat Embryol 98: 207-14,1993. [3] Farina Lipari E and Valentino B. Immunohistochemical research on oxytocin in the hypothalamic accessory nuclei. It J Anat Embryol 100:189-193,1995. [4] Farina Lipari E, Lipari D, Gerbino A, Di Liberto D, Bellafiore M, Catalano M, Valentino B. The hypothalamic magnocellular neurosecretory system in the developing rats. Eur J Histochem 45:163- 168, 2001. [5] Farina Lipari E, Lipari D, Bellafiore M, Anzalone R, Cappello F, Valentino B. Presence of atrial natriuretic factor in normal and hyperplastic human prostate and its relationship with oxytocin localization Eur J Histochem 45:133-138, 2002. [6] Farina Lipari E, Lipari D, Dieli F, Valentino B. Atrial natriuretic peptide secretion during development of the rat supraoptic nucleus. Eur J Histochem 49: 379-384, 2005. [7] Farina Lipari E, Lipari A, Dieli F, Valentino B. Presence ANP in the suprachiasmatic nucleus in developing rat. It J Anat Embryol 112: 19-25, 2007. [8] Guthowska J, Jankowski M, Lambert C, Mukaddam-Daher S, Zingg H, McCann S. Oxytocin releases atrial natriuretic peptide by combining with oxytocin receptors in the heart. Proc Natl Acad Sci (USA) 94:11704-9,1997. [9] Pasqualino A, Peri G, Farina Lipari E. Osservazioni sui neuroni ipotalamici accessori. Atti Accademia Scienze Lettere ed Arti di Palermo. Serie V, Vol. VIII, 85-91,1988. [10] Peri G, Farina Lipari E, Lipari D. Osservazioni sullo sviluppo di alcuni nuclei ipotalamici. Atti Accademia Scienze Lettere ed Arti di Palermo. Serie V, Vol.XII, 15-34, 1991-92. [11] Valentino B, Peri G, Gerbino A, Buscemi M, Lipari A, Basile I, Farina Lipari E. ANP presence in the choroid plexus in the lateral ventricles in the developing rat. It J Anat Embryol 110, Suppl 1: 114, 2005. [1]

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IMAGING ECOGRAfICO DEL CORDONE OMBELICALE UMANO


[Echographic Imaging of the human umbilical Cord] Riccardo Mandracchia1, Annamaria Mauro, Luana Lipari1, Federico Romano2, Antonino Perino2, Massimo Midiri3 e Aldo Gerbino1
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Dipartimento di Medicina Sperimentale (DI.ME.S) - Sezione di Istologia ed Embriologia Arcangelo Pasqualino di Marineo- 2Clinica Ostetrica e Ginecologica - 3Dipartimento di Biotecnologie mediche e Medicina Legale - Sezione di Scienze Radiologiche - Universit degli Studi di Palermo (I)

Parole chiave: Cordone ombelicale, Imaging, Arteria ombelicale unica Key words: Umbilical cord, Imaging, SUA Riassunto. Il cordone ombelicale un condotto organico che assicura i rapporti vascolari tra madre e feto. Esso costituito da una vena e due arterie ombelicali, spiralizzate intorno allasse maggiore venoso e circondati da tessuto mucoso maturo (gelatina di Wharton). Tale struttura rivestita dalla membrana amniotica (ectoblasto dellamnios), tenacemente adesa. La lunghezza, a termine, varia in media dai 50 ai 60 cm. Ad oggi lecografia si pone in maniera quasi esclusiva nel controllo per immagini della gravidanza normale e nellindividuazione delle deviazioni patologiche. Ecograficamente il cordone ombelicale ben evidente a 8 settimane, epoca in cui comincia la sua crescita in lunghezza e larghezza. Il Color-Doppler ne semplifica la visualizzazione rendendo agevole alloperatore il controllo dei tre vasi. Le alterazioni del cordone ombelicale comprendono: brevit assoluta o relativa; lunghezza eccessiva; cisti; ematomi; tumori; trombosi; presenza di arterie soprannumerarie; completa assenza del cordone ombelicale; persistenza della vena ombelicale destra; varici. Lanomalia pi frequente costituita dalla presenza di unarteria ombelicale unica (SUA), con frequenza di circa l1% delle gravidanze singole e del 5% delle plurime. Abstract. Human umbilical cord is an organic culvert that assures the vascular relationships between mother and fetus. It is constituted from a vein and two umbilical arteries. Two arteries bundle up themselves around to the venous axis. This vascular structure is covered from the Whartons jelly and amniotic membrane. The length, at term, is comprised between 50 cm and 60 cm. Today the echography is used, nearly exclusively, in the control for images of the normal pregnancy and in the location of pathological shunting lines. In the echographic study the umbilical cord is obvious to 8 weeks, age in which begins its increase in length and width. The Color-Doppler helps the operator in the visualization and control of the three vessels. The alterations of the umbilical cord comprise: absolute or relative brevity; excessive length; cysts; hematomas; tumors; thrombosis; presence of other arteries; absence of the umbilical cord; persistence of the right umbilical vein; varicous vein. The more frequent anomaly is the presence of a single umbilical artery (SUA), with frequency of approximately 1% of the single and 5% of the multiple pregnancies.

Introduzione Lesigenza di evitare lesposizione a radiazioni ionizzanti, il rapido sviluppo tecnologico che ha consentito lesame in tempo reale, la qualit e la quantit di informazioni disponibili
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Riccardo Mandracchia, Annamaria Mauro, Luana Lipari, Federico Romano, Antonino Perino, Massimo Midiri e Aldo Gerbino

ed il costo non elevato hanno reso sempre pi esclusivo luso dellecografia nel controllo per immagini della gravidanza normale e nellindividuazione delle deviazioni patologiche. Modernamente sono obsolete metodiche radiologiche di uso frequente in passato quali lesame diretto delladdome, la pelvimetria, la placentografia e lamniosgrafia. In questi ultimi anni, inoltre, trovano spazio, nella diagnostica per immagini fetale, tecniche quali il 3D imaging e la Risonanza Magnetica (RM). Il 3D imaging un sistema che consente una scansione di tipo volumetrico che differisce dalla classica acquisizione B-mode per la quantit di tessuto in esame. Una delle principali applicazioni dellecografia tridimensionale la visualizzazione della faccia del feto ma possibile ottenere anche unottimale rappresentazione delle piccole parti fetali, degli arti e degli organi genitali. La RM si affiancata in questi ultimi anni allecografia con una intensa attivit di sperimentazione. La RM ha in comune con lecografia la possibilit di ottenere immagini multiplanari e la potenziale non nocivit, ma consente inoltre di effettuare studi con ampio campo di vista offrendo la visualizzazione completa del feto e della cavit uterina, con maggiore dettaglio anatomico e senza le eventuali influenze negative legate allobesit materna o alla scarsa risoluzione di contrasto dellecografia. Per quanto riguarda lo studio del cordone ombelicale, ad oggi, lapproccio quasi esclusivamente utilizzato quello ecografico. Lesame ecografico viene effettuato con sonde transaddominali (TA), convex o settoriali da 3,5 MHz, bidimensionali o volumetriche e, nel I trimestre di gravidanza, possono essere utilizzate sonde vaginali (TV) da 5,6-7,5 MHz, bidimensionali o volumetriche. Le sonde TA richiedono, nel I trimestre, un adeguato riempimento vescicale, mai reso eccessivo, per evitare di comprimere il diametro antero-posteriore uterino con conseguente allungamento della cavit. Quando la vescica poco repleta e lutero antiversoflesso, il suo fondo risulta molto vicino alla parete, pertanto si possono avere echi di riverbero che interferiscono con lesame rendendolo poco chiaro. La sonda vaginale, ricoperta da un condom o da un dito di guanto in cui stato preventivamente introdotto un gel per ultrasuoni, va posizionata nel fornice posteriore consentendo una migliore risoluzione in quanto la distanza tra la superficie della sonda e la zona da esaminare si riduce ad un quarto (o un quinto) rispetto alla distanza tra sonda TA e zona da esaminare. La visione, pertanto, risulta essere pi precisa perch la perdita di definizione, anche in caso di ingrandimento dellimmagine, molto modesta. La sonda endovaginale inoltre non richiede riempimento vescicale prima dellesame; permette unottima definizione particolarmente in caso di retroversione uterina, obesit materna e consente, con maggiore precocit, una eventuale diagnosi che pu spingersi anche ad una settimana rispetto allimpiego della sonda TA. Tuttavia le pazienti non sempre sono disposte a sottoporsi allesame ecografico con sonda TV, pur essendo oramai ampiamente dimostra172

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to che tale metodica non ha ripercussioni sullandamento della gravidanza. Il cordone ombelicale, o funicolo, costituisce il trait dunion tra feto e placenta. Esso ha una lunghezza i 50-60 cm a termine di gravidanza ed in genere il suo diametro di 15-20mm. Esso costituito da: vasi ombelicali; tessuto gelatinoso lasso con elementi cellulari sparsi e con ampie lacune: la cosiddetta Gelatina di Wharton; guaina amniotica. Il cordone ha una struttura assai peculiare per il fatto di essere formato da tre vasi (due arterie ed una vena) lunghi e privi di ramificazioni che, invece di decorrereparallelamente come in altri organi, hanno un decorso elicoidale detto, anche se impropriamente, spiralizzato. Le arterie si avvolgono di solito alla vena proteggendo la sua sottile parete dalle compressioni, ma pu accadere anche che la vena si avvolga intorno alle arterie aumentrando cos le possibilit di una sua compressione. La Gelatina di Wharton insieme ad un efflusso regolato da un apparato similsfinteriale (anello ambelicale prima e dotto venoso o di Aranzio poi) fa assomigliare il cordone, sia istologicamente che funzionalmente, ad un organo erettile, mentre la presenza di spiralizzazioni lo rendono turgido. Tale architettura ha una funzione emodinamica precisa di pompa peristaltica che favorirebbe il ritorno venoso al cuore del feto. Non possono infine non ricordarsi le propriet biochimiche del cordone, consistenti nella sintesi e liberazione di potenti sostanza vasoattive e coagulanti, la cui azione si esplica al momento del suo taglio prevenendo cos il dissanguamento del neonato. Ecograficamente il cordone ombelicale ben evidente a 8 settimane, epoca in cui comincia la sua crescita in lunghezza e larghezza. In letteratura sono riportati studi che mettono in rapporto la sua lunghezza allet gestazionale, ma nella pratica clinica la valutazione di questo rapporto non ha trovato applicazione perch non facilmente riproducibile. Recentemente stata proposta la misurazione del diametro del cordone ombelicale come possibile indice prognostico nella prima met della gravidanza, analogamente alla valutazione effettuabile nella seconda met. Il cordone si presenta come una struttura modicamente ecogenica, nastriforme, pulsante, che fluttua nel liquido amniotico. In condizioni di liquido amniotico regolare se ne ottengono immagini longitudinali, oblique e trasversali. Le sezioni trasversali, ottenute a partire dalla 14 settimana, permettono ovviamente un riconoscimento agevole dei tre vasi che lo compongono: la vena, che si presenta con un calibro nettamente superiore rispetto alle due arterie disposte a spirale. Il Color-Doppler ne semplifica la visualizzazione rendendo agevole alloperatore il controllo dei tre vasi: la differenza di colore tra la vena e le due arterie dipende dalla differente direzione del flusso rispetto alla sonda (convenzionalmente si utilizzano le gradazioni dal blu al rosso).
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Linserzione placentare, evidenziabile con facilit, a forma di U o V, posta, pi frequentemente, in prossimit del centro del piatto coriale della placenta, ma pu dislocarsi verso il margine di esso, senza con questo assumere particolare significato patologico. Al contrario, nellinserzione velamentosa, il funicolo si inserisce sulle membrane ad una certa distanza dal margine placentare ed i vasi funicolari decorrono per un tratto pi o meno lungo nellinterstizio tra corion ed amnios prima di raggiungere il bordo della placenta. Quindi lVCI rappresenta un pericolo molto grave perch i vasi possono rompersi al momento della ritira delle membrane ed il feto morire per dissanguamento. Inoltre nellinserzione velamentosa, i vasi ombelicali privi del rivestimento protettivo della Gelatina di Wharton possono subire essere compressi dalla parte presentata o da altre parti del corpo fetale; una compressione duratura pu essere causa di alterazioni circolatorie in grado di determinare una sofferenza fetale di gravit variabile con una mortalit fetale, durante il travaglio di parto, del 50%. Si stima che lincidenza di tale anomalia sia pari all1% circa nelle gravidanze singole, mentre raggiunge il 9% nelle gravidanze gemellari. Le anomalie associate si riscontrano nel 6-8% dei casi. Includono: atresia esofagea, uropatia ostruttiva, dislocazione congenita dellanca, trisomia 21, singola arteria ombelicale, spina bifida. Si parla invece di inserzione velamentosa previa o di vasa previa quando i vasi attraversano la zona del polo inferiore dele membrane amniocoriali. In tal caso il pericolo per il feto notevole (anche se i vasi non sono localizzati nellarea dellorifizio uterino interno). Infatti affinch il feto possa fuoriuscire dallutero seguendo le vie naturali, necessario che le membrane si rompano a livello del polo inferiore del sacco , e la rottura pu quindi interessare uno o pi vasi previ. La mortalit perinatale in tale emergenza ostetrica oscilla tra il 75 ed il 100% dei casi. Lincidenza di 2:10000 gravidanze e si associa a placenta bilobata, lobo succenturiato. La detection rate ecografia, mediante lutilizzo di sonde TV, del 90% circa. All8 settimana, in seguito alla ridotta capacit delladdome fetale, occupato dal fegato e dai reni, le anse intestinali, migrando verso il cordone ombelicale, determinano una erniazione fisiologica (onfalocele fisiologico). Il persistere di queste anse intestinali al di fuori della parete addominale oltre la 12 settimana assume significato patologico probante per un onfalocele o gastroschisi. In ambedue i casi alleco si osserva una matassa di visceri che protrude dalla parete addominale e si affonda nel liquido amniotico. Lonfalocele caratterizzato da un difetto del territorio centrale della parete addominale, che interessa lombelico e comporta la fuoriuscita dei visceri rivestiti dal peritoneo, dalla gelatina di Warthon e dalla membrana amniotica. La gastroschisi si differenzia dallonfalocele in quanto linserzione del cordone ombelicale alla parete addominale normale, mentre i visceri fuoriescono lateralmente (quasi sempre a destra dellombelico) senza essere rive174

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stiti dalle membrane. La diagnosi differenziale deve quindi tener conto della caratteristica localizzazione della fuoriuscita dei visceri dalla parete addominale, che nel caso di onfalocele situata alla base del cordone ombelicale, oltre che del rivestimento delle membrane intorno ai visceri erniati. Nella gastroschisi, invece, linserzione del cordone ombelicale risulta essere normale. LEco-Color-Doppler, in questo caso, agevola la diagnosi differenziale in quanto evidenzia, istantaneamente, il decorso dei vasi nel cordone e, quindi, la loro localizzazione. Il ritardato ritorno delle anse intestinali allinterno della cavit addominale segno prognostico sfavorevole. Infatti i feti in cui la fisiologica regressione in cavit addominale dei visceri avviene con ritardo, alla 13 settimana, possono successivamente andare incontro a volvoli. Misurazione ecografia del cordone, note di tassonomia fisiopatologica La lunghezza del cordone proporzionale allattivit motoria del feto e pu essere misurata soltanto nel primo trimestre di gravidanza; dalla 6 alla 12 settimana ha una precisa correlazione con let gestazionale. necessario un esame prolungato, preferibilmente con sonda TV e Color-Doppler, in quanto occorre visualizzare il cordone nella sua interezza, quando esso disteso pressoch completamente. LOligoidramnios, la Sindrome da briglie amniotiche, la Gemellarit, alcune malformazioni, sono tutte condizioni in cui si ha una brevit assoluta o relativa del cordone con riduzione della normale attivit motoria del feto. Queste anomalie, per lo stesso motivo, possono determinare il minore avvolgimento a spirale dei vasi, evidenziabile agli ultrasuoni, specie con il Color-Doppler. Nei casi in cui i vasi decorrono pressoch paralleli, stato riscontrato un aumento del rischio di morbilit fetale: parto prematuro, sofferenza fetale in travaglio, malformazioni e/o cromosomopatie fetali. Al contrario una lunghezza eccessiva pu comportare la formazione di veri nodi, di trombi, di giri intorno al collo ed agli arti fetali, prolasso e previet del funicolo la cui diagnosi pu essere facilitata dal Color-Doppler. Limportanza della diagnosi dipende dallepoca gestazionale: la presenza di multipli giri di funicolo o di previet impone una vivace attenzione nella condotta ostetrica. I veri nodi sono relativamente frequenti e, nel corso della gravidanza, non sono correlati ad una particolare frequenza di mortalit e morbilit fetali. Sono difficili da diagnosticare ecograficamente anche con il Color-Doppler. I falsi nodi invece non hanno alcuna importanza clinica e sono espressione di varicosit della vena o di un accumulo di gelatina di Warthon che comporta una localizzata dilatazione di varia entit. Le cisti derivano da residui del dotto onfalomesenterico, dallallantoide (cisti allantoidee) o da inclusione amniotica ed appaiono ecograficamente come formazioni tondeggianti, a margini netti, ipo-anaecogene, adese al funicolo e regrediscono frequentemente alla fine del primo trimestre.
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Gli ematomi sono rari, secondari a lesione della vena ombelicale: da trazione, compressione del funicolo tra tessuti materni e fetali o da alterazione della parete venosa; pi spesso sono iatrogeni, successivi a cordocentesi e sono localizzati prevalentemente in prossimit della inserzione addominale con alto rischio per il feto. Ecograficamente appaiono come aree rotondeggianti ipo-anecogene di diversa grandezza. I tumori del cordone comprendono gli angiomi, gli angiomixomi, i miosarcomi, i teratomi ed i dermoidi; sono estremamente rari e localizzati generalmente in prossimit dellinserzione placentare. Gli angiomi appaiono come noduli ad ecogenicit omogenea o mista, spesso associati a polidramnios, aumento della-FP ed idrope fetale. La trombosi, anchessa molto rara, dovuta a fenomeni di compressione sul cordone ed responsabile frequentemente di morte endouterina. Anomalie delle arterie ombelicali (Quadri ecografici) Una delle anomalie vascolari pi frequenti costituita dalla presenza di unarteria ombelicale unica (SUA), con frequenza dello 0,2-1,1% nella gravidanza singola e del 6-11% nella multipla. Generalmente secondaria allatrofia di una delle due arterie e, nel 30-60% dei casi, associata a malformazioni fetali. Esistono due forme di SUA, una elicoidale ed una diritta. una condizione abbastanza comune nei gemelli, nella gravidanze con diabete franco, in associazione con cordoni lunghi e placente piccole. Frequentemente associata ad altre patologie: IUGR, malformazioni fetali, aneuploidie, morte fetale. Le anomalie del sistema organico nei bambini nati vivi associate ad arteria ombelicale unica sono: muscoloscheletriche, urogenitali, gastrointestinali, cardiovascolari, respiratorie e del sistema nervoso centrale. Inoltre, la SUA stata riscontrata nel 6-11% dei bambini con anomalie cromosomiche, principalmente trisomia 13 e 18; tuttavia il la diagnosi ecografica di singola arteria ombelicale non indicazione al cariotipo fetale a meno che non si riscontrino altre anomalie fetali oppure ancora ci si trovi dinnanzi ad un IUGR. necessario verificare la presenza di SUA in un lungo tratto del cordone ombelicale in quanto la fusione delle due arterie, in unico vaso in prossimit dellinserzione placentare, un evento frequente che, comunque, viene considerato come una variante fisiologica. La diagnosi semplice con il Color-Doppler dalla 14 settimana circa, e, per i motivi precedentemente esposti, imperativo un esame ecografico accurato e completo del feto per escludere eventuali altre malformazioni. In caso di SUA ed assenza di anomalie, la flussimetria fetale non evidenzia modifiche sostanziali. La presenza di arterie soprannumerarie evento raro e deriva dalla persistenza dellarteria vitellina. La completa assenza del cordone ombelicale, infine, unanomalia estremamente rara, sempre letale per il feto, dovuta a malformazione delle pieghe embrionali, per cui i visceri
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addominali, e talora quelli toracici, sono esteriorizzati, senza membrane di protezione e legano il corpo fetale direttamente alla placenta. Una forma molto meno grave, ma simile, la Sindrome da cordone ombelicale corto, in cui il funicolo, molto breve, si pu trovare associato ad onfalocele, estrofia vescicale, alterazioni del rachide ed ano imperforato. Anomalie della vena ombelicale (Quadri ecografici) Durante lembriogenesi tre sistemi di vene si connettono con il cuore: le cardinali (anteriore, media e posteriore) che drenano sangue dal corpo, le vene vitelline connesse con il sacco vitellino, le ombelicali destra e sinistra che comunicano con la placenta. La vena ombelicale sinistra confluisce nel tratto infraepatico della vena vitellina sinistra, prima che questa si connetta con la vena cava inferiore per, successivamente, dare origine al dotto venoso. Levoluzione fisiologica prevede latrofia della vena ombelicale destra intorno alla 7 settimana. Per fattori non ancora identificati, pu avvenire la trombosi della vena ombelicale sinistra che determina la persistenza della vena ombelicale destra: per la maggior parte dei casi comunica regolarmente con il dotto venoso e con la vena porta di destra mentre raramente si immettono direttamente in cava inferiore od in atrio destro. La diagnosi ecografica agevole: la colecisti appare in posizione mediana e compresa tra la vena ombelicale e lo stomaco; la curvatura della connessione ombelico-portale diretta verso lo stomaco invece che verso il fegato. Il riscontro di questa anomalia deve indurre ad un attento esame fetale per la contemporanea presenza, in circa il 20% dei casi, di altre malformazioni (sindrome asplenica, displasie renali, anencefalia, difetti interventricolari, truncus arteriosus, arteria ombelicale singola), soprattutto quando la vena ombelicale oltrepassa il circolo portale. Altra patologia associata alla vena ombelicale quella varicosa. una rara anomalia della vena ombelicale che si verifica con maggiore frequenza nel tratto intra-addominale. Il difetto di parete di natura evolutiva e si riscontra frequentemente nel terzo trimestre. Anche qui la diagnosi agevole e si effettua mediante la misurazione in B-mode della sezione massima del tratto ectasico (massimo diametro vena ombelicale a termine 10 mm) e mediante utilizzo di Color-Power Doppler che consente di rilevare turbolenze vascolari od eventuali formazioni trombotiche. Il rischio di mortalit perinatale elevato per cromosomopatie, idrope e malformazioni. La formazione di un trombo influenza in senso negativo la prognosi. Il management prevede un frequente monitoraggio fetale per controllare le dimensioni e leventuale trombizzazione della varice e le variazioni di pre-carico mediante Doppler venoso. La coesistenza di altre malformazioni rappresenta unindicazione allesame del cariotipo fetale.

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1. Inserzione Velamentosa del C.O. (Color Doppler); 2. Normale paio di arterie ombelicali confermate dalla adiacenza alla vescica (Color Doppler); 3. C.O. (Color Doppler); 4. Arteria ombelicale unica; 5. Inserzione del C.O. (Power Doppler); 6. Inserzione del C.O. (Color Doppler); 7. Inserzione Velamentosa del C.O. (Power Doppler); 8. Inserzione Velamentosa del C.O.: imaging ecografico; 9. C.O.: tre vasi, due arterie ed una vena; 10. C.O.: tre vasi, due arterie ed una vena.

Imaging ecografico nello studio del Cordone ombelicale umano

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[23] Nyberg DA, Mack LA. Abdominal wall defects. In: Nyberg D.A., Mahony B.S., Pretorius D.H. Diagnostic ultrasound of fetal anomalies. Publisher 1990; Inc. Cap. 11:395-429. [24] Sen C. The use of first trimester ultrasound in ruotine practice. J Perinat Med 2001; 29(3):212-221. [25] Stehling MK, Mansfield P, Ordidge RJ et al. Echo-planar imaging of the human fetus in utero. Magn Reson Med 1990; 13:314-318. [26] Strong TH, Elliott JP, Radial TG. Non-coiled umbilical blood vessels: A new marker for the fetus at risk. Obstet Gynecol 1993; 81:409-411. [27] Timor-Tritsch LE, Rottem S. Normal and abnormal fetal anatomy in the first 15 weeks of pregnancy using transvaginal ultrasound. In: Diagnostic ultrasound applied to obstetric and gynecology; Sabbagha R.E., ed. Lippincott 1994. [28] Hall DR, Odendaal HJ, Kirsten GF, Smith J, Grove D. Expectant management of early onset, severe preeclampsia: perinatal outcome. BJOG 2000: 107(10):1258-64. [29] Makikallio K, Vuolteenaho O, Jouppila P, Rasanen J. Association of severe placental insufficiency and systemic venous pressure rise in the fetus with increased neonatal cardiac troponin T levels. Am J Obstet Gynecol 2000: 183(3):726-31. [30] Marsal K. Rational use of Doppler ultrasound in perinatal medicine. J Perinat Med 1994: 22(6): 463-74. [31] Ghezzi F, Raio L, Gunter Duwe D, Cromi A, Karousou E, Durig P. Sonographic umbilical vessel morphometry and perinatal outcome of fetuses with a lean umbilical cord. J Clin Ultrasound 2005 Jan;33(1):18-23. [32] Raio L, Cromi A, Ghezzi F, Passi A, Karousou E, Viola M, Vigetti D, De Luca G, Bolis P. Hyaluronan content of Whartons jelly in healthy and Down syndrome fetuses. Matrix Biol 2005 Apr;24(2):166-74. [33] Hasegawa J, Matsuoka R, Ichizuka K, Sekizawa A, Farima A, Okai T. Velamentous cord insertion into the lower third of the uterus is associated with intrapartum fetal heart rate abnormalities. Ultrasound Obstet Gynecol 2006 Apr;27(4):425-9. [34] Predanic M, Perni SC, Chasen ST, Baergen RN, Chervenak FA. Ultrasound evaluation of abnormal cord coiling in second trimester of gestation in association with adverse pregnancy outcome. Am J Obstet Gynecol 2005 Aug;193(2): 387-94. [35] Cromi A, Ghezzi F, Di Naro E, Siesto G, Bergamini V, Raio L. Large cross-sectional area of the umbilical cord as a predictor of fetal macrosomia. Ultrasound Obstet Gynecol 2007 Nov;30(6):861-6.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

EXPERIMENTAL COLITIS: CHEMICAL AND BACTERIAL INDUCTION


[Colite sperimentale: induzione chimica e batterica] Inaya Abdallah Hajj Hussein,, Rania Tohme, Kassem Barada, Mostafa Hassan Mostafa, Angelo Leone5, Jean-Noel Freund, Rosalyn A. Jurjus, Walid Karam4 and Abdo Jurjus
Faculty of Medicine, American University of Beirut, Beirut, Lebanon - Faculty of Science, Beirut Arab University, Beirut, Lebanon - INSERM 682, Strasbourg, France - 4 Institute National De Pathologie, BeirutLebanon - 5 Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, Facolt di Medicina e Chirurgia, Universit degli Studi di Palermo, Italia

Key words: Colitis, E. coli, Iodoacetamide, IBD model Parole chiave: Colite, E. coli, Iodoceacetamide, modello IBD Abstract. Well-controlled models of ulcerative colitis (UC) constitute an important tool to elucidate the pathophysiologic processes implicated in its development. This study reports a novel model of colitis induced in rats using a combination of chemical (iodoacetamide) and enteropathogenic E. coli (EPEC). Male SpragueDawly rats (n=158) were divided into four groups inoculated intrarectally on a weekly basis with 4 different combinations: (a) 1% methylcellulose (MC), (b) 100 l of 6% iodoacetamide (IA) in 1% MC, (c) 200 l containing 4 x 108 colony factor units (CFU) of EPEC, (d) combined treatment of (IA) followed by bacteria (B) after 2 days. Thirty days post treatment, each of the four groups was divided into two subgroups; the inoculation was stopped for one subgroup and the other subgroup continued with biweekly inoculation till the end of the experiment. Colitis was evaluated by the clinical course of the disease, the macroscopic and microscopic alterations, activity of myeloperoxidase (MPO), and by TNF-a gene expression. Results showed findings indicative of UC in the combined treatment (IA+B) as well as the IA continued treatment: slow rate of increase in body weight, diarrhea, and bloody stools, high colonic ulcer score, as well as histological alteration characteristic of UC in human indicating an extensive inflammatory reaction. Across the experimental duration, the MPO activity was consistently elevated and the TNF-a gene expression upregulated compared to the control. In conclusion, the chronic experimental ulcerative colitis model reported in the present study resembles, to a great extent, the human model. It is reproducible with characteristics indicative of chronicity. Riassunto. Sommario: Modelli di Colite Ulcerosa (UC) costituiscono uno strumento importante per elucidare i meccanismi fisiopatologici implicati nel suo sviluppo. Questo studio descrive un nuovo modello di colite indotta nel ratto utilizzando una combinazione di sostanze chimiche e batteriche (IODOACETAMIDE, E. COLI) (EPEC). Ratti maschi (SPRAGUR-DAWLY) N=158. Sono stati divisi in 4 gruppi. Veniva effettuata inoculazione intrarettale ogni settimana utilizzando quattro diverse combinazioni: (a) 1% metilcellulosa (MC), (b) 100l al 6% di iodoacetamide (IA) in 1% di MC, (c) 200l contenente 4 x 180 (CFU) colonie di EPEC, (d) trattamento combinato di (IA) seguito da batteri (B) dopo due giorni. Trenta giorni dopo il trattamento ogni gruppo stato diviso in 2 sottogruppi; Un subgruppo non stato pi inoculato mentre laltro subgruppo veniva inoculato settimanalmente fino alla fine dellesperimento. La Colite stata valutata seguendo il decorso della malattia, osservando le modificazioni macroscopiche e microscopiche, lattivit della mieloperossidasi (MPO), e lespressione genica del TNF-a. I risultati mostrano dati indicativi della colita ulcerosa da trattamento com-

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binato (IA+B) e anche in quella con trattamento continuato di IA: basso aumento di peso corporeo, diarrea, feci snguinolente, alte colonie batteriche e modificazioni istologiche tipiche della colite ulcerosa umana con una estesa reazione infiammatoria. Lungo tutta la durata del trattamento lattivit della MPO stata molto elevata nonch lespressione genica del TNF-a se paragonata al controllo. In conclusione la Colite Ulcerosa sperimentale rappresenta un modello che somiglia molto al quello umano ed riproducibile con caratteristiche proprie di cronicit.

Introduction It is well established that ulcerative colitis (UC) is an inflammatory condition of the gastrointestinal tract (GIT) resulting from interrelated genetic and environmental factors, especially bacteria which cause mucosal barrier disruption thus exposing the mucosal immune system directly to luminal bacteria and bacterial products (Fiocchi 2006). The clinical manifestations of colitis include nutrient malabsorption, bloody diarrhea and weight loss (Dotan et al. 2002, Farrel and Peppercorn 2002). Histopathologically, UC is considered as a mucosal disease that usually involves all (20% of the cases) or part (80% of the cases) of the colon with mild inflammation. The mucosa is erythematous and has a fine granular surface that looks like sandpaper (Jankey and Price 1969). In more severe disease, the mucosa is hemorrhagic, edematous, and ulcerated with invasion of inflammatory cells in the epithelium and lamina propria giving rise to irregular mucosal surface and distorted crypts architecture. The course of the disease shows periods of remission where the mucosa may appear normal, however, patient with recurrent attacks may develop atrophic featureless mucosa. In rare cases, patients with fulminant disease can develop toxic colitis or megacolon where the bowel wall thins out and the mucosa is severely ulcerated, leading to adhesions and to perforation (Fiocchi 1998). For many years, researchers have been addressing the question as to whether a specific pathogen could cause inflammatory bowel disease (IBD) (Campieri and Gionchetti 2001). Attempts made, so far, to find a causative bacterial strain for IBD, and particularly for UC, have been unsuccessful. Over the past years, evidence accumulated implicating endogenous luminal bacteria in the pathophysiology of UC, especially that areas of highest bacterial concentration and diversity are found in the colon. Much attention has been paid to the role of E. coli in the onset of UC, as E. coli is the predominant facultative anaerobic gram negative species of the normal intestinal flora. They play an important role in promoting the stability of the intestinal microbial flora and in maintaining the normal intestinal physiology (Campieri 2001). Besides commensal strains, certain clones of E. coli, possess virulent properties that cause disease in humans. More recently, it has been suggested that a particular subtype of E. coli could play a pathogenic role in UC (Rath 2003). Studies on mucosal adhesion of pathogenic bacteria in UC revealed an enhanced adhesion of E. coli
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isolates (from stool specimens and rectal biopsies from UC patients) to buccal epithelial cells causing mucosal damage similar to those of enteropathogenic E. coli (EPEC) (Nazli et al. 2004; Schuppler et al. 2004; Yu and Kaper 1992). Adherence of EPEC strains into the intestinal mucosa is a very complicated process and produces dramatic effects in the ultrastucture of the cells resulting in reduced tight junction density and leaky epithelial barrier (Goosney 1990; Nougaryrede 2003; Muzo-Moons M 2004). More than 60 different experimental models of IBD have been developed in the past two decades. Data collected confirmed the need of the presence of normal enteric flora to develop experimental colitis (Cash and Hooper 2005; Macpherson and Harris 2004). However, none of the developed models mimics precisely the human disease (Hoffmann et al. 2003; Jurjus et al. 2004). Therefore, more studies are needed to further develop this area of research. The iodoacetamide-induced acute UC model in rat developed by Satoh et al. (Satoh et al.1997) has been extensively studied in our laboratory from different angles (Prez-Navarro et al. 2005; Mourad et al. 2001; Jurjus et al. 2006). In this model, an array of morphological and functional alterations has been described, however, the model lacked chronicity. This study

Figure 1. Time frame and schedule of the experimental design. 185

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reports the success in inducing a chronic UC model using the combined synergistic effect of iodoacetamide, a sulfhydryl group blocker, and enteropathogenic E. coli (EPEC), a strain with adhesion property, instilled repetitively in the descending colon of rats. Materials and methods Animals: A total of 158 adult male Sprague-Dawley rats, weight range 200+25 g, were used in this experiment in accordance with the criteria set for care and use of animals by the Institutional Animal Care and Use Committee at the American University of Beirut. Animals were housed in rack mounted cages, with a maximum of 10 rats per cage, and kept on a 12 hours light/dark cycle in a controlled temperature and humidity room. Standard laboratory pelleted formula and tap water were provided ad libitum. Induction of experimental colitis: The rats were randomized into 4 groups: (i) the methylcellulose treated (MC) control group (n= 37), the animals were inoculated intrarectally on a weekly basis with 100 l of 1% methylcellulose (MC) the vehicle (Sigma, M-0512, USA,) as scheduled in Figure 1; (ii) The iodoacetamide-treated (IA) group (n=42), in this group the rats were inoculated with 100 l of 6% iodoacetamide (IA) (Sigma, I-6125, USA) dissolved in methylcellulose based on the previously described experimental model by Satoh in 1997; (iii) The bacteria-treated (B) group(n=37), this group was inoculated with 200 ml suspension containing 4x108 colony factor unit of EPEC; (iv) The combined treatment group (n=42), a combination of iodoacetamide and bacteria (IA+B); this group was inoculated intrarectally on a weekly basis with the same doses of IA followed by bacteria after 48 hours. Experimental colitis was induced by regular weekly intracolonic inoculation, 7 cm proximal to the anal verge using 2 mm diameter polyethylene tubing. On day 30, however, each group was split into 2 subgroups. In one subgroup inoculation of the different treatment continued as preplanned and in the second subgroup it was discontinued. Three rats from each group and later subgroup (after 30 days), at each time point, were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg/kg body weight) and sacrificed on days 3, 7, 14, 21, 42, 56, and 70. The collected tissues were stored at 80 C for analysis Clinical course assessment: The different groups were characterized using the various criteria commonly reported in the literature to describe inflammatory bowel disease, in general, and ulcerative colitis,
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Table 1. Criteria for macroscopic grading of the experimental colitis model

in particular. The parameters used in this study covered reported signs and symptoms. The animals were observed on a daily basis and checked for diarrhea, loose stools, gross rectal bloody stools, or any other gross abnormalities. The weight of each animal was registered on a weekly basis to check for weight loss after induction of colitis. Such observations were reported as numerical scores (see table 1). Macroscopic assessment: Observation and evaluation of inflammation were performed according to reported and modified criteria by Jurjus et al. (2006) for colonic changes, Table 1. Parameters like diarrhea, hyperemia, adhesions, ulceration and megacolon were assessed to describe the inflammatory status. Each colon was assigned, in a double blind way, a score on a scale ranging from 0 (normal) to 15 (maximal activity of colitis) indicating ulcerations and severe inflammation of the colon.
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Table 2. Criteria for microscopic grading of a chronic model of experimental colitis

Microscopic assessment: The descending colon was removed and immersed in cold phosphate buffer (PBS) pH 7.4. One cm piece was removed from the colon proximal to the ulcer site, immediately immersed in 10% buffered formalin and processed for routine light microscopy according to standard procedures. Serial 5m sections were cut and stained with hematoxylin and eosin (H&E) and Periodic Acid Schiff (PAS) using standard methods. The microscopic alterations were checked according to criteria adapted and modified from Jurjus et al. (2006), Table 2, and a numerical score of colonic changes was reported accordingly. Histological grades, ranging from 0 (normal) to 18 (intense inflammatory reaction), represent the numerical sum of scoring criteria. The scoring was based on observations made by 2 independent researchers on six sections from each colon. Thus, the scores reported represent the average of 36 readings (2 observers, 6 sections per animal and 3 animals per time point). These changes were photographed and reported as numerical scores.
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Colonic inflammation assessment by myeloperoxidase activity (MPO): The assessment of MPO activity in the mucosa scrapings was, therefore, carried out as a quantitative marker for granulocytic infiltration in the colonic tissue. Protein concentration and quantification were determined in the mucosal scrapings of the descending colon using the protein assay reagent kit (Bio- Rad). One unit of MPO activity was defined as the quantity of enzyme able to convert 1 mmol/min of hydrogen peroxide at 25 oC as defined by Zheng et al. (1996). The results were expressed as MPO units per gram of wet tissue. Analysis of mRNA and protein expression: Reverse Transcription-PCR (RT-PCR). Total RNA was extracted from colonic tissues by TRIR (Invitrogen) reagent. RNA was resuspended in RNase free water quantified and subjected to RT-PCR reaction using RT-PCR kit; Reddy Mix Version (Abgene, Promega). RT-PCR was performed in a final volume of 25 l containing 12.5 l Reddy mix that contain (optimize reaction buffer, dNTP mix, MgCl2 and DNA polymerase), 0.5 l of 25 pmol specific primers that have already been described for TNF-a, and -actin, 0.5 l of reverse transcriptase and 1 g of RNA was reverse transcribed at 48C for 30 minutes, and heating them for 2 minutes at 94C. Then 45 cycles of PCR for TNF- a and 25 cycles for actin using the following conditions; denaturation of 94C for 30 s, annealing temp of 55 C for 60 s and extension temperature of 68 C for 2 min, followed by a final extension step at 68C for 7 min. The primers were TNF-a sense, 5- AGA ACT CCA GGC GGT GTC C- 3. TNF-a antisense, 3- GAT TCC TGT GGG GAC TCC C T- 5 (484bp). -actin sense, 5- AAC CCT AAG GCC AAC CGT GAA AAG- 3; -actin antisense, 3- ATA CAA CGG GAT CTG AAG CTC G - 5 (540bp). PCR products were separated in 1.5 % agarose gel electrophoresis and visualized by ethilium bromide staining (1 g/ml ). DNA product sizes were estimated relative to 100 bp DNA ladder. Control reaction were run to rule out contamination of RNA with genomic DNA, in which reverse transcriptase was omitted from the reaction mixtures. Transcripts were normalized to the corresponding -actin band and expressed as arbitrary density units. Western Blot Analysis: Protein isolation was carried out by lysing colonic mucosal scrapings in 1.5 ml homogenization buffer (NaCl 11.7g/l, MgCl2-6H2O 1g/l, EDTA 0.76 g/l KCl 0.37g/l, Tris 24.2 g/l, pH 7.4) using polytron homogenizer on ice. Then, the homogenates were subjected to 30 sec sonication and centrifugation at 12,000 rpm for 10 min at 4C. The protein content of the supernatant was quantified by Bio-Rad Reagents. The protein samples diluted in sample buffer 2X (10% glycerol, 5% betamercaptoethanol, 4% SDS, 125mMTris-HCL 1M pH 6.8 and few traces of bromophenol blue) were loaded as 40 g/lane in 12% SDS-acrylamide
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gels, separated by electrophoresis, and electrotransfered to nitrocellulose membranes (Bio-Rad). To detect the specific protein, the TNF-a Rabbit polyclonal anti-rat (Chemicon) antibody was used at the concentrations recommended by the manufacturers. Equal loading of the protein samples was confirmed by parallel Western blots for GAPDH. The intensities of the protein bands were quantitated by image scanning the X-ray films and measuring each band by Image J software (NIH imaging software). Correction was done by subtracting for level background and normalizing against GAPDH protein level. Data or Statistical Analysis: Statistical significance of differences between treatment and control groups was determined by the Students t test. Where applicable, p-values were reported for the 3 independent comparisons Differences were considered statistically significant for p< 0.05. Values are presented as the mean SE. Results Data coming from this study showed that the synergistic effect of repetitive intracolonic instillation of iodoacetamide and EPEC, as outlined earlier, resulted in a chronic model of experimental colitis whereby the inflammatory process was sustained for the total period of the experiment, a hundred days. 1. Induction and clinical Course 1.1. Changes in animals weight: As shown in Figure 2A and B the rats in the control, bacteria or iodoacetamide-treated groups had a trend of increase in weight significantly higher than the rates recorded in rats treated with the combined treatment (iodoacetamide and bacteria) in both continued and discontinued injection subgroups. The rats in the combined treatment group (IA+B) had significantly (p<0.005) the lowest rate of increase in weight gain. It was 0.15 g/d compared to 1.83 g/d for control, 1.14 g/d for iodoacetamide, and 1.48 g/d for bacteria. Similar results were also obtained with discontinued injection subgroups 0.13 g/d in the combined treatment (IA+B) compared to 1.5 g/d in the normal, 1.37 g/d in the IA and 1.45 g/d with the bacteria. Such a decrease in weight gain is in support of chronic UC, a phenomenon not obtained in the iodoacetomide-treated animals once inoculations were discontinued after four weeks. Furthermore, two way ANOVA analysis for two experimental parameters (i.e. treatments and days) detected the presence of significant differences among the different experimental groups (p<0.005) as well as among the various treatment intervals (p<0.005) for their effect on rat body weight.

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Figure 2. Change in rats average weight in various experimental groups, in the continued (A) and discontinued (B) injection subgroups. r = represents, the correlation coefficient of weight change vs time. All readings were statistically significant (p<0.005). 191

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1.2. Inspection of the Stools Throughout the experimental duration, control animals were having normal beaded compact stools. Similarly, all the animals injected with the bacteria suspension of EPEC alone did not exhibit abnormal stools except in some cases 1 or 2 days post inoculation whereby loose stools were observed. In contrast, the iodoacetamide-treated group showed a pattern whereby the diarrhea consistently reappeared for 2 to 3 days post instillation of IA then changed later to loose stools. However, when the animals were divided into the continued and discontinued subgroups: the iodoacetamide-treated discontinued injection subgroup exhibited a pattern similar to the control group starting 1 week following the last IA injection and continued as such for the rest of the experimental duration. The iodoacetamide-treated continued injection subgroup maintained to show diarrhea for the first 2 to 3 days after the injection in almost all animals then loose stools and sometimes beaded compact stools for the rest of the week until the following injection. On the other hand, the animals in the combined treatment group (IA+B) both continued and discontinued subgroups, developed symptoms and signs in a very consistent manner. They all appeared sick and had severe diarrhea and bloody stools at some stages of the experiment, a feature rarely seen in the iodoacetamide-treated discontinued subgroup. These symptoms may be correlated with and resulted in the lowest rate of weight increase observed in combined treatment group (IA+B). 1.3. Macroscopic Findings 1.3.1. MC-treated group (Control Group) The findings were considered as the normal baseline. The reported data are averages of three observations per time point per group. Rats in the control did not show any inflammatory sites in the descending colon, however, the whitish site of injection, 0.5 cm in diameter, was easily identified. The average ulcer score was 2 0.9 out of 15 indicating the absence of ulcer formation (Figure 3). This observation was noted althrough the experimental duration in both continued and discontinued subgroups, (Figure 4A). 1.3.2. B-treated Group The findings in this group were similar to the control group except for occasional vasodilatation and a white granulomatous site of inoculation (Figure 4B) with an overall average ulcer size 0.5 cm. The overall average score for this group was 2.6 0.96 out of 15 as illustrated in Figure 3. 1.3.3. IA-treated Group In this group, perianal redness was sometimes noticed prior to sacrifice. Ascending and transverse colon were invariably normal. At most time points, inflammation was restricted to the descending colon including sometimes the jejunum around the site of injection.
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Figure 3. Macroscopic assessment. Overall average colonic score at different time intervals of the various experimental groups both in the continued and discontinued injection subgroups. (Readings represent average of days 3,7,14,28,42,56, and 70 in each group).

There was obvious dilatation of many blood vessels and hyperemia around the site of the injection and many times adhesions (Figure 4C). The size of the injection site was 1 to 1.5 cm. The overall average score of mucosal damage for the various time points in the continuous injection subgroup was 7 0.9 out of 15, while the overall average core value in the discontinued injection subgroup was 5.2 1.6 out of 15, correlating with less severe symptoms than the continued subgroup (Figure 3). 1.3.4. IA+B-treated Group Perianal redness was consistently observed accompanied by stains of stools and blood. Palpating protruding structures through the abdominal wall of the rat prior to surgery was clearly noticeable. The dilated descending colon was severely inflamed. The animals exhibited multiple abdominal adhesions over the descending colon with extensive generalized dilatations of abdominal vessels althrough the duration of the experiment (Figure 4D). Sometimes megacolon was also observed in this group and at times toxic megacolon was seen particularly in the continuous injection subgroup. It is important to note that the descending colon was hyperemic with vasodilatation starting from day 3 and throughout the
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experimental duration (Figure 4D). The mucosa was always thick with a larger ulcer (2-3 cm2). The overall average score was 13.5 1.4 for the continued and 12 1.8 for the discontinued injection subgroups (Figure 3). In brief, the ulcer score in the control group was significantly lower than that in the iodoacetamidetreated group (p<0.005) and the combined treatment group (P<0.005) but not significantly different from the bacteria-treated group. Moreover, there was significant difference in ulcer score between the iodoacetamide and the bacteria-treated groups (p<0.005) as well as between the combined treatment and the iodoacetamide group (p<0.05). Furthermore, the score combined treatment group was significantly higher than that in the bacteria-treated animals (p<0.05). 2. Microscopic Findings: This model was further characterized by assessing the microscopic alterations focusing mainly on the descending colon especially proximal to the site of inoculation and its surrounding and compared them with commonly reported colonic alterations in chronic ulcerative colitis cases. All layers of the colon were thoroughly studied and reference was made to normal intestinal mucosa taken from control animals. 2.1. MC and B-treated Groups The mucosal architecture of the MC group was considered as normal. There was no ulceration of the epithelial lining. The crypts and the lamina propria inflammatory infiltrate were also considered as normal baseline (Figure 4 E). The overall histologic average was 2 1.2 (Figure 5) with no significant difference between the continued and discontinued injection subgroups. A similar description was ascribed to the B-treated group with an average score of 1.89 1.1(Figures 4F and 5). 2.2. IA-treated Group The iodoacetamide-treated continued injection subgroup showed focal mucosal ulceration and depletion of epithelial lining for about 1 cm (Figures 4G and 5). In addition, there was a massive infiltration of inflammatory cells and increase in the gut-associated lymphoid tissues. The crypts were partially destroyed, hypertrophied, surrounded by inflammatory cells infiltrate and edema (Figure 4G). The mucosa and submucosa were filled with cells such as neutrophils, lymphocytes, macrophages, eosinophils, and clusters of mast cells. The overall average histologic score for IA-treated animals was 12 1.35. However, when the treatment with IA was stopped, the tissues regained their normal like status to a great extent starting 1 week after discontinuing the injections. The epithelial lining was almost back to normal. Less inflammatory infiltrate, scattered mast cells and no vasodilatations were observed. The overall histologic score for IA-treated discontinued injection subgroup animals was 9 1.
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2.3. Combined (IA+B)-treated Group The inflammation was very severe proximal to the site of injection whereby all four layers of the colon wall were frequently affected. Histological examination revealed the

Figure 4. Representative photographs of macroscopic (upper row) and microscopic (lower row) findings on day 70 according to treatment category MC, B, IA, IA+B. Figures (A) and (B) depict views of a normal colon, however, in colons of B-treated note the granulomatous whitish site of multiple inoculation of the EPEC (). (C) note the vasodilatation and enlargement of the colon (forceps) in the IA treated rats. (D) note adhesions (*) hyperemia and vasodilatation of an enlarged colon and a darkened site reflecting blackish mucosa () in the IA+B treated animals. (E&F) show normal microscopic appearance of the colon in the MC and B-treated groups. Well aligned parallel crypts () continuous epithelial lining () normal muscularis mucosa and lamina propria infiltration by cells(*). (G) shows cryptitis (), infiltration of inflammatory cells (+) and edema (*) in the submucosal with thinning of the muscularis mucosa and vasodilatation(). (H) depicts obvious crypts deformities and bifurcation (), cryptitis(), extensive inflammatory cells infiltrate (+) and edema (*). 195

Inaya Abdallah Hajj Hussein, Rania Tohme, Kassem Barada, Mostafa Hassan Mostafa, Angelo Leone, Jean-Noel Freund, Rosalyn A. Jurjus, Walid Karam, and Abdo Jurjus

Figure 5. Microscopic Assessment: Overall average histological score in the various experimental groups both in the continued and discontinued injection subgroups. (Readings represent average of days 3,7,14,28,42,56, and 70 in each group).

presence of a diffuse distortion in the mucosal architecture with marked crypt atrophy and extensive infiltration of mononuclear cells (Figure 4H). The mucosal surface was irregular, villiform and heavily infiltrated by various types of inflammatory cells exceeding the submucosa. The crypts reached a pathological state compared to their control counterparts by exhibiting prominent loss of crypt parallelism and irregularity in crypt size, spacing and shape. In addition, cryptitis and crypt abscesses were present (Figure 4H). The mast cells were clustered in the submucosa. The sites affected by inflammation were edematous, ulcerated, and devoid of any glands. Furthermore, the muscularis mucosae were gradually thinning out or absent. The surface of the ulcer defect was covered with large cellular debris (necrotized tissue and products of inflammation). This was the case throughout the duration of the experiment period in both subgroups. The overall histologic score was the highest in the continued injection subgroup, with an average of 16.2 0.9 and 15.4 1.7 for the discontinued injection subgroup (Figure 5). 3. Myeloperoxidase Activity: The determination of the MPO activity was considered as a good estimate of the intestinal inflammatory status. This activity was consistently the highest in the combined (IA+B) treatment group with regular continuous inoculation at all time points from day 7 to day 30 and maintained this profile of high activity up to the last day of the experiment (Figure 6).
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Figure 6. Average MPO activity in the various experimental groups in the continued (A) and discontinued (B) injection subgroups. (Readings represent average of days 3,7,14,28,42,56, and 70 in each group). P<0.005.

One week following induction of inflammation, the MPO activity in the combined (IA+B) treatment group reached an average of 41 2.2 U/g tissue which was significantly higher than the respective control values (11.7 2.1, p < 0.005) and almost twice the values of the bacteria-treated group (28.5 2.8, p < 0.005) and those of IA-treated alone (23.2 7.7, p < 0.005). On the other hand, the MPO activity in the IA (23.2 7.7) and B (28.5 2.8) treated animals were 2fold higher than that in the MC-treated group (Figure 6). A pattern similar to that observed at day 7 was also noted at days 14, 21, 28, 42, 56 and 70, respectively (Figure 6). In general, in the continued injection experiment the MPO activity score in the control group was significantly lower than all the other groups (p<0.005) but not significantly different from the bacteriatreated group. Furthermore, the combined treatment group was
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significantly higher than that in the bacteria-treated animals (p<0.05). Even in the discontinued injection subgroups, there was a significant difference in MPO activity score between the control group and the combined treatment group (p<0.05) In brief, the combined treatment group had a significantly higher MPO activity score than the iodoacetamidetreated groups (p<0.05) (Figure 6). 4. Inflammatory Signaling: TNF-a Expression TNF-a is a prototypical proinflammatory cytokine secreted by activated macrophages, monocytes and chronically activated T lymphocytes in the intestine. It is well documented as being a key regulatory factor in various inflammatory processes involved in the pathogenesis of ulcerative colitis. Upregulation of the proinflammatory cytokine TNF-a is an upstream modulator of COX-2 increased expression during ulcerative colitis. These data are similar to several studies that have reported an increased TNF-a expression at both mRNA and protein levels in patients with IBD. In this study, levels of TNF-a mRNA expression were assessed as arbitrary density units (ADU) in relation to the endogenous level of b- actin. 4.1 TNF-a mRNA Expression On day 7, the increased expression of TNF-a mRNA reached 7-to-8 fold in the combined treatment (IA+B) group when compared to the other groups (Figure 7a). On day 28 there was a significant transient rise of TNF-a expression in the IA-treated group reaching 7-fold higher than that in the B-group, slightly higher than that in the combined (IA+B) group which was slightly downregulated (Figure 7b). On day 56 there was a transient overall decline in the expression of the TNF-a mRNA in the continued injection subgroup followed by an elevation in the levels of the transcript at day 70 post-treatment (Figure 7c and f). However, in the discontinued injection subgroup, there was a time-dependent decrease in the TNF-a levels following day 28 to day 70 post-treatment, and, on day 56, levels of TNF-a mRNA decreased in all groups (Figure c to f). The combined treatment (IA+B) continued injection subgroup maintained a relatively higher level of the TNF-a mRNA compared to IA-treated or the B-treated and MC-treated control groups. However, the discontinued injection subgroup exhibited expression of mRNA below the detection limits (Figure 7c and d). During the course of the experimental duration, the increase in the TNF-a mRNA reached its peak in the combined treatment (IA+B) group both (in the continued and discontinued injection subgroups) except for the transient rise in the iodoacetamide-treated group at day 28 (Figure 8b). However, it is important to note that after stopping the inoculation, the levels of TNF-a mRNA expression decreased in the discontinued compared to the continued injection subgroup but it remained at all time points more expressed in the combined treatment (IA+B) subgroup (Figure 8).
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Figure 7. Representative Photoghraphs from three independent experiment of TNF-a mRNA expression in the descending colon in the various experimental groups at selected time points. Band intensity was adjusted for the corresponding b-actin, and values were expressed as arbitrary density units (ADU). 199

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Figure 8. Expression of TNF-a mRNA in the descending colon in the various experimental groups both in the continued (A) and discontinued (B) injection subgroups, in response to treatment at all time points. 200

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Figure 9. Representative Photographs of TNF-a protein expression in the descending colon in the various experimental groups at selected time points. Band intensity was adjusted for the corresponding GAPDH, and values were expressed as arbitrary density units (ADU). 201

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Figure 10. Expression of TNF-a protein in the descending colon in the various experimental groups both in the continued (A) and discontinued (B) injection subgroups, in response to treatment at all time points. 202

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4.1.2. TNF-a Protein Expression Western blot analysis of TNF-a protein expression showed that at 7 days post treatment, all groups exhibited close levels of TNF-a protein. In both MC and B-treated groups the protein levels were 2-fold lower than the protein levels in the IA-treated and the combined treatment (IA+B) groups (Figure 9). Across the experimental duration, TNF- a protein expressed the highest levels in the combined treatment (IA+B) continued injection subgroup (Figure 9 a to f and 10A), whereas in the discontinued injection subgroup all animals had decreased expression following the cessation of the injections (Figure 9B). It is worth mentioning that the combined treatment (IA+B) discontinued injection subgroup maintained relatively the highest values of TNF-a protein levels. In general, TNF-a protein and mRNA expression levels correlated across the experimental groups and subgroups indicating an effect of the treatment on both post-transcriptional and post-translational processes of TNF-a. Discussion For the past three decades, numerous models of UC, with a variable range of clinical manifestations similar to those observed in human IBD, have been published, however, none of these resembled closely enough the clinical entity of human UC (Jurjus et al. 2004). Indirect evidence for the interaction between luminal flora and the immune system exists from studies using such animal models mostly based on disruption of the immunoregulatory mechanisms (Cash and Hooper 2005; Macpherson and Harris 2004; Strober et al. 2002) The results of the present study demonstrate that the present experimental UC model, using a SH blocker and an enteropathogenic E. coli, resembles the human situation and is reproducible with characteristics indicative of chronicity. The clinical course of the induced disease for almost a hundred day and the major symptoms of loss in body weight (Figures 2A and B), diarrhea and rectal bleeding are among the characteristics of our model. The clinical profile of colitis was further supported by macroscopic findings (Figure 3 and 4) and histological alterations (Figure 4 and 5) typical of chronic ulcerative colitis in human (Salem and Truelove1965; Crawford and Kumar 2003; Zheng et al. 2000). Moreover, the analysis of colonic tissue myeloperoxidase activity showed a consistent elevation of activity in the combined IA+B treatment group, an observation indicative of severe inflammatory processes in the mucosa of the descending colon (Figure 6) (Zheng et al.1996). Analysis of body weight gain across the various experimental groups and subgroups revealed that rats treated with a combination of IA+B exhibited the lowest rate of growth per day in both the continued and the discontinued injected subgroups compared to MC, IAtreated discontinued injection subgroup and B treated group. In addition, the IA continued injection subgroup behaved similarly to combined treatment continued injection subgroup
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but with a higher overall growth rate. Once the injection for the IA-treated subgroup was discontinued (day 28), the growth rate increased again. Moreover, the combined treatment group showed an undulating course of increase and decrease in weight, which might indicate periods of relapse and remission of the disease. Such a decrease was consistent in both continued and discontinued combined treatment subgroups as well as in the iodoacetamide-treated continued injection subgroup. On the other hand, the iodoacetamide-treated subgroup with discontinued injection behaved like the controls after stopping the injection (Figure 2B). The low weight gain (e.g. in the IA+B group) is due probably to malabsorption to a defective barrier. This barrier could not be restored appropriately and there may be persistent activation of mucosal inflammation (Mayer 2000) and malabsorption of nutrients in addition to diarrhea affecting the physiology of other parts of the gut. Actually, changes in weight gain in the experimental groups (IA+B) were paralleled by increased rates of diarrhea, loose and bloody stools, and sometimes swollen abdomen and megacolon. The outcome of abdominal inspection showed that, the combined treatment group exhibited tenderness of the descending colon as well as various degrees of megacolon. These observations provide further support to the eligibility of our experimental protocol in inducing UC and its validity as a novel animal model for the disease. Besides, the IAtreated animals of the continued injection subgroup showed similar findings but were not as consistent, in particular with the megacolon. This distinct feature was not encountered in the other B-treated or MC control groups. The clinical course of the combined treatment induced UC was further supported by the reported high range of scores for macroscopic alterations. These included a generalized vasodilatation, adhesions, enlargement of the descending colon and ulcer formation (Figure 4). This observation suggests that the continuous stimulation by iodoacetamide is necessary for maintaining the inflammatory process. In this case, the score range in the iodoacetamide-treated and combined treatment indicates respectively a moderate to severe inflammation in the descending colon (Figure 4C and D) and is consistent with commonly reported clinical findings in UC (Podolsky 2002; Satoh et al.1997). In general, the gross observations reported in the combined treatment group were more consistent and more characteristic of chronic UC. There was an extended hyperemia with adhesions and megacolon, ulceration, vasodilatation, and a particular redness in the nearby segment of the jejunum. The overall score was between 10-15 in the combined IA+B treatment animals with continued injection subgroup treatment, while the score in the discontinued injection subgroup ranged from 9-14. Therefore, despite discontinuing the injections in one of the subgroups, the inflammatory process maintained its course, a point of major interest for further investigation.
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Furthermore, the microscopic findings in the descending colon revealed the presence of a plethora of changes in the histology, particularly in the combined IA+B treatment group, followed by the IA-treated group with continuous injection (Figure 4). Such changes were mostly in the mucosa. This observation is in line with the common description of UC as a disease of the mucosa. However, in few cases, in inflammation the combined IA+B treatment group was very severe and in some cases all four layers of the colon (the mucosa, submucosa, musculosa, and serosa) were altered. Histological assessment of colonic damage showed that althrough the entire duration of the experiment; the combined IA+B treatment group had the highest histologic score, in both continued and discontinued subgroups (between 8 to 16). These changes were less pronounced in the iodoacetamide groups and were minimal to lacking in the B-treated and MC-treared control groups (Figure 5). Therefore, changes in the microscopic structure of the combined treatment subgroups mimicked the changes commonly encountered in severe cases of UC (Dotan and Mayer 2002; Farrel and Peppercorn 2002; Terashima et al. 2001). These include extensive hyperemia and loss of epithelial lining, ulceration of the mucosa, severe depletion of goblet cells, loss of crypts and sometimes crypts abscess, cryptitis, with dense inflammatory cell infiltration, severe dilatation of several blood vessels, loss or thinning of muscularis mucosa, and invasion by lymphoid tissues (Satoh et al.1997; Szabo et al.1981). In addition, MPO activity which gives a reproducible and qualitative estimate of inflammation, and its determination reflects the average level of inflammation, in the mucosa (Lu et al. 2003) could serve as a quantitative index of disease severity. At the site of injury, MPO content was utilized as a marker of the quantity of neutrophils, which had migrated to the lesion site during the infections. For this reason, interpretation must be tempered by the concept that MPO level reflect the minimum number of blood neutrophils which must have reached the lesion to contribute their MPO, and that the measurement may represent an underestimate of the total migration of neutrophils to the lesion. The combined treatment exhibited the highest MPO activity in the descending colon (Figure 6). It is therefore, clear that the degree of inflammation was the highest in the combined IA+B treatment group; in particular the subgroup with continued injection. This inflammatory activity was maintained at a much higher rate than in the control, B-treated or the IA-treated groups. However, it is important to note that once injection was discontinued, the combined IA+B treatment subgroup maintained its highest activity compared to the other groups. This observation provides further support of the notion that the continuous inflammatory process was expressed in the combined treatment group regardless of continued or discontinued injections. Once again, these findings go in line with the characteristics of chronic UC (Dotan and Mayer 2002; Farrel and Peppercorn 2002). Further characterization of the studied model was carried out at the molecular level to evaluate the expression of the inflammatory signaling. The alteration in the inflamma205

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tory signals was evaluated by analyzing the mRNA and protein expressions of TNF-a, a proinflammatory cytokine which can induce the production of COX-2 and COX-2 protein expression in the experimental groups. It is well established that TNF-a, a proinflammatory cytokine released from monocytes and macrophages, play an important role in ulcerative colitis (Buset 1996; Borudy et al. 2003; Kidd 1997). It is worth noting that increased local expression of TNF-a is of prime importance in driving chronic inflammation and mediating tissue injury (Nazli et al. 2004). There is significant correlation between the production of TNF-a and the severity of UC. High levels of proinflammatory cytokines in the mucosa lead to the excessive production of matrix degrading enzymes by gut fibroblasts, loss of mucosa integrity and ulceration. In UC, high levels of TNF-a have been documented in the lamina propria of IBD patients and increased TNF-a mRNA and protein expressions have been detected in the intestinal mucosa of IBD particularly associated with tissue injury (Buset 1996; Borudy et al. 2003). It is the most important mediator of the response to Gram-negative bacteria and also plays a role in the immune response to other microorganisms (Ishigura 1990). Accumulating evidence indicates that either enteric bacteria or immune dysfunctions play a pivotal pathogenic role (Amati et al. 2003; Rogler and Andus 1998). Endotoxins or lipopolysaccharides (LPS), derived from the outer membrane of Gram negative enteropathogenic E.coli interact with CD14 on the surface membrane of mononuclear cells, thus triggering a signal cascade that leads to the production and release of TNF-a, strongly involved in the pathogenesis of ulcerative colitis (Bing et al. 1998). In this respect, both TNF-a and LPS may represent a putative therapeutic target for the treatment of UC (Kusugami et al. 1995; Cohen et al. 1996; Van Damme et al. 2001; Goncalves et al. 2001). Results of the present study demonstrated that the mucosal scrapings of the colon contained significantly elevated levels of TNF-a mRNA and protein in combined treatment (IA+B) both subgroups and at all time points compared to control (Figure 7-10). Occasional rise of TNF-a mRNA expression was noted, however, in the IA-treated group (Figure 7b and f, and days28 and 70). The deficits leading to chronic mucosal inflammation can be grouped into two; defects in pathways involved in immune regulation and defects in pathways involved in barrier function of the epithelium (Fries et al.1999; Nazli et al. 2004; Schuppler 2004). Models of disrupted barriers function have been demonstrated to develop mucosal inflammation (Buset 1996; Nazli 2004; Schuppler et al. 2004). Chemical agents such as acetic acid, dextran sodium sulfate, and iodoacetamide, can injure the epithelium. In a normal animal all means of chemical injuries result in a transient inflammatory process. Repair mechanisms are natural and a stable mucosal barrier is re-established. Indeed, a leading concept in the pathogenesis of chronic intestinal inflammation is the break in mucosal tolerance, an
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active process by which an injurious immune response is prevented, suppressed or shifted to a non-injuring class of immune reaction (Nazli et al. 2004). The intestine is in permanent contact with billions of bacteria belonging to the normal intestinal flora, food protein, and potentially pathogenic bacteria and has to discriminate and define selective action towards pathogenic and non-pathogenic components. Mucosal tolerance exists in order to prevent an immune response against the bodys own bacteria that would otherwise give rise to chronic intestinal inflammation. EPEC colonize the intestinal mucosa and, by subverting intestinal epithelial cell cytoskeleton function, produce a characteristic histopathological feature. EPEC with invasive properties exert their main impact on the host by causing gross destruction of the epithelial architecture and tight junction proteins. Thus the regular presence of the chemical in addition to the EPEC could create an optimal environment for chronic UC to develop and sustain (Nazli et al. 2004; Porras et al. 2004). With increasing evidence of alteration in colonic microecology, which might result in overgrowth of potentially pathogenic bacteria, interest is growing in the potentially beneficial effects of manipulating the microbial environment in the intestine through the use of probiotics. Most recently, it has been proposed that treatment with probiotic agents might outcompete pathogenic bacteria and prevent their adherence, increase in the intestinal IgA immune response, enhance mucins expression and was able to prevent the development of colitis in animal models (Campieri and Gionchetti 2001; Farrel 2002). Concluding Commentary: The hypothesis of this work was based on several lines of indirect evidence implicating the normal gut flora in the pathophysiology of inflammatory bowel disease (Nazli et al. 2004; Schuppler 2004). These data were coupled with increased evidence that epithelia under metabolic stress from a chemical or bacterial agent could get to a status of chronic inflammation. In the present model, iodoacetamide, a Sulfhydryl group blocker was the chemical agent instilled in the descending colon of the rats followed by a load of the bacterial agent enteropathogenic E. coli. Consequently, the analysis of the different relevant parameters (clinical pictures, histological changes, and cytokine) were consistent and supported the objectives of the study be it the induction of a chronic experimental UC model with partial characterization. The gathered evidence indicates that the defects of intestinal permeability induced by iodoacetamide may facilitate the passage of LPS derived from the Gram-negative enteropathogenic E.coli or other enteric bacterial flora into the circulation. It is speculated that enhanced intestinal permeability, probably caused by the inflammation induced by iodoacetamide, could precede and prepare the ground for the development of UC in the rats especially that, an inflamed colon is a site of increased intestinal permeation as described
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in patients suffering from UC. In conclusion, developing animal models to study the human ulcerative colitis is critical for elucidating the molecular pathways by which this disease develops and progresses to advanced stages. Indeed, identifying the molecular pathways initiating this pathological disorder may allow us to gain an insight into the strategy (ies) that can lead to IBD prevention. Furthermore, the present study may permit the characterization of molecular and morphologic effects of the disease that may either enhance our ability to develop therapeutic agents with higher efficacy or lesser side effects in humans. Furthermore, outcome of such a study could result in an eventual clinical assessment of agents that target molecular pathways known to play a role in the initiation of the IBD in this subset of subjects, specifically those asymptomatic individuals at high-risk for a later development of the disease.

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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Current Opinion in Gastroenterology 22: 30-43, 2006. Dotan I, and Mayer L. Immunopathogenesis of inflammatory bowel disease. Current Opinion in Gastroenterology 18: 421-427, 2002. Farrel RJ, and Peppercorn MA. Ulcerative Colitis. Lancet, 359: 331- 40, 2002. Jankey N, and Price L. Small intestinal histochemical and histological changes in ulcerative colitis. Gut 10: 267-269, 1969 Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 115: 182-205, 1998. Campirei M. and Gionchetti P. Bacteria as the cause of ulcerative colitis. Gut, 48: 132-135, 2001. Rath C. Role of commercial bacteria in chronic experimental colitis: lessons from HLA-B27 transgenic rat. Pathology 70: 131-138, 2003. Nazli A, Yang PC, Jury J, Howe K, Watson JL, Soderholm JD, Sherman PM, Perdue MH, and McKay DM. Epithelia under metabolic stress perceive commensal bacteria as a threat. Am J Pathol 164(3): 947-957, 2004. Schuppler M, Lotzsch K, Waidmann M, and Autenrieth IB. An abundance of Escherichia coli is harbored by the mucosa-associated bacterial flora of interleukin-2-deficient mice. Infect Immun 72(4): 1983-1990, 2004. Yu J, and Kaper JB Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol 6(3): 411-417, 1992. Goosney DL, Knoechel DG, and Finlay Brett. Enteropathogenic E. coli, Salmonella, and Shigella: Masters of Host Cell Cytoskeleton Exploitation, Emerging Infectious Diseases 5: 1990. Nougaryde JP, Fernandes PJ and Donnenberg MS. Adhesion pf enteropathogenic Escheria coli to host cells. Cellular Microbiology 5: 359, 2003. Muzo-Moons MM, Schneeberger EE, and Hecht GA. Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells. Cellular Microbiology 6: 783-793, 2004. Cash H, and Hooper L. Commensal bacteria shape intestinal immune system development. ASM News 71: 77-84, 2005. Macpherson AJ, and Harris NL. Interactions between commensal intestinal bacteria and the immune system. Nature Rev Immunol 4: 478-485, 2004. Hoffmann CJ, Pawlowski N, Khl A, Hhne W, and Zeitz M.. Animal models of inflammatory Bowel Disease: An overview. Pathology 70: 121-130, 2003. Jurjus AR, Khoury NN, and Reimund JM, Animal models of inflammatory bowel disease. J Pharmacol Toxicol Methods 50: 81-92, 2004. Satoh H, Sato F, Takami K, and Szabo S. New ulcerative colitis model induced by sulfhydryl blockers in rats and the effects of antiinflammatory drugs on the colitis. Japanese Journal of Pharmacology 73: 299-309, 1997. Prez-Navarro R, Ballester I, Zarzuelo A, and Snchez D. Disturbances in epithelial ionic secretions in different experimental models of colitis. Life Sciences 76: 1489-1501, 2005. Mourad FH, Abo Rashed N, Barada K, Homeidan F, and Nassar CF. Inhibitory effect of chemical colitis on jejunal water absorption: role of vasoactive intestinal peptide (VIP) and nitric oxide (NO). Gut 4g (Suppl 3): A1178, 2001.

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[21] Zheng L, Wang SX, and Cui DY. A simple method of assessing inflammatory bowel disease. Zhongguo Yaolixue Tongba 12: 468-469, 1996. [22] Strober W, Fuss IJ, and Blumberg RS. The immunology of mucosal models of inflammation. Annu Rev Immunal 20: 495-549, 2002. [23] Salem SN, and Truelove SC. Small-intestinal and gastric abnormalities in ulcerative colitis. British Medical Journal 1: 827-831, 1965. [24] Crawford JM, and Kumar V. The oral cavity and the gastrointestinal tract. Basic Pathology. Tth ed. W.B. Saunders Company, the Curtis Center Independence Square West Philadelphia, Pennsylvania; pp.543-590, 2003. [25] Mayer L. The development of animal models of inflammatory bowel disease. Intern Rev Immunol 19: 77-90, 2000. [26] Gordon J, Stappenbeck TS, and Hooper LV. Commensal Bacteria make a difference. Trend Microbial 11(4): 150-151, 2003. [27] Hooper LV. Bacterial contributions to mammalian gut development. Trends Microbiol 12: 129-134, 2004. [28] Podolsky DK. Inflammatory bowel disease. New England Journal of Medicine, 347: 417-429, 2002 29. Farrel RJ, and Peppercorn MA. Ulcerative Colitis. Lancet 359: 331- 40, 2002. [29] Terashima S, Hoshino Y, Kanzaki N, Kogure M, and Gotoh M. Ulcerative duodenitis acompanying ulcerative colitis [Case Reports]. Journal of Clinical Gastroenterology 32: 172-175, 2001. [30] Szabo S, Trier JS, and Frankel PW. Sulfhydryl compounds may mediate gastric cytoprotection. Science 214: 200-202, 1981. [31] Lu J, Wang A, Ansari S, Hershberg RM, and McKay DM. Colonic bacterial superantigens evoke an inflammatory response and exaggerate disease in mice recovering from colitis. Gastroenterology 125(6): 1785-1795, 2003. [32] Buset M. Inhibition of human colonic epithelial cell proliferation in vivo and invitro modulation of the phenotype by extracellular matrix. Proc Natl Acad Sci (USA), 93: 7717-7722, 1996. [33] Borody T, Warren EF, Leis S, Surace R, and Ashman MA. Treatment of ulcerative colitis using fecal bacteriotherapy. Journal of Clinical Gastroenterology 37(1): 42-47, 2003. [34] Kidd PM. Glutathione: Systemic Protectant against Oxidative and Free Radical Damage. Alternative Medicine Review 2(3): 155-176, 1997. [35] Ishigura Y. Mucosal proinflammatory cytokine production correlates with endoscopic activityof ulcerative colitis. Journal of Gastroenterology 34: 66-74, 1999. [36] Amati L, Caradonna L, Leandro G, Magrone T, Minenna M, Faleo G, Pellegrino NM, Jirillo E, and Caccavo D. Immune abnormalities and endotoxemia in patients with ulcerative colitis and in their first degree relatives: attempts at neutralizing endotoxin-mediated effects. Current Pharmaceutical Design 9: 1937-1945, 2003. [37] Rogler G, and Andus T. Cytokines in inflammatory Bowel Disease. World Journal of Surgery 22: 382-389, 1998. [38] Bing X, Crusuis J, Meuwissen S, and Pena A. Inflammatory Bowel Disease: definition, epidemiology, etiologic aspects, and immunogenetic studies. World Journal of Gastroenterology 4: 446- 458, 1998. [39] Kusugami K, Fukatsu A, Tanimoto M, and Shinoda M. Elevation of interlukin-6 in inflammatory bowel disease is macrophage and epithelial cell- dependent. Digestive Diseases Science 40: 949-959, 1995. [40] Cohen MC, and Cohen S. Cytokine functions. A study in biologic diversity. American Journal of Clinical Pathology 105: 589-598, 1996. [41] Van Damme N, De Keyse F, Demetter P, Beaten D, Mielants H, and Verbruger G. The proportion of Th1

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[42] [43] [44] [45] [46]

cells, which prevail in gut mucosa, is decreased in inflammatory bowel syndrome. Clinical and experimental immunology 125: 383-390, 2001. Goncalves N, Ghaem-Maghami M, Monteleone G, Frankel G, Dougan G, Lewis D, Simmons C, and MacDonald T. Critical role for tumor necrosis factor alpha in controlling the number of luminal pathogenic bacteria and immunopathology in infectious colitis. Infection and Immunity 69: 6651-6659, 2001. Fries W, Mazzon E, Squarzoni S, Martin A, Martines D, Micali A, Sturniolo GC, Citi S, and Longo G. Experimental colitis increase small intestine permeability in the rat. Laboratory Investigation 79: 49- 57, 1999. Nazli A, Yang PC, Jury J, Howe K, Watson JL, Soderholm JD, Sherman PM, Perdue MH, and McKay DM. Epithelia under metabolic stress perceive commensal bacteria as a threat. American Journal of Pathology 164: 947-957, 2004. Schuppler M, Lotzsch K, Waidmann M, and Autenrieth IB. An abundance of Escherichia coli is harbored by the mucosa-associated bacterial flora of interleukin-2-deficient mice. Infection and Immunity 72: 1983-1990, 2004. Porras M, Martin MT, Soler M, and Vergara P. Intestinal motor disorders associated with cyclical bacterial overgrowth in a rat model of enteritis. American Journal of Physiology - Gastrointestinal and Liver Physiology. 287: 58-64, 2004.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

THE CHRONICAL GASTRITIS HELICOBACTER PYLORI CORRELATED: IMMUNOHISTOCHEMICAL BEHAvIOUR Of NITRIC OXIDE SYNTHASE ISOfORMS IN RATS STOMACH
[La gastrite cronica Helicobacter pylori-correlata: comportamento immunoistochimico delle isoforme dellossido nitrico sintetasi nello stomaco del ratto] Maria Laura Uzzo
DI.ME.S. Dipartimento di Medicina Sperimentale - Sezione di Istologia ed Embriologia - Facolt di Medicina e Chirurgia. Universit degli Studi di Palermo (I)

Key words: Helicobacter pylori, NOS, Gastritis, Immunohistochemistry Parole chiave: Helicobacter pylori, NOS, Gastrite, Immunoistochimica Abstract. Recent non extensive studies indicate that Nitric Oxide (NO), a novel intercellular messenger, may play a role in pathogenesis of gastric lesions by Helicobacter pylori (Hp). Aim of this study is to evaluate the immunohistochemical distribution and modification of NO synthases (cNOS - iNOS) in experimentally induced gastritis by Hp in rats. Riassunto. Studi recenti hanno dimostrato il ruolo svolto dallOssido nitrico (NO) nella patogenesi delle lesioni gastriche causate dallHelicobacter pylori (Hp). Questo studio si prefissato lobiettivo di valutare il comportamento immunoistochimico delle varie isoforme NOS in ratti infettati sperimentalmente da HP in cui veniva quindi indotta una gastrite cronica.

Introduction The documentation furnished in 1983 by B.Y. Marshall and Y. Warren, which isolated, for the first time, from bioptic fragments of gastric mucosa, taked by patient affected by gastritis, in the laboratory of microbiology of the Royal Perth Hospital in western Australia, a spiraliform and flagellated bacterium, the Helicobacter pylori, produced in the scientific community the effect of a shock, but promoted an explosion of studies directed to establish the correlations among Helicobacter pylori and the digestive illnesses. These studies have established by now that this microorganism is the cause of the most greater part of the cases of chronic gastritis and that it has involved in the 95% of pathogenesis of the duodenal ulcers and of the 80% of the gastric ulcers. Such acquisitions have forced to rewrite in the more adjourned manuals of gastroenterology the chapters concerning the epidemiology and the therapy of the ulcerous illness, that, today must be considered an infectious illness which, as such, it sets as therapeutic problem the eradication of the Hp. The results of the this therapy has dramatically modified the natural history of the duodenal and gastric
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ulcerous illness: the old affirmation not acid, not ulcer ratified in 1910 by the Croatian physician Karl Swartz it has to be integrated by the verification today that if it is not present in the gastric mucosa also the Hp, the ulcer is not formed. The peptic ulcer, therefore, must be considered not more today as an acid correlated illness, but as an acid-pylori correlated illness: in other words the infection by Hp causes the mucosal inflammation and this is the presupposition to develop, with the acid presence, the ulcerous lesion. Numerous studies show that the prevalence of the infection increases with the age. In the western countries, the percentage of the infected population gradually raises from the infancy up to the 60 year-old age. The positiveness of the serum tests for Helicobacter pylori is rare in children, but is around the 20% of the individuals with age around the 40 years and in the 50% of the subjects with superior age to the 60. The prevalence of the infection in the most elderly subjects often results understimated, because the serum tests in the subjects in this band of age can give false-negative results. The actual rates of infection in the populations of the western countries could be motivated hypothesizing that the acquisition of the bacterium occurs every year in 1% of the population. However, this acquisition is not completely correct. In fact, more accurate studies have shown as the rate of acquisition of new infections, in the western populations, is around the 0,3-0,5% for year. In reality a decrease in the prevalence of the infection, rather rapid, in the western countries is observed. Therefore the tallest prevalence of the infection in the most elderly people is due to the effect cohort. In other words, the elderly people of our days have a taller prevalence of infection because they live in a period in which it was more probable to contract the infection from Helicobacter pylori [1,2]. Microbiological characteristics The Helicobacter pylori belong to Helicobacter genus, that also includes the kinds mustelae, felis and muridarum that dont infect the man and the Gastrospirillum hominis. The genus pylori is the only one in degree to colonize the human stomach. The Hp is a Gram-negative bacterium of form curve or spiral that under conditions to it unfavorable (es.in vitro) change its own bent structure in coccoid form. The dimensions vary among 2 and 6m. It is provided of 4-6 flagella originated to one of the poles of the cell, that assure motility, and of a coat that covers both the bacterial body as the flagella that results useful in the phase of colonization [3,4]. The cultivation of the Helicobacter results rather difficult to take place because of its demands nutritional, and of its weakness. It is necessary therefore the use of grounds of transport and selective grounds for the growth of the strain both with agar how liquid. Stewart or Portagerm-pylori for the transport maintaining the collecting to 4-10 C up to the moment of the seeding.
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Agar chocolate or Agar blood as non selective grounds and Skirrow or Brussels Campylobacter medium of Glupczynski as selective for the growth to be used in microaerofilia to 37C. The colonies appear after 3-7 days, they are of varying dimensions among 0,5-1mm and they appear translucent. It doesnt ferment the carbohydrates, it doesnt produce indolo and it doesnt reduce the nitrates, it is catalase and oxidase positive. As it regards the maintenance of the strain, the Hp can be preserved desiccating the bacterial suspensions on cotton, directly from the liquid state [5]. Pathogenetic mechanism of the tissutal damage However much has been verified by now; the infection by Hp is associated to active chronic gastritis, peptic ulcer and gastric neoplasia. The exact pathogenetic mechanism with the bacterium induces the mucosal damage is not still known. It must be clarified, first, that the Hp infects the most greater part of the people in which provokes a chronic gastritis, but only a small percentage (20%) of the infected subjects will develop a peptic ulcer and only in a least percentage will develop a gastric cancer or a cells B lymphoma lower degree of wickedness [3]. An explanation of this phenomenon could reside in the existence of bacterial strains with different degree of pathogenicity, endowed with potential ulcerogens or carcinogenic. Such hypothesis, suggested by numerous clinical-epidemiological observations, that doesnt exclude the incidence of extrinsic factors (smoke) and of physiological factors indirectly modified by the same infection (acid secretion), is ascribed to the genetic variability of the Hp, broadly documented by studies of molecular biology, that could be responsible of the discriminated expression of different factors of virulence first mentioned [6,7]. With this pathogenetic vision regarding the expression of different factors of virulence from genetically different strains of Hp, in the last times a considerable interest is noticed towards the immunopathogenetic role that the mediators of the mucosal inflammation can develop in the cellular answer to the infection by Hp. Has been shown how numerous proinflammatory citokines (IL-1, TNF -a, IL-8) can be released by mucosal tissue infected by Hp (epithelial cells); they could matter in the induction and perpetuation of the mucosal damage [8]. A third pathogenetic possibility in the determinism of the mucosal lesions Hp mediated, recently advanced, foresees the intervention of a new molecule from the contradictory functional personality: the nitroxide (NO) [9,10,11]. For the purpose to clarify the meaning of the nitroxidergic pathogenesis of the tissutal damage from Hp I hold necessary to premise some considerations on the biochemical characteristics and on the functional roles of the NO, limiting the intervention of this molecule
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to the field of the immune defense in progress of inflammatory trials of bacterial nature, since the infection from Hp induces a local answer that hesitates in an increase of the mucosal production of IgG and IgA, with a strong infiltration of neutrophils. The biosynthetic way that brings to the formation of the NO is based on the activation of an endocellular enzyme: the Nitroxide Sinthetase (NOS), that cathalizes the synthesis of the Nitric Oxide making hydroxylation the nitrogen of the terminal group guanidinic of the L-arginine to form NO and citrulline. NO acts on the cell target activating a molecular target: the guanilate soluble ciclase (sGC) which stimulates in the cell target the synthesis of GMPciclico (cGMP). It founds therefore among the rising cell of NO and the cell target a street of segnaletic trasduction (NO/cGMP signalling pathway) whose activation stimulates or inhibites biological effects according to the molecular mechanism primed on the effector by the same cGMP, will be realized. In reality the NOS is represented by a family of three isoformes: the nNOS the eNOS and the iNOS. The nNOS and the eNOS, originally considered expressed exclusively respectively from nervous cells and from endothelial cells, but subsequently showed present immunohistochemically in other cytotypes of epithelial and muscular nature, are constitutive (cNOS) and they produce small quantities (picomoles) of NO for brief periods of time [12,13,14]. The iNOS is expressed from macrophages immunostimulated following induction during inflammatory processes, from cytokine (IL-1, IL-2, IL-8, TNF -a) or bacterial endotoxin (LPS) or interferon, in such cases the iNOS produces great uncontrolled quantities (nanomoli) for a lot of times. This isoform is responsible of the bactericidal and tumoricidal effects that NO develops towards bacterial extraneous or neoplastic cells, but it can be responsible of the citotossic effects that NO as such or as peroxinitritic anion (ONNOO) it is able, in pathological conditions, to expound towards the self cells. Under this aspect NO has been summoned in the determinism of tissutal lesions that represents the histopathological substratum of many inflammatory events that are developed in many organic parenchima (lung, nervous system, chronic inflammatory illnesses of the digestive apparatus = IBD). With the intent to examine in the detail and in more systematic way the matter, we have experimentally conducted a study on the existing relationships among experimentally induced gastritis by HP in rat and production of NO, appraising immunohistochemically the behavior of the three isoformes of the NOS (nNOS, eNOS and iNOS) on fragments of gastric wall in rats sacrificed to various intervals of time by the beginning of the experimental bacterial inoculation [15,16]. Materials and methods We have used 28 male Wistar rats (weight ~200 gr.) distributed in two groups: The first group, constituted of 20 rats, received for five days, by gastric probe, a Helico216

The chronical gastritis helicobacter pylori correlated: immunohistochemical behaviour of nitric oxide synthase isoforms in rats stomach

bacter pylori suspension (ATCC 43504 1ml/die), concentrated 106 - 107 cells/ml and diluted 1:4, prepared in nutrient broth (BT) from culture in blood Agar medium or Columbia Agar enriched with horse blood. The second group (control group), constituted of 8 rats, received for five days, by gastric probe, a solution (1ml/die) prepared similarly from non inoculated plates. Beginning from ten days after suspension administration, the rats, five by five with two control rats, under penthobarbital anesthesia, were sacrificed during four different sessions, placed at intervals of one week. Specimens taken from antropyloric and oxintic region and from prestomach were fixed in Bouin mixture and embedded in paraffin. The sections were processed by avidin biotin standard with AEC as chromogen, with the use of a rabbit polyclonal antibody against nNOS and mouse monoclonal antibodies against eNOS and iNOS. The sections were finally covered with a hydrosoluble medium. We have observed the sections with Nikon microscope equipped by system for color image processing (Lucia M). Images were stored on magnetooptic support. Results Control rats The nNOS immunoreactivity, in accordance with physiological and biochemical data, histochemically confirmed in our previous studies, is localized in a discrete subpopulation of myenteric and submucosal ganglionic neurons (Meissner and Auerbach plexuses), intensely immunoreactive and morphologically similar to Dogiel type I neurons. The luminal mucosal epithelium displays very weak reactivity. In addition in the glandular epithelium, nNOS immunoreactivity is exclusively confined, in the basal portion of tubular adenomeri. The middle and proximal regions of glandular epithelium and neuroendocrine cells, (excluding several antral region cells very reactive) are areactive. Smooth muscle cells of the muscular tonaca exhibit scattered and weak immunoreactivity. Several immunoreactive macrophages in the chorion and submucosa were found; the meaning of this reactivity is uncertain (Fig.1). The eNOS immunoreactivity was found in the wall of submucosal microvessels, specially in the endothelium. Prestomach mucosal epithelium, gastric luminal and glandular epithelium are areactive (Fig. 2). The iNOS immunoreactivity was found only in mucosal and submucosal macrophages (Fig. 3). Infected rats The nNOS immunoreactivity, already in the animals sacrificed after one week some more in those sacrificed subsequently, compared to the controls, appears more intensely
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[1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

Figures 1-8: [1] Control rat: weak nNOS immunoreactivity in basal layer of glandular epithelium. Ob.10x - [2] Control rat: eNOS reactivity only limited in luminal mucosal epithelium. Ob. 10x - [3] Control rat (10days): All the tissue is negative for iNOS except a weakly reactivity in some intrachorial histiocytes. Ob.20x - [4] Infected rat (18days): wide nNOS immunoreactivity in glandular epithelium (mostly in basal cells) and in luminal epithelium. Neuroendocrine D cells strongly immunoreactive. Ob 10x - [5] Infected rat (18days): Intense nNOS immunoreactivity in some nitroxidergic gangliar neurons of submucosal plexus and in intermuscular nervous fibers. nNOS reactivity in parietal muscular cells. Ob.40x - [6] Infected rat (18days): Intense nNOS immunoreactivity in nitroxidergic neurons of myenteric plexus. Immunoreactivity of parietal myocells and intermuscular fibers. Ob 40x - [7] Infected rat (18days): eNOS immunoreactivity in luminal epithelium extended to the medium apical area of glandular epithelium. Submucosal vessels immunoreactive. Ob.10x - [8] Infected rat (18days): Submucosal vessels eNOS immunoreactive. Ob.20x.

and widely distributed, in the whole thickness of glandular epithelium particularly in the basal region of tubular adenomeri. Intense immunoreactivity showed in a large amount of mucosal neuroendocrine cells mostly morphologically and immunohistochemically identified such as somatostatinergic paracrine cytotypes (Fig. 4-5). Intense nNOS immunoreactivity is present also in some myenteric and submucosal ganglionic neurons and in their intermuscular fibers. Finally are found several reactive macrophages in the chorion and submucosa (Fig. 6). The eNOS immunoreactivity appears, according to expectations, in the wall of submucosal microvessels, but surprisingly the glandular epithelium, especially in sacrificed animals after 31st day, shows intense immunoreactivity, such as the parietal smooth muscle cells. Another likewise finding is the intense eNOS immunoreactivity in the stratified squamous epithelium of prestomach and in the intrachorial and submucosal macrophages (Fig. 7-8-9).
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The chronical gastritis helicobacter pylori correlated: immunohistochemical behaviour of nitric oxide synthase isoforms in rats stomach

[9]

[10]

[11]

[12]

[13]

Figures 9-13: [9] Infected rat (25 days): Intense eNOS immunoreactivity in basal layer of prestomach epithelium. Ob. 40x - [10] Infected rat (10 days): Weak increase of iNOS immunoreactivity in glandular and luminal epithelium. Intrachorial and submucosal macrophages iNOS immunoreactive. Ob.10x - [11] Infected rat (18days): Increase of iNOS immunoreactivity in adenomeri of basal layer of glandular epithelium. Intrachorial and submucosal macrophages iNOS strongly immunoreactive Ob. 20x - [12] Infected rat (25 days): Further increase of iNOS immunoreactivity in tubular adenomeri of basal layer of glandular epithelium. Ob. 20x - [13] Infected rat (25 days): Microgranular aspect of iNOS immunoreactivity irregularly distributed in tubular adenomeri. Ob. 40x.

Maria Laura Uzzo

The iNOS immunoreactivity is peculiarly present in the intrachorial and submucosal macrophages and in the glandular epithelium (basal region) of the animals for a long time treated (Fig. 10-11-12-13). Conclusions Our results provide the immunohistochemical evidence that, in our experimental model, NO produced in excess in gastric mucosa is involved in the pathogenesis of the cellular damage in Hp gastritis (cellular cytotoxicity and apoptosis), likely through the formation of peroxinitrite free radical. By the way we hypothesized a possibility of a induction by Hp, not only (as expected) on iNOS but also on nNOS and eNOS. In the present study we demonstrate, for the first time, that Hp stimulates a strong expression of the three different enzymatic isoforms: nNOS, eNOS and iNOS immunoreactivity, with respect to the controls, appears in fact significantly increased. On this matter its useful to precise something: Our immunohistochemical data furnish, consequently, the evidence, apparently paradoxical, that nNOS and eNOS, both considered only constitutive isoforms could be induced by appropriate stimuli and in this case up-regulated. Other recent researches support this eventuality in several conditions and in the near future it will be possible to identify the different NOS making a note of the nature of stimuli able to evoke its expression. Its allowed to affirm that Hp infection also stimulates expression of eNOS immunoreactivity in several not usually eNOS containing-cells: our data just demonstrate that eNOS immunoreactivity, differently from control rats, is present not only in endothelial cells but also in luminal and glandular epithelial cells, in submucosal and intrachorial macrophages, in smooth muscle cells and finally in squamous epithelium of prestomach. Another important result of our study regards the intense nNOS immunoreactivity revealed in somatostatinergic neuroendocrine cells in infected animals. Everybody knows that antral mucosa plays a key role in the regulation of gastric acid secretion via release of the gastrin by G cells. The release of somatostatin from D cells exerts a negative inhibitory control on gastrin release [7], demonstrated that Hp infection provokes a decrease of somatostatin in the antral mucosa and a related increase of gastrinic level. The loss of inhibition on gastrin release due to the somatostatin decrease during Hp infection may be an important factor in the duodenal ulcer pathogenesis. Today the mechanism by which Hp causes a depletion of somatostatine is not clarified [7]. The massive production of NO in somatostatinergic cells revealed by strong nNOS immunoreactivity, could fit into pathogenetic events previously reported: NO produced at high rates by citokines induction and Hp cytotoxic antigens may play an autocrine inhibitory control on the D cells, decreasing somatostatin release; the increased gastrin release explains the acid gastric hypersecretion in the duodenal ulcer Hp related.
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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] Stachura J. et al. Helicobacter pylori from duodenal ulcer patients expresses inducible nitric oxide synthase immunoreactivity in vivo and in vitro. J Phisiol Pharmacol, 1996, 47:1, 131-5 Lamarque D, Tran Van Nhieu J, Breban M. What are the gastric modifications induced by acute and chronic Helicobacter pylori infection? Gastroenterol Clin Biol. 2003 Mar;27(3 Pt 2):391-400. Review. French. Szepes Z. et al. Purified lipopolysaccharide from Helicobacter pylori provokes epithelial cell injury and induction of nitric oxide synthase in rat duodenum: inhibition of cytotoxicity by superoxide dismutase. Proc. DDW Washington 1997. Crespo A, Suh B. Helicobacter pylori infection: epidemiology, pathophysiology, and therapy. Arch Pharm Res. 2001 Dec; 24(6): 485-98. Review. Owen RJ. Bacteriology of Helicobacter pylori. Baillieres Clin Gastroenterol. 1995 Sep; 9(3): 415-46. Review. Nam KT, Oh SY, Ahn B, Kim YB, Jang DD, Yang KH, Hahm KB, Kim DY. Decreased Helicobacter pylori associated gastric carcinogenesis in mice lacking inducible nitric oxide synthase. Gut. 2004 Sep; 53(9): 1250-5. Moss SF et al. Effect of Helicobacter pylori on gastric somatostatin in duodenal ulcer disease. Lancet 1992, 340: 930-932. Felley CP et al. Expression of inducible nitric oxide synthase and interleukin-8 in human stomach infected with Helicobacter pylori. Proc. DDW Washington 1997. Lamarque D et al. Induction of nitric oxide synthase by an extract of Helicobacter pylori (Hp) in rat duodenum. Proc. DDW San Diego 1995 n. 311. Maliakkal RJ et al. Nitric oxide levels in gastric juice of patients with Helicobacter pylori infection. Proc. DDW San Diego 1995 n. 344. Maliakkal RJ et al. Serum nitric oxide levels before and after eradication of Helicobacter pylori infection - a prospective study. Proc. DDW San Diego 1995 n. 343. Moncada et al. Nitric oxide, physiology, patophysiology and pharmacology. Pharmacol Rew 43: 109,1991. Wong A et al. Expression of inducible nitric oxide synthase and cyclooxygenase-2 and modulation by omeprazole in Helicobacter pylori gastritis. Proc. DDW Washington 1997. Chaturvedi R, Asim M, Lewis ND, Algood HM, Cover TL, Kim PY, Wilson KT. L-arginine availability regulates inducible nitric oxide synthase-dependent host defense against Helicobacter pylori. Infect Immun 2007 Sep; 75(9): 4305-15. Kim H. Oxidative stress in Helicobacter pylori-induced gastric cell injury. Inflammopharmacology 2005; 13(1-3): 63-74. Review. Wang YF, Guo CL, Zhao LZ, Yang GA, Chen P, Wang HK. Effect of Helicobacter pylori infection on gastric mucosal pathologic change and level of nitric oxide and nitric oxide synthase. World J Gastroenterol 2005 Aug 28; 11(32): 5029-31.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

TWENTY YEARS HISTORY Of IMMUNOHISTOCHEMICAL MAMMALS DENTAL PULP STUDIES


[Ventanni di storia sullo studio immunoistochimico delle polpe dentali di mammiferi] *Angelo Leone, **Roselyn A. Jurjus, *Aldo Gerbino, *Luana Lipari, *Annamaria Mauro and *Maria Buscemi
Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia - Facolt di Medicina e Chirurgia, Universit di Palermo (I) - ** Department of Human Morphology, Faculty of Medicin, American University of Beirut, Beirut, Lebanon

Key words: Dental pulp, Orthodontic treatment, Neurotransmitters Parole chiave: Polpa dentale, Trattamento ortodontico, Neurotrasmettitori Summary. After a long preliminary study, our histology laboratory has worked in collaboration with the Department of Periodontology of the University of Palermo, studying the activity of Acetylcholinesterase and some neuropeptides (CGRP, Substance P, Enkephalins, Endorphins) on human dental pulp in experimental conditions; in the last few years we co-operated with Winchmore Hill Dental Practice in London (UK) to study the expression of Nitric Oxide Synthase (NOS) using immunohistochemistry method in orthodontic treated dental pulps and now we are begginning a collaboration with the American University of Beirut. The study has been carried out on samples taken from orthodontically treated and non-orthodontically treated teeth. Control pulps came from premolars extracted for orthodontic reasons; treated pulps were taken from premolars subjected to middle-intensity forces for orthodontic treatment and then extracted. Our results show that specific Cholinesterase Activity (AchE) remained unchanged in all the cases we observed, while there was a decrease in the presence of neuropeptides until they disappeared completely after seven days. It may be hypothesized that the cholinergic innervation is not stimulated by middle-intensity forces, which are however effective for the release of the neuropeptides involved in the delicate mechanisms causing pain. The results on NOS expression suggest a close correlation between the duration of the orthodontic traction and the expression of the above-mentioned neurotransmitter (NO). Riassunto. Dopo un lungo periodo di studi preliminari il nostro gruppo ha collaborato con il Dipartimento di Parodontologia dellUniversit di Palermo saggiando lattivit acetilcolinesterasica e di alcuni neuropeptidi (CGRP, Sostanza P, Enkefaline, Endorphine) nella polpa dentale umana in condizioni sperimentali. Negli ultimi anni abbiamo collaborato con la Clinica Dentale Winchmore Hill di Londra (UK), per studiare la nitrossido sintetasi (NOS) nelle polpe dentali umane ortodonticamente trattate e non, attraverso metodi immunoistochimici e da qualche tempo abbiamo intrapreso una collaborazione con lUniversit Americana di Beirut. Le polpe controllo provenivano da premolari estratti per motivi ortodontici, le polpe trattate provenivano da premolari sottoposti a forze medie per un determinato periodo di tempo e successivamente estratti. I nostri risultati dimostrarono che lattivit acetilcolinesterasica non varia in tutti i reperti, mentre lespressione dei neuropeptidi decresceva fino a scomparire allincirca al settimo giorno. Si pu ipotizzare che linnervazione colinergica non stimolata da forze di media intensit, questultime avrebbero, invece, effetto nel rilascio dei neuropeptidi coinvolti nel delicato meccanismo del dolore. Il risultato dellespressione di NOS suggerisce una stretta correlazione tra la durata del trattamento ortodontico e lespressione del nitrossido (NO).

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Introduction In 1987 Walker JA Jr. et al. [1] in a article published in Am.J.Orthod. Dentofacial. Orthop. indicate that orthodontic force mobilizes at least one neuropeptidergic pathway in the human tooth pulp. In 1989 Robinson Q.C. et al. [2] for the first time measured b-endorphinlike immunoreactivity (BE-LI) in human dental pulp. In a interesting paper about the effect of traumatic occlusion on CGRP and SP immunoreactive nerve fibre morphology in rat molar pulp and periodontium, Kvinnsland I. et al. [3] indicate that the nerve changes in some cases might be transient. Italian authors, [4] also pubblished a article titled Immunohistochemical localization of endothelin-like immunoreactivity in human tooth germ and mature dental pulp. Their findings suggest that vascular endothelium may be the only source of endothelin in human dental tissues and it was also proposed that endothelin released in mature tooth may partecipate in the regulation of the pulp blood flow. Effectively they do not clarify the role of endothelin in developing tissue but they suggest that endothelin may be involved in tooth development. Wang D. et al. [5] quantified catecholamines in normal human dental pulp which results were very helpful for the future studies. Rodd HD et al. [6] of the School of Clinical dentistry of the University of Sheffield, published on Arch oral Biol (2002) a comparative immunohistochemical analysis of the peptidergic innervation of human primary and permanent tooth pulp, this work has revealed connection with nociception, inflammation and healing. During these years our group developed some research over human dental pulp in different conditions and the results were discussed in numerous conferencies throughout Europe [7,8,9,10,11,12,13] having as the aim the treatment of pain. In fact the treatment of pain thus becomes a central theme in odontoiatric practice and even if pain is known to be the expression of an individual response, and is therefore a difficult parameter to measure with accuracy, it is interesting to recognize and quantify the biochemical activities involved in the responses to pain stimuli. To this regard we have performed heir research aiming to detect the presence of neuromediators in the various tissues of the tooth and in closely related district. Several neuromediators have been observed on the human dentogingival interface [14], probably associated with autonomous sensory functions. Research on the cat (Wahkisaka et al., 1986) [15], using immunofluorescence techniques, has also shown the presence of CGRP in the molar pulp. Other researches have studied the CGRP- containing fibres in rat molar pulp with microabscess formations [16] observing that the CGRP-containing fibres modify in relation to the pathological situations and probably play a protective role as regards the dental pulp. A notable contribution has been given (Casasco et al., 1990) [17] by an immunohistochemical study on the peptidergic innervation of human dental pulp that provides a rich
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Twenty years History of Immunohistochemical mammals dental Pulp Studies

[1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

Figures 1-8: [1] Human dental pulp. AchE, Control, Nervous fibres running parallel,100x - [2] Human dental pulp. AchE,Treated 7 days, Nervous trunk, 160x - [3] Human dental pulp. AchE,Control, Nervous fibres spreding out along the vessel wall, 100x - [4] Human dental pulp. Sub P, Treated 4 days, Perivascular reactivity, 100x - [5] Human dental pulp. nNOS, Control, Parenchymal cells and vessels walls are positive, 40x - [6] Human dental pulp. nNOS, Treated 6-month: Vessel walls and parenchymal cells are positive, 20x - [7] Human dental pulp. iNOS, Treated 3-month, Vessel wall are positive, 40x - [8] Human dental pulp. H.E. Treated 14-month, 40x.

map of neuromediators that can be observed in this territory (CCK, CGRP, C-PON, L-Enk, M-Enk, NPK, PHI, Somatostatine, SP, VIP). In relation to the above data in the literature and also to our own research on chemical mediators in various organs [18,19,20,21,22], we initiated an experimental study on human dental pulp. Meterials and metods In particular we assessed the presence and the possible variations of Acetycholinesterase activity, as an expression of cholinergic innervation, and of some neuropeptides (CGRP, Sub P, Met-Enk., Endorphin) in human dental pulp extracted from teeth subjected to middle-intensity orthodontic forces by using two groups of dental pulps: GROUP A: control pulps removed from teeth extracted for orthodontic reasons;
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GROUP B: treated pulps originated from teeth subjected to orthodontic forces caused by the application of elastic chains developing a weight of 250 g, in order to subject the teeth to middle-intensity stimulation and to study its effect in different time-periods; extraction was effected between one hour and seven days after application of the chains. The pulps came from children of both sexes aged between 11 and 13 years. All the teeth were premolars extracted for orthodontic reasons following Carbocaine anaesthesia. After extraction the teeth were cut by a high-speed cutter under a water-jet. The pulps were removed and placed in fixatives suitable for histochemical and immunohistochemical research: Karnowski fixative for research on acetylcholinesterase activity, and Bouin for immunohistochemical investigation of neuropeptides. Karnovsky fixed pulps: Karnovsky fixative consists of sodium maleate buffer, pH 6 (10 ml), with the addition of formol calcium (10 g CaCI in 100 ml H20, pH 7) and saccharose 0.8 M (10 ml). The pulps were kept in fixative at 4C for 12 hours and then placed for one hour in saccharose. After cryostat freezing at -20C, 20 thick sections were made and refixing was performed for 15' at 4C, and the pulps were incubated in the Karnovski medium (Martinez-Rodriguez IC-24 model, 1964) [23]. Incubation at 37C was effected for 5 hours. The sections were quickly dehydrated and mounted in balsam. The histochemical control trials were performed by incubating some sections with specific AchE inhibitors (Iso-OMPA) and with physiostigmin, a ChE inhibitor. Bouin fixed pulps: the pulps were deparaffinized with toluene plus histosol for 10, followed by two changes with 100 alcohol. A solution of methanol with added hydrogen peroxide was placed on the sections in order to block endogenous peroxidase activity. This was followed by two passages in 95 alcohol dehydration and washing for 5 in saline buffer. The sections were placed on slides and kept in a moist chamber where the various solutions stratified. The primary antibody, diluted as necessary in Tris 0.05 M buffer with Azide plus 1 BSA (bovine serum albumin), was placed on the sections using an Eppendorf micropipette. The slides in the closed moist chamber remained in incubation for 24 h at room temperature. After washing in Tris for 5, the second layer was applied, consisting of IgG antirabbit sheep (1:250 anti-rabbit serum) kept in contact for 15 at room temperature. This was followed by TBS washing for 5 and contact with 1:250 PAP (goat, peroxidase-antiperoxidase complex) for 30 at room temperature. Brief TBS washing (three changes) was followed by a incubation in diaminobenzidine (0.04 in non-saline Tris) with the addition of two drops of Perhydrol. The antigen-antibody complex used was polyclonal and the reaction was realized on human tissues, appearing as a reddish-brown staining. Immunohistochemical control trials were performed with the same techniques and timing in organs known to be
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positive. The immunohistochemical control trials were performed by incubating the same sections in absence of the primary antibody. Results Tables 1, 2, 3 and 4 summarize the observations conducted on the preparations subjected to histochemical and immuno-histochemical techniques. AchE: with regard to the acetylcholesterase activity investigated as an expression of the presence of cholinergic terminals, our control specimens showed a cholinergic distribution consistent with the data reported in the literature (Pohto et al., 1968) [24] and observed with same techniques. Only the finding concerning incubation is not consistent, as an incubation of 18 hours is described but not the temperature at which it was performed. In our specimens we detected (Fig.1) the presence of several small nervous trunks running parallel out of the radicular portion. These trunks wind their way into the middle part of the pulp, massing particularly in a central zone. Under high magnification (Fig.2), it is possible to observe the single cholinergic nervous fibres, which near the vessels, (Fig.3) spread out along the vessel wall. In the distal zone, just below the odontoblasts, the nervous trunks terminate in a dense network. The specimens originating from treated pulps subjected to the same techniques did not show any variation. CGRP and Sub P: these two neuropeptides were observed in traumatic occlusion in the periodontium and the premolar in the two-month-old rat. A wide variety of mechanism are responsible for their presence: from the possible role of maintenance, proliferation and modification of the responsiveness of the odontoblasts to the functional involvement of the pulpar vascularization, secondary to iatrogenic or microenvironmental insults.Our specimens indicated a positivity due to thin intrapulpar fibres and perivascular fibres (Fig.4) which remain unmodified after one hour of treatment.Four days after application of elastic chains it was still possible to find many very fine intrapulpar fibres, and perivascular fibres reactivity was unmodified. This occurred both in the case of CGRP-positive fibres and in those containing Sub P, which showed in controls and after 1 h a sub-odontoblastic positivity. Enkephalins and Endorphins: also with regard to these neuromediators it was possible to observe reactivity in the form of perivascular fibres and of some intraparenchymal fibres, especially endorphin-positive fibres. No variations were found after 1 h of treatment compared to controls after 24 h it is possible to note absence of parenchimal fibres. The immunohistochemical data were almost identical in the days following to CGRP and Sub P. There was total absence of reactivity 7 days after treatment. Ours research on the orthodontic treated human dental pulp progressed studying the expression of nNOS and iNOS in order to develop our understanding of NOS immunorec227

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tivity in pulp during orthodontic treatment and so, having a more complete image about histochemical process in dental pulp during orthodontic treatment. We dinstinguished this materials between groups A and B: GROUP A: control pulps removed from bicuspids extracted for orthodontic reasons; GROUP B: pulps removed from teeth subjected to orthodontic forces using the STRAIGHT WIRE technique, with Nickel Titanium and stainless steel archwires. The teeth, all upper or lower bicuspids, were extracted from patients of both sexes, aged 11-13, under anaesthesia with carbocaine. The extractions were performed 7 days, 14 days, 3 months, 6 months and 14 months after the beginning of the orthodontic treatment. All pulps were then placed in Bouins fxative for immunohistochemical analysis and stained using haematoxylin-eosin for morphological control. After fixation the pulps were embedded in paraffin and 7/^m-thick sections were cut. We used anti-nNOS and anti-iNOS polyclonal antibodies (Transduction Laboratories) for the immunohisto-chemical assay and the Ultrastain Polyvalent Strept ABC-HRP Kit for the detection. All sections were mounted using Dako Faramount Aqueous Mounting Medium. Immunohistochemical reactivity for nNOS: CONTROL PULPS (i.e., pulps of untreated, intact teeth extracted for orthodontic reasons): nNOS was detected in odontoblasts, vessels walls and in the treated dental pulp cells as well Fig. 5. TREATED PULPS: - after 6 month traction nNOS was detected both in vessels and in the parenchymal tissue (Fig. 6); - after 14 month traction nNOS had the same localisation as in previous cases. Immunohistochemical reactivity for iNOS: CONTROL PULPS: the absence of iNOS expression was related to the well-preserved pulpal structure TREATED PULPS (e.g. subjected to orthodontic forces): - after 3 month traction vessel walls were found positive for iNOS; - after 6 month traction iNOS immunohistochemical reactivity was detected in nervous fibres - after 14 month traction increased reactivity was found, reaching its peak in odontoblasts and at the vascular and nervous levels. The pulpal structure appears well-preserved on optical microscopy.

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Table of antybodies Antibody to CGRP (syntethetic, rat) Sub P (syntethetic, human) Met 5 Enkephaline (syntethetic, bovine) b-Endorphine (syntethetic, human) Type Rabbit Source Amersham Diluition 1:300

Rabbit Polyclonal Rabbit Polyclonal Rabbit Polyclonal

UCB-Bioproducts

1:200

UCB-Bioproducts

1:200

UCB-Bioproducts

1:200

Conclusion A recent study [25] on cat tooth pulp showed that the afferent nerves play an important role in the pulps haemodynamic response to chemical and experimental procedures in the teeth. It has been amply demonstrated that feline and human dental pulp are richly innervated by myelinic afferent nerves (A fibres) and amyelinic afferent nerves (B fibres), and that the amyelinic fibres contain Sub P, neurokinin A and CGRP [26, 27]. It has been shown that high electric stimulation of cat dental pulp activates the C fibres and causes the release of P substance, determining vasodilatation in cat dental pulp [28]. These effects are not reported after A fibre stimulation. In the EE-stained sections that we used as a morphological reference we did not observe any case of vasodilatation, which is a frequent occurrence during philogistic phenomena recorded in collateral studies. In relation to the afferent C fibres, it is possible that the stimuli to the control pulps and to those subjected to chains in our samples were not sufficient to trigger either the vasomotor cholinergic mechanism or the supposed, but not yet proven, afferent role of the cholinergic fibres running down into the dentinal tubules. In fact it has been hypothesized that some stimuli may be propagated along the odontoblastic intratubular prolongation and thus be transmitted to the nervous fibre, even if the exact modality is still unknown. The constant acetylcholinesterase reactivity in our specimens therefore brings no support to this theory, although we realize the necessity of checking results after greater stimulation. The problem remains open and merits further experimentation with other techniques.
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TABLE 1 Control pulps AchE CGRP SUB P Met-ENK b-End Cholinergic fibres present in all the pulp. Large-calibre trunks, thion subodontoblastic fibres Small intrapulpar and perivascular positive fibres Vascular fibres. Intraparenchymal fibres. Sub-odontoblastic diffyuse positivity. Thin perivascular fibres. Intraparenchymal and vascular fibres.

TABLE 2 Treated pulps 1 hour AchE As control 3 days As control 7 days As Control

We believe it is likely that in the pulp there is a vasomotor mechanism caused by strong stimuli which affects the microenvironment until it modifies the pulps hydrodynamics, with consequent mechanical stimulation also of the odontoblastic prolongations and considerable pain, thus bringing into play the odontoblasts as receptor cells. We have observed the vasomotor cholinergic mechanism in research on the organs of various mammals [29]. Sub P- and CGRP-positive fibres play a regulatory role in the blood flow, as already shown. This finding is confirmed by our studies, as both CGRP and Sub P appear to act in functional equilibrium to low-intensity stimuli in a different role from that of the cholinergic fibres in relation to the intensity, and probably also to the quality, of the stimulus. TABLE 3 -Treated pulps 1 hour CGRP Sub P As control As control 4 days Increased reactivity perivascular fibres Rare perivascular fibres 7 days No reactivity No reactivity

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Table 4 Treated pulp 1 hour Met-E NK As control 24 hours 7 days

No parenchimal fiNo reactivity bres, perivasal fibres present

It should also be remembered that CGRP seems to be localized in the cholinergic fibres; the colocalization of Sub P, which is a strong vasodilator, has also been demonstrated [15]. It might therefore be thought that the fibres contain several mediators that alternate their functional role in relation to the intensity of the stimulus. It is also known that the P substance is responsible for the pain mechanism and that its activity is modulated by the release of enkephalins and endorphins. These substances were observed in the control pulps, but not in the pulps subjected to 7 days treatment. Some researchers [30] have shown by microanalysis that the quantity of enkephalins reduces in relation to the passing of the days and the level of the forces applied. One might therefore hypothesize an impoverishment of the neuromediators, as in fact is shown by our immunohistochemical findings, or a reduction in concentration that is not observable. The complexity of the innervation of the pulp, in which it has been possible to demonstrate the presence of adrenergic fibres and the intricate relationships with the neuromediation mechanisms, represents a challenge for the understanding of the activities of this territory, which is now regarded as a functional unit in its own right (the pulpdentine complex). For that reason it seems interesting, to refer our preliminar finding of a diffuse sub-odontoblastic reactivity of sub P. The data suggested a possible paracrine role of odontoblast. It is not improbable, even if the hypothesis requires valid confirmation, that the odontoblasts may have an as yet unknown neuropeptide secretory role, which is plausible if we consider their ectomesenchymal origin. The immunohistochemical analysis of treated and untreated pulps showed that nNOS was expressed in all pulps, including control and treated samples. This finding is consistent with what other authors have already observed in several tissues where the nNOS enzyme is an essential metabolite in NO synthesis [31,32]. On the other hand, an increase in iNOS immunohistochemical detection was apparent in pulps from orthodontically treated teeth, indicative of pulp stress. After three months a fairly small quantity of enzyme indicated mild inflammation, which became more and more apparent with the increase of orthodontic forces in time, as shown by the images

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and results shown here. These findings would easily lead us to the conclusion that the application of increased orthodontic forces causes growing stress in treated pulps. Recently Di Nardo et al. [33] have dimonstrated that NOS play a significant role in the pathogenesis of pulpitis. Our aim is to evaluate whether this stress is mainly due; to the application of forces of greater intensity rather than to the duration of orthodontic traction itself. As a future direction, we plan to check the pulp response to long-term weak orthodontic traction and compare it to the resistance of the pulpal tissue to stronger forces applied for a shorter amount of time. In case of no pulp injury after weak force application for a long time, teeth movement should also be evaluated to establish whether a longer treatment using weak traction would be a more suitable option, avoiding pulp problems due to the application of high-intensity orthodontic forces.

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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] Walker JA JR, Tranzer FS, Harris EF, Wakelyn C, Desiderio DM. The enkephalin response in human tooth pulp to orthodontic force. Am J Orthod Dentofacial Orthop. 1987;92(1):9-16. Robinson QC, Kilmar JT, Desiderio DM, Harris Ef, Fridland G. Immunoreactive evidence of beta-endorphin and methionineenkephalin-ArgGly-Leu in human tooth pulp. Life Sci1989; 45 (11):987-99. Kvinnsland I, Heyeraas KJ. Effect of traumatic occlusion on CGRP and SP immunoreactive nerve morphology in rat molar pulp periodontium. Histochemistry1992;97(2):111-20. Casasco A, Calligaro A, Casasco M, Springall DR, Tenti P, Marchetti C, Poggi P, Polak JM. Immunohistochemical localization of endothelin-like immunoreactivity in human tooth germ and mature dental pulp Anat Embryol 1991; 183(5):515-20. Wang D Wang Z, Zhao L, Zhang Q. Quantitative study of cateholamines in normal human dental pulp. Hua Xi Kou Qiang Yi Xue Za Zhi, 1998;16(2): 132-3,137. Rodd HD, Boissonade FM. Comparative immunohistochemical analysis of the peptidergic innervation of human primary and permanent tooth pulp. Arch Oral Biol 2002;47(5):375-85. Buscemi M, Gerbino A, Tessitore V, Leone A, Dangelo M, Giuliana G. Studio immunoistochimico preliminare sulla distribuzione di alcuni neuropeptidi sulla polpa umana in diverse condizioni. 1992, Giardini Naxos, I Congresso Nazionale GISOB, 30 aprile-2 maggio 1992, vol. 1, p. 19. Buscemi M, Gerbino A, Tessitore V, Dangelo M, Giuliana G. A preliminary istochemical and immunohistochemical study on the human dental pulp. Experimental situation. The Ninth European Anatomical Congress 1992; Krakow, Poland, vol. 1, p. 39. Gerbino A, Tessitore V, Leone A, Valentino B, Buscemi M, DAngelo M, Giuliana G. Distribuzione dellinnervazione colinergica nelle polpe dentali in condizioni sperimentali Atti 46 Covegno nazionale della Societ Italiana di Anatomia, Santa Margherita Ligure 4-7 Ottobre 1992 (1), p. 133. Giuliana G, Buscemi M. Osservazioni sperimentali su alcuni neuromediatori pulpari ( Studio prelminare) XXIII Congresso nazionale S.I.O.C.M.F. 1992 Bologna. Gerbino A, Leone A, Patel M, Uzzo ML, Buscemi M. Observation et distribution de loxyde dazote (NO) dans la pulpe dentaire humaine. 45e Congrs du GIRSO 2001, Bruxelles, vol. 1, p. 7. Leone A, Uzzo ML, Buscemi M, Gerbino A. Etudes prliminaire sr la distribution de la Cathepsine D dans la pulpe dentaire humaine sous traitement orthodontique. 46me Congrs du GIRSO, 2002 ParedesPortugal, vol. 1, p. 12. Buscemi M, Gerbino A, Valenza V. Cholinesterase activities in dental papilla of newborn rats. Eur Journal Histochem 1995, 39. Suppl 1. Luthman J, Johansson O, Ahlstrom U, Kvint S. Immunohistochemical studies of the neurochemic markers, CGRP, Enkephalin, Galanin, -MSH, NPY, PHI, Proctolin, PTH, Somatostatin, SP, VIP, Tyrosine, Hidroxilase and neurofilament in nerves and cells of the human attached gingival. Arch Oral Biol 1988; 33:149. Wakisaka S, Ichkawa H, Nischikava S, Matsuo S, Takano Y. Akai M The distribution and origin of calcitonine gene related peptide-containing nerve fibres in felin dental pulp. Histochemistry 1986; 86:585. Taylor PE, Byers MR, An immnumoctochemical study of morphological reaction of nerves containing calcitonin gene related peptide to microabscess formation and healing in rat molars. Arch Oral Biol 1990;35:629. Casasco A, Calligaro A, Casasci M, Springalls DR, PolaK J.M, Poggi P, Marchetti C Peptidergic nerves in human dental pulp. An immunocytochemical study. Histochemistry 1991; 95: 115. Buscemi M, Costa V. Ricerche istochimiche sullinnervazione adrenocolinergica della tuba uterina uma-

[15] [16] [17] [18]

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na. Bol Soc Ita Biol Sper 1975;51:352. [19] Buscemi M. Studio immonoistochimico dellinnervazione adrenocolinergica della lingua in alcuni mammiferi Ric Med 1988;11:1. [20] Tessitore V, Buscemi M, Gerbino A, Franchina F, Lipari D. CGRP and Substance P in the pineal gland. Atti 43 Congr. Soc. Ital Anat 1988; Giardini Naxos (I), p. 256. [21] Tessitore V, Buscemi M, Gerbino A, Bonaventura G, Franchina F. An immunohistochemical investigation of regional distribution and role of calcitonine-gene- related peptide (CGRP) in mammals brain. Neurosc. Letters 1990;39:212. [22] Tessitore V, Bonaventura G, Buscemi M, Franchina F, Gerbino A. A Immunohistochemical distribution and role of calcitonine gene-realated peptide (CGRP) in mammals Gep system. Atti XIV Congr Soc Ital Histoch 1991, Siena (I). [23] Martinez-Rodriguez R, Riba Soto A, Moja Mangas J. Demontrastration de la actividad acetilcolinesterasica con un nuevo metodo histoquimico.Trabajos del Insituto Cajal de Investigaciones Biologicas 1964, 56:27. [24] Pohto P, Antila R. Acetycolinesterase and noradrenaline in the nerves pulpes. Acta Odont Scand 1968;26:641. [25] Gazelius B, Edwall B, Olgart L, Lundberg JM, Hokfelt T. Fisher J. Vasodilatatory effects of coexistence of calcitonine generelated peptide (CGRP) and substance P in sensory neurons of cat dental pulp Acta Physio. Scand 1987, 98:1. [26] Olgart L, Hokfelt T, Nilsson G. Pernow B.Localization on substance P-like immunoreactivity in nerves in the tooth pulp. 1977a, Pain, 4: 153. [27] Olgart L, Gazelius B, Brodin E, Nilsson G.. Release of of substance P-like immunoreactivity from the dental pulp. Acta Physiol Scand, 1977b, 101: 510. [28] Gazelius B, Olgart L,. Vasodilatetion in the dental pulp produced by electrical stimulation of the inferior alveolar in the cat. Acta Physiol Scand, 1980, 108,181. [29] Buscemi M, Gerbino A, Lipari D, Peri G. Indagini istochimiche sullinnervazione colinergica di vari distretti mesenterici in alcuni mammiferi. Ric Med 1985 10: 533-540. [30] Walker JA, Tanzer F, Harris EF, Wakelyn C, Desiderio DM. The enkephaline response in human tooth pulp to ortodontic force. Am J Orthod Dentofacial Orthop 92:9. [31] Degnim AC, Nagayama D H. Nitric Oxide and the puklmonary artery smooth muscle cell. S Pediatr Surg. 1996,5:3,160-4. [32] Harren M, Schonefelder G, Paul M, Horak. High expression of inducible nitric oxide synthase correlates with intestinal inflammation of interleukin-2-deficient mice. NY Acad Sci 1998, 210-5. [33] Di Nardo F, Lohinal Z, DArcangelo C, De Fazio PE, Speranza L, De Lutiis MA, Patruno A, Grilli A. Felaco M. Nitric synthase in healthy and inflamed human dental pulp. J Dent Res, 2004; 83 (4) : 312-6.

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Urogenital tract

Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

CARCINOMA EREDITARIO DELLOvAIO


[Hereditary Ovarian Cancer] Loredana Bruno1#, Sergio Rizzo1#, Valentina Cal1, Mario Federico1, Rita Passantino1, Rita Ferla1, Giuseppe Cicero1, Eliana Gulotta3, Michele Frazzetta4, Maria Buscemi2, Aldo Gerbino2, Viviana Bazan1 e Antonio Russo1
1

Dipartimento di Discipline Chirurgiche ed Oncologiche, Centro di Riferimento Regionale per la Caratterizzazione Biomolecolare e lo Screening dei Tumori Ereditari, Universit di Palermo - 2 Sezione di Istologia ed Embriologia Dipartimento di Medicina Sperimentale - 3 Dip. GENURTO - 4 Dip. di Discipline Chirurgiche ed Oncologiche - # Entrambi gli autori hanno contribuito egualmente a questo lavoro.

Parole chiave. Carcinoma Ereditario dellOvaio; HOC; HBOC; BRCA; HNPCC; MMR; Test Genetico; Chirurgia Profilattica Key words. Hereditary Ovarian Cancer; HOC; HBOC; BRCA; HNPCC; MMR; Genetic Testing; Prophylactic Surgery Sommario. Circa il 10% dei carcinomi ovarici (CaOv) sono ereditari ed associati ad una predisposizione genetica autosomica dominante ad alta penetranza. Sono state descritte tre principali sindromi ereditarie: Carcinoma Ovarico Ereditario Sito-specifico (HOC, Hereditary Ovarian Cancer), HBOC (Hereditary Breast and Ovarian Cancer), HNPCC (Hereditary Non Poliposis Colorectal Cancer, sindrome di Lynch II); la prima di queste si ritiene sia una variante della sindrome HBOC. Mutazioni germinali nei geni BRCA si riscontrano nel 90% di tutti i tumori ovarici ereditari di tipo epiteliale mentre il rimanente 10% causato da mutazioni nei geni MLH1 ed MSH2, che rapppresentano i geni di suscettibilit per lHNPCC. Il test genetico viene offerto ad ognuno dei membri delle famiglie affette dalle tre sindromi sopra indicate. La maggior parte dei carcinomi ovarici associati a mutazioni nei geni BRCA sono di stadio avanzato e di tipo sieroso ad alto grado. Lo screening raccomandato per i carcinomi ovarici nelle donne ad alto rischio prevede semestralmente ecografia pelvica e dosaggio del CA125 sierico. La salpingo-ooforectomia bilaterale sembra poter ridurre significativamente il rischio di carcinoma ovarico nei portatori di mutazione nei geni BRCA. Listerosalpingo-ooforectomia dovrebbe esser proposta alle donne affette da HNPCC. Abstract. At least 10% of ovarian tumors are hereditary and associated with highly penetrant, autosomal, dominant genetic predisposition. Three clinical manifestations of hereditary ovarian cancer have been identified: site-specific ovarian cancer (HOC, Hereditary Ovarian Cancer), HBOC (Hereditary Breast and Ovarian Cancer syndrome), HNPCC (Hereditary Non Poliposis Colorectal Cancer, Lynch II syndrome). BRCA germline mutations account for more than 90% of all hereditary epithelial ovarian tumors whereas most of the remaining 10% are caused by MLH1 and MSH2 mutations, which are susceptibility genes of HNPCC. Genetic testing is available for each of the three hereditary syndromes described. Most ovarian cancer associated with germline BRCA mutations are advanced stage and high grade serous carcinomas. Recommendations for OC surveillance in high risk women include transvaginal pelvic ultrasound with color doppler and serum CA125 every six months. Bilateral salpingooophorectomy appears to be effective to reduce the risk of ovarian cancer in BRCA mutation carriers. Hysterosalpingo-oophorectomy should be considered in HNPCC women.

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Loredana Bruno, Sergio Rizzo, Valentina Cal, Mario Federico, Rita Passantino, Rita Ferla, Giuseppe Cicero, Eliana Gulotta, Michele Frazzetta, Maria Buscemi, Aldo Gerbino, Viviana Bazan e Antonio Russo

1. Introduzione Nel mondo occidentale il Carcinoma dellOvaio (CaOv) rappresenta una delle principali malattie ginecologiche ad alta mortalit, la quarta causa di morte per tumore nelle donne, con una incidenza di circa 192.000 nuovi casi ogni anno [1]. I metodi di screening ad oggi inadeguati e lassenza di sintomi significativi determinano la diagnosi di tali tumori in stadio avanzato e conseguentemente un ridotto tasso di sopravvivenza. Non ci sono importanti fattori di rischio ambientali ad eccezione di una prolungata esposizione agli estrogeni nel corso della vita. Lunico fattore di rischio realmente significativo la storia familiare di carcinoma ovarico. Il rischio nel corso della vita di sviluppare un carcinoma ovarico aumenta dall1.6% nella popolazione generale al 4% quando un parente di I grado affetto da CaOv e al 7% in presenza di due parenti di I grado affetti [2, 3]. Laggregazione familiare dei casi di CaOv rappresenta un valido strumento per identificare le famiglie ad alto rischio di sviluppare tale neoplasia. Il rischio di tumore familiare diminuisce con lincremento dellet della madre o della sorella che hanno sviluppato la malattia, infatti stato stabilito che il rischio relativo di cancro ovarico al di sotto dei 55 anni 5.2 e dopo i 55 anni si abbassa a 3.4 [4]. Tale rischio raddoppia nelle famiglie con una parente di I grado affetta da CaOv e subisce un aumento del 50% quando vi un parente di I grado affetto da carcinoma alla mammella e/o allovaio [5]. Let alla diagnosi di cancro della mammella, dellovaio o della prostata nei genitori ha un effetto minore sul rischio di CaOv nelle figlie. Nelle donne con una forte storia familiare di CaOv, questo viene diagnosticato in giovane et rispetto ai casi sporadici. Lassociazione tra il rischio di tumore dellovaio e di altri tumori, quali carcinoma della mammella, della prostata, del colon-retto dipende quindi dalla storia familiare e dallet alla diagnosi ma anche dai sottotipi istologici di tumore ovarico e dal tipo di predisposizione genetica [6]. 2. Sindromi associate al carcinoma ovarico di tipo ereditario La maggior parte dei CaOv sono di tipo sporadico mentre il 5-10% sono ereditari ed associati ad una predisposizione genetica autosomica dominante ad alta penetranza. La scoperta di alterazioni in geni di suscettibilit sta alla base della consulenza oncogenetica, che permette di individuare, tra i soggetti ad alto rischio di CaOv, i portatori di mutazioni germinali in questi geni [7-9]. Sebbene il CaOv di tipo sieroso rappresenti listotipo pi comune, anche altri istotipi possono presentarsi nelle diverse sindromi in cui vi un aumentato rischio di sviluppare un CaOv [10].
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Il processo di carcinogenesi in questi tumori, analogamente agli altri, il risultato dellacquisizione di mutazioni genetiche, che si sommano a quelle germinali nel caso di donne con familiarit di CaOv. Nelle famiglie con storia familiare di CaOv, i membri affetti da altre neoplasie quali il carcinoma alla mammella o al colonretto, hanno un rischio pi alto di sviluppare un CaOv. I pazienti con storia familiare di CaOv possono essere classificati schematicamente in tre principali sindromi: Carcinoma Ereditario dellOvaio Sito-specifico (HOC, Hereditary Ovarian Cancer), Carcinoma Ereditario della Mammella e dellOvaio (HBOC, Hereditary Breast and Ovarian Cancer), Carcinoma dellOvaio Ereditario associato alla Sindrome di Lynch II (HNPCC, Hereditary Non Poliposis Colorectal Cancer). Questultima caratterizzata da cancro colonrettale ed un incrementato rischio di tumori allendometrio, ovarici, gastrici, pancreatici e del tratto biliare [11]. Il contributo delle mutazioni nei geni BRCA (BReast CAncer) quello che determina in misura maggiore le prime due sindromi. Il 90% dei casi di CaOv ereditario sono dovuti a mutazioni in questi geni, la rimanente parte attribuibile a mutazioni nei geni MLH1 e MSH2 che sono i geni di suscettibilit della sindrome di Lynch II [12-15]. Inoltre esistono altre sindromi familiari minori che predispongono al CaOv e si manifestano con una frequenza dell1% rispetto al totale: la sindrome di Gorlin, lOsteocondromatosi e la sindrome di Peutz-Jeghers [16-18] (Tabella 1).

2.1. Carcinoma Ereditario dellOvaio Sito-specifico (HOC) Nella sindrome del CaOv Sito-specifico (HOC) si riscontrano famiglie in cui vi la presenza di due o pi parenti di I o di I e di II grado con CaOv di tipo epiteliale (Figura 1).
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Loredana Bruno, Sergio Rizzo, Valentina Cal, Mario Federico, Rita Passantino, Rita Ferla, Giuseppe Cicero, Eliana Gulotta, Michele Frazzetta, Maria Buscemi, Aldo Gerbino, Viviana Bazan e Antonio Russo

Figura 1. Albero genealogico di una famiglia con sindrome del Carcinoma Ovarico Ereditario (HOC). CaOv: Carcinoma Ovarico.

In tali famiglie, il rischio di cancro allet di 70 anni di circa il 5%, tre volte pi alto se paragonato alla popolazione generale (1,6%) [2]. stato ipotizzato che questa sindrome rappresenti una variante della HBOC piuttosto che una sindrome a se stante. Un recente lavoro sembra confermare questa ipotesi dal momento che nessun gene di suscettibilit stato identificato esclusivamente per il CaOv; pertanto questa sindrome viene ad oggi considerata una manifestazione particolare della sindrome HBOC in cui vi una prevalenza di casi di CaOv [19]. 2.2. Carcinoma Ereditario della Mammella e dellOvaio (HBOC) In tale sindrome, le famiglie affette presentano nellalbero geneaologico membri con carcinoma della mammella (CaM) e dellovaio (Figura 2). LHBOC caratterizzata da una precoce et di insorgenza di CaM associato a CaOv a qualsiasi et, carcinoma alla mammella bilaterale, CaM e/o CaO nella stessa persona e CaM maschile [20-23]. Nei casi di CaM e CaOv i geni di suscettibilit possono essere trasmessi sia per via materna che paterna. Laggregazione di pi casi di CaOv e di CaM, e la precoce et di insorgenza hanno sin da subito fatto pensare ad una predisposizione genetica nelle famiglie affette da questa sindrome. I due geni di suscettibilit che sono stati associati al cancro ovarico di tipo epiteliale sono i geni BRCA1 e BRCA2. Entrambi vengono trasmessi in maniera autosomica dominante ad alta penetranza [24, 25]. La genetica di suscettibilit: i geni BRCA. Il gene BRCA1 (OMIM 113705), il primo dei due geni ad essere stato isolato, intorno al 1994, localizzato nel cromosoma 17q21 e ri240

Carcinoma Ereditario dellOvaio

Figura 2. Albero genealogico di una famiglia con sindrome del Carcinoma della Mammella e dellOvaio Ereditario (HBOC). CaM: Carcinoma della Mammella; CaOv: Carcinoma Ovarico.

sulta costituito da 24 esoni di cui 22 codificanti per una fosfoproteina di 1863 amminoacidi (aa) con peso molecolare di 220 Kd. Lesone 11 del gene ha notevoli dimensioni e codifica per il 60% della proteina. Il gene BRCA2 (OMIM 600185), scoperto circa un anno dopo, e localizzato nel cromosoma 13, pi grande del gene BRCA1: risulta costituito da 27 esoni di cui 26 codificanti per una proteina di 3418 aa. Entrambi i geni mostrano una elevata omologia strutturale e non sono simili ad altri geni conosciuti [24-26]. Le proteine BRCA hanno una localizzazione nucleare, sono espresse in una variet di tessuti ed hanno un ruolo dominante nel meccanismo di riparo del danno al DNA. Altri meccanismi in cui le proteine sono coinvolte sono: il controllo dei checkpoint del ciclo cellulare, lubiquitinazione delle proteine ed il rimodellamento della cromatina. Svolgendo queste funzioni sono considerati geni oncosoppressori [27] (Figura 3). Mutazioni nei geni BRCA e carcinoma ovarico. Mutazioni nei geni BRCA sono state ampiamente descritte in famiglie affette da carcinoma alla mammella e/o allovaio. In particolare, le mutazioni in BRCA1 avvengono nel 75% delle famiglie affette da cancro ovarico di tipo ereditario. Le mutazioni patogenetiche includono piccole delezioni, inserzioni, o mutazioni puntiformi che portano alla formazione di codoni di stop e conseguentemente alla formazione di proteine tronche non funzionali. Le mutazioni identificate non ricadono allinterno di hot spots ma sono distribuite lungo lintero gene. Anche riarrangiamenti germinali sono stati descritti nei geni BRCA in famiglie ad alto rischio di CaM e CaOv [28, 29]. Il rischio cumulativo nellarco della vita di CaOv epiteliale associato alle mutazioni nei geni BRCA, va dal 40-50% per il gene BRCA1 al 20-30% per il gene BRCA2. Nel comples241

Loredana Bruno, Sergio Rizzo, Valentina Cal, Mario Federico, Rita Passantino, Rita Ferla, Giuseppe Cicero, Eliana Gulotta, Michele Frazzetta, Maria Buscemi, Aldo Gerbino, Viviana Bazan e Antonio Russo

Figura 3. Geni BRCA1 e BRCA2. I rettangoli grigi rappresentano gli esoni il cui numero corrispondente indicato sotto ciascun box. Viene mostrata la localizzazione delle mutazioni "founder" associate a rischio d'insorgenza di cancro ovarico. Inoltre, la figura indica le regioni dei geni BRCA pi frequentemente coinvolte nell'insorgenza del CaOv.

so, questi due geni provocano dal 3 al 12 % di tutti i carcinomi ovarici [21, 30]. Tutte le mutazioni nei due geni BRCA e le rispettive frequenze, che sono state identificate a partire dalla scoperta dei due geni, sono riportate in un database internazionale denominato Breast Cancer Information Core Database (BIC; http//research.nhgri.nih.gov/bic). Gli studi pi recenti sulle mutazioni nei geni BRCA hanno focalizzato la loro attenzione sullassociazione tra la localizzazione delle mutazioni ed il rischio di carcinoma ovarico. Lipotesi stata confermata dalla scoperta che mutazioni nella porzione compresa tra i nucleotidi 2401-4190 del gene BRCA1 aumentano il rischio di CaOv mentre sembrano diminuire il rischio di CaM [31]. Le mutazioni in BRCA2 sono responsabili del 35% dei CaM di tipo ereditario ma conferiscono un rischio minore (10-20%) di sviluppare carcinomi dellovaio. Entrambi i geni sono coinvolti anche nel cancro al seno maschile (6%). Tutte le casistiche sottolineano un leggero incremento (6-14%) del rischio di sviluppare anche altri tipi di cancro come carcinoma del colon-retto, della prostata ed del pancreas [32]. Per il gene BRCA2 recentemente stato osservato che le mutazioni comprendenti la porzione 4075-6503 dellesone 11 sono associate ad un incrementato rischio di CaOv. Tale regione dellesone 11 stata pertanto definita Ovarian Cancer Cluster Region (OCCR) [33, 34]. Non esistono mutazioni in omozigosi per il gene BRCA1 mentre al contrario esistono delle rare patologie quali lanemia di Fanconi ed il tumore di Wilms dovute a mutazioni in omozigosi del gene BRCA2 [35]. Questa caratteristica conferma che, nonostante lelevata omologia dei due geni, esistono differenze funzionali tra le proteine .
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Mutazioni founders nei geni BRCA. La scoperta che specifiche mutazioni sono presenti in alcune popolazioni e gruppi etnici ha condotto molti studiosi allidentificazione di mutazioni founder [36]. Lesempio pi rappresentativo di founder mutation nei geni BRCA1/2 rappresentato dalle mutazioni identificate nella popolazione degli ebrei Ashkenazi [37-47]; il rischio di OC nei portatori delle tre founder mutation rispettivamente del 14%, 33% e 20% [38, 46]. Esistono due mutazioni founder identificate nella popolazione Norvegese, la 1675delA, e la 1135insA, che sono state riscontrate con unalta frequenza, 2.1% e 0.8% rispettivamente, anchesse in casi di CaOv [48]. Altra mutazione founder associata al rischio di cancro ovarico stata identificata nel gene BRCA2 (BRCA2-999del5) riscontrata con una percentuale compresa tra 6 e 7.9% in casi di cancro ovarico della popolazione Islandese [49, 50]. Infine, un recente lavoro condotto su 283 casi di cancro ovarico nellUK e negli USA sembra confermare lipotesi che la mutazione ins6KbEx13 del gene BRCA1 una founder mutation dellUK; tale mutazione infatti stata riscontrata solo nei casi del Regno Unito dove stata riscontrata con una frequenza del 6% [31] (Tabella 2).

2.3. Carcinoma dellOvaio Ereditario associato alla Sindrome di Lynch II (HNPCC) La sindrome di Lynch II, denominata Hereditary Non-Polyposis Colorectal Cancer (HNPCC), comprende, nei membri di una famiglia affetta, diversi tipi di carcinomi insieme a quello del colonretto tra cui cancro ovarico, endometriale, urogenitale, pancreatico e delle vie biliari [13, 14] (Figura 4). Una donna appartenente ad una famiglia con sindrome Lynch II ha un rischio di sviluppare il CaOv aumentato di circa tre volte rispetto alla popolazione generale, ma in realt tale rischio dipende dalla frequenza della malattia nei parenti di I e II grado [51]. Tale sindrome trasmessa in maniera autosomica dominante nei geni implicati nel
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Loredana Bruno, Sergio Rizzo, Valentina Cal, Mario Federico, Rita Passantino, Rita Ferla, Giuseppe Cicero, Eliana Gulotta, Michele Frazzetta, Maria Buscemi, Aldo Gerbino, Viviana Bazan e Antonio Russo

Figura 4. Albero genealogico di una famiglia con sindrome del Cancro Colonrettale Non-Poliposico Ereditario (HNPCC). CaOv: Carcinoma Ovarico; CRC: Cancro Colonrettale; End: Cancro Endometriale.

meccanismo del riparo al danno del DNA, denominato sistema del mismatch repair (geni MMR) [52]. Ad oggi pochi studi ci consentono di analizzare i casi di cancro ovarico in queste famiglie HNPCC, ma quello che si evince la precoce et di insorgenza, infatti let media alla diagnosi 42.7 anni (Watson P, Gynec Oncol 2001). Tuttavia, rispetto ai casi di cancro ovarico sporadico che insorge intorno ai 50 anni, let di insorgenza non differisce particolarmente poich in in donne con predispozione genetica raro osservare casi di cancro ovarico al di sotto dei 45 anni [53]. Watson e lInternational Collaborative Group hanno riscontrato che la maggior parte dei CaOv nelle famiglie con HNPCC di tipo epiteliale, sono differenziati o moderatamente differenziati e sono di stadio FIGO I e II alla diagnosi. Carcinomi endometriali sincroni sono stati riportati nel 21.5% dei casi. Le caratteristiche clinico-patologiche pi favorevoli di questi tumori, rispetto agli sporadici, rendono possibile un trattamento chirurgico radicale [54]. La genetica di suscettibilit: i geni MMR. Il 70% dei portatori di mutazione nelle famiglie affette da HNPCC hanno mutazioni nei geni hMSH2 e hMLH1. Altri geni quali hPMS1, hPMS2 e hMSH6 sono coinvolti in misura minore, ma anche le loro mutazioni si manifestano ad alta penetranza [55-57]. Tali geni sono coinvolti nei processi di riparo al DNA e sono responsabili del riparo dei mismatch dei nucleotidi durante la replicazione del DNA impedendo cos la propagazione di alterazioni geniche [58]. I geni MMR sono localizzati in 5 diversi cromosomi e codificano per proteine che agiscono sottoforma di eterodimeri che lavorano in sequenza per riconoscere e riparare i mismatch del DNA. La proteina MSH2 forma eterodimeri con MSH6 e MSH3; tali eterodimeri
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sono in grado di riconoscere e legare il mismatch durante le fasi S o G2 del ciclo cellulare. MLH1 con PMS1 o PMS2 forma eterodimeri che sono coinvolti nel taglio del filamento contenente il mismatch e nel reclutamento degli enzimi che provvedono al conseguente riparo [59]. Le alterazioni del gene MSH2 bloccano la formazione di eterodimeri direttamente coinvolti nel riparo al danno del DNA, mentre MSH6 e MSH3 hanno funzioni sovrapponibili e quindi alterazioni in uno di tali geni non impediscono il normale funzionamento del pathway[60]. Lalterazione di tale pathway determina nella cellula uninstabilit delle sequenze dei microsatelliti nel DNA (MSI), che comporta un rapido accumulo di mutazioni e una conseguente trasformazione maligna [61]. Donne affette da sindrome di Linch II con mutazioni nel gene MSH2 hanno approssimativamente il 12% di rischio nel corso della vita di avere un tumore allovaio [62]. Alcuni studi si sono indirizzati alla scoperta di possibili aplotipi di questi geni che, in misura maggiore rispetto alle mutazioni, potrebbero contribuire alla suscettibilit per il CaOv[63]. Infatti, diversi comuni polimorfismi nei geni MMR possono essere associati alla variazione del rischio di cancro. Song et al. in uno studio condotto su 1531 casi di CaOv invasivo rispetto a 2570 controlli, hanno dimostrato che uno specifico single nucleotide polymorphism (SNP) del gene PMS2 da un rischio aumentato di CaOv, mentre due SNPs di MSH6 e MSH3 sembrano avere un effetto protettivo recessivo, anche se le frequenze genotipiche delle varianti non risultavano significative [64]. Mentre le mutazioni dei geni BRCA sono identificate pi frequentemente in carcinomi ovarici di tipo sieroso, le mutazioni dei geni MMR invece sono riscontrate prevalentemente in vari istotipi di carcinoma ovarico [10]. Inoltre, alcuni casi di carcinoma ovarico sieroso e indifferenziato presentano mutazioni nel gene TP53, mentre altri di tipo endometrioide hanno mostrato il 90% di overespressione di BCL2 e instabilit dei microsatelliti, ed altri di tipo mucinoso presentano il 40-50% delle mutazioni di ki-RAS [65]. Le linee guida per le famiglie con HNPCC sono da riferirsi ai criteri di Amsterdam [66]. Negli individui che rientrano in questi criteri viene effettuato il test genetico a partire da un campione di sangue mediante sequenziamento diretto dei geni di suscettibilit, MLH1 ed MSH2. Se la storia familiare soddisfa criteri meno stringenti, come quelli di Bethesda, vengono eseguiti dei test di pre-screening quali lanalisi dellinstabilit dei microsatelliti (MSI) e limmunoistochimica (IHC) [67, 68]. Tali analisi necessitano del tessuto tumorale del membro pi giovane allinterno della famiglia senza effettuare lanalisi mutazionale del DNA che risulterebbe inutile in termini di denaro e tempo.
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Limmunoistochimica valuta la presenza o lassenza delle proteine MLH1, MSH2, MSH6 e PMS2 mediante specifici anticorpi e non ci consente di predire un loro difetto, quale la formazioni di proteine tronche o riarrangiamenti genomici [69]. Lassenza della proteina suggerisce quale dei geni MMR analizzare. Purtroppo, in caso di mutazioni di senso, lIHC non risulta efficace nella diagnostica molecolare, infatti la proteina potrebbe non essere funzionale ma essere rilevata dallanticorpo. Pertanto spesso in famiglie affette da HNPCC con rischio di CaOv viene associato allIHC anche lanalisi dellinstabilit dei microsatelliti come test di pre-screening [67]. Il set utilizzato di markers microsatellitari lo stesso di quello utilizzato per i tumori sporadici, che mostrano uninstabilit del 10-15% dei casi rispetto al 85%-92% di quelli familiari. Ci dimostra che spesso nelle famiglie HNPCC per valutare il rischio di tumore ovarico potrebbe risultare necessario lanalisi dellMSI [70, 71]. I tumori sono definiti ad alta MSI se almeno il 30% dei markers risultano positivi, con bassa MSI se questi sono meno del 30%. Se nessuno dei markers mostra instabilit il tumore risulta stabile [72]. Tale metodo ha una sensibilit pari al 93% per i portatori di mutazione nei geni MMR, ma, paragonato allimmunoistochimica, non offre indicazioni su altri geni che potrebbero essere alterati. Nelle famiglie con una forte storia familiare per il tumore del colon e il CaOv consigliato comunque lo screening mutazionale almeno dei principali geni, qualsiasi sia il risultato dellanalisi microsatellitare, poich spesso nelle famiglie con HNPCC selezionate utilizzando i criteri di Bethesda, sono presenti meno casi di carcinoma al colon-retto disponibili per lanalisi dei microsatelliti rispetto agli altri casi di carcinomi correlati compreso lovaio. 2.4. Altre sindromi ereditarie predisponenti al carcinoma ovarico Un incrementato rischio di sviluppare un carcinoma ovarico ereditario dovuto ad un gruppo di sindromi minori, quali la Sindrome di Gorlin, la Sindrome di Peutz-Jeghers, lOsteocondromatosi o Sindrome di Ollier. Tali sindromi sono imputabili a specifici geni di suscettibilit, le cui mutazioni possono determinare un particolare istotipo di CaOv. La Sindrome di Gorlin o del carcinoma nevo-basocellulare (NBCCS) una malattia autosomica dominante a penetranza pressocch completa in cui il gene coinvolto PTCH1, costituito da 24 esoni, che codifica per una glicoproteina che inibisce proteine coinvolte nella crescita e nel differenziamento cellulare. Mutazioni di questo gene avviano il processo di cancerogenesi probabilmente attraverso lalterazione della normale via apoptotica. Tale malattia causa carcinomi cellulari multipli, cheratocisti odontogeniche e pits palmo246

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plantari e pu causare fibrosarcomi ovarici. La Sindrome di Peutz-Jeghers una rara malattia autosomica dominante legata ad alterazione del gene STK11 che codifica per una chinasi serina-treonina. caratterizzata da amartomi gastrointestinali e da pigmentazione maculare melanotica delle mucose, delle labbra, delle dita, dei piedi. La Sindrome di Ollier, legata alla alterazione dei geni EXTs, caratterizzata da multiple esostosi in genere benigne ma con possibile evoluzione sarcomatosa. Per i membri affetti da queste sindromi, il rischio di sviluppare un CaOv durante la loro vita inferiore al 2% [10, 16-18]. 3. Rischio cumulativo eredo-familiare Il rischio nel corso della vita di sviluppare il CaOv in famiglie con predisposizione genetica varia in rapporto alla suscettibilit nelle famiglie per le mutazioni nei geni BRCA o nei geni MMR. Il rischio pi alto sembra quello conferito dalle mutazioni nel gene BRCA1. Il rischio per le donne appartenenti a famiglie con HNPCC circa il 12%, mentre arriva sino al 50% nel caso in cui nelle famiglie vi sia un portatore di mutazione del gene BRCA1 [73]. Le mutazioni in BRCA1 sono associate ad un rischio di CaOv stimato tra 28% e 44% nel corso della vita comparato al rischio nella popolazione generale dell1.6%. Il Breast Cancer Linkage Consortium ha stabilto che il rischio cumulativo di cancro ovarico in famiglie che risultano portatrici di mutazione nel gene BRCA2 pi basso rispetto a BRCA1 e risulta dello 0.4% al di sotto dei 50 anni e del 27% a 70 anni. Inoltre frequente osservare casi di CaOv con suscettibilit ai geni MMR e al gene BRCA1 intorno ai 45 anni, mentre la maggior parte dei CaOv associati a mutazioni nel gene BRCA2 sembrano avvenire dopo i 50 anni [21]. Le mutazioni nei geni BRCA inoltre conferiscono un rischio aumentato di cancro ovarico in donne gi affette da CaM e tale valore sembra aumentato di almeno 10 volte rispetto alle donne che non sono portatrici di mutazioni (Tabella 3).

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4. La consulenza oncogenetica La consulenza oncogenetica un processo di comunicazione che offre al paziente la possibilit di comprendere loccorrenza o il rischio di ricorrenza di una malattia genetica allinterno dei membri di una famiglia. Nellambito delle patologie tumorali ereditarie essa rivolta a pazienti affetti da tumore o ai loro familiari con sospetta predisposizione genetica allo sviluppo di specifiche neoplasie ricorrenti nella famiglia [74]. La consulenza oncogenetica si svolge presso centri competenti e prevede un approccio multidisciplinare rappresentato da diverse figure professionali, quali il genetista, il medico oncologo e lo psicologo; ciascuno di questi specialisti svolge un ruolo ben determinato allinterno del programma di consulenza. Le donne a rischio di sviluppare una neoplasia allovaio vengono sottoposte durante la consulenza prima allanalisi della storia familiare attraverso la ricostruzione dellalbero genealogico e successivamente alla valutazione del rischio personale [75] (Figura 5). Valutazione del rischio. Il calcolo del rischio effettuato durante la consulenza oncogenetica, in quanto condizione predisponente, assume un duplice ruolo, da un lato va a quantificare la probabilit che lindividuo si ammali, dallaltro la vulnerabilit dellindividuo stesso. Tra i problemi della consulenza genetica ed in misura maggiore del test genetico vi sono lo stress psicologico, a cui tali individui vengono sottoposti, lansia relativa al risultato e alla loro aspettativa di vita, la perdita della privacy ed i cambiamenti nella dinamica familiare. Per tali motivazioni, negli ultimi anni, stata riconosciuta limportanza del supporto psicologico, durante e dopo la consulenza genetica, al paziente ed ai suoi familiari [76, 77]. Oggi, la valutazione del rischio nei pazienti con CaOv, viene eseguita mediante specifici modelli matematici, quali BRCApro, Shattuck-Eidens, Couch e Myriad. Tali modelli utilizzano dati di prevalenza, penetranza, frequenza di mutazioni nei geni BRCA1/2, numero degli affetti e dei sani nei membri della famiglia, dellet alla diagnosi ed infine del gruppo etnico di appartenenza [78-81]. Il risultato ottenuto dal calcolo del rischio mediante il software BRCApro un valore percentuale: gli individui, che risultano avere un valore superiore al 10%, sono considerati ad alto rischio di essere portatori di una mutazione germinale e vengono sottoposti al test genetico. Tale test comprende lanalisi molecolare nei geni di suscettibilit per la patologia considerata. Ricostruzione dellalbero genealogico e criteri di eleggibilit. La ricostruzione della storia familiare del paziente (probando), rappresenta un momento fondamentale del processo di counsulenza oncogenetica, in quanto in base alla comprensione dellalbero genealogico, gli specialisti potranno fare una diagnosi pi corretta, prevedere una prognosi
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Figura 5. Diagramma del trattamento delle donne ad alto rischio dinsorgenza di Carcinoma Ovarico Geni BRCA: geni BReast CAncer; Geni MMR: geni MisMatch Repair; CaM: Carcinoma della Mammella; CaOv: Carcinoma Ovarico; EPT: Ecografia Pelvica Transvaginale; a: anno.
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Tumori dellovaio, mammella, colon, endometrio, pancreas e del tratto biliare

Loredana Bruno, Sergio Rizzo, Valentina Cal, Mario Federico, Rita Passantino, Rita Ferla, Giuseppe Cicero, Eliana Gulotta, Michele Frazzetta, Maria Buscemi, Aldo Gerbino, Viviana Bazan e Antonio Russo

pi accurata e, quindi, consigliare il consultante sulle decisioni da prendere. La ricostruzione dellalbero genealogico prevede che le informazioni clinico-patologiche di ogni membro della famiglia vengano raccolte per almeno tre generazioni a partire dal probando. Inoltre la storia della famiglia deve contenere sia quella paterna che materna, poich la maggior parte dei geni coinvolti nel rischio di sviluppare il carcinoma dellovaio sono egualmente ereditati (trasmissione autosomica). Dal momento che il CaOv ereditario identificato mediante lanalisi della storia familiare, necessario che le informazioni ottenute durante la costruzione dellalbero geneaologico siano molto accurate, pertanto buona norma verificare quanto riportato dal paziente visionando i referti anatomo-patologici, i certificati di morte o i registri tumori. Qualora il genetista abbia identificato la possibilit di un disordine genetico nella famiglia, il probando decider se effettuare o meno il test genetico. Tale decisione verr presa liberamente dal soggetto a rischio, che una volta informato dei vantaggi di sottoporsi ad un test genetico, ma anche dei limiti di ci, firmer il consenso informato allesecuzione del test [82]. Dallanalisi della storia familiare, il genetista ricever le informazioni necessarie per attribuire al probando una specifica sindrome nella famiglia. Le famiglie con HOC sono riconosciute da una aggregazione alta di casi di carcinomi allovaio tra le donne di una famiglia che abbiano sviluppato il tumore al di sotto dei 50 anni. Mentre le famiglie con HBOC sono riconosciute dalla copresenza di membri affetti da carcinoma alla mammella e allovaio con insorgenza precoce e da membri affetti da entrambe le patologie, da carcinoma alla mammella maschile o dalla presenza di due o pi membri della famiglia di I grado, affetti da carcinomi sia allovaio che alla mammella al di sotto dei 50 anni [1]. Le famiglie HNPCC sono di solito identificate clinicamente dallanalisi della storia familiare di carcinoma al colon-retto piuttosto che CaOv [53]. Tali famiglie soddisfano principalmente i criteri di Amsterdam, in cui deve essere esclusa la poliposi adenomatosa familiare, e viene considerato il cancro al colon-retto e quelli ad esso correlati in tre parenti di I grado oppure in due generazioni successive, di cui almeno uno con diagnosi prima dei 50 anni. In alcune famiglie, specie quelle pi piccole, vengono adottati i criteri meno stringenti di Bethesda, che includono i criteri di Amsterdam, ma prevedono linclusione di probandi con diagnosi di carcinoma al colon-retto al di sotto dei 60 anni ed inoltre comprendono i parenti di II grado che abbiano avuto tumori correlati alla sindrome di Lynch [66, 68]. I criteri che vengono utilizzati dal genetista per definire donne ad alto rischio di CaOv ereditario sono validi per entrambe le sindromi, considerando, per, solo membri di I grado della famiglia, nel modo seguente:
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due o pi donne con cancro ovarico nei membri di I grado a qualsiasi et; un individuo con cancro ovarico a qualsiasi et e uno con cancro alla mammella sotto i 50 anni; un individuo con cancro ovarico a qualsiasi et e due parenti con cancro alla mammella diagnosticato sotto i 60 anni; un individuo affetto portatore di mutazione in uno dei geni di predisposizione al cancro ovarico; tre membri della famiglia con cancro al colon-retto, di cui almeno uno con diagnosi prima dei 50 anni e un caso di cancro ovarico. Esistono dei limiti allapplicazione dei suddetti criteri di selezione; una di esse dipende dal fatto che la maggior parte delle famiglie sono piccole e ci rende difficile la comprensione dei meccanismi di ereditariet. Il tutto si complica nel caso in cui vi siano famiglie con molti membri maschi, poich risulta difficile identificare i portatori di mutazione, dal momento che questi tramanderanno la mutazione germinale senza manifestarla. Ad ogni modo questi sono gli unici strumenti che un genetista possiede per individuare le famiglie ad alto rischio di sviluppare il CaOv [53]. Test genetico per i geni BRCA o MMR. Il test genetico per lindividuazione di mutazioni germinali sta entrando sempre pi nella pratica clinica nei servizi di diagnostica clinica. Unico e grande limite del test che il risultato indica la probabilit e non la certezza di sviluppare un cancro; non tutti gli individui con un test positivo per mutazioni nei geni BRCA o nei geni MMR svilupperanno la malattia. Il test genetico consiste nel sequenziamento automatico diretto di tutti gli esoni codificanti i geni BRCA1/2 e delle regioni di confine esone-introne. Inoltre recenti ricerche hanno portato alla scoperta di nuove tecniche per lidentificazione del 5-10% delle alterazioni, dovute a larghe delezioni coinvolgenti uno o pi interi esoni, che non possono essere rilevate mediante sequenziamento automatico a causa della presenza dellallele wild type [83]. Nel caso di famiglie HNPCC che soddisfano i criteri di Amsterdam possibile eseguire direttamente il sequenziamento dei geni di suscettibiliti sul DNA estratto del sangue periferico del paziente affetto. Tuttavia per le famiglie che soddisfano i criteri di Bethesda si utilizzano dei test di pre-screening per identificare il gene alterato. Un parente affetto il cui tumore mostra unalta MSI ha unalta probabilit di presentare mutazioni nei geni MMR e si procede, quindi, allanalisi mutazionale di questi ultimi. Lanalisi dellMSI, nonostante abbia una sensibilit del 93% nei portatori di mutazione patogenica nei geni MMR, da unidea del difetto del sistema MMR senza per fornirci altre indicazioni. Se non viene identificata alcuna mutazione nel membro affetto sconsigliato il testing agli altri membri della famiglia, poich il risultato sarebbe inconclusivo (risultati falsi negativi inficiati dalla possibile presenza di altri geni MMR sconosciuti).
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Alcuni autori suggeriscono che nelle famiglie con una forte storia familiare per tumore al colon-retto e al carcinoma ovarico consigliabile comunque eseguire lo screening mutazionale almeno dei principali geni MMR, qualsiasi sia il risultato delle analisi microsatellitare. Spesso infatti nelle famiglie HNPCC selezionate con i criteri di Bethesda sono presenti meno casi di tumore al colon-retto disponibili allanalisi dei microsatelliti rispetto ad altri casi di tumori correlati compreso quelli allovaio [67]. Se viene rilevata una determinata mutazione in un membro affetto allora viene suggerito il counselling agli altri membri sani della famiglia, che decideranno di eseguire il test genetico per la ricerca della singola mutazione. Ci ovviamente risulta il pi grande vantaggio del test genetico, ma questultimo ha anche grandi limiti, poich esistono pi di centinaia di differenti mutazioni nei geni di suscettibilit al cancro ovarico e spesso risulta complicato identificare tutte le mutazioni germline nellintero gene analizzato con le tecniche correnti, infatti dagli studi di popolazione, ad oggi, in meno del 50% delle famiglie vengono identificate le alterazioni genetiche. Tale fase detta pre-test prevede che tutti coloro che aderiscono al test genetico conoscano esattamente i potenziali vantaggi e svantaggi legati ad esso. Solo dopo possono decidere di firmare il consenso informato ed essere pronti a ricevere il risultato del test genetico che avr ripercussioni etico-legali e psicologiche per la famiglia a cui appartiene. Infatti una piccola parte delle famiglie a rischio decide di non eseguire il test genetico e sceglie di effettuare solo lo screening clinico di sorveglianza per il cancro ovarico. Coloro che invece scelgono di eseguire il test seguiranno programmi di sorveglianza specifici non solo dal punto di vista clinico ma anche chirurgico [82, 84]. Interpretazione del test genetico. La consulenza genetica post-test consiste nella comunicazione del risultato del test genetico e da lopportunit di un nuovo momento di incontro tra lo specialista ed il paziente. Tale evento necessario per approfondire e/o ribadire ulteriormente i concetti di test genetico e di rischio genetico sia per il paziente che per i membri della sua famiglia [82]. Attualmente non esiste un test genetico con una sensibilit del 100%, conseguentemente per i casi di forte aggregazione familiare non si pu escludere la presenza di una alterazione genetica. Il risultato del test genetico pu essere, positivo, negativo o talvolta non informativo. Un risultato positivo si ha quando una mutazione deleteria nei geni di suscettibilit al cancro ovarico viene identificata nel probando della famiglia analizzata. Per mutazioni deleterie si intendono tutte quelle che portano alla formazione di una proteina funzionalmente inattiva, ad esempio mutazioni frameshift dovute a piccole inserzioni o delezioni che portano alla formazione di una proteina tronca. Un risultato positivo
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per il test genetico implica la ricerca della stessa mutazione in tutti i membri della famiglia e lattuazione di un piano di sorveglianza adeguato. Infatti, ogni membro della famiglia ha una probabilit pari al 50% di avere ereditato la mutazione. Naturalmente la ricerca di una mutazione nota, pi rapida e meno dispendiosa dal punto di vista economico. Un risultato negativo del test genetico indica che non stata identificata alcuna alterazione genetica. Linterpretazione di un risultato negativo dipendente dal caso specifico: se una mutazione stata identificata nella famiglia ed il paziente considerato non la possiede, allora per esso va considerato un rischio pari a quello della popolazione generale, il test viene allora definito veramente negativo. Se invece, un risultato negativo ottenuto in una paziente con una forte storia familiare, con tale risultato non si pu escludere la presenza di altre alterazioni germinali sconosciute [84]. Un risultato non informativo si ottiene quando viene identificata una unknown variant, ovvero unalterazione genetica di significato biologico sconosciuto. A questa categoria appartengono tutte le mutazioni missenso o le varianti identificate in porzioni non codificanti del gene, per le quali non stata ancora dimostrata una chiara associazione con la patologia. Per la loro caratterizzazione sono necessari studi biochimico/funzionali allo scopo di poter identificare limportanza clinica di tali varianti e nella stima del rischio. A tali studi si aggiungono quelli riguardanti lanalisi di segregazione della mutazione nei membri affetti e in quelli sani della famiglia [85]. Comunque il programma di consulenza genetica non termina con la comunicazione del risultato del test al paziente, infatti il consulente genetico deve sempre essere disponibile per rispondere ad ulteriori dubbi insorti nel paziente dopo aver conosciuto lesito del proprio test. Inoltre, necessario monitorare il paziente a rischio ed i suoi familiari, perch la storia di tumori in tali famiglie continuamente in evoluzione. Infine, tenuto conto della rapida evoluzione della ricerca medica, i pazienti devono essere incoraggiati a ricontattare periodicamente il consulente per ricevere le pi recenti informazioni circa i progressi in questo settore. 5. Associazioni clinico-istopatologiche e prognosi Il pattern dettagliato delle caratteristiche istopatologiche nelle donne con carcinoma ovarico portatrici delle mutazioni di BRCA, confrontate con le donne con carcinoma ovarico non portatrici di queste mutazioni genetiche, ancora oggi non completamente definito dal momento che, nella maggior parte degli studi finora condotti, il numero di casi stato relativamente basso. La maggior parte dei dati disponibili riguarda i carcinomi associati a mutazioni germinali di BRCA1 dato che queste, nelle pazienti con questo tumore, sono quattro volte pi frequenti rispetto alle mutazioni di BRCA2 (Tabella 4).
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Circa il 94% dei carcinomi ovarici che si sviluppano in pazienti con mutazioni di BRCA1 sono di istotipo sieroso rispetto al 60% di quelli sporadici [86]. Listotipo mucinoso poco rappresentato nelle portatrici di mutazioni di BRCA1: possibile che mutazioni di questo gene non siano coinvolte nello sviluppo di questo sottotipo di carcinoma ovarico. Tuttavia, per quanto occasionalmente, carcinomi mucinosi invasivi e borderline sono stati riportati anche nelle portatrici di mutazioni di BRCA1 [2, 87]. Le frequenze dei carcinoma ovarico del tipo endometrioide e del tipo a cellule chiare sono uguali o leggermente inferiori nelle portatrici di mutazioni di BRCA1 rispetto ai controlli, rappresentando pertanto una frazione non indifferente di neoplasie in queste pazienti. Piuttosto rari sono invece il carcinoma a cellule transizionali, il carcinoma squamoso, i sarcomi ed il disgerminoma. I carcinomi dellovaio associati a mutazioni di BRCA1 e soprattutto di BRCA2 sono di grado istologico pi alto e presentano una maggiore componente solida rispetto ai tumori sporadici. Essi, inoltre, risultano caratterizzati da iperespressione e/o mutazione nel gene Tp53 (60% vs 30%), mentre lespressione di HER sovrapponibile a quella dei carcinomi dellovaio non associati a queste mutazioni genetiche. Carcinomi dellovaio associati a mutazioni di BRCA1 e di BRCA2 presentano caratteristiche morfologiche ed immunofenotipiche simili, differentemente dai carcinomi mammari associati a queste mutazioni genetiche. Le principali differenze tra carcinomi dellovaio associati a mutazioni di BRCA1 e di BRCA2 sono il pi basso rischio di insorgenza di carcinoma nelle portatrici di mutazioni di BRCA2 e linsorgenza del tumore, in queste donne, in et pi avanzata. La pi alta frequenza dellalto grado e delliperespressione e/o mutazione di p53, noti fattori prognostici sfavorevoli, nei carcinomi dellovaio associati a mutazioni di BRCA, hanno sollevato il sospetto che questi tumori abbiano una prognosi peggiore. In realt alcuni studi hanno evidenziato una prognosi migliore tra le portatrici di BRCA1 affette da carcinoma ovarico avanzato rispetto a una serie di casi sporadici confrontabili per stadio (sopravvivenza media 77 vs 29 mesi e 91 vs 54 mesi) [87]. Lo stesso risultato stato ottenuto da unindagine nazionale condotta in Israele tra pazienti con mutazioni dei geni BRCA [88] e in uno studio giapponese in pazienti trattate con cisplatino [89]. Al contrario, altri due studi non hanno rilevato alcun vantaggio di sopravvivenza tra le pazienti affette da carcinomi ovarici ereditari rispetto a quelle affette da carcinomi sporadici [90, 91]. Ci sono almeno due possibili spiegazioni alla sopravvivenza pi lunga delle pazienti con carcinoma dellovaio associato a mutazioni di BRCA. Sebbene questi tumori si manifestino con caratteristiche chirurgiche e patologiche sovrapponibili a quelle dei casi sporadici di, le loro propriet biologiche potrebbero rendersi responsabili di un decorso clinico pi indolente, probabilmente come conseguenza di un pi basso tasso di divisione cellulare. Alternativamente, i carcinomi dellovaio associati a mutazioni di BRCA potrebbero rispondere pi favorevolmente alla chemioterapia, una eventualit che ben si accorda
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con la dimostrazione in questi tumori di un pi lungo intervallo libero da malattia dopo la chemioterapia primaria rispetto ai controlli. Questa ipotesi sarebbe, inoltre, sostenuta dalla funzione espletata dalle proteine BRCA: esse sono coinvolte nei meccanismi di riconoscimento e riparazione dei danni al DNA e nellassemblaggio del fuso mitotico. Linattivazione delle proteine BRCA causerebbe una maggiore sensibilit delle cellule neoplastiche ovariche agli agenti citotossici che agirebbero producendo addotti al DNA come il cisplatino; viceversa proteine BRCA1 funzionalmente attive sarebbero necessarie affinch gli agenti antimicrotubuli come i taxani possano esercitare la loro azione citocida [89]. Queste osservazioni, al momento sostenute da studi in vitro, potrebbero avere un chiaro risvolto clinico considerato che entrambi i farmaci, da soli o in associazione, vengono comunemente utilizzati nelle varie fasi della neoplasia.

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6. Strategie di Prevenzione nei gruppi ad alto rischio 6.1. Screening ginecologico Esplorazione pelvica bimanuale. Tra la popolazione generale non ha n la specificit n la sensibilit per identificare efficacemente il carcinoma ovarico. Non esistono dati sullutilit clinica dellesplorazione pelvica bimanuale nelle donne con una predisposizione genetica al carcinoma ovarico. Esame colpocitologico. Pu occasionalmente rivelare la presenza di cellule maligne di provenienza ovarica, ma non viene ritenuto una metodica di screening per il carcinoma ovarico praticabile, dato che possiede una sensibilit limitata al 10-30% [92]. CA125 sierico. I dati relativi allo screening della popolazione a rischio per una predisposizione genetica al carcinoma ovarico con il dosaggio del Ca125 sierico sono limitati. Uno studio su 180 donne ad alto rischio riporta un aumento del Ca125 in 4 su 8 carcinomi epiteliali ovarici, ma soprattutto in stadio avanzato (25% al I-II stadio vs 75% al II stadio) [93]. Un altro studio condotto su 1.502 donne con familiarit positiva per carcinoma ovarico (e sospetta predisposizione genetica in un terzo dei casi), il Ca125 era in grado di migliorare il valore predittivo positivo dellecografia pelvica transvaginale (dal 12,7% al 42,9%), ma a scapito della sensibilit (da 100% a 43%) [73]. In un altro studio condotto in donne ad alto rischio per carcinoma ereditario della mammella/ovaio o carcinoma ereditario non polipoide del colon-retto (HNPCC), il marcatore risultava alto nell11% dei casi; in premenopausa lincremento si associava con patologie pelviche benigne (endometriosi, adenomiosi, miomi), mentre in menopausa non venivano evidenziate immagini sospette allecografia di approfondimento [94]. Ecografia pelvica transvaginale (EPT). superiore allecografia transaddominale nella diagnosi preoperatoria delle masse annessiali [95]. Entrambe le tecniche sono meno specifiche nelle donne in premenopausa rispetto a quelle in postmenopausa per le modificazioni delle ovaie durante il ciclo mestruale. I valori predittivi positivo e negativo della EPT sono superiori nelle donne con familiarit positiva per carcinoma ovarico e possono venire migliorati dallaggiunta delle tecniche di color Doppler, almeno tra le donne in postmenopausa [96, 97]. I dati sui potenziali benefici della EPT in donne ad alto rischio sono limitati. In uno studio sono state riscontrate anormalit nel 3,8% delle EPT, di cui solo il 5% (3 su 61) erano risultate riferibili a carcinomi ovarici (2 al I stadio e 1 al II stadio) [98, 99]. In un altro studio tutti i 5 carcinomi ovarici trovati in un gruppo di 180 donne a rischio erano stati correttamente diagnosticati dalla EPT, ma solo uno era al I stadio, mentre gli altri 4 erano al IV [93]. Test di screening combinato. Nella popolazione generale la sensibilit della
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combinazione di uno screening basato sul dosaggio del Ca125 e lEPT dellordine dell80-100%, ma il valore predittivo molto basso. Pertanto basandosi solo sul valore del Ca125 tra lo 0,1-0,6% di donne sane subirebbero un intervento inutile, mentre tale percentuale sarebbe ancora maggiore utilizzando la sola EPT come metodica di screening (0,1% a 4,4%). Uno studio clinico randomizzato riporta che 6 su 10.000 donne inviate allo screening hanno un carcinoma ovarico correttamente diagnosticato (di cui solo la met a uno stadio con possibilit di cura) e 24 subiscono un intervento non necessario [100]. Questo studio suggerisce che la combinazione dei due metodi pu anticipare la diagnosi di neoplasie ovariche e che lanticipo diagnostico pu tramutarsi in un miglioramento della sopravvivenza. Nello stesso tempo, per, esso dimostra le difficolt e la spesa che si devono affrontare per condurre un programma di screening per una malattia cos rara. La situazione potrebbe essere diversa in un gruppo di pazienti ad alto rischio per la mutazione dei geni BRCA1 e BRCA2, dove la maggiore incidenza della malattia potrebbe spostare significativamente il calcolo dei costi-benefici dello screening. 6.2. Contraccettivi orali La possibilit di ridurre lincidenza di neoplasie nelle sindromi ereditarie attraverso strategie di chemioprevenzione una delle possibilit pi intensamente investigate dalla clinica. La possibilit, infatti, di ridurre il rischio globale di sviluppare un tumore da parte di un soggetto predisposto senza lobbligo di ricorrere alla demolizione chirurgica o ad altri interventi invasivi , data let dei pazienti interessati, un obiettivo primario. Nella popolazione generale lassunzione di contraccettivi orali si associa con una riduzione del rischio del 40-50%, che aumenta con la durata di assunzione e persiste per 10-15 anni dopo la sospensione [101, 102]. Lo stesso effetto stato osservato anche in donne con familiarit positiva per carcinoma ovarico [103] e tra le portatrici di mutazioni dei geni BRCA1 e BRCA2 [104]. Tuttavia, uno studio retrospettivo su unampia coorte di pazienti affette da carcinoma ovarico fa supporre che leffetto protettivo non riguardi le portatrici di mutazioni di BRCA1, ma solo le non portatrici [105]. Inoltre, deve essere tenuto presente che unaltro studio riporta una maggiore incidenza di carcinoma mammario tra le portatrici di mutazioni di BRCA1/2 che avevano assunto contraccettivi orali per oltre 48 mesi (OR 7,8 p 0,004) [106]. Questi dati devono essere valutati con estrema cautela date le esigue dimensioni del campione, ma alla luce di tali incertezze alcuni autori ritengono che lutilizzo di contraccettivi orali non possa essere raccomandato come opzione per ridurre il rischio di carcinoma ovarico nelle portatrici di mutazioni di BRCA1/2 [19, 107].
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6.3. Modificazione dei fattori di rischio non-genetici Laver avuto almeno una gravidanza a termine riduce il rischio di carcinoma ovarico tra le donne della popolazione generale. Le donne che hanno avuto un figlio hanno un rischio del 45% inferiore rispetto alle nullipare e ulteriori gravidanze sembrano diminuire ulteriormente il rischio del 15% [108]. Al contrario, sebbene i dati siano limitati, tra le portatrici di mutazioni di BRCA uno studio caso-controllo fa supporre che il rischio di carcinoma ovarico cresca con il numero di figli: ogni parto risulta associato con un incremento del 40% del rischio fino al quinto figlio, mentre le gravidanze successive hanno un effetto protettivo [109]. Gravidanze tardive hanno un effetto protettivo e ogni intervallo di 5 anni associato con una riduzione del rischio del 18%. Pertanto, le donne che hanno tutti i loro figli dopo i 30 anni e le nullipare formano il gruppo a minor rischio (RR 0,30). Anche in un altro studio caso controllo laver partorito in et avanzata ha un effetto protettivo rispetto al carcinoma ovarico, ma solo fra le donne con una familiarit positiva [110]. Aver allattato almeno un figlio riduce il rischio di carcinoma ovarico tra la popolazione generale. Non esistono invece dati sulle donne con una predisposizione ereditaria al carcinoma ovarico. 6.4. Chirurgia profilattica Annessiectomia profilattica (Prophylactic Salpingo-Oophorectomy, PSO). Viene generalmente riservata a donne con mutazione accertata dei geni BRCA1 e BRCA2. I dati sullefficacia della procedura sono pochi e di bassa qualit, essendo derivati da studi retrospettivi condotti su piccole coorti di pazienti. Vi accordo sul fatto che la protezione non sia completa dal momento che una carcinosi peritoneale pu svilupparsi nel 1,8-10,7% delle pazienti ad alto rischio sottoposte ad annessiectomia profilattica, probabilmente in relazione alla comune origine embriologica del peritoneo e dellepitelio di rivestimento ovarico [111-113]. A tale proposito bisogna comunque considerare che un esame accurato delle ovaie asportate profilatticamente pu rivelare piccoli foci di carcinoma che erano inizialmente sfuggiti allesame istopatologico; pertanto possibile che alcuni dei carcinomi peritoneali di queste casistiche rappresentino in realt la disseminazione metastatica di carcinomi ovarici non diagnosticati al momento del primo intervento [114, 115]. Inoltre nel calcolo rischio/beneficio dellannessiectomia profilattica, sono da prendere in considerazione le potenziali morbilit e mortalit connesse alla chirurgia [116], i rischi di una menopausa precoce sul sistema cardiovascolare [117] e sullosteoporosi [118] e quelli collegati a una HRT a lungo termine [119]. Nel 1995 il National Institute of Health (NIH), sulla base degli studi fino ad allora pubbli258

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cati in cui lefficacia della annessiectomia profilattica veniva derivata dal follow up di casistiche di donne considerate ad alto rischio ma non sottoposte al test genetico, raccomandava che le donne portatrici di mutazioni fossero sottoposte ad annessiectomia profilattica dopo il completamento del loro piano familiare o comunque dopo i 35 anni di et [120]. Al contrario il Cancer Genetic Studies Consortium concludeva che i dati erano insufficienti a esprimere raccomandazioni a favore o contro lannessiectomia profilattica per ridurre il rischio di carcinoma ovarico [121]. Successivamente alla pubblicazione di questi documenti, diversi studi di analisi decisionale hanno esaminato limpatto dellovariectomia e della mastectomia profilattica sul rischio di carcinoma dellovaio e della mammella in portatrici di mutazioni dei geni BRCA [122-124]. Nel pi recente di essi (van Roosmalen MS et al.) lannessiectomia profilattica eseguita a 30 anni di et si associa a un guadagno in termini di sopravvivenza attesa di 5,3-9,5 anni in relazione alla penetranza della mutazione; inoltre si introduce leffetto protettivo nei confronti del carcinoma mammario conferito dallannessiectomia profilattica in premenopausa, che stato confermato anche tra le portatrici di mutazioni dei geni BRCA [125]. Pi recentemente sono stati pubblicati alcuni studi caso-controllo condotti su portatrici di mutazioni BRCA1/2 sottoposte ad annessiectomia profilattica oppure follow up clinicostrumentale [126-129] (Tabella 5). In uno studio caso-controllo retrospettivo (Rebbeck et al.), condotto su 551 portatrici di mutazioni di BRCA1 o BRCA2, 259 donne sono state sottoposte a ovariectomia e 292 a controlli clinico-strumentali per un follow up medio di 8 anni. Nel primo gruppo sono state riscontrate 6 neoplasie ovariche al I stadio (2,3%) al momento dellannessiectomia profilattica, mentre 2 donne (0,2%) hanno sviluppato una neoplasia sieroso-papillare peritoneale dopo 3,8 e 8,6 anni dallintervento. Nel gruppo sottoposto a follow up sono state invece registrate 58 neoplasie (19,9%). Pertanto, escludendo i casi diagnosticati al momento dellintervento, lannessiectomia profilattica ha ridotto lincidenza di neoplasia dellepitelio celomatico del 96% (IC 95% 0,01-0,16). Inoltre, lannessiectomia risultata anche ridurre del 53% il rischio di carcinoma mammario rispetto ai controlli tra le donne che non erano state sottoposte anche a mastectomia profilattica (IC 95% 0,29-0,77) [127]. Kauff et al. hanno condotto uno studio prospettico su 170 portatrici di mutazioni di BRCA1 o BRCA2, di cui 98 sottoposte ad annessiectomia profilattica e le restanti a follow up clinico-strumentale. Durante 24,2 mesi di follow up, nel primo gruppo sono stati riscontrati 3 carcinomi mammari e 1 carcinoma dellepitelio celomatico peritoneale, mentre fra le donne sottoposte a follow up si sono sviluppati 8 carcinomi mammari, 4 carcinomi ovarici e 1 carcinoma dellepitelio celomatico peritoneale. Considerando complessivamente le neoplasie mammarie e ginecologiche, lannessiectomia risultata conferire una riduzione
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del rischio pari al 75% (IC 95% 0,08-0,74) [126]. Il terzo studio (Scheuer et al.) consiste in una raccolta prospettica delle neoplasie diagnosticate in un gruppo di 251 portatrici di mutazioni di BRCA1 o BRCA2. Tra le 90 donne sottoposte ad annessiectomia profilattica sono state riscontrate una neoplasia ovarica (stadio IC) e una neoplasia tubarica (stadio IA) al momento dellintervento, mentre fra le donne sottoposte a follow up sono stati riscontrati un carcinoma peritoneale e 3 carcinomi ovarici al I e II stadio dopo un follow up medio di 24,8 mesi [128]. Il pi ampio studio prospettico sulle portatrici di mutazioni di BRCA1 o BRCA2 sottoposte a PSO ha evidenziato una riduzione del rischio dell80%. Il rischio cumulativo di sviluppare un carcinoma peritoneale primitivo nei ventanni successivi allintervento profilattico stato valutato del 4,3% [129]. Complessivamente, questi dati confermano lefficacia della annessiectomia profilattica nel ridurre il rischio di carcinoma dellepitelio celomatico peritoneale e la possibilit di asportare neoplasie al I stadio non sospettate sulla base delle indagini clinico strumentali al momento della chirurgia profilattica. Questultimo dato sottolinea limportanza di sottoporre i tessuti asportati a una dettagliata analisi istopatologica alla ricerca di neoplasie occulte che potrebbero richiedere trattamenti aggiuntivi. Per quanto concerne laccettazione della procedura, le portatrici di mutazioni dei geni BRCA di nazionalit americana, canadese ed europea sono pi inclini a sottoporsi allannessiectomia profilattica che alla mastectomia profilattica (50% vs 8-28%) [130, 131]. Inoltre, la maggior parte degli studi riporta maggior soddisfazione per la decisione presa e un miglioramento della qualit di vita in coloro che scelgono la chirurgia profilattica rispetto a coloro che optano per un follow up clinico-strumentale. La maggior parte delle portatrici a cui viene proposta HRT dopo lannessiectomia profilattica, si sottopone ad essa [132-134]. Riguardo il possibile aumento del rischio di neoplasia mammaria associato alla somministrazione di HRT in portatrici di mutazioni dei geni BRCA sottoposte a PSO, uno studio caso-controllo sembra non confermare questa possibilit [125]. In questo studio, infatti, lannessiectomia profilattica in portatrici non sottoposte a mastectomia profilattica si associava a una riduzione significativa del rischio di carcinoma mammario (RR 0,53 dopo 5 anni e RR 0,33 dopo 10 anni) anche tra le utilizzatrici di HRT. In caso di intervento elettivo la via laparoscopica preferibile, essendo gravata da un tasso di complicazioni del 9%, che sono serie solo nell1-2% dei casi [135]. Alcuni autori suggeriscono a tal fine di eseguire anche listerectomia al fine di asportare la porzione istmica della tuba, sebbene la quota di neoplasie che insorgono in questa sede sia sconosciuta e nonostante questo atteggiamento sia verosimilmente destinato a ridurre laccettabilit della procedura, oltre ad aumentarne rischi e costi [136].
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Legatura delle tube di Falloppio. In uno studio prospettico associata a una riduzione del rischio di carcinoma ovarico del 33% tra la popolazione generale [137]. Anche uno studio caso-controllo tra donne con mutazioni di BRCA1 o BRCA2 dimostra una riduzione significativa del rischio (OR 0,39) in quelle sottoposte a legatura tubarica [138]. Annessiectomia profilattica in corso di isterectomia. stato calcolato che tra le donne della popolazione generale al di sopra dei 40 anni sottoposte a isterectomia siano necessarie 400 annessiectomie profilattiche per prevenire un caso di carcinoma ovarico. La riduzione dellincidenza del 10% del carcinoma che verrebbe a verificarsi, considerando solamente gli Stati Uniti, determinerebbe la prevenzione di 2.300 nuovi casi allanno [139]. Poich il beneficio proporzionale allincidenza della neoplasia, la rimozione delle ovaie in corso di isterectomia in un gruppo di donne ad alto rischio si tradurrebbe in un maggior numero di neoplasie ovariche prevenute [116]. Inoltre, per quanto concerne i rischi di una menopausa precoce conseguente allannessiectomia, bisogna considerare che, entro 2 anni dallisterectomia eseguita in premenopausa, nel 30% delle donne si verificano sintomi da carenza estrogenica e una riduzione della massa ossea anche se le ovaie vengono preservate [140]. Poich in portatrici di mutazioni di BRCA aumenta anche il rischio di carcinoma delle salpingi, queste devono essere asportate interamente nel corso di un intervento profilattico. Isterectomia profilattica. Listerectomia semplice associata con una riduzione del rischio di carcinoma ovarico tra la popolazione generale [137]. Le pazienti operate per via laparotomica per altre indicazioni vengono pi frequentemente sottoposte anche ad
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annessiectomia profilattica rispetto a quelle operate per via vaginale [141]. Ci dipende dallestrema facilit e rapidit dellannessiectomia nel corso di una laparotomia, mentre la via vaginale presenta maggiori difficolt tecniche specialmente nelle donne giovani. Per questo motivo alcuni autori suggeriscono di eseguire interventi vaginali laparoassistiti che garantiscono anche una migliore esplorazione della cavit addominale e una pi agevole rimozione degli annessi [135]. Le portatrici di mutazioni geniche correlate della sindrome del carcinoma del colonretto non polipoide ereditario (HPNCC), dovrebbero ricevere una consulenza circa le possibili misure preventive. A coloro che non intendono avere altri figli o che vengono sottoposte a una chirurgia per il carcinoma colon-rettale, dovrebbe essere offerta lopzione di una isterectomia profilattica (oltre allannessiectomia profilattica) per ridurre il rischio di carcinoma endometriale.

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7. Punti Chiave 1. Il principale fattore di rischio per lo sviluppo del CaOv rappresentato da una forte familiarit. 2. Sono state descritte tre sindromi ereditarie associate al CaOv: HOC (Hereditary Ovarian Cancer), HBOC (Hereditary Breast and Ovarian Cancer), HNPCC (Hereditary Non Poliposis Colorectal Cancer); la prima si ritiene una variante di HBOC. 3. HOC e HBOC sono associate a mutazioni nei geni BRCA1 e BRCA2. 4. HNPCC associata a mutazioni nei geni MMR. 5. Ai pazienti con anamnesi suggestiva di CaOv ereditario dovrebbero essere offerti counseling e test genetico. 6. La maggior parte dei CaOv associate a mutazioni BRCA sono di tipo sieroso. 7. Lo screening raccomandato prevede semestralmente ecografia pelvica e dosaggio del CA125 sierico. 8. Le donne ad alto rischio di CaOv dovrebbero sottoporsi a salpingo-ooforectomia profilattica dopo aver completato le gravidanze, sebbene la chirurgia non protegga completamente dal rischio di carcinoma peritoneale primitivo. 9. Listerosalpingo-ooforectomia dovrebbe esser proposta alle donne affette da HNPCC.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

RILIEvI IMMUNOISTOCHIMICI DELLA ORfANINA fQ NELLAPPARATO GENITOURINARIO DEL RATTO


[Immunohistochemical study of orphanin FQ in rats urogenital tract ]
Giuseppe Bonaventura
DI.ME.S. - Sezione di Istologia ed Embriologia Arcangelo Pasqualino di Marineo - Facolt di Medicina e Chirurgia - Universit degli Studi di Palermo (I)

Parole chiave: Orfanina, Immunoistochimica, Apparato urogenitale Key words: Orphanin, Immunohistochemistry, Urogenital tract Abstract. Nociceptin/Orphanin FQ (OFQ) is a recently discovered endogenous neuropeptide that structurally resembles an opioid peptide. In the central nervous system this peptide is implicated in the nociception. More recently its documented distribution in other peripheral structures suggests a possible role for OFQ in regulation of other physiological functions. On this subject the mouse vas deferens is proposed as available bioassay for nociceptin. It therefore seems interesting to evaluate immunohistochemical expression of this neuropeptide in distinct segments of rat male urogenital tract. The results obtained supply the morphohistochemical basis to demonstrate that Orphanin plays a neurocrine control on motility and a paracrine control on secretion in examined urogenital apparatus segments. Riassunto. Orphanin FQ (Nociceptin) un neuropeptide endogeno scoperto di recente strutturalmente simile ai peptidi oppioidi. Nel sistema nervoso centrale questo peptide implicato nella nocicezione. Recentemente la sua documentata distribuzione in altri distretti ha suggerito un possibile ruolo per OFQ nella modulazione di altre funzioni fisiologiche. Su questo argomento il vaso deferente di topo proposto come modello sperimentale valido per la orfanina. Pertanto sembrato interessante valutare lespressione immunoistochimica di questo neuropeptide in diversi segmenti dellapparato genitourinario del ratto. I risultati ottenuti forniscono la base morfologica per dimostrare che lorfanina gioca un controllo di tipo neurocrino sulla attivit motoria ed uno di tipo paracrino sulla attivit secretiva nei segmenti dellapparato urogenitale di ratto esaminati.

Introduzione La orfanina FQ (N/OFQ) un neuropeptide endogeno identificato inizialmente nel SNC, che presenta analogie strutturali con i neuropeptidi oppioidi, implicati, come noto, nella nocicezione, donde la qualifica alternativa di nocicettina ad esso attribuito e la conseguente assimilazione del suo ruolo alla nocicezione. Nel corso del procedimento di clonazione dei recettori degli oppioidi (, e k) era stato previamente isolato nel 1994 un recettore ad essi parzialmente affine e perci qualificato come ORL-1 (Opioid-Receptor-Like) [1,2,3], rimasto orfano del suo ligando naturale per circa 1 anno, fino a quando la nocicettina non venne identificata nel 1995 da Meunier [4] come ligando endogeno del recettore ORL-1,

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per questo ribattezzato come recettore NOP. Una esaustiva rassegna sulla distribuzione immunoistochimica della Nocicettina/Orfanina nel SNC stata realizzata nel 1999 da Neal e coll. [5]; essa rivela che il neuropeptide ha in questo distretto una distribuzione assai estesa, il che lascia presumere che esso sia implicato non soltanto nei complessi e discussi fenomeni nocicettivi ma anche nei processi di apprendimento e di memoria, nellansia, nellepilessia, nellanoressia nervosa e nel bilancio energetico [6,7,8,9]. Il sistema N/OFQ-recettore NOP rappresenta un interessante bersaglio farmacologico per lo sviluppo di nuovi ligandi selettivi agonisti o antagonisti, che da un lato offrono nuove prospettive terapeutiche, dallaltro definiscono ulteriori ruoli di tale sistema in varie situazioni patofisiologiche [10,11,12,13]. Pi recentemente, al di l dei neuroeffetti centrali, numerose evidenze sperimentali enfatizzano alcune azioni periferiche, di particolare interesse terapeutico, che la nocicettina/ orfanina, attivando il suo recettore NOP, esplica al di fuori del SNC, specie nellambito degli apparati cardiovascolare, genitourinario, gastroenterico e respiratorio [14,15,16,17,18,19, 20]. Le suesposte implicazioni funzionali dellorfanina a livello di tali distretti periferici ci hanno stimolato ad esaminarne la sua distribuzione in tali sedi. Lo studio iniziato con il mappaggio immunoistochimico del neuropeptide su alcuni segmenti dellapparato genitourinario del ratto (vescichette seminali, dotto deferente, pene, ghiandole bulbouretrali e vescica). Materiali e metodi Sono stati impiegati 10 ratti tipo Wistar, trattati in accordo con la Convenzione di Helsinki sullutilizzo degli animali nella ricerca biomedica, e sacrificati previa anestesia (50 mg/kg di Nembutal somministrato per via endoperitoneale). Sono stati prelevati il pene, le ghiandole bulbouretrali, le vescichette seminali, i dotti deferenti e la vescica urinaria. I campioni fissati in liquido di Bouin ed inclusi in paraffina sono stati sezionati e processati per la tecnica immunoistochimica mediante EnVision+System HRP con AEC come substrato (Dako Cytomation), utilizzando un anticorpo policlonale anti Orfanina FQ (Chemicon AB5515). Contemporaneamente i controlli negativi sono stati realizzati su sezioni adiacenti processate in base allo stesso protocollo omettendo il passaggio dellantisiero primario. Tutti i campioni sono stati studiati mediante fotomicroscopio Leica DMLD. Risultati Ghiandole bulbouretrali: Lepitelio degli adenomeri mucosi tubulo-alveolari rivela discreta immunoreattivit per la nocicettina, che si presenta uniformemente distribuita negli adenomeri dellintera formazione ghiandolare. La reattivit citoplasmatica dei singoli ci272

Rilievi immunoistochimici della orfanina FQ nellapparato genitourinario del ratto

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Figures 1-6: [1] Vescichette seminali di ratto. Forte ma discontinua immunopositivit dellepitelio ghiandolare pseudostratificato. Ob. 10x - [2] Vescichette seminali di ratto. Immunopositivit di cellule epiteliali maggiormente evidente nel settore citoplasmatico medio-apicale. Ob. 20x - [3] Ghiandole bulbouretrali di ratto. Discreta immunoreattivit dellepitelio degli adenomeri mucosi. Contrasto nucleare con ematossilina di Mayer 1%. Ob. 10x - [4] Ghiandole bulbouretrali di ratto. Tronchicini nervosi immunoreattivi nel connettivo perighiandolare. Contrasto nucleare con ematossilina di Mayer 1%. Ob. 20x - [5] Dotto deferente di ratto. Intensa ma non uniforme immunoreattivit a carico dellepitelio pseudostratificato mucosale. Contrasto nucleare con ematossilina di Mayer 1%. Ob. 20x - [6] Pene di ratto. Forte immunopositivit dellepitelio prepuziale. Ob. 20x.

totipi secernenti mostra un aspetto cribroso relativo alla presenza di vescicole di secreto mucoso. Nel connettivo perighiandolare si apprezzano tronchicini nervosi immunoreattivi. Dotto deferente: Lepitelio pseudostratificato mucosale mostra intensa ma disomogenea immunopositivit per la orfanina. Il corion non rivela alcuna specifica immunoreattivit. Vescichette seminali: Lepitelio ghiandolare pseudostratificato mostra assai intensa immunoreattivit non uniformemente distribuita nei vari territori dellorgano. Nei citotipi epiteliali la immunopositivit maggiormente concentrata nel settore citoplasmatico medio-apicale. Pene: Intensa immunoreattivit per la nocicettina estesa a tutti i citotipi dellepitelio prepuziale. Vescica: Elevata immunopositivit alla orfanina nellepitelio di transizione mentre debolmente reattiva appare la sviluppata ed articolata compagine muscolare. Conclusioni I reperti da noi ottenuti forniscono per la prima volta una prova immunoistochimica convincente che la orfanina, ritenuta finora esclusivamente espressa dai neuroni centrali,
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[7]

[8]

[9]

Figures 7-9: [7] Pene di ratto. Intensa reattivit orfaninergica a carico delle cellule dellepitelio prepuziale. Ob. 40x - [8] Vescica di ratto. Intensa reattivit orfaninergica a carico delle cellule dellepitelio di transizione. Ob. 20x - [9] Vescica di ratto. Intensa reattivit orfaninergica a carico delle cellule dellepitelio urinifero. Ob. 40x.

anche prodotta, al di fuori del SNC, in distretti periferici sia nervosi (nervi) sia in citotipi di natura diversa appartenenti ad alcuni segmenti dellapparato genitourinario del ratto. I nostri risultati pertanto possono costituire una base morfoistochimica per una possibile interpretazione di nuovi ruoli svolti dallorfanina in tali distretti. La immunopositivit evidenziata a carico di tronchi nervosi decorrenti nel connettivo tra gli adenomeri delle ghiandole bulbouretrali fa supporre che la orfanina intervenga in questo organo nel controllo neurocrino della funzione di elaborazione e/o dismissione del secreto. La robusta immunopositivit esibita dalle cellule secernenti delle ghiandole bulbouretrali e delle vescichette seminali testimonia la capacit orfaninergica di tali citotipi e autorizza a ritenere che la orfanina possa essere implicata con meccanismo autocrino e/o paracrino nella traduzione di segnali coinvolti nella modulazione delle varie fasi del ciclo secretorio. La intensa immunoreattivit manifestata dai citotipi epiteliali di rivestimento (dotto deferente e pene) suggerisce una implicazione certa dellorfanina che interviene con meccanismo autocrino/paracrino nella prestazione funzionale dei relativi citotipi che al presente non risulta compiutamente chiarita. La immunopositivit riscontrata a carico dellepitelio di transizione vescicale rivela la capacit orfaninergica di tale distretto che deve sicuramente correlarsi a prestazioni funzionali assai specializzate ed esclusive dellepitelio mucosale vescicale. Ci sembra interessante puntualizzare in proposito che uno studio micronelettronico sullepitelio vescicale del ratto compiuto da Nesci e Tessitore [21] ha rilevato equivalenti ultrastrutturali a carico dei citotipi dellepitelio di transizione e modalit di rapporti assai intimi tra lo strato epiteliale basale e capillari sottoepiteliali, fortemente indicativi per lesistenza di fenomeni di assorbimento e/o di eliminazione di materiale fluido. La mucosa vescicale non costituirebbe pertanto una struttura inerte posta al confine tra spazio cavitario vescicale e tessuti sotto274

Rilievi immunoistochimici della orfanina FQ nellapparato genitourinario del ratto

coriali, ma una lamina bitissutale funzionalmente attiva e come tale permeabile ad alcuni liquidi. Tali fenomeni di trasmigrazione presuppongono ovviamente un ordinato svolgersi di eventi sequenziali endocellulari che debbono riconoscere un fine controllo che potrebbe essere svolto proprio dalla orfanina, la quale prodotta dagli epiteliociti mucosali si comporterebbe da regolatore autocrino-paracrino dei processi assorbitivi e/o di eliminazione di materiali fluidi. La discreta immunoreattivit dellorfanina a carico delle componenti muscolari vescicali suggerisce che essa possa esercitare un controllo sullattivit motoria dellorgano vescicale. Le nuove strategie terapeutiche, che fanno uso di orfanina e di suoi analoghi, adottate di recente per il trattamento di disordini motori funzionali che interessano le basse vie urinarie [22,23,24,25], sembrano confortare la validit del ruolo funzionale dellorfanina da noi supposto in tale ambito.

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Bibliografia [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] Bunzow JR, Saez C, Mortrud M, Bouvier C, Williams JT, Low M, Grandy DK. Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a mu, delta or kappa opioid receptor type. FEBS Lett 1994;347:284288. Lachowicz JE, Shen Y, Monsma FJ, Sibley DR. Molecular cloning of a novel G protein coupled receptor related to the opiate receptor family. Journal of Neurochemistry 1995;64:3440. Wang JB, Johnson PS, Imai Y, Persico AM, Ozenberger BA, Eppler CM, Uhl GR, cDNA cloning of an orphan opiate receptor gene family member and its splice variant. ??????????? Meunier JC, Nociceptin/orphanin FQ and the opioid receptor-like ORL1 receptor. Eur J Pharmacol 1997;340:115. Neal CR, Jr, Mansour A, Reinscheid R, Nothacker HP, Civelli O, Watson SJ, Jr. Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat. J Comp Neurol 1999;406:503547. Jenck F, Moreau JL, Martin JR, Kilpatrick GJ, Reinscheid RK, Monsma FJ Jr, Nothacker HP, Civelli O, Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress. Proc Natl Acad Sci (USA) 1997;94:1485414858. Pomonis JD, Billington CJ, Levine AS. Orphanin FQ, agonist of orphan opioid receptor ORL1, stimulates feeding in rats. Neuroreport 1996;8:369371. Sandin J, Georgieva J, Schott PA, Ogren SO, Terenius L. Nociceptin/orphanin FQ microinjected into hippocampus impairs spatial learning in rats. Eur J Neurosci 1997;9:194197. Gutierrez R, Leff P, Anton B. Inhibition of kindling development by nociceptin/orphanin FQ. Society for Neurosciences 1998. p. 536.18. Cal G, Guerrini R, Rizzi A, Salvadori S, Regoli D. Pharmacology of nociceptin and its receptor: a novel therapeutic target. Br J Pharmacol 2000 Apr;129(7):1261-83. Review. Carr G, Rizzi A, Guerrini R, Barnes Ta, Mcdonald J, Hebbes Cp, Mela F, Kenigs Va, Marzola G, Rizzi D, Gavioli E, Zucchini S, Regoli D, Morari M, Salvadori S, Rowbotham Dj, Lambert Dg, Kapusta Dr, Calo G. [(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 (UFP-102), a highly potent and selective agonist of the nociceptin/ orphanin FQ receptor. J Pharmacol Exp Ther 2005 Mar;312(3):1114-23. Cal G, Guerrini R, Rizzi A, Salvadori S, Burmeister M, Kapusta Dr, Lambert Dg, Regoli D. UFP-101, a peptide antagonist selective for the nociceptin/orphanin FQ receptor. CNS Drug Rev 2005 Summer;11(2):97-112. Review. Arduin M, Spagnolo B, Cal G, Guerrini R, Carr G, Fischetti C, Trapella C, Marzola E, Mcdonald J, Lambert Dg, Regoli D, Salvadori S. Synthesis and biological activity of nociceptin/orphanin FQ analogues substituted in position 7 or 11 with Calpha,alpha-dialkylated amino acids. Bioorg Med Chem 2007 Jul 1;15(13):4434-43. Champion HC, Kadowitz PJ. Nociceptin, an endogenous ligand for the ORL1 receptor, has novel hypotensive activity in the rat. Life Sci 1997. 60:241245.PL. Giuliani S, Tramontana M, Lecci A, Maggi CA. Effect of nociceptin on heart rate and blood pressure in anaesthetized rats. Eur J Pharmacol 1997;333:177179. Yazdani A, Takahashi T, Bagnol D, Watson SJ, Owyang C. Functional significance of a newly discovered neuropeptide, orphanin FQ, in rat gastrointestinal motility. Gastroenterology. 1999;116:108117. Osinski Ma, Brown Dr. Orphanin FQ/nociceptin: a novel neuromodulator of gastrointestinal function? Peptides 2000 Jul; 21(7):999-1005. Review. Fischer A, Forssmann WG, Undem BJ. Nociceptin-induced inhibition of tachykinergic neurotransmission

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[14] [15] [16] [17] [18]

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in guinea pig bronchus. J Pharmacol Exp Ther 1998;285:902907. [19] Shah S, Page CP, Spina D. Nociceptin inhibits non-adrenergic non-cholinergic contraction in guinea-pig airway. Br J Pharmacol 1998;125:510516. [20] Giuliani S, Lecci A, Tramontana M, Maggi CA. The inhibitory effect of nociceptin on the micturition reflex in anaesthetized rats. Br J Pharmacol 1998;124:156672. [21] Nesci E, Tessitore V. Osservazioni al microscopio elettronico ed a scansione sullepitelio di transizione della vescica urinaria. Biologica Latina 1969 vol. XXII fasc. 4: 235-255. [22] Lazzeri M, Cal G, Spinelli M, Guerrini R, Beneforti P, Sandri S, Zanollo A, Regoli D, Turini D. Urodynamic and clinical evidence of acute inhibitory effects of intravesical nociceptin/orphanin FQ on detrusor overactivity in humans: a pilot study. J Urol 2001 Dec;166(6):2237-40. [23] Lazzeri M, Cal G, Spinelli M, Guerrini R, Salvadori S, Beneforti P, Sandri S, Regoli D, Turini D. Urodynamic effects of intravesical nociceptin/orphanin FQ in neurogenic detrusor overactivity: a randomized, placebo-controlled, double-blind study. Urology 2003 May;61(5):946-50. [24] Lazzeri M, Cal G, Spinelli M, Malaguti S, Guerrini R, Salvadori S, Beneforti P, Regoli D, Turini D. Daily intravesical instillation of 1 mg nociceptin/orphanin FQ for the control of neurogenic detrusor overactivity: a multicenter, placebo controlled, randomized exploratory study. J Urol. 2006 Nov;176(5):2098-102. [25] Lazzeri M, Spinelli M. The challenge of overactive bladder therapy: alternative to antimuscarinic agents. Int Braz J Urol 2006 Nov-Dec;32(6):620-30. Review.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

AN IMMUNOHISTOCHEMICAL STUDY ON THE CB1 RECEPTOR EXPRESSION IN RAT SALIvARY GLANDS


[Studio immunoistochimico sullespressione dei recettori CB1 nelle ghiandole salivari del ratto] Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore
DI.ME.S. Dipartimento di Medicina Sperimentale - Sezione di Istologia ed Embriologia Arcangelo Pasqualino di Marineo, Facolt di Medicina e Chirurgia, Universit degli Studi di Palermo (I)

Key words: CB1 receptor, Immunohistochemistry, Endocannabinoids, Salivary glands Parole chiave: Recettore CB1, Immunoistochimica, Endocannabinoidi, Ghiandole salivari Abstract. Recent studies have demonstrated that endocannabinoids plays a central role in regulation of food intake and energy balance in rodents and humans, by interaction with neuronal CB1 receptor in feeding centres of hypothalamus. However the presence of CB1 receptor was discovered recently in other peripherical tissues. Knowing the important role of the salivary glands in digestion, the aim of the present study is to investigate the CB1 receptor immunohistochemical expression and distribution in the major salivary glands of rats. Our results indicate that CB1 receptor is exclusively expressed in the ducts epithelia. Our findings provide the morphological basis to display that endocannabinoids acting through the CB1 receptor affecting salivary glands function with autocrine-paracrine-endocrine mechanism. Riassunto. Recenti evidenze sperimentali documentano che gli endocannabinoidi svolgono nei roditori e nelluomo un ruolo centrale nella regolazione dellassunzione alimentare e del bilancio energetico, attivando il recettore neuronale CB1 nei centri ipotalamici della fame. La presenza del recettore CB1 stata tuttavia recentemente dimostrata in alcuni tessuti periferici. Consapevoli del ruolo delle ghiandole salivari nei processi digestivi, abbiamo svolto uno studio immunoistochimico sulla espressione del CB1 nelle ghiandole salivari maggiori del ratto. I nostri risultati documentano lespressione del CB1 esclusivamente negli epiteli duttali e forniscono la base morfoistochimica per suggerire che gli endocannabinoidi, attivando il recettore CB1, influenzano con meccanismo autocrino, paracrino e endocrino la funzione delle ghiandole salivari.

Introduction Recent studies demonstrate that endocannabinoids (anandamide, arachidonilglicerol, noladine, virodamine, N-arachidonildopamine) play in rodents and humans a central hypothalamic orexigenic role in the regulation of food intake and energy homeostasis, by interaction with neuronal CB1 receptor in the feeding centres of hypothalamus [1,2,3,4,5,6,7]. Nevertheless the presence of CB1 receptor, considered since its first identification as brain specific, that is just elaborated by the central neurons, discovered very recently in other peripheral tissues, outside central nervous system (adipose organ, scheletal mus-

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cles, hepatocytes, enteric nervous system) [8,9,10]. Our previous studies indicate that CB1 receptor is widely distributed in the mucosal and neural structures of gastroenterical tract and in endocrine cells of pancreas, districts that play a key role in the regulation of nutritional and glicemic balance [11]. Knowing about the primary role of the salivary glands in food intake and digestion (prima digestio fit in ore), we have undertaken an immunohistochemistry study on the CB1 receptor expression and distribution in the major salivary glands of the rat. Material and methods We have used 10 Wistar rats treated in according to the Helsinki Convention on the animal use in the biomedic research. The rats have been sacrified upon anaesthesia with Nembutal administered via endoperitoneal. The major salivary glands (parotid, submandibular, sublingual) are been excited and their fragments have been fixed in Bouin liquid and then included in paraffin wax. The obtained sections have been processed with Anti-CB1 (Biosource Europa S.A.) and revealed with En-Vision + System HRP with AEC as substratum. Negative controls were prepared by replacing the first antibody solution with PBS or incubating the sections with the primary antiserum saturated with the homologous antigen. Results The mayor salivary glands of the rat are, as its known, compound tubuloacinose structures. The duct section is represented by interlobular excretory ducts, striated salivary ducts and preterminal ducts, whose epithelium could present mucous differentiation. In the three salivary glands examined, CB1 receptor immunoreactivity is present exclusively in the excretory ducts, in striated salivary ducts, in intercalated ducts and in preterminal ducts. The acini are negative or really much weakly positives (parotid gland). Parotid gland: the CB1 immunoreactivity, limited exclusively to the ducts, appears dishomogeneously distributed: just some cells of ductular epithelium are strongly positives, others are negatives or weakly positives. In general, CB1 immunoreactivity presents a widespread granular distribution or is widely cytoplasmatic; sometimes is strictly perinuclear (Fig.1). The serous acini are substantially negative. Submandibular and sublingual glands: presented similar staining in the immunoreactive structures thats why they are assimilated in the description of the results. In the parenchymal areas with exclusively mucous secretion, the epithelium of the ex282

An immunohistochemical study on the CB1 receptor expression in rat salivary glands

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Figures 1-7b: [1] Parotid gland: CB1 immunoreactivity in ductular epithelia not homogeneously distributed. The acini are not reactive . Ob.20x - [2] Sublingual gland: strong CB1 immunoreactivity in some epithelial cells of intercalated ducts. The acini are not reactive Ob.40x - [3] Submandibular gland: intense CB1 microgranular immunoreactivity in some epithelial cells of intercalated ducts. The acini are not reactive Ob.40x - [4] Sublingual gland: strong CB1 immunoreactivity in some epithelial cells of striated duct. The acini are not reactive Ob.40x - [5] Submandibular gland: in the parenchymal areas with exclusively mucous secretion the CB1 immunoreactivity is not present in the acini; in the periacinar stroma small cells strongly immunoreactive were observed. Ob.40x - [6] Sublingual gland: in the parenchymal areas with mucous and serous secretion intense CB1 immunoreactivity is observed in scattered cells in epithelia of preterminal ducts (neuroendocrine cells?). The acini are not reactive Ob. 20x - [7a-b] Submandibular gland: in the parenchymal areas with mucous and serous secretion CB1 immunoreactivity is present in scattered cells in the epithelia of preterminal duct (neuroendocrine cells?). The acini are not reactive Ob. 20x (7a) Ob. 40x (7b).

cretory ducts and of the salivary striate ducts, present CB1 immunoreactivity not extended to every cells: in some of them was not detected immunoreactivity, in other intense staining was found with granular or diffuse distribution (Fig. 2-3-4). The acini not express CB1 receptor immunoreactivity but among them its possible to see small cells immunoreactive (Basket cells?, immune cells?) (Fig.5). In the parenchymal areas with mucous and serous secretion the serous acini are not immunoreactive. The big mucous cells of preterminal ducts epithelia show a very weak immunoreactivity, but intense CB1 immunoreactivity is expressed, in the same duct, by small cells (endocrine cells?) (Fig.6-7a,7b). Excretory and intercalated ducts epithelia shows intense but not uniform immunoreactivity. The acini not express CB1 receptor immunoreactivity.
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Conclusion Our immunohistochemical findings demostrate for the first time, that CB1 receptor is expressed by different structures of the mayor salivary glands of the rat, so that we consequently presume that the endocannabinoids, in addiction to their central orexigenic hypothalamic role have a direct peripheral control on the salivary glands function. In particular, the exclusive immunohistochemical expression of the CB1 receptor in the ducts of the salivary glands of the rat, suggest that the endocannabinoids dont control directly the first phase of the elaboration of secretion by the acini immunohistochemically negative, but they regulate with autocrine-paracrine mechanism and, on some aspects probably with endocrine mechanism, the function of the ductal epithelial cells implicated, not only in carrying the salivary secretion but also in the modification of the saliva through the reabsorption of the electrolytes and the production of proteins [12]. This functional perspective is made plausible by a previous experimental observations regarding the stimulation induced by delta 9-THC that is able to modify the salivary flux [13]. A previous study of farmacological nature also agrees with this data, bacause indirectly document the CB1 expression in the rat parotid under stimulation with anandamide, that induces AMP accumulation, amylase release and Na-K-ATPase inhibition [14]. Foregain biochemical and microanalytic researches, have documented the production of endocannabinoids (anandamide and 2-arachidonilglicerol) in the salivary glands of an arthropod ectoparasite [15]. This reinforce more the direct intervention of the endocannabinoids on the salivary glands function.

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References [1] [2] [3] Di Marzo V, Matias I. Endocannabinoid control of food intake and energy balance. Nat Neurosci 2005 May;8(5):585-9. Cota D, Woods SC. The role of the endocannabinoid system in the regulation of energy homeostasis. Curr Opin Endocr Diab 2005 12, 338-351. Cota D, Marsicano G, Tschp M, Grbler Y, Flachskamm C, Schubert M, Auer D, Yassouridis A, ThneReineke C, Ortmann S, Tomassoni F, Cervino C, Nisoli E, Linthorst AC, Pasquali R, Lutz B, Stalla GK, Pagotto U.The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis. J Clin Invest 2003 Aug;112(3):423-31. Pagotto U, Vicennati V, Pasquali R The endocannabinoid system in the physiopathology of metabolic disorders. Horm res 2007 67 Suppl 1: 186-90. Komorowski J, Stepie H. The role of the endocannabinoid system in the regulation of endocrine function and in the control of energy balance in humans. Postepy Hig Med Dosw (online) 2007;61:99-105. Elmquist JK, Elias CF, Saper CB. From lesions to leptin: hypothalamic control of food intake and body weight. Neuron 1999 Feb;22(2):221-32. Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS. Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 1999 Feb;20(1):68-100. Pertwee RG. Evidence for the presence of CB1 cannabinoid receptors on peripheral neurons and for the existence of neuronal non CB1 cannabinoid receptors. Life Sci 1999 Vol65, Nos. 6/7, 597-605. Pinto L, Capasso R, Di Carlo G, Izzo AA. Endocannabinoids and the gut. Prostaglandins Leukot Essent, Fatty Acids 2002 Feb-Mar; 66(2-3):333-41A. Storr M, Sibaev A, Marsicano G, Lutz B, Schusdziarra V, Timmermans JP, Allescher HD. Cannabinoid receptor type 1 modulates excitatory and inhibitory neurotransmission in mouse colon. Am J Physiol Gastrointest Liver Physiol 2004 Jan; 286(1):G110-7. Tessitore V, Uzzo ML, Spatola G, Cucco D, Bonaventura G. 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 vol. 111 suppl.2, 256. Riva A, Testa-Riva F, Del Fiacco M, Lantini MS. Fine structure and cytochemistry of the intralobular ducts of the human parotid gland. J Anat 1976 Dec; 122(Pt 3):627-40. Mc Connell WR. A study of the effect of delta 9- tetrahydrocannabinol (delta 9-THC) on mammalian salivary flow. J Pharmacol Exp Ther 1978 206, 567-573. Busch L et al. Expression and biological effects of CB1 cannabinoid receptor in rat parotid gland. Biochemical Pharmacology 2004 68, 1767-1774. Fezza F et al. Endocannabinoids and related fatty acid amides, and their regulation, in the salivary glands of the lone star tick. Biochimica et Biophysica Acta 2003 1663, 61-67.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

THE RECENT LITERATURE ABOUT MICRONUCLEI. CONSIDERATIONS AND RELIEfS


[La letteratura recente sui micronuclei. Considerazioni e rilievi] Luana Lipari1, Marcello Ciaccio2, Francesco Burruano3 and Silvia Tortorici3
Dipartimento di Medicina Sperimentale - Sezione di Istologia ed Embriologia Arcangelo Pasqualino di Marineo 2Cattedra di Biochimica Clinica - 3Dip. Discipline Chirurgiche e Oncologiche e Dip. Scienze Stomatologiche G. Messina - Sez. di Chirurgia Speciale Odontostomatologica, Facolt di Medicina e Chirurgia, Universit degli Studi di Palermo (I)
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Key words: DNA damage, Chromosome, Genetic toxicology Parole chiave: Danno del DNA, Cromosoma, Tossicologia genetica Abstract. The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays is one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured realibly. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumors and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity wich can be achieved with relative ease within one system. The basic principles and methods of the CBMN assay are described and areas for future development identified. Riassunto. Lo studio del danno del DNA a livello cromosomico parte essenziale della tossicologia genetica perch la mutazione cromosomica un importante evento nella carcinogenesi. La metodica del micronucleo uno dei metodi preferiti per la valutazione del danno cromosomico in quanto consente di misurare la perdita cromosomica ed il danno cromosomico. Poich i micronuclei possono esser espressi solo in cellule che hanno completato la divisione nucleare stato sviluppato un nuovo metodo che consente didentificare in tal modo le cellule bloccando la loro citochinesi attraverso lutilizzo della citocalasina-B (Cyt-B), un inibitore dellassemblaggio dei microfilamenti. La metodica applicata a vari tipi cellulari per monitorare il danno genetico nella popolazione, eseguire screening di agenti chimici potenzialmente genotossici e per

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specifici scopi quali la predizione della radiosensibilit dei tumori e la variazione inter-individuale nella radiosensibilit. Il blocco della citochinesi (CBMN) pu fornire, utilizzando semplici criteri morfologici, le seguenti misure di genotossicit e citotossicit: la rottura cromosomica, la perdita cromosomiale, il riarrangiamento cromosomico (ponti nucleoplasmici), linibizione della divisione cellulare, la necrosi e lapoptosi. Lutilizzo di sonde molecolari consente di distinguere la perdita di cromosomi dalla rottura degli stessi ed, ancora, pu venire efficacemente misurata la non-disgiunzione in cellule binucleate non micronucleate. Altres, la tecnica CBMN fornisce multiple e complementari misure di genotossicit e citotossicit che possono esser ottenute con relativa facilit allinterno di un unico sistema. Sono qui descritti i principi ed i metodi base del CBMN ed il loro impiego in future aree dapplicazione.

Introduction The level of genetic integrity of human populations is increasingly under threat due to industrial activities that result in exposure to chemical and physical genotoxins. Other factors that can influence genetic damage include lifestyle factors (e.g, diet), various medical therapies, and climatic changes (e.g., increased exposure to ultraviolet radiation due to depletion of atmospheric ozone). It is therefore important to be able to determine what is an acceptable level of genetic damage in a human population, identify individuals who could be hypersensitive to selected genotoxins, effectively screen new chemicals that are relased into the environment, determine the level of increase in genetic damage in a population following a major accident, and routinely monitor individuals who are occupationally exposed to agents that can be detrimental at the genetic level. A number of methodologies have been developed over the past 20 years, varying from cytogenetic techniques that can give a broad assessement of mutagenic events to adduct assays that are designes to detect exposure to specific agents. No one technique can satisfy all the requirements identified above, and it is now realized that a variety of methods can be combined to provide an effective screening system. As part of effort in this area of research , it has been involved in the development of micronucleus (MN) assays in human lymphocytes to measure both whole chromosome loss and chromosome breaks. The MN methodology is simple and allows rapid and assessment of cells, thus making it an economical procedure to implement on a large scale. MN assays can only be effective as quantitative biological dosimeters if one can identify those cells that have divided after exposure because only dividing cells can express micronuclei. *** The observation that chromosome damage can be caused by exposure to ionising radiation or carcinogenic chemicals was among the first reliable evidence that physical and

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chemical agents can cause major alterations to the genetic material of eukaryotic cells. Altough the understanding of chromosome structure is incomplete, evidence suggest that chromosome abnormalities are a direct consequence and manifestation of damage at the DNA level, for example, chromosome breaks may result from unrepaired double strand breaks in DNA and chromosome rearrangements may result from misrepair of strand breaks in DNA. It also recognised that chromosome loss and malsegregation of chromosomes are an important event in cancer and ageing and that they are probably caused by defects in the spindle, centromere or as a consequence of undercondensation of chromosome structure before metaphase. In the classical cytogenetic techniques, chromosomes are studied directly by observing and counting aberrations in metaphase.This approach provides the most detailed analysis, but the complexity and laboriousness of enumerating aberrations in metaphase preparations has stimulated the development of a simpler system of measuring chromosome damage. It was proposed that an alternative and simpler approach to assess chromosome damage in vivo was to measure micronuclei (MNi), also known as Howell-Jolly bodies to haematologists, in dividing cell populations such as the bone-marrow. The micronucleus assay in bone-marrow and peripheral blood erytrocytes is now of the best estabilished in vivo cytogenetic assays in the field of genetic toxicology, however, it is not a technique that is applicable to other cell populations in vivo or in vitro and methods have since been developed for measuring MNi in a variety of nucleated cells in vitro. MNi are expressed in dividing cells that either contain chromosome breaks lacking centromeres (acentric fragmentes) and/or whole chromosome that are unable to travel to the spindle poles during mitosis. At telophase, a nuclear envelope forms around the lagging chromosome and fragments, which then uncoil and gradually assume the morphology of an interphase nucleus with the exception that they are smaller than the main nuclei in the cell, hence the term micronucleus. MNi, therefore, provide a convenient and realible index of both chromosome breakage and chromosome loss. Because MNi are expressed in cells that have completed nuclear division they are ideally scored in the binucleated stage of the cell cycle. Occasionally nucleoplasmic bridges between nuclei in a binucleated cell are observed. These are probably dicentric chromosomes in which the two centromeres were pulled to opposite poles of the cell and the DNA in the resulting bridge covered by nuclear membrane. Thus, nucleoplasmic bridges in binucleated cells provide an additional and complemantary measure of chromosome rearrangement, which can be scored together with the micronucleus count. It is evident from above that MNi can only be expressed in dividing eukaryotic cells. In other words, the assay cannot be used efficiently or quantitatively in non-dividing cell populations or in dividing cell populations in which the kinetics
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of cell division is not well understood or controlled. Consequently, there was a need to develop a method that could distinguish between cells that are not dividing and cells that are undergoing mitosis within a cell population. Furthemore, because of the uncertainty of the fate of MNi following more than one nuclear division it is important to identify cells that have completed one nuclear division only. These requirements are also necessary because cells divide at different rates in vivo and in vitro depending on the various physiogical, genetic and micronutrient conditions. Several methods have been proposed based on stathmokinetic, flow cytometric and DNA labelling approaches but the method that has found most favour due to its simplicity and lack of uncertainty regarding its effect on base-line genetic damage is the cytokinesis-block micronucleus (CBMN) assay. In the CBMN assay, cells that have completed one nuclear division are blocked from performing cytokinesis using cytochalasin-B (Cyt-B) and are consequently readily identified by their binucleated appearance. Cyt-B is an inhibitor of actin polymerisation required for the formation of the microfilament ring that constricts the cytoplasm between the daughter nuclei during cytokinesis. The use of Cyt-B enables the accumulation of virtually all dividing cells at the binucleate stage in dividing cell populations regardless of their degree of synchrony and the proportion of dividing cells. MNi are then scored in binucleated cells only, which enables reliable comparison of chromosome damage between cell populations that may differ in their cell division kinetics. The method was initially developed for use with cultured human lymphocytes, but has now been adapted to various cell types such as solid tumour and bone-marrow cells. Furthermore, new developments have also occurred that allow MNi originating from whole chromosome to be distinguished from MNi originating from chromosome fragments, the conversion of excision-repaired sites to MNi within one cell division, the use of molecular probes to identify non-disjunction events in binucleated cells and the integration of necrotic and apoptotic cells within the CBMN assay. It has recently been proposed that the micronucleus assay be used instead of metaphase analysis for genotoxicity testing of new chemicals. Criteria for selecting binucleated cells wich can be scored for micronucleus Frequency The cytokinesis-blocked cells that may be scored for MN frequency should have the following characteristics: The cells should be binucleated; The two nuclei in a binucleated cell should have intact nuclear membranes and be situated within the same cytoplasmic boundary;
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The two nuclei in a binucleated cell should be approximately equal in size, staining pattern and staining intensity; The two nuclei within a BN cell may be attached by a fine nucleoplasmic bridge which is no wider than 1/4th of the nuclear diameter; The two main nuclei in a BN cell may touch but ideally should not overlap each other. A cell with two overlapping nuclei can be scored only if the nuclear boundaries of each nucleus are distinguishable; The cytoplasmic boundary or membrane of a binuclaeted cell should be intact and clearly distinguishable from the cytoplasmic boundary of adjacent cells. Criteria for scoring micronuclei MNi are morphologically identical to but smaller than nuclei. They also have the following characteristics: The diameter of Mni in human lymphocytes usually varies between 1/16th and 1/3rd of the mean diameter of the main nuclei wich corresponds to 1/256th and 1/9th of the area of one of the main nuclei in a BN cell, respectively; MNi are non-refractile and they can therefore be readily distinguished from artefact such as staining particles; MNi are not linked or connected to the main nuclei; MNi may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary; MNi usually have the same staining intensity as the main nuclei occasionally staining may be more intense. Spontaneus Micronucleus frequencies in human populations Spontaneus or baseline MN frequencies in cultured human lymphocytes provide an index of accumulated genetic damage occurring during the lifespan of circulating lymphocytes. The half-life and mean lifespan of long-lived T-lymphocytes has been estimated to be 3 years and 4 years, respectively. The observed genetic instability may also reflect accumulated mutations in the stem cell lineage from which the mature lymphocytes originate.The type of mutations that could contribute to spontaneous micronuclei include mutations to kinetochore proteins, centromeres and spindle apparatus that could lead to unequal chromosome distribution or whole chromosome loss at anaphase and unrepaired DNA strand breaks induced endogenously or as a result of environmental mutagens, which may result in acentric chromosome fragments. Studies using kinetochore antibodies to identify whole chromosome suggest that approximately 50% of spontaneously occurring micronuclei are the consequence of whole chromosome loss and the rest are presumably
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derived from acentric chromosome fragments. For the purpose of biological dosimetry, the spontaneous MN frequency refers to the incidence of MN observed in the absence of the environmental risk or exposure that is being assessed. The spontaneous MN frequency of a population has to be established to determine acceptable normal values as well as providing baseline data for those situations when spontaneous MN frequency for individuals is not known before exposure. The population database should enable an estimate of the induced MN frequency (i.e, observed minus expected spontaneous frequency) to be determined after an exposure accident and given the establishment of dose-response parameters, an exposure dose can be calculated. The population database should span every decade up to 90 years and include a minimum of 20 individuals of each sex per decade. Ideally, this database should be updated regularly (every 2 years) to take into account the possible contribution of changing lifestyle factors as well as changes in technique. The database should also be used to identify lifestyle factors that can contribute significantly to the micronucleus index. During an International Atomic Energy Agency-sponsored Research Coordination meeting for Radiation Biological Dosimetry held in Rio de Janeiro in June 1990, it was agreed that a compilation and comparison of baseline MN frequency data from various countries would be a useful exercise because it could enable a normal range of baseline values to be established as well as identify possible factors. Analysis of review The aim of biomonitoring studies in the humans exposed to genotoxicants is primarily to identify individuals or populations at the risk of developing chronic diseases [1]. It is well documented that the frequency of chromosomal damages in circulating lymphocytes is a relevant biomarker of health risk in humans, reflecting both early biological effects of exposure to genotoxic carcinogens and individual susceptibility [2]. The fact that the presence of MN occurs as a result of chromosome damage and reflects the degree if genetic instability was reported by numerous authors [3,4,5]. The cytokinesis-block micronucleus (CBMN) assay is a preferred method for measuring MN in the cultured cells. This test can be used for the study of cellular and nuclear dysfunction caused by in vitro or in vivo ageing, micronutrient deficiency or excess, genotoxin exposure and genetic defects in genome [6]. Several studies have studied the use of MN , for example in a study published on September 2007 it was evaluated the frequency of MN by using CBMN assay in cord blood lymphocytes in mewborns who born in the city located in central Serbia in 2006 that is seven years after extensive environmental pollution, with damage to soil, water and air. The mean frequency of MN found was 4.73 per 1,000 binucleated cells. The results of 41 newborns of both genders (20 males and 21 females) did not show
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a significant difference in the MN frequency in the newborns relating to gender (p> 0.05). This difference based on gender was found in the results of other researchers, who reported that gender could be important determinant for this differentiation only in adults [7,8,9,10]. The authors evaluated two factors such as mothers age and cigarette smoking as potential factors that could modify the values of MN. No significant difference was observed in the frequency of MN among the newborns whose mothers aged 19-29 and 30-40 (p>0.05). Similar results were reported [11] who studied the effect of maternal age on chorionic villus micronuclei (CVMN) and did not find the correlation between CVMN frequency and maternal age. There was no significant difference between the newborns who in utero exposed to maternal cigarette smoking and the unexposed newborns (p>0.05). The effect of smoking was evaluated in a large number of biomonitoring studies on exposed or non exposed populations. In the literature, data on peripheral blood lymphocyte are most frequent. Di Giorgio et al. (1994) [12] showed positive results, but large number of reports did not find any correlation between MN and smoking. Thus [13] applying the pooled re-analysis of 24 databases from the Human Nucleus (HUMN) international collaborative project, confirmed that smorkers did not experience the increase in MN frequency. Effect of maternal smoking on the newborn MN was studied by a few authors. Results are in agreement with earlier results who investigated the effect of maternal smoking on the frequency of chromosome aberrations in first trimester chorionic villus cells. They reported statistically non significant differences between non-smoking and smoking mothers.It was noticied that both male and female newborns whose mothers few cigarette smokers had non significantly lower mean frequency of the MN in comparison to mean frequency in the newborns whose mothers were non-smokers. This finding is in an agreement with the results reported by Gourabi and Mozdarani (1998) and Rothfuss et al. (1998) [14,15] They concluded that a few cigarette per day might stimulate cell-protective response, causing an apparent lowering in the Mn frequency. Comparing the newborns born in three different time periods, 12 month, 18 month and seven years after pollution, a decrease in MN frequency was observed. The MN values significantly decreased in newborns born in 2006 in comparison to newborns born 12 month after the pollution ( at the end of 2000). The decrease in MN frequency corresponded to substantial changes in environment in this city during these periods. Many studies reported that genetic damage and exposure to pollutants increased the MN frequency in children. Wronka et al. (2000) [16] detected a relationship between the MN frequency in buccal epithelium cells and exposure to air pollutants in two different sample of children. Very recently, Neri et al. (2006) [17], applying meta-analysis, showed that exposure to airborne pollutants, soil and drinking water contaminants mostly increased chromosome aberration and MN levels in children. Results show that reduction of environmental pollution decreased the number of
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Figure Micronucleus in a dividing nucleated cell. Micronuclei originate from either lagging chromosomes or chromosome fragments at anaphase. Micronuclei are readily identified because they are morphologically identical to but smaller than the main nuclei. A non dividing cell is unable to expresse its chromosome damage as micronuclei. Cells that have completed one nuclear division can be accumulated and identified as binucleate cells by adding cytochalasin-B, a cytokinesis blocking agent. Micronuclei are then scored in binucletated cells only.

binucleated cells containing more than 1 MN/1,000 CB cells. Only 6 mewborns out of 41(14.6%) showed more than 1 MN/1,000 CB cells in respect to 7/22 newborns born in the beginning of 2000 (31.8%), and 2/14 newborns born at the end of 2000 (14%). Analysis of variance and greater inter-individual variation also demonstrated relationship between the MN frequencies and the level of environmental pollution in four different periods in this city (F=6.95, p=0.000). If it was accepted that micronuclei in circulating lymphocytes are a relevant biomarker for biological effects of exposure to genotoxins, it can speculate that decrease of MN frequency in cord blood lymphocytes of newborns born seven years after environmental pollution, corresponds to substantial changes in environment in city. Environmental pollution is the potential risk for human health, and detection of micronuclei in cord blood lymphocytes is an important method for identification of transplacental mutagens in evalution and protection of childrenshealth. Future developments It is evident that the in vitro micronucleus assay has evolved into a robust assay for genetic damage with applications in ecotoxicology, nutrition, radiation sensitivity testing both for cancer risk assessment and optimisation of radiotherapy, biomonitoring of human populations and importantly testing of new pharmaceuticals and agrichemicals. There is little doubt that there is a need for an automated scoring system for quicker and more reliable
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data acquisition which would ideally be based on the scoring of slides also prepared for visual scoring- this should enable consistent results to be obtained that are not influenced by inter-individual and temporal variability of human scores. For this goal to be achieved it is essential that scoring criteria are well developed and that a robust slide preparation protocol be put in place and that slide preparations be permanent so that they can be reexamined visually if necessary. Currently image analysis systems have been developed for automated scoring of micronuclei in mammalian cells but these systems do not take account of other important events such a necrosis, apoptosis and cytostasis which are essential for the correct interpretation of the result obtained. In the future we should expect to have an automated system that can score realibly the various end-points possible with the cytokinesis-block micronucleus assay. Finally, it is also essential to keep abreast of more recent developments in understanding of micronucleus formation and events that may alter expression of this end-point. Some notable example are: the formation of micronuclei as a result of gene amplification in which the cell eliminates excess amplified DNA directly from the nucleus, during S phase, into a micronucleus produced by nuclear budding the observation that treatment with specific mitotic spindle inhibitors may cause mitotic slippage leading to polyploidy nuclei and micronuclei and therefore implicating that it may be useful to score not only MNi in binucleated cells but also MNi in monucleated cells in cytokinesis-blocked cultures the possible elimination of micronucleated cells and micronuclei by apoptosis. All of above points to the fact that the full potential of the in vitro cytokinesis-block micronucleus assay is readily achievable once all the morphologicalend-points of cytotoxicity, cytostasis and DNA damage are integrated into the system. It was originally described the phenomenon of field cancerisation to account for their observation of multifocal squamos cells carcinoma in the upper aerodigestive tract or observation about pre tumoral lesions (e.g leukoplachia). Second or third primaries developed in 7-30% of head and neck squamos cell carcinoma (HNSCC) patients. The main risk factor besides the individual-determined, endogenous susceptibility is the chronic abuse of tobacco and alcohol. Biomarkers are instruments of individual tumor prevention and help to detect high-risk patients. They should allow statements concerning environmental and occupational exposition and further give information on the status of susceptibility. Biomarkers are divided in three groups: the first to define the exposure to carcinogenic agents, the second to show biological effects on the target tissue and the third to give information about the individual susceptibility. In this examination the micronuclei (MN) assay was used as a tool to estimate the individual risk of tumor disease in the upper ae297

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rodigestive tract. Ideally, this biologic marker shows the degree of field cancerisation in the upper aerodigestive tract depending on the smoking and drinking habits of the examined person. Despite the well-known influence of alcoholic beverages as co-carcinogenic factors, it could not show any statistically significant relation between alcohol consumption and the development of MN. This can partly be explained by a likely understatement of the individual alcohol consumption by tumor patients at interview. On the other hand, the significant influence of tobacco abuse showed a direct effect on carcinogenesis of the upper aerodigestive tract in comparison to the minor effect of alcohol. The analysis of additional risk factor showed only a weak significance on the development of MN when an exposure to organic solvents exists. The MN frequency seems to be a suitable procedure in combination with the other biomarkers for cancer risk assessment in the upper aerodigestive tract. Hence, specific biomarkers on cytogenetic end points may help establishing preventive measures to reduce cancer risks but they will not allow any statement as to whether or when a malignant change may happen. It becomes more significant for pre-screening programs of high-risk groups, especially for those cancers such as HNSCC and other tumors or pre-tumors lesions, whose etiology can be strongly influenced by environmental genotoxic exposures.

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References [1] Bonassi S, Bolognesi C, Abbondadolo A, Barale R, Bigatti P, Cimurri L, Dalpra L, De Ferrari M, Forni A, Lando C, Padovani P, Pasquini R, Stella M & Puntoni R. (1995), Influence of sex on cytogenetic end points:evidence from a large human sample and review of the literature. Cancer Epidemiol 4, 671-679. Price PJ, Gregory EA & Skeen PC. (1978), Ritalin, Benzedrine and Dexedrine do not transform F 1706 rat cells Cancer Lett 5, 345-349. Rosssner P, Bavarova H, Ocadlikova D, Svandova E & Sram RJ. (2002), Chromosomal aberrations in peripheral lymphocytes of children as biomarkers of environmental exposure and life style. Toxicol. Lett 134, 79-85. Mateuca R, Lombaert N, Aka PV, Decordier I & Kirsh-Volders M. (2006), Chromosomal changes: induction, detection methods and applicabilit in human biomonitoring. Biochimie 88, 1515-1531. Zalacain M, Sierrasesumaga L, Larrannaga C & Patinno-Garcia A. (2006) Effects of benzopirene-7,8diol-9,10-epoxide (BPDE) in vitro and of maternal smoking in vivo on micronuclei frequencies in fetal cord blood. Pediatr Res 60, 180-184. Fenech M. (2006), Cytokinesis-block micronucleus assay evolves into a cytome assay of chromosomal instability, mitotic dysfunction and cell death. Mutat. Res 600, 58-66. Barale R, Chelotti L, Davini T, Del Ry S, Andreassi M G, Ballardin M, Bulleri M, He J, Baldacci S, Di Pede F, Gemignani F & Landi S. (1998), Sister chromatid exchange and micronucleus frequency in human lymphocytes of 1.650 subjects in an Italian population: II Contribution of sex, age and lifestyle. Environ. Mol. Mutagen 31, 228-242. Bolognesi C, Lando C, Forni A, Landini E, Scarpato R, Migliore L & Bonassi S. (1999), Chromosomal damage and ageing: effect on micronuclei frequency in perpheral blood lymphocytes. Age and ageing 28, 393-397. Bonassi S, Fenech M, Lando C, Lin YP, Ceppi M, Chang WP, Holland N, Kirsh-Volders M, Zeiger E, Ban S, Barale R, Bigatti MP, Bolognesi C, Jia C, Di Giorgio M, Ferguson L, Fucic A, Lima OG, Hrelia P, Krishnaja AR, Lee TW, Migliore L, Mikhalevich L, Mirkova K, Mosesso P, Mulle WU, Odagiri Y, Scarfi MR, Szabova E, Vorobtosova I, Vral A & Zjino A. (2001), Human Micro Nucleus Project: International database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I Effect of laboratory protocol, scoring criteria and host factors on the frequency of micronuclei. Environ. Mol. Mutagen 37. 31-45. Pedersen M, Vinzents P, Petersen JH, Kleinjans JCS, Plas G, Kirsh-Volders M, Dostal M, Rossner P, Beskid O, Sram RJ, Merlo DF & Knudsen LE. (2006), Cytogenetic effects in children and mothers exposed to air pollution assessed by the frequency of micronuclei and fluorescence in situ hybridization (FISH): A family study in the Czech Republic. Mutat. Res 28, 112-120. Cui YQ, Dong ZW, Liu SB, Zhang SC, Wang Y & Ji XY. (1990), Assessment of the mutagenic effect of maternal factors on human chorionic villi by micronucleus test. Prog Clin Biol Red 340, 217-222. Di Giorgio C, De Meo MP, Laget M, Guirad H, Botta A & Dumenil G. (1994), The micronucleus assay in human lymphocytes: screening for inter-individual variability and application to biomonitoring. Carcinogenesis 15, 313-317. Bonassi S, Neri M, Lando C, Ceppi M, Lin YP, Chang WP, Holland N, Kirsh-Volders M, Geiger E, Fenech M & HUMN collaborative group (2003), Effect of smoking habit on the frqyency of micronuclei in human lymphocytes :result from the Human MicroNucleus project. Mutat Res 543, 155-166. Gourabi H & Mozdarani H. (1998), A cytokinesis-blocked micronucleus study of the radioadaptive re-

[2] [3] [4] [5] [6] [7]

[8] [9]

[10]

[11] [12] [13] [14]

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[15] [16] [17] [18] [19] [20]

sponse of lymphocytes of individuals occupationally exposed to chronic doses of radiation. Mutagenesis 13, 474-480. Rothfuss A, Dennog C & Speit G. (1998), Adaptive protection against the induction of oxidative DNA damage after hyperbaric oxygen treatment. Carcinogenesis 19, 1913-1917. Wronka I, Schmager J & Borowiecka A. (2000), The population research on incidence of micronuclei in the cells of oral epithelium. Scripta Periodica III, pp 3. Neri M, Ugolini D, Bonassi S, Fucic A, Holland N, Merlo DF. (2006), Childrens exposure to enviromental pollutants and biomarkers of genetic damage II. Results of a comprehensive literature search and meta-analysis. Mutat Res 612, 14-39. Fenech M. (1993), The cytokinesis-block Micronucleus Technique and its application to genotoxicity studies in human populations Envir Health Perspectivies Supplements Vol. 101 (Suppl. 3): 101-107. Fenech M. (2000), The in vitro micronucleus technique Mutat. Res 455, 81-95. Olivera M-D, Darko G, Slobodan A, Marija B, Sladjana U, Dragoslav M. (2007), J Exp Med 213, 231-239.

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Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007

PARENTAL RECOGNITION Of OvERWEIGHT AS A HEALTH PROBLEM IN SCHOOL-AGE CHILDREN LIvING IN AN INNER-CITY NEIGHBORHOOD Of PALERMO, ITALY: A CROSS-SECTIONAL STUDY
[Percezione del problema delleccesso di peso nellinfanzia da parte di genitori residenti nel centro storico di Palermo]

Antonino Bianco1, Caterina Mammina2,4, Marianna Bellafiore3,4, Giuseppe Battaglia1, Felicia Farina3,4 and Antonio Palma3,4*
PhD School in Motor Activity Sciences; 2Department of Hygiene and Microbiology G. DAlessandro, Section of Hygiene; 3Department of Experimental Medicine, Section of Anatomy; 4Faculty of Exercise and Sport Sciences, *Corresponding author -University of Palermo (I)
1

Key words: Physical activity, Overweight, Childhood, Parental perception, Obesogenic factors Parole chiave: Attivit fisica, Eccesso di peso, Et evolutiva, Percezione dei genitori, Ambiente obesogenico Abstract. Objective. The objective of this study was to evaluate how parents living in Palermo, southern Italy, perceive their childrens weight status and role of physical activity and what believe about contributing and counteracting factors within the childhood overweight crisis. Methods. A cross-sectional survey was performed on a convenience sample of parents of 6-13 year-old children who attended grades 1, 3 and 5 of primary and grades 1 and 3 of secondary, respectively, public schools. The schools were located in an inner urban district of Palermo, Italy, characterized by low to medium income residents. Thirteen schools were selected. Parents were asked for coming to school and participating to the investigation. The survey was administered in the spring of 2006. For descriptive analysis, frequencies, crosstabulation and chi-square statistics were calculated. Role of specific demographic and social characteristics education, gender, age class and BMI - of respondents was assessed by multivariate logistic regression analysis. Results. Three hundred eleven parents completed the questionnaire. Eighty-three percent believed that overweight in childhood is a serious health hazard, but one third still interpreted the childrens overweight as an expression of health. The most significant contributors to childhood overweight were thought to be junk food and beverages (78.0%) and fast food (63.2%), followed by lack of exercise in school curriculum (48.7%). Beliefs about responsibilities for combating childhood obesity significantly varied according to education level. Conclusions. Parents perceptions of their own childrens weight status are mainly influenced by education of parents. Given that a large proportion of parents apparently fail to recognize overweight is a health problem and attribute themselves with the responsibility for combating the childhood obesity crisis, schools and media should develop strategies to help these parents correct their misperceptions. Moreover, public support for environmental changes could effectively rise with the increasing public awareness that many interrelated obesogenic factors in the modern environment are playing a key role. Riassunto. Obiettivi specifici. Lobesit considerata la patologia nutrizionale pi frequente dellet evolutiva nei Paesi industrializzati. La percezione da parte dei genitori non solo del problema ma anche della complessit della rete causale (ambiente obesogenico) determinante per le strategie di prevenzione. Lobiettivo dello studio stato quello di valutare in un campione di genitori del centro storico di Palermo la percezione

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del problema, del ruolo dellattivit motoria e delle convinzioni sui fattori contribuenti e sui soggetti che hanno la responsabilit di combattere lobesit infantile. Metodi. Lindagine stata condotta con la somministrazione di un questionario ai genitori di bambini di 6-13 anni. Risultati. 311 genitori hanno completato il questionario. L83,0% risultato ritenere che lobesit infantile sia un grave problema di Sanit pubblica. Tuttavia, un terzo ha dimostrato di considerare ancora il sovrappeso unespressione di salute. I fattori contribuenti pi significativi sono stati identificati nei cibi e bevande industriali (78,0%), fast-food (63,2%) e mancanza di attivit motoria nella scuola (48,7%). apparso evidente anche che il 97,0% considera i genitori stessi come i soggetti con la massima responsabilit nel combattere lobesit infantile, ma il pattern di risposta apparso significativamente influenzato dal livello di istruzione. In generale, le abitudini alimentari godevano di pi largo consenso come fattori obesogenici rispetto alla sedentariet e alla scarsa attivit fisica. Inoltre, le madri pi istruite sono risultate significativamente pi consapevoli del ruolo negativo di un limitato dispendio energetico e hanno riconosciuto pi frequentemente la responsabilit di industrie alimentari, scuola e servizi sanitari. Conclusioni. Il supporto da parte dellopinione pubblica ai cambiamenti dello stile di vita crescente, ma potrebbe avere maggiore peso ed efficacia se ci fosse una pi diffusa consapevolezza dei numerosi e intercorrelati fattori obesogenici che stanno giocando un ruolo chiave nel configurare lo stile di vita dominante.

Introduction Obesity is now considered to be the most prevalent nutritional disease of children and adolescents in western countries [1,2]. Persistency into adulthood and the association with morbidity are the most relevant problems of childhood obesity [1,2]. Previous studies in Europe have shown that the southern countries, including Italy, have the highest prevalence [3]. The results of a recent study show that prevalence of overweight and obesity in 5-year-old Italian children is 24%. In particular, one of six children, independently from sex and BMI references used (national, International Obesity Task Force, and CDC), was overweight. The prevalence of obesity averages 8% [4]. The prevalence of overweight and obesity in the Sicilian schoolchildren is one of the highest ever reported: prevalence of overweight and obesity in a cohort of randomly selected 48,897 11-15-year-old Sicilian children is nearly 40% at age 11 and over 25% at age 15 [5]. Excessive weight during childhood stems from several interacting factors, including poor diet and exercise habits [6]. There is a growing agreement between public health experts that the so-called toxic environment plays a crucial role in the childhood obesity epidemic, by social and economic forces that encourage the consumption of excess of energy and discourage energy expenditure [6,8]. The causal chain of the crisis is complex and, accordingly, interventions to prevent and control overweight in childhood have met with limited success [6]. Parents have an important role to play in preventing weight-related problems in their children. In particular, parental perceptions of their childrens weight status may affect the family choices, regarding weight, eating, and exercise [9,12]. Thus, it is not surprising that behaviour
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modification programs that involve parents, particularly mothers, have more of an impact than those that do not [2,10]. Moreover, awareness of the multiple interplaying obesogenic factors in the modern lifestyle is required to support changes in the physical, social, economical and political environment so that a healthy lifestyle can become the default [13]. The objective of this study was to evaluate how parents, living in southern Italy, perceived their childrens weight status, the role of physical activity and beliefs about contributors and parties having responsibility for counteracting the overweight and obesity crisis. Association with specific socio-demographic factors was also investigated. Methods Sample. We conducted a cross-sectional survey on a convenience sample of parents of 6-13 year-old children who attended grades 1, 3 and 5 of primary and grades 1 and 3 of secondary, respectively, public schools. This education level is compulsory in Italy. The schools were located in an inner urban district of Palermo, Italy, characterized by low to medium income residents. Thirteen schools were selected. Parents were asked for coming to school and participating to the investigation by a written invitation from teachers, who had been previously trained about the content and the objectives of the investigation and the information to be given. The survey was administered in the spring of 2006. Questionnaire and procedures A structured questionnaire was developed for parents to investigate perception of weight excess as a problem in childhood, role of physical activity and beliefs about contributors and parties having responsibility for counteracting the obesity crisis. The questions about these last issues were adapted from Evans WD et al , 2005[9]. Answers were not mutually excluding. The questionnaire was self-administered to the parents at school in the presence of a trained component of the working group who assisted collection of the data according to a standardized procedure. Verbal consent of participants was elicited and confidentiality was warranted. The questionnaire included a section containing some information about socio-demographic characteristics of respondents (gender, age in years, race/ethnicity, marital status, relationship with the pupil, number of family components, family education level) and self-reported anthropometric measures. Race/ethnicity was self-reported by participants, who were categorized as Italian and non-Italian, due to small numbers. Marital status was self-reported and categorized as married, divorced/separated, cohabitating, never married. Relationship with the pupil was
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self-reported and categorized as mother, father and other. Family education level was assessed in the questionnaire as reported by the respondent for both mother and father. In the analysis education level was entered as a categorical variable: low = less than high school; high = high school diploma, more than a high school degree. Height and weight were self-reported by participants. Body Mass Index (BMI) was calculated as weight (kg) divided by height squared (m2). Standard thresholds were firstly used to classify individuals as normal/underweight (BMI <25), overweight (BMI = 25-30) and obese (BMI >30); then collapsed into two categories (normal and overweight) due to small numbers. Parents responded to the items about perception of excess of weight as a problem, physical activity and health threats, contributors and agents responsible for intervening against childhood obesity, using three option, yes, no and dont know. EpiInfo software ver.6.0 (CDC, Atlanta, GA, US) and Stata software ver. 9.2 (Statacorp LP, Texas, US) were used for data management and analysis. For descriptive analysis, frequencies, cross-tabulation and chi-square statistics were calculated. To assess whether belief about contributing factors and responsibility for intervention against excess of weight varied by specific demographic and social characteristics of respondents, multivariate logistic regression analysis was conducted. Independent variables included education, gender, age class and BMI. Odds ratio (OR) and 95% confidence intervals (CIs) are reported. In all analyses, differences were considered statistically significant at P 0.05. Results Sample characteristics Three hundred-eleven (20% approximately) of the 1,500 parents who were invited to fill out the questionnaire went to school and completed it. Table 1 summarizes socio-demographic characteristics of respondents. Median age of respondents was 37.0 years (range 23 67). Eighty-three % of respondents were female and more than 90% were of Italian nationality. This is likely due to the anticipated difficulties in compiling the questionnaire by the non-Italian parents. Marital status and number of family members are consistent with the most prevalent family patterns in Italy. Only 37% of mothers and 30% of fathers, respectively, had at least 9 years of education. Frequencies of both family composition and education level were consistent with those obtained from nationally representative samples of the Italian population and from census data [14]. Of interest, 35% of the respondents refused to report or did not know the education level of father. Cross-tabulation analysis was carried out to
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examine potential association between the respondents who reported the education level of father versus those who did not report it and demographic characteristics, such as education of mother and number of components of the family. No significant association was found. Thus, the subsample of respondents who did not report the education of father was not different from the larger portion of respondents who did report it. Self-reported BMI of participants was also consistent with data published from the Italian Institute of Statistics about the population of southern Italy [14]. Perception of childhood obesity, contributing factors and responsibility for fighting weight gain Our results revealed that 83.3% of participants believed that overweight in childhood is a serious health hazard, such as underage smoking and drinking. No significant association was detected with age and gender. Mothers with a high education level embraced this belief with a statistically significant higher frequency (high vs low educational level, 93.9 vs 77.5%, P < 0.001). Relationship with the father education level revealed a similar answer pattern (high vs low educational level, 92.4 vs 78.0%, P = 0.002). Nonetheless, one third approximately of participants interpreted the overweight in childhood as an expression of health. No significant association was found with age and gender. Again, high education level of parents was significantly associated with a lower prevalence of this perception (mother, high vs low educational level, 15.5 vs 44.1%, P < 0.001; father, high vs low educational level, 18.3 vs 45.1%, P < 0.001). The most significant contributors to childhood overweight were thought to be junk food and beverages (78.0%) and fast food (63.2%), followed by lack of exercise in school curriculum (48.7%). Interestingly, there was a relatively low prevalence of positive answers to questions about contribute of viewing TV 2 hours per day (30.6%) and using PC, videogames or Internet 1 hours per day (26.0%). Ninety-seven percent of participants considered parents to have the most responsibility for fighting childhood obesity. Between 16.4 and 26.0% of participants only attributed some responsibility to any parties external to the family, such as food companies, school, public health or government. Pattern of responses did not varied significantly by age and gender of participants. These data are summarized in Table 2. Role of physical activity In constructing the survey, some questions were proposed to specifically test awareness of respondents about role of physical activity in childhood overweight and their support in favour of physical activity-based interventions. Consistently high prevalence rates of po305

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sitive answers, between 71.7% and 92.6%, were obtained in favour of increasing school and extra-school physical activity (Table 3). Restricting the amount of time spent with TV, PC or Internet could positively contribute to health and wellbeing in childhood for 79.8% of respondents (Table 3). No significant differences were detected by age and gender. Influence by socio-demographic characteristics of parents Table 4 presents results of cross tabulation analysis conducted to examine potential association between education level of mother and father and perceptions about contributors and responsibilities in childhood overweight. More educated mothers were significantly more likely to recognize as contributing factors not only junk food and beverages and fast food, but also lack of exercise in school curriculum, lack of places to exercise and lack of security in the neighbourhood. Moreover, more educated mothers were significantly more likely to attribute some responsibility to food companies, TV and Internet advertising, health care services and government. In no cases was fathers education level significantly associated with identification of some contributing factor or responsible subject. Logistic regression revealed that a lower mothers education level was negatively and significantly associated with perception as a health threat to childhood of junk food and beverages, lack of exercise at school, lack of places to exercise and lack of security. Furthermore, mothers low education was negatively associated in a statistically significant manner with attribution of responsibility to fight overweight to food companies, school and healthcare services. Being overweight did not have a considerable effect; the effect was significantly negative for junk food and beverages only within contributors and significantly positive for healthcare services only within parties involved in combating childhood weight gain. No reporting education level of father was significantly and positively associated only with identification of junk food and beverages as contributing factor. These results are illustrated in Table 5. Conclusions The literature points consistently toward parents as the parties having a key role in the prevention of weight-related problems in childhood [9,12]. They are generally seen as the first role model of a healthy lifestyle from both eating habits and physical exercise standpoints. Accordingly, interventions addressing knowledge and practices of parents, rather than children, are more likely to be successful [2,10]. However, several studies suggest that many parents are unaware of overweight of their children or do not consider it as a health threat [15,16]. Furthermore, considerable evidence suggests that diet and lifestyle of children are strongly influenced by multiple and interplaying social, cultural, economic
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and political layers, that call for novel prevention approaches [7,13]. Its not astonishing, hence, that delegations from 48 countries at a conference organized by the World Health Organization Regional Office for Europe in October 2006 agree on changes to be made on both sides of the energy balance, i.e. nutrition and physical activity [17]. It has been also recognized that fighting weight excess, especially in childhood, requires an ecological and transdisciplinary approach, including social-cultural, economical and political actions at the international, national and local level [17]. In our study, perceptions and beliefs about childhood weight excess of a sample of parents living in an inner neighborhood of a large urban centre of southern Italy, such as Palermo, were investigated. Ability to recognize in the environment obesogenic agents and entities that influence childhood weight excess and association with some socio-demographic variables were also analyzed. Not surprisingly, a large majority of participants were mothers. Data about education suggest that, although participation to the survey was on a voluntary basis, the final sample of participants was not skewed toward a higher education level. However, influence of a more acute attention to health problems of childhood, whether spontaneous or driven by a more favourable informational micro-environment, cannot be excluded. Within the survey participants, awareness of overweight as a serious health hazard for children appears to be diffuse, accounting for more than 80%, but simultaneously one third of parents is continuing to think at the overweight as an indicator of wellbeing and health. Paradoxically, overweight children may appear better nourished and be perceived as good eaters, hence causing their parents less concern than the thinner ones. Interestingly, a statistically significant positive association was evident between a high parents education level and perception of overweight as a health problem. Conversely, more educated parents consider at a significantly lower frequency an overweight child as a healthy child. Our data are consistent with those of previous studies, showing that mothers with a low education more frequently are likely to misidentify overweight status of their children and, when correctly categorize them, do not consider this as a health concern [9,15]. A high percentage of the survey participants agreed on the positive contribution to education and health of childhood of more time and relevance to physical activity as well as of less recreational time spent with PC, Internet, TV or videogames. Moreover, the respondents overwhelmingly favoured requiring more physical activity in school, though fewer favoured extra-school physical activity at ones own expense. Nevertheless, within the proposed list of possible contributors the eating style related options proved to be largely more established as obesogenic factors than sedentary pursuits and reduction in physical activity. Consistently with findings by other Authors, more educated mothers were significantly more like to be aware of the unhealthy role of a limited energy expenditure in the
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weight-related problems of childhood [9,15]. The majority of respondents identified the parents as the parties having most responsibility for intervention aimed at reducing weight excess in children. Parents feeding style and, more generally, lifestyle has a crucial role in their childrens lives well into adolescence [18,19]. However, there is a growing agreement within public health experts on the influential role of the so-called toxic environment and the need of reversing the obesitygenerating environment by economic and politic interventions and broad changes of many present ways of life [7,8,13]. Again, within the survey participants, more educated mothers were significantly more likely to attribute responsibility for combating childhood obesity to food companies, school and healthcare services. This survey confirms that parents should better understand the interplay of environmental and familial influences on the obesity epidemic, and hence should be appropriately empowered. But, consistent with other studies, in low education contexts parental perception about the causal chain of the childhood obesity crisis is likely skewed toward a failure of personal responsibility [13] Moreover, balance between the two categories of obesogenic factors, those encouraging the consumption of excess energy and those discouraging energy expenditure, is likely distorted in favour of the first ones. Given low education level as an indicator of low income, our study suggests that a poor perception of external influences is probably more established in low income households, where their pressure is expected to be more effective [20,23]. There are some limitations in this study. First, the study population consisted of a convenience sample of eligible parents, leading to selection bias. In addition, the low response rate and the characteristics of the study population, including predominately inner-city inhabitants of Italian nationality, may limit the generalization of the results. Moreover, the self-reported nature of the data and the cross-sectional study design need to be considered when interpreting the results. Parents perceptions of their own childrens weight status is largely influenced by education. Recognition of physical activity support may heighten the parents role in preventing overweight and obesity. Given that a large proportion parents of overweight children fail to recognize that their child has a weight problem and identify themselves as having most responsibility in combating childhood overweight, schools and media should develop strategies to help these parents correct their misperceptions [24]. Public support for environmental changes could effectively rise with the increasing public awareness that many interrelated obesogenic factors in the modern environment are playing a key role. Additional knowledge about public awareness may help health care providers to raise better understanding of health issues related to childhood obesity and support for effective solutions.
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Acknowledgements The authors are indebted to Dr. Maria Antonietta Fig, AUSL6, for contributing to the planning of the study and contacting the schools. They are also grateful to the Director of the Distretto 10, AUSL 6, for his technical support. The study has been supported by grants from the Italian Ministry of University (ex 60%) to FF and AP.

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Parental recognition of overweight as a health problem in school-age children living in an inner-city neighborhood of Palermo, Italy: a cross-sectional study

Antonino Bianco, Caterina Mammina, Marianna Bellafiore, Giuseppe Battaglia, Felicia Farina and Antonio Palma

Parental recognition of overweight as a health problem in school-age children living in an inner-city neighborhood of Palermo, Italy: a cross-sectional study

Antonino Bianco, Caterina Mammina, Marianna Bellafiore, Giuseppe Battaglia, Felicia Farina and Antonio Palma

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] Dietz WH, Robinson TN. Clinical practice. Overweight children and adolescents. N Engl J Med 2005; 352: 2100-2109. Hardy LR, Harrell JS, Bell RA. Overweight in children: definitions, measurements, confounding factors, and health consequences. J Pediatr Nurs 2004;19: 376-384. Lobstein T, Frelut ML. Prevalence of overweight among children in Europe. Obes Rev 2003;4:195200. Maffeis C, Consolaro A, Cavarzere P, Chini L, Banzato C, Grezzani A, Silvani D, Salzano G, De Luca F, Tato L. Prevalence of Overweight and Obesity in 2- to 6-year-old Italian Children. Obesity 2006; 14: 765-769. Baratta R, Degano C, Leonardi D, Vigneri R, Frittitta L. High prevalence of overweight and obesity in 11-15-year-old children from Sicily. Nutr Metab Cardiovasc Dis 2006; 16: 249-255. Poskitt EM. Tackling childhood obesity: diet, physical activity or lifestyle change? Acta Paediatr 2005; 94:396-8. Ball K, Timperio AF, Crawford DA. Understanding environmental influences on nutrition and physical activity behaviors: where should we look and what should we count? Int J Behav Nutr Phys Act. 2006; 3: 33. Lustig RH. The skinny on childhood obesity: how our western environment starves kids brains. Pediatr Ann 2006; 35:898-902, 905-907. Evans DW, Finkelstein EA, Kamerow DB, Renaud JM. Public perceptions of childhood obesity. Am J Prev Med 2005; 28: 26-32. Golan M, Crow S. Parents are key players in the prevention and treatment of weight-related problems. Nutr Rev 2004; 62: 39-50. Howard KR. Childhood overweight: parental perceptions and readiness for change. J Sch Nurs 2007; 23: 73-79. Gustafson S, Rhodes R. Parental correlates of physical activity in children and early adolescents. Sports Medicine 2006, 36:79-97. Schwartz MB, Brownell KD. Actions necessary to prevent childhood obesity: creating the climate for change. J Law Med Ethics 2007; 35:78-89. Italian Institute of Statistics. [http://www.istat.it] Baughcum AE, Chamberlin LA, Deeks CM, Powers SW, Whitaker RC. Maternal perceptions of overweight preschool children. Pediatrics 2000;106:1380-1386. Campbell MW, Williams J, Hampton A, Wake M. Maternal concern and perceptions of overweight in Australian preschool-aged children. Med J Aust 2006;184: 274-277. Brug J. The European charter for counteracting obesity: a late but important step towards action. Observations on the WHO-Europe ministerial conference, Instanbul, November 15-17, 2006. Int J Behav Nutr Phys Act 2007; 4:11. Faith MS, Scanlon KS, Birch LL, Francis LA, Sherry B. Parent-child feeding strategies and their relationships to child eating and weight status. Obes Res 2004;12:1711-1722. Kalakanis LE, Goldfield GS, Paluch RA, Epstein LH. Parental activity as a determinant of activity level and patterns of activity in obese children. Res Q Exerc Sport 2001; 72: 202-209. Gordon-Larsen P, Nelson MC, Page P, Popkin BM. Inequality in the built environment underlies key health disparities in physical activity and obesity. Pediatrics 2006; 117: 417-424. Jain A, Sherman S, Chamberlin L, Carter Y, Powers S, Whitaker R. Why dont low-income mothers worry

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about their preschoolers being overweight? Pediatrics 2001;107:11381146. [22] Moore LV, Diez Roux AV. Associations of neighborhood characteristics with the location and type of food stores. Am J Public Health 2006, 96: 325-331. [23] Zenk SN, Schulz AJ, Israel BA, James SA, Bao S, Wilson ML. Fruit and vegetable access differs by community racial composition and socioeconomic position in Detroit, Michigan. Ethn Dis 2006, 16: 275-280. [24] Etelson D, Brand DA, Patrick PA, Shirali A. Childhood obesity: do parents recognize this health risk? Obes Res 2003 Nov; (11): 1362-8.

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Analitical index and Contributors

Adhesion molecules. 22, 37, 40, 41, 71 Adipose organ. 51, 52, 281 Adjusted residual. 129, 132 Airway inflammation. 17, 19, 20, 22 Airways. 17, 18, 19, 20, 21, 22, 23, 24, 30, 31, 37, 38, 39, 41, 42, 47, 53 Alveoli. 54 Alzheymer disease. 111 Amniotic membrane. 171 Amyotrophic lateral sclerosis. 111 Aneurysm. 98, 100, 101 ANF (v. Atrial natriuretic factor) Angioblast. 70 Angiogenesis. 70, 147, 152 ANP (v. Atrial natriuretic factor) Aorta. 98, 99, 100, 101 Aortic aneurysm. 97 Apoptosis. 21, 23, 117, 121, 220, 297 Asthma. 18, 20, 21, 32, 33, 38 Atherosclerosis. 61, 62, 70 Atrial natriuretic factor. 47, 48, 49, 63, 145, 148, 152, 153, 156, 158, 159, 165, 166, 167 Attentive behaviour. 137 Basement membrane. 31, 147 BENI (Brachiury Expression Nuclear Inhibitors). 12 Bloody diarrhea. 184 Brain. 51, 52, 118, 121, 281 BRCA. 237, 239, 241, 242, 243, 247, 251, 253, 254, 255, 259, 260, 261, 263 Bronchi. 52 Bronchial epithelial damage. 17 Bronchial epithelium. 17, 52, 53 Bronchial tree. 31 Cannabinoids. 51, 52 Carbon monoxide. 19 Carcinogenesis. 111 Cardiomyopathy. 61, 62, 65 Catecholamines. 118, 224 Caudate nucleus. 118 CD105. 149 CD31. 70 CD34. 63, 71 CD73. 149 CD90. 149 Central nervous system. 52, 54, 117, 271, 274, 281

Chaperones. 109, 110, 112 Chaperonins. 109, 110 Chaperonopathy. 109, 111 Chaperonotherapy. 109, 112 Childhood. 301 Cholinesterase. 223 Choroid plexus. 165, 167 Chromosome. 289, 290, 291, 294, 295 Chronic heart failure. 69, 72 Chronic obstructive pulmonary disease. 29, 30, 32, 33 Cigarette smoke. 29, 30, 31, 32, 33 Ciliated epithelium. 21 Cluster analysis. 129, 131 Colitis. 183, 184, 187, 203, 207 Collagen. 31, 32, 97, 146, 147, 149, 151 Collagenase. 31 Colon. 184, 186, 187, 188, 192, 193, 194, 195, 203, 204, 205, 206, 207 Coronary artery. 62, 63 COX-2. 206 Cytokeratin. 149 Cytokine. 198, 206, 215, 216 Dental pulp. 223, 224, 225, 227, 228, 229 Diarrhea. 187, 192, 203, 204 DNA damage. 289 Dopamine. 117, 118, 137 Edema. 18 Elastase. 31, 32, 39, 40, 41 Elastic fibres. 99 Elastin. 32, 97, 151 Embryo. 70 Emphysema. 30 Endocannabinoid. 51, 52, 53, 54, 281, 284 Endocardial fibrosis. 62 Endocardium. 62, 74 Endorphin. 223, 227, 230 Endothelial cells. 69, 70, 71, 72, 73, 74, 98, 100, 151, 153, 154, 156, 159, 220 Endothelin. 224 Endotoxin. 206 Endurance training. 17, 37 Enkephalin. 223, 227, 231 Enteric nervous system. 51, 52 Eosinophil. 17, 19, 20, 37, 38, 39, 40, 41, 42, 194

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Analitical index and Contributors

Extracellular matrix. 29, 30, 31, 32, 97, 98, 99, 152, 158 Fibroblasts. 29, 32, 33, 69, 99, 100, 101, 149, 156, 206 GABA. 118 Gastric cancer. 215 Gastritis. 213, 215, 216, 220 Gastrointestinal tract. 184 Gelatinase. 31, 32, 33, 34 Genetic disease. 61 Genetic Testing. 237, 251 GLUT (Glucose Transporter). 13 Glycosaminoglican. 145, 146, 147 Goblet cell. 23, 205 Golgi apparatus. 148 Granulocyte. 23 Gut. 204 Gut-associated lymphoid tissue. 194 Haloperidol. 137, 138, 139, 140 Heart. 61, 62, 63 Helicobacter pilori. 213, 214, 215, 216 Hematopoietic tissues. 70 Hepatocyte. 51, 52, 151, 282 Hereditary Ovarian Cancer. 237 Hsp10. 109, 110, 111, 112 Hsp60. 109, 110, 111, 112 Hsp70. 112 Human umbilical cord. 145, 154, 155, 156, 158 Huntington disease. 111 HUVEC. 70, 71, 74, 151, 153, 158, 160 Hyaluronic acid. 147 Hydrogen peroxide. 69, 72, 73, 74, 75 Hypertension. 61, 62 Hypotalamus. 153, 165, 281 Idiopathic Dilated Cardiomyopathy. 61 IgA. 207, 216 IL-1. 215, 216 IL-2. 216 IL-6. 41, 42 IL-8. 19, 22, 215, 216 Induced sputum. 17, 20, 21, 23, 37, 38, 39, 40, 41, 42 Inflammatory bowel disease. 184, 186, 207 Iodoacetamide. 183, 186, 190, 192, 194, 198, 204, 207

Jejunum. 192, 204 Lamina mucosa. 53 Large bowel. 111 Leukocyte. 20, 22, 23 Leukocytosis. 37, 41 Lipoplysaccharide. 206 Liver. 151 Lung. 19, 29, 30, 31, 32, 33, 34, 47, 48, 51, 52, 53, 54, 216 Lymphocytes. 17, 20, 37, 38, 39, 63, 194, 198, 293 Macrophages. 20, 30, 32, 99, 151, 194, 198, 206, 216, 217, 218, 220 Marathon swimming. 37 Mast cells. 20, 194 Megacolon. 184, 187, 193, 204 Mesenchyme. 147 Mesoderm. 70 Metalloproteinase. 29, 31, 33, 34, 97 Methacholine. 20 Micronucleus. 289, 291, 293, 297 Mitochondria. 64, 109, 148, 149 MMP-12. 32, 34 MMP-14. 31, 32 MMP-2. 29, 31, 32, 33, 34, 97, 98, 99, 100, 101, 145, 151, 152, 155, 156, 158, 159 MMP-3. 31 MMP-8. 34 MMP-9. 31, 32, 34, 97, 98, 99, 145, 152, 156, 158, 159 Monocytes. 31, 206 Morphogenesis. 30 Multivariate analysis. 129 Muscle cells. 101 Myeloperoxidase. 183, 189, 196, 203 Myocarditis. 62, 63, 65 Myocardium. 62, 63 Myocyte. 62, 63, 64 Myocytolysis. 63 Myofibroblasts 149 Nanog. 149 Nasal cavity. 47, 48 Neurogenesis. 117, 122 Neuropeptide. 165, 223 Neuroprotection. 117 Neurotransmitter. 223

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Analitical index and Contributors

Neutrophils. 17, 19, 20, 30, 31, 32, 38, 39, 40, 41, 194 Neutrophilia. 37, 41, 42 Nigrosomes. 119 Nitric oxide. 69, 71, 72, 213, 215, 216 Nitrogen dioxide. 19 Nociceptin. 271, 272 Nodose ganglion. 53 NOS. 71, 72, 74, 97, 99, 101, 145, 151, 154, 155, 158, 213, 216, 218, 220, 223, 227, 228, 231 Nutrient malabsorption. 184 Obesogenic factors. 301 Oct-4. 149 Orphanin. 145, 156, 158, 160, 271, 272, 273, 274, 275 Overweight. 301 Oxidative stress. 31, 69, 71, 72, 73, 74, 121 Oxytocin. 145, 153, 156, 158, 159, 160, 165, 166, 167 p38. 19 p53. 112 Pancreas. 282 Paraventricular nucleus. 153, 165, 166 Parkinson disease. 117 Parental perception. 301 Parotid gland. 282 Peptic ulcer. 214, 215 Physical activity. 301 Pleura. 47, 48 Pneumocyte. 51, 53, 54 Prophylactic Surgery (PSO). 258 Prostate. 111 Proteinase inhibitor. 29, 32, 34 Proteoglican. 146, 147 Putamen. 118 RANTES. 19, 22 Reactive nitrogen species. 69, 72 Reactive oxygen species. 32, 69, 72, 120 Remodelling. 17, 20, 21 Reticulocyte. 40, 41 Salivary glands. 51, 52, 281, 282, 284 Skeletal muscle. 51 Smooth muscle. 19, 53, 69, 71, 148, 149, 217 Somatostatin. 220, 225 Squamous cells. 40, 69

Stem cells. 149, 150 Steroids. 20 Stochastic analysis. 129, 131 Stomach. 214 Striated muscle. 52 Striatum. 118 Sublingual gland. 282 Submandibular gland. 282 Substance P. 148, 223, 229 Substantia nigra. 117, 118 Supraoptic nucleus. 153, 165, 166 TIMP-2. 33, 98 T-Pattern analysis. 129, 133 Trachea. 47, 48, 52 Troponin T. 63 Ulcerative colitis. 183, 184, 186, 194, 198, 203, 208 Umbilical cord. 145, 146, 147, 148, 150, 156, 158, 171 Urogenital tract. 271, 272, 274 Uterine exocervix. 111 Valve disease. 61 Vasopressin. 148, 165, 166 VEGF. 72 Vimentin. 149 VIP. 48, 148 vWF. 70, 148 Weight loss. 184, 187 Whartons jelly. 145, 146, 148, 149, 150, 155, 156, 159, 171, 173 Wiebel & Palade bodies. 148 Zonulae occludens. 148

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Anaitical index and Contributors

Anzalone R., 29, 69 Ardizzone N., 109 Barada K., 183 Battaglia G., 301 Bazan V., 237 Bellafiore M., 301 Bellia V., 17 Benigno A., 117 Bianco A., 301 Bonanno A., 17, 37 Bonaventura G., 51, 271, 281 Bonsignore M.R., 17, 37 Bruno L., 237 Bucchieri F., 109 Burruano F., 289 Buscemi M., 145, 223, 237 Cal V., 237 Campanella C., 109 Cappello F., 61, 109 Carbone M., 29, 69 Carini F., 79 Casarrubea M., 129, 137 Chimenti L., 17, 37 Ciaccio M., 289 Ciaccio G.M., 69 Cicero G., 237 Conway de Macario E., 109 Corrao S., 29, 69 Crescimanno G., 137 Cucco D., 51, 281 David S., 109 Di Felice V., 61 Di Giovanni G., 117 Di Stefano A., 69 Farina F., 29. 69 Farina-Lipari E., 47, 165 Federico M., 237 Ferla R., 237 Frazzetta M., 237 Freund J.N., 183 Geraci F., 11 Gerbino A., 145, 171, 223, 237 Gervasi M., 29 Giannuzzi P., 69 Giudice G., 11

Gulotta E., 237 Haij Hussein I.A., 183 Hassan Mostafa M., 183 Jurjus A., 183 Jurjus R.A., 183, 223 Karam W., 183 La Rocca G., 29, 69 Leone A., 145, 183, 223 Licciardi A., 117 Lipari A., 165 Lipari D., 47, 51 Lipari L., 171, 223, 289 Lo Iacono M., 29 Lo Piccolo C., 79 Loria T., 29, 69 Macario A.J.L., 109 Magno F., 29, 69 Mammina C., 301 Mandracchia R., 171 Marcian V., 109 Marino-Gammazza A., 109 Mauro A., 145, 171, 223 Merendino A., 109 Messina P., 79 Midiri M., 171 Mikuz G., 61 Morici G., 17, 37 Palma A., 301 Passantino R., 237 Patern A., 17, 37 Peri G., 47, 109 Perino A., 171 Pizzo F., 69 Profita M., 37 Ribbene A., 109 Riccobono L., 37 Rizzo S., 237 Romano F., 171 Russo A., 237 Scardina G.A., 79 Sconzo G., 11 Sergi C., 61 Sorbera F., 137 Spatola G.F., 51, 97, 281 Steiner H.J., 61

323

Tessitore V., 51, 281 Tohme R., 183 Tortorici S., 289 Turturici G., 11

Uzzo M.L., 51, 213, 281 Valentino B., 47, 165 Valenza V., 79 Zummo G., 29, 61, 69, 109

324

CONTENTS
5 9 11 15 17 Preface Guest contribution GASTRULATION IN SEA URCHIN AND AMPHIBIAN EMBRYOS
Giuseppina Turturici, Fabiana Geraci, Gabriella Sconzo and Giovanni Giudice

Airway cells EXERCISE-INDUCED CHANGES IN AIRWAY CELLS


Laura Chimenti, Giuseppe Morici, Alessandra Patern, Anna Bonanno, Vincenzo Bellia, and Maria R. Bonsignore

29

New perspectives on the roles of proteinases and lung structural cells in the pathogenesis of chronic obstructive pulmonary disease
Giampiero La Rocca, Rita Anzalone, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Marco Gervasi, Melania Lo Iacono, Giovanni Zummo and Felicia Farina

37

AIRWAY CELLS IN SWIMMERS: A CASE REPORT AND A REvIEW Of THE LITERATURE


Giuseppe Morici, Laura Chimenti, Anna Bonanno, Loredana Riccobono, Mirella Profita, Alessandra Patern and Maria R. Bonsignore

47 51

PRESENZA DEL fATTORE NATRIURETICO ATRIALE NELLAPPARATO RESPIRATORIO


Biagio Valentino, Diego Lipari, Giovanni Peri e Elvira Farina Lipari

IMMUNOHISTOCHEMICAL EXPRESSION Of CB1 CANNABINOIDS RECEPTOR IN RAT LUNG


Giuseppe Bonaventura, Daniela Cucco, Diego Lipari, Giovanni Francesco Spatola, Maria Laura Uzzo e Vincenzo Tessitore

59 61

Heart, microcirculation Morphological features of the Idiopathic Dilated Cardiomyopathy


Consolato Sergi, Francesco Cappello, Hans-Joerg Steiner, Valentina Di Felice, Giovanni Zummo and Gregor Mikuz

69

HYDROGEN PEROXIDE IN ENDOTHELIUM: MULTIfACETED ROLES IN CELLULAR STRESS AND SIGNALLING


Rita Anzalone, Giampiero La Rocca, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Francesca Pizzo, Giuseppina Maria Ciaccio, Antonino Di Stefano, Pantaleo Giannuzzi, Felicia Farina and Giovanni Zummo

325

79 97

Metodiche microperfusionali e sepsi


Francesco Carini, Chiara Lo Piccolo, Giuseppe Alessandro Scardina, Pietro Messina e Vincenzo Valenza

Immunohistochemical expression of iNos and matrix metalloproteinases MMP-2 and MMP-9 in aortic aneurysms
Giovanni Francesco Spatola

107 Cellular stress 109 Chaperonology: a novel research field for Experimental Medicine in the XXI century
Francesco Cappello, Fabio Bucchieri, Sabrina David, Claudia Campanella, Anna Ribbene, Antonella Marino-Gammazza, Nella Ardizzone, Anna Merendino, Vito Marcian, Giovanni Peri, Everly Conway de Macario, Alberto J.L. Macario and Giovanni Zummo

115 Brain, behavior 117 The Substantia Nigra Dopaminergic Neurons


Arcangelo Benigno, Attilio Licciardi and Giuseppe Di Giovanni

129 Multivariate analysis as tool for the study of behavior


Maurizio Casarrubea

137 MODIfICAZIONI INDOTTE DALLALOPERIDOLO SUL COMPORTAMENTO ATTENTIvO DEL RATTO


Giuseppe Crescimanno, Maurizio Casarrubea e Filippina Sorbera

143 Organogenesis, Clinical Embryology 145 Human umbilical cord at term. Immunohistochemical expression of MMP-2, MMP-9, Orphanin fQ and others peptides involved in the regulation of vascular tone
Annamaria Mauro, Maria Buscemi, Angelo Leone and Aldo Gerbino

165 Ontogenesi dei peptidi vasopressina, ossitocina, ANP (Peptide Atriale Natriuretico) in alcuni nuclei ipotalamici in embrioni di ratto
Elvira Farina Lipari, Alessio Lipari e Biagio Valentino

171 Imaging ecografico del Cordone ombelicale umano


Riccardo Mandracchia, Annamaria Mauro, Luana Lipari, Federico Romano, Antonino Perino, Massimo Midiri e Aldo Gerbino

181 Inflammatory process 183 Experimental colitis: chemical and bacterial induction
Inaya Abdallah Hajj Hussein, Rania Tohme, Kassem Barada, Mostafa Hassan Mostafa, Angelo Leone, Jean-Noel Freund, Rosalyn A. Jurjus, Walid Karam and Abdo Jurjus

326

213 THE CHRONICAL GASTRITIS HELICOBACTER PYLORI CORRELATED: IMMUNOHISTOCHEMICAL BEHAvIOUR Of NITRIC OXIDE SYNTHASE ISOfORMS IN RATS STOMACH
Maria Laura Uzzo

223 Twenty years History of Immunohistochemical mammals dental Pulp Studies


Angelo Leone, Roselyn A. Jurjus, Aldo Gerbino, Luana Lipari, Annamaria Mauro and Maria Buscemi

235 Urogenital tract 237 Carcinoma Ereditario dellOvaio


Loredana Bruno, Sergio Rizzo, Valentina Cal, Mario Federico, Rita Passantino, Rita Ferla, Giuseppe Cicero, Eliana Gulotta, Michele Frazzetta, Maria Buscemi, Aldo Gerbino, Viviana Bazan e Antonio Russo

271 Rilievi immunoistochimici della orfanina fQ nellapparato genitourinario del ratto


Giuseppe Bonaventura

279 Oral 281 AN IMMUNOHISTOCHEMICAL STUDY ON THE CB1 RECEPTOR EXPRESSION IN RAT SALIvARY GLANDS
Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore

287 Stein, Physical activity 289 The recent literature about micronuclei. Considerations and reliefs
Luana Lipari, Marcello Ciaccio, Francesco Burruano and Silvia Tortorici

301 Parental recognition of overweight as a health problem in school-age children living in an inner-city neighborhood of Palermo, Italy: a cross-sectional study
Antonino Bianco, Caterina Mammina, Marianna Bellafiore, Giuseppe Battaglia, Felicia Farina and Antonio Palma

317 Analitical index and Contributors

327

Finito di stampare nel mese di dicembre 2007 presso le Officine Tipografiche Aiello & Provenzano Bagheria, Palermo