Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Università Campus Bio-Medico di Roma Corso di dottorato di ricerca in Immunologia Oculare XXII ciclo anno 2007 Fisiopatologia, diagnosi, qualità della vita, stadiazione e nuove strategie di trattamento della cheratocongiuntivite primaverile (VKC) Marta Sacchetti Coordinatore Prof Stefano Bonini Tutore Dott Alessandro Lambiase 26 Febbraio 2010 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Abstract La cheratocongiuntivite primaverile (VKC) è una patologia allergica cronica che colpisce prevalentemente i bambini caratterizzata da intensi sintomi oculari con recrudescenza stagionali spesso associate a coinvolgimento corneale. Tale malattia è ancora scarsamente caratterizzata dal punto di vista della patogenesi, della diagnosi, dell’impatto sulla qualità della vita e del trattamento. La VKC è una patologia ad eziologia allergica caratterizzata da una risposta immunitaria di tipi T helper 2 (Th2). L’immunità innata svolge un ruolo nella patogenesi della VKC come è stato evidenziato in recenti studi, tra cui un nostro studio in cui è stato dimostrato un aumento delle cellule natural killers nella congiuntiva di pazienti con VKC rispetto ai controlli. Nostri dati preliminari indicano anche un coinvolgimento degli ormoni sessuali nella patogenesi della VKC. L’inquadramento diagnostico della VKC pone delle difficoltà, in quanto i bambini sperimentano limitazioni nella qualità della vita, dovuta alla malattia, e il quadro clinico presenta ampia variabilità nell’arco dell’anno. L’impatto della VKC sulla qualità della vita nel bambino non era mai stata valutata, abbiamo quindi creato e validato un questionario specifico per bambini con VKC (il QUICK© test ). Inoltre, poiché non esistono classificazioni della severità della VKC, abbiamo effettuato uno studio retrospettivo su 207 casi di VKC, i dati sono stati analizzati con il metodo CART, al fine di ricavare un algoritmo decisionale da applicare nella pratica clinica, di proporre una stadiazione della severità e individuare i fattori di rischio per un peggiore esito visivo. Dal punto di vista terapeutico, attualmente i farmaci di scelta della VKC sono i colliri antiallergici topici, nelle fasi lievi e moderate, e gli steroidi topici nelle fasi di acuzie, efficaci ma non privi di effetti collaterali, anche gravi. C’è quindi l’esigenza di terapie alternative, che consentano un miglior controllo della malattie con minori effetti collaterali. In uno studio multicentrico abbiamo dimostrato l’efficacia della Ciclosporina A in collirio nel ridurre il numero delle recrudescenze della VKC. 2 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. INDICE 1. Introduzione…………………………………………………………………………..5 2. Caratteristiche cliniche e inquadramento diagnostico della VKC………………...7 3. Patogenesi della cheratocongiuntivite primaverile……………………………….13 3.1 Fattori genetici……………………………………………………………………………….13 3.2 Fattori ormonali……………………………………………………………………………..14 3.3 Studio preliminare sul ruolo deglio romoni sessuali nella patogenesi della VKC…16 4. Fisiopatologia ……………………………………………………………………….19 4.1 Mediatori nella VKC ……………………………………………………………………….19 4.2 Cellule nella VKC……………………………………………………………………..…….21 5. Trattamento della cheratocongiuntivite primaverile…………………………….24 5.1 Studio multicentrico randomizzato controllato in doppio cieco per la valutazione dell’efficacia del trattamento con ciclosporina A in collirio nella prevenzione delle recidive dell’infiammazione nella VKC……………………………………………………………….27 5.2 Studio aggiuntivo per valutare l’efficacia della ciclosporina per il trattamento delle fasi acute della VKC………………………………………………………………………….31 5.3 Trattamento chirurgico della cheratocongiuntivite primaverile ………………..35 6. Conclusioni………………………………………………………………………….36 7. Bibliografia…………………………………………………………………………..37 8. Lavori allegati ……………………………………………………………………..48 8.1 Sacchetti M, Baiardini I, Lambiase A, Aronni S, Fassio O, Gramiccioni C, Bonini S, Bonini S.Development and testing of the quality of life in children with vernal keratoconjunctivitis questionnaire.Am J Ophthalmol. 2007 Oct;144(4):557-63. 8.2 Bonini S, Sacchetti M, Mantelli F, Lambiase A.(2007)Clinical grading of vernal keratoconjunctivitis.Curr Opin Allergy Clin Immunol;7(5):436-41. 3 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 8.3 Lambiase A, Normando EM, Vitiello L, Micera A, Sacchetti M, Perrella E, Racioppi L, Bonini S, Bonini S (2007): Natural killer cells in vernal keratoconjunctivitis. Mol Vis 13: 1562–1567. 8.4 Marta Sacchetti, Alessandro Lambiase, Flavio Mantelli,Velika Deligianni, Andrea Leonardi, Stefano Bonini. Tailored Approach to the Treatment of Vernal Keratoconjunctivitis. Ophthalmology IN PRESS 8.5 Iovieno A, Lambiase A, Sacchetti M, Stampachiacchiere B, Micera A, Bonini S.(2008) Preliminary evidence of the efficacy of probiotic eye-drop treatment in patients with vernal keratoconjunctivitis.Graefes Arch Clin Exp Ophthalmol.246(3):435-41. 4 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 1. Introduzione La cheratocongiuntivite primaverile (VKC) è una patologia allergica cronica che colpisce prevalentemente i bambini, caratterizzata da intensi sintomi oculari quali prurito, lacrimazione, fotofobia e arrossamento, associati ad infiammazione della congiuntiva bulbare e tarsale. (Bonini 2000) L’origine allergica di questa condizione è largamente accettata, ma l’eziologia e la patogenesi sono ancora scarsamente caratterizzate. Recentemente, sono stati condotti numerosi studi sulle cellule e i mediatori coinvolti nell’infiammazione che hanno evidenziato come la patogenesi della VKC è molto più complessa di una semplice reazione da ipersensibilità di tipo I, che caratterizza altre forme di congiuntivite allergica, più comuni e meno severe. Ad oggi però, il ruolo di una possibile predisposizione genetica e/o dei fattori ambientali coinvolti nell’insorgenza, nella progressione e nella risoluzione di tale condizione non sono stati ancora chiariti. Sebbene sia definita “primaverile” la VKC ha spesso un andamento cronico durante tutto l’anno con recrudescenze stagionali, in primavera ed in estate, caratterizzate da un peggioramento della sintomatologia ed un frequente interessamento corneale che va dalla cheratite puntata superficiale a vere e proprie ulcere corneali con compromissione della funzione visiva.(Leonardi 2002, Bonini 2000) Tale decorso, associato ad un’importante variabilità della severità della sintomatologia nel corso dell’anno, comportano una compromissione della qualità della vita del bambino con VKC e richiedono un importante impegno nella gestione e nell’approccio al paziente con VKC, volto ad un controllo ottimale dei sintomi e i segni sia durante l’anno, ma soprattutto nelle fasi di acuzie. I farmaci antiallergici topici disponibili per il trattamento della VKC si sono dimostrati efficaci nel controllo dei sintomi nelle forme lievi e moderate della malattia, laddove le fasi di acuzie dell’infiammazione, richiedono l’uso di steroidi topici, efficaci ma non scevri di effetti collaterali anche gravi e permanenti quali il glaucoma e la cataratta. C’è quindi lì esigenza di 5 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. valutare l’ efficacia di nuove terapie per il controllo della malattia, quali la Ciclosporina A in collirio, che consentano un buon controllo della malattia, e minori rischi di effetti collaterali. 6 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 2. Caratteristiche cliniche e inquadramento diagnostico della VKC La cheratocongiuntivite primaverile ha una maggiore prevalenza in zone di clima temperato, l’area del Meditarreneo, l’Africa centrale e occidentale, il Medio Oriente, il Giappone, il sucontinente Indiano e il Sud America. La VKC colpisce più frequentemente i bambini di sesso machile con un rapporto maschi/femmine di 4:1 to 2:1 in diversi studi, caratteristicamente, tale differenza non si mantiene nelle forme che si presentano in età adulta, in cui il rapporto maschi/femmine non mostra sostanziali differenze. (Neumann et al. 1959; Bonini et al. 2000, Bielory 2000, Lambiase 2009). La VKC insorge prima dei 10 anni, l’età media di insorgenza è tra 4 e 6 anni, e in genere risolve dopo la pubertà, presentando quindi un durata media tra 4 e 10 anni. (Bielory 2000; Leonardi 2002a). Nella sua forma tipica, la VKC si presenta con intensi sintomi oculari quali prurito, iperemia, fotofobia e lacrimazione. Le principali caratteristiche cliniche della VKC sono elencate in tabella (Bonini S 2000) 7 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Nella maggior parte dei pazienti (98%), la VKC è bilaterale, anche se piccole differenze di gravità tra i due occhi sono comuni (Bonini et al. 2000). L’andamento della malattia mostra spesso un esordio stagionale ma, nell’evoluzione, può diventare perenne dopo pochi anni. In circa un quarto dei pazienti con VKC la malattia è presente durante tutto l'anno, senza alcuna remissione, fin dall'esordio (Bonini et al. 2000). I pazienti con VKC presentano tipicamente diversi episodi di infiammazione attiva durante tutto l'anno, durante tali riacutizzazioni la sintomatologia si aggrava notevolmente a causa dell’infiammazione oculare che frequentemente si accompagna ad un interessamento corneale, caratterizzato da alterazioni epiteliali, quali epiteliopatia puntata, fino a vere e proprie ulcere corneali a scudo, formazioni di placche e neovascolarizzazione corneale. (Allansmith 1988; Buckley 1988). L’insorgenza di ulcera corneale è riportata nel 3-11% dei pazienti con VKC,e nel, 6% dei pazienti affetti da VKC causa una riduzione del visus dovuto esiti corneali. (Neumann et al. 1959; Cameron 1995; Tabbara 1999, Bonini 2000). Un’altra caratteristica della VKC è la secrezione di muco iper-denso, filamentoso e appiccicoso, che provoca intenso fastidio e aggrava la sintomatologia. Durante le riacutizzazioni, si assiste anche alla comparsa di punti di trantas al limbus, depositi di eosinofili degradati e detriti di cellule epiteliali.(Figura 1) 8 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Figura 1. Durante le riacutizzazioni dell’infiammazione della VKC si osserva un frequente intereassamento corneale con puntata superficiale (A) o vere e proprie ulcere conrneali (B), associate a inetnsa iperemia congiuntivale (C), papille congeste (C e F), punti di trantas (D) e inensa secresione di muco iperdenso (E, F) 9 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. La reazione papillare congiuntivale è un’altra caratterista della VKC, ma tende a permanere anche nelle fasi di quiescenza. Le papille nella VKC sono tipicamente di grandi dimensioni (> 1 mm), più frequentemente localizzate al tarso superiore, altre volte possono interessare il limbus. (Figura 2) Figura 2. Reazione papillare limbare moderata (A) e severa (B) e reazione papillare tarsale superiore mederata 2+( C) e severa 3+ (D). In base alla localizzazione delle papille, la VKC viene classificata in tarsale, limbare o mista (Verin et al. 1999). Nelle fasi di riacutizzazione, le papille diventano congeste, iperemiche e intervallate a muco rappreso. Bonini et al. (2000) ha classificato le papille tarsali superiori come segue: (1) Grado 0: nessuna reazione papillare. (2) Grado 1 +: papille rare, di 0,2 millimetri, sparse nella congiuntiva tarsale o intorno al limbus. (3) Grado 2 +: papille di 0,3-1 millimetri sulla congiuntiva tarsale o al limbus. (4) Grado 3 +: papille di 1-3 mm tutte le sulla congiuntiva tarsale o per 360° intorno al limbus. 10 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. (5) Grado 4 +: papille di più 3 millimetri sulla congiuntiva tarsale o formazione gelatinosa al limbus che coinvolge la cornea periferica. La dimensione delle papille correla direttamente con la persistenza e il peggioramento dei sintomi nel follow-up a lungo termine (Bonini et al. 2000). (Figura 3) Figura 3. Curva di sopravvivenza dei pazienti con VKC in base al grado di severità dlle papille tarsali (Bonini S 2000) La sintomatologia oculare, e soprattutto il prurito e la fotofobia, possono variare da lieve a intensi ed essere aggravati da l'esposizione al vento, polvere, luce intensa, caldo o lo sforzo fisico associato con la sudorazione. Tale esagerata iper-reattività a stimoli non-specifici come il calore, sole e del vento durante la fase attiva della VKC ha suggerito il coinvolgimento dell’infiammazione neurogenica nella patogenesi della VKC (Bonini, 1992a). L’intensa sintomatologia, la forte variabilità di manifestazioni cliniche della VKC, e il fatto che la VKC colpisce prevalentemente i bambini nell’età evolutiva, ha spinto il nostro gruppo a indagare l’impatto della VKC sulla qualità della vita. Poiché non esistevano strumenti specifici per valutare la qualità della vita nella VKC, un questionario specifico per bambini con la cheratocongiuntivite primaverile (il QUICK© test ), è stato creato e validato su una popolazione di bambini con VKC da 5 a 12 anni. (Sacchetti M 2007-lavoro allegato) 11 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Non ci sono precisi criteri diagnostici per questa malattia. Iperemia, prurito, fotofobia, lacrimazione e secrezione mucosa sono sintomi tipici della VKC, ma non specifici, in quanto sono comuni anche ad altre malattie della superficie oculare quali la cheratocongiuntivite atopica, le blefarocongiuntiviti, l’occhio secco. Le papille giganti sulla congiuntiva tarsale superiore e al limbus sono caratteristiche tipiche della VKC. La diagnosi si basa quindi sulle caratteristiche cliniche, l’età, la familiarità,l’andamento della malattia, e sulla presenza dei tipici segni e sintomi clinici, con l’inevitabile conseguenza che i casi lievi o atipici possono sfuggire alla diagnosi. Nonostante la disponibilità di nuove conoscenze sulla patogenesi della VKC, nessun test clinico o marker diagnostico di laboratorio è stato sviluppato per supportare la diagnosi nei casi atipici o per predire il corso di questa malattia. (Bonini et al. 2000) La mancanza di criteri diagnostici standard e di un linguaggio comune tra gli oculisti per quanto riguarda la gravità della VKC rende più difficile diagnosticare e trattare questa malattia. Bonini et al. (2007) ha proposto una classificazione clinica delle fasi cliniche della VKC per aiutare i medici ad utilizzare un linguaggio comune nella diagnosi e nella gestione della VKC e per consentire una più omogenea la selezione dei pazienti per sperimentazioni cliniche. (Bonini 2007-lavoro allegato) 12 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 3. Patogenesi della cheratocongiuntivite primaverile 3.1 Fattori genetici La caratteristica distribuzione geografica, e la persistenza di incidenza della VKC nelle popolazioni emigrate suggerisce la possibilità di una predisposizione genetica alla base della patogenesi della VKC. A sostegno di tale ipotesi, uno studio ha dimostrato che la VKC è più comune tra individui di origine asiatica e africana che vivono in Svezia (Montan et al. 1999). Finora, però, nessun fattore genetico predisponente è stato identificato perla VKC. Inoltre, la costante e maggiore presenza di eosinofili nel sangue, lacrime e nel raschiato congiuntivale, l'espressione di una moltitudine di mediatori e citochine, così come la predominanza delle cellule CD4 a livello locale nella VKC suggeriscono che tale malattia potrebbe appartenere ad un fenotipo di upregulation del cluster di geni di citochine sul cromosoma 5q. Il cluster di geni di citochine, attraverso i suoi prodotti come la interleuchina (IL) -3, -4, -5 e il GM-CSF, regola la prevalenza di cellule T helper di tipo 2 (Th2), la crescita e la funzione dei mastociti e degli eosinofili così come la produzione di immunoglobuline (Ig) E nella VKC (Bonini et al.1995a). La presenza di familiarità per malattie allergiche come asma, rinite, eczema, orticaria e è stata riportata nel 49% dei pazienti affetti da VKC (Bonini et al. 2000) suggerendo una predisposizione allo sviluppo di malattie atopiche in questi pazienti. Inoltre, un terzo dei pazienti con VKC presenta malattie atopiche associate (Bonini et al. 2000, Tuft et al.1989), tra cui la più comune è l’asma. Anche il cheratocono, altra condizione ad ereditarietà multifattoriale, sembra avere una maggiore incidenza nei bambini con VKC (circa 15%) (Iqbal et al. 2003). 13 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 3.2 Fattori ormonali La predilezione per il sesso maschile e la risoluzione dopo la pubertà suggeriscono inoltre un ruolo degli ormoni sessuali nello sviluppo della VKC. Uno studio di Bonini del 1995 evidenziava un aumento significativo nell’espressione dei recettori degli estrogeni e del progesterone nella congiuntiva di bambini con VKC, rispetto ai controlli sani. (Bonini et al. 1995b) L’influenza degli ormoni androgeni ed estrogeni sul sistema immunitario è stata evidenziata da numerosi studi epidemiologici, clinici e sperimentali. Infatti, è stata evidenziata una diversa prevalenza delle malattie allergiche tra i sessi. In generale i bambini sono più frequentemente affetti da eczema atopico, asma e congiuntivite allergica rispetto alle bambine, e tale rapporto si inverte dopo la pubertà, con maggiore frequenza di malattie atopiche nelle donne rispetto agli uomini.(W Chen 2008) È stato dimostrato che gli estrogeni sono in grado di stimolare la sintesi di anticorpi potenziando l’immunità umorale. Inoltre le cellule mastocitarie esprimono in Recettore alfa per gli estrogeni, che stimola la degranulazione mastocitaria. (Cutolo M 2004, Narita S 2007). Il progesterone sembra essere in grado di potenziare la produzione di IgE, ma studi dimostrano anche la sua azione di soppressione del rilascio di istamina. (Mitchell VF 207, Vasiadi M 2006) L’azione degli androgeni nelle patologie atopiche è ancora poco chiara. Il pregnenolone, il deidroepiandrosterone (DHEA) e il DHEA-solfato (DHEAS), causano rapida degranulazione mastocitaria in vitro. (Malkin CJ 2003). Studi in modelli animali di allergia hanno evidenziato che il DHEA è in grado di sopprimere l’ infiammazione allergica delle vie aeree suggerendo che l’effetto soppressivo del DHEA sia associato ad una downregulation della risposta Th2 (Yu CK 2002). È stato inoltre dimostrato che la somministrazione di DHEA sopprime l’aumento delle IgE del siero e dei livelli di IL-6 in un modello animale di eczema atopico. (Sudo N 2001). In un altro studio, i livelli di DHEA sierici in pazienti adulti di sesso maschile (di età compresa tra 1930 anni) con eczema atopico sono risultati significativamente inferiori a quelli dei controlli sani 14 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. di sesso maschile della stessa età. (Tabata N 1997) Tuttavia, poiché non sono noti i recettori ormonali specifici per il DHEA, resta da stabilire se l'effetto del DHEA sia mediato attraverso la sua maggiore conversione in testosterone e in estradiolo negli uomini rispetto alle donne. Il testosterone sembrerebbe avere un effetto inibitorio della rispostaallergica, infatti, non influenza il rilascio di istamina in colture cellulari di mastociti in vitro (Pitton C 1988) e, in un modello animale di rinite allergica, il testosterone ha evidenziato un effetto inibitorio nella produzione di IgE. In vivo, non sono state dimostrate differenze di genere tra i livelli di testosterone e i risultati dei test cutanei positivi o la presenza di malattie atopiche.(Uekert 2006) Androgeni ed estrogeni esercitano inoltre una significativa influenza sulla salute e il benessere della superficie oculare e degli annessi oculari. Gli steroidi sessuali agiscono modulando molteplici aspetti anatomici e fisiologici della ghiandola lacrimale e del Meibomio,di congiuntiva cornea. Si ritiene che gli steroidi sessuali siano coinvolti nello sviluppo e/o la progressione di molte patologie del segmento anteriore, come l’occhio secco, intolleranza alle lenti a contatto, le cheratocongiuntiviti allergiche, la neoangiogenesi corneale, malattie autoimmuni. (Sullivan D 2004) È opinione ormai accettata che il meccanismo d’azione degli steroidi sessuali a livello oculare sia dovuto prevalentemente alla formazione locale -intracrina- di androgeni e estrogeni da precursori ghiandola surrenale, quali il DHEA, il DHEA-S e l’androstenedione. Infatti, gli esseri umani ei primati sono gli unici in possesso di ghiandole surrenali, che secernono DHEA e DHEA-S, che vengono poi convertiti in potenti androgeni, quali, il testosterone e il diidrotestosterone (DHT) ed estrogeni dagli enzimi della steroidogenesi a livello periferico. (Labrie F 1995) Questo processo consente agli organi bersaglio di aggiustare la sintesi e il metabolismo degli steroidi sessuali in base alla necessità locale. (Figura 4) 15 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Figura 4 Enzimi steroidogenici unami nei tessuti periferici intracrini. (Labrie 1998) Un recente lavoro ha evidenziato che le cellule della superficie oculare e degli annessi oculari esprimono l'RNA messaggero degli enzimi necessari alla sintesi e il metabolismo degli steroidi sessuali e dei recettori degli androgeni evidenziando come i tessuti oculari siano bersaglio dell’azione degli androgeni.(Schirra F. 2006, Rocha EM 2000) 3.3 Studio preliminare sul ruolo degli ormoni sessuali nella patogenesi della VKC I livelli sierici dei principali ormoni sessuali (LH, FSH, E1, E2, progesterone, DHEA, DHEA-S, testosterone totale, testosterone libero, DHT, SHBG) sono stati valutati in 14 bambini pre-puberi (maschi, età <11 anni, LH<1 UI/l, Testosterone <200 ng/100 ml) con VKC (età media 7,3±2,5 anni) e paragonati ad un gruppo di controllo (N=74, età media 8,3±1,8anni) con le stesse caratteristiche di età e sesso (maschi, età <11 anni, LH<1 UI/l, Testosterone <200 ng/100 ml). I nostri dati preliminari mostrano un aumento significativo dei livelli sierici di DHEA-S (p=0,002), Estrone (p=0,005) e Testosterone libero (p=0,001) e una riduzione significativa di DHT (p=0.001) e della sex hormones binding globulin (SHBH) (p=0,004) rispetto ai controlli. (Fugura 5) 16 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Tali risultati evidenziano una disregolazione degli ormoni sessuali nei bambini con VKC, con una riduzione del DHT, il maggiore ormone androgeno attivo in età pre-pubere, che potrebbe contribuire alla patogenesi della malattia. Figura 5. I bambini con VKC mostrano un aumento significativo dei livelli sierici di DHEA-S, Estrone e Testosterone libero e una riduzione significativa di DHT e SHBH rispetto ai controlli. Inoltre, dati preliminari derivanti dal microarray effettuato su citologie ad impressione congiuntivale in bambini con VKC (n=3), mostrano una alterazione anche a livello locale 17 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. dell’espressione della SHBG, che risulta ridotta a livello congiuntivale, rispetto ai controlli sani (n=3). Mostrano inoltre un aumento dell’espressione del 17-beta-steroido-deidrogenasi 2 (17BHSD-2) e una riduzione dell’espressione della 5-alfa-reduttasi 3, che partecipa al metabolismo del DHT. Tali dati, se confermati dalla real-time PCR, mostrerebbero un impairment del tono androgenico a livello locale nelle VKC. 18 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 4. Fisiopatologia Nonostante la familiarità per atopia, i livelli sierici di IgE totali e specifiche, il numero più elevato di mastociti ed eosinofili, e la risposta favorevole alla terapia anti-allergica - tutte caratteristiche delle malattie allergiche - siano state osservate anche nella VKC (Bielory 1992; Abelson 1993), la patogenesi di questa malattia sicuramente molto più complessa di una semplice reazione di ipersisensibilitàdi tipo I. infatti, circa il50% dei pazienti con VKC presenta negatività ai test allergometrici cutanei (Bonini et al. 2000). La forte abbondanza di citochine Th2, l'aumentata espressione dei loro recettori e la scarsità di cellule T helper di tipo 1 (Th1) nelle lacrime e nel siero di pazienti con VKC confermano il ruolo cruciale svolto dalla risposta Th2 nell'insorgenza e la perpetuazione dell’ infiammazione osservata nella VKC. Inoltre, il sistema immunitario, sistema nervoso ed endocrino sembrano interagire uno con l'altro nella patogenesi della VKC (Bonini et al. 2004). 4.1 Mediatori nella VKC Citochine e chemochine Le cellule T helper (CD4), i mastociti e gli eosinofili che infiltrano la congiuntiva nella VKC sono le principali fonti di citochine in questa malattia. In particolare, le cellule T isolate a livello congiuntivale nella VKC mostrano un profilo Th2 (Calder et al. 1999). I livelli di citochine Th2, vale a dire IL-4 e IL-5, sono stati trovati elevati nei pazienti VKC (Calder et al. 1999; Leonardi et al. 1999a). Inoltre, un aumento dell’ espressione di mRNA per le citochine di tipo Th2 è stata osservata a livello tissutale delle VKC (Metz et al. 1997). I livelli sierici di IL-4 e i livelli lacrimali di IL-4 e IL -5 sono più elevati nei pazienti con VKC rispetto ai controlli. È interessante notare che, IL-2, interferone (IFN)-gamma e fattore di necrosi tumorale (TNF)-beta, le principali citochine secrete da Th1, non sono aumentate nella VKC (Leonardi, 2002°, Bonini 2003). Questi risultati confermano che VKC ha principalmente un profilo di tipo Th2 (Uchio et 19 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. al.2000; Fujishima et al. 2002). Le chemochine (CC), sono potenti attivatori e chemioattrattanti, sono prodotte non solo dalle cellule infiammatorie, ma anche da cellule epiteliali attivate, fibroblasti e cellule endoteliali vascolari nella congiuntiva. Le chemochine sono coinvolti nel normale traffico e reclutamento dei leucociti durante la risposta infiammatoria. Le chemochine sono raggruppate in sottofamiglie dette CXC, CC, C e CX3C (Abu El-Asrar et al. 2001a). Elevati livelli di eotassina sono stati trovati nel muco dai pazienti VKC, inoltre, i livelli eotassina correlavano significativamente con la percentuale di eosinofili nelle lacrime questo suggerisce che l’eotassina possa essere responsabile del reclutamento degli eosinofili nella VKC (Leonardi et al. 2003a). L’eotassina, associata a MCP e RANTES, sono altamente espressi nei tessuti limbari e possono essere responsabili della massiccia infiltrazione degli eosinofili nella VKC. L’IL-8 è attivamente secreta dai macrofagi e dalle cellule epiteliali nella VKC è un chemoattrattante e un attivatore di polimorfonucleati. Essa svolge un ruolo cruciale nella la migrazione delle cellule infiammatorie, quali polimorfonucleati ed eosinofili (Miyoshi 2001).Il recettore delle chemiochine (CXCR) -3 è anche notevolmente aumentato ed espresso abbondantemente sui linfociti T nella congiuntiva dei pazienti VKC attiva, suggerendo un ruolo nella regolazione del reclutamento di linfociti nella congiuntiva dei pazienti con VKC (Abu ElAsrar et al. 2001a). Mediatori infiammatori L’Istamina è un importante mediatore infiammatorio nelle congiuntivit allergiche, rilasciata da mastociti e basofili attivati. La concentrazione lacrimale di istamina è aumentata nei pazienti con VKC rispetto ai controlli sani (Abelson et al. 1980). Inoltre, i pazienti con VKC dimostrano una iper-reattività congiuntivale non-specifica se provocati con test di provocazione congiuntivale con istamina (Bonini et al. 1992a). Anche le Metalloproteinasi (MMP) e gli inibitori delle MMP (TIMP) sono coinvolti nella patogenesi della VKC, i livelli lacrimali di pro-MMP-1 e pro-MMP-9 sono 20 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. significativamente aumentati nei pazienti con VKC rispetto ai soggetti di controllo. L’attività di MMP-9 è correlata in modo significativo con il coinvolgimento della cornea e la formazione delle papille giganti (Leonardi et al. 2003b,c). Fattori di crescita Diversi fattori di crescita, come il fattore di crescita epidermica(EGF), il fattore di crescita dei fibroblasti (FGF), il Trasformingrowth factor beta-1 (TGFb-1) sono stati aumentati nella VKC. Questi fattori inducono la crescita dei fibroblasti e la produzione di procollagene (Leonardi et al. 1998). Recentemente, i recettori per il fattore di crescita nervoso (NGF) sono stati dimostrati nella congiuntiva dei pazienti con VKC attiva. Alti livelli plasmatici del NGF sono stati descritti nella VKC in correlazione con il numero di mastociti congiuntivali, suggerendo che i fattori neurali possono avere un ruolo nella patogenesi della VKC (Lambiase et al. 1995). È interessante notare che, la sostanza P - un neuropeptide con ben nota attività su cellule del sistema immunitario -è aumentata nel plasma e nelle lacrime dei pazienti con VKC (Fujishima et al. 1997; Lambiase et al. 1997). 4.2 Cellule nella VKC I mastociti, le cellule T, gli eosinofili e i macrofagi sono coinvolti nella patogenesi della VKC. I mastociti sono una caratteristica costante nei tessuti congiuntivali della VKC. Il notevolmente aumento del numero dei mastociti trovati in campioni di papille giganti tarsali suggerisce un ruolo attivo di queste cellule nell’anomalo metabolismo del tessuto connettivo osservato nella VKC. Queste cellule esprimono Fc [epsilon] RI sulla loro superficie cellulare, che consente loro di legare le IgE (Church 1997). Il legame con le IgE specifiche per allergeni risultati nel rilascio di mediatori pro-infiammatori, quali istamina, proteasi, prostaglandine D2e leucotrieni C4. Questi mediatori sono responsabili del prurito, iperemia, lacrimazione e chemosi nelle congiuntiviti allergiche (Church 1997). I mastociti liberano anche citochine, tra cui IL-4. Le citochine liberate dai mastociti sono responsabili dell'avvio dell’ infiammazione allergica, con 21 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. conseguente infiltrazione di eosinofili. IL-4 svolge un ruolo chiave dell'infiammazione allergica attraverso l’attivazione e la crescita delle cellule T, l'induzione della produzione di IgE dalle cellule B, l'aumento di molecole di adesione e la modulazione della differenziazione di tipo Th2, che è essenziale per la reazione allergica. (Fukagawa et al. 2000). Triptasi e chimasi, indicatori dell'attivazione dei mastociti, sono aumentati nelle lacrime delle VKC e sono stati proposti come markers di severità della VKC (Tabbara 2001). Gli eosinofili sono una caratteristica costante nelle lacrimale e nella citologia congiuntivale delle VKC. Infatti, circa il 50-90% delle cellule nelle lacrime durante la fase attiva della VKC sono eosinofili (Leonardi, 2002a). Gli eosinofili, insieme con i mastociti, sono le principali cellule effettrici dell’infiammazione oculare nella VKC e risultano aumentati in modo significativo nelle lacrime, nella circolazione periferica e nei tessuti congiuntivale provenienti da pazienti con VKC. (Trocmé et al.1989, 1993; Leonardi et al. 1995). Gli eosinofili attivati rilasciano citochine, chemochine, leucotrieni e proteine come MBP, ECP, la per ossidasi eosinofila (EPO) e la proteina eosinofila X / neurotossina (EPX) (Tomassini et al. 1994; Leonardi 2002a). I livelli sierici e lacrimali di ECP e EPX sono più alti in pazienti VKC rispetto ai soggetti normali (Bonini et al. 1992a). I livelli di ECP lacrimale correlano positivamente con segni clinici e sintomi e si riducono dopo trattamento con desametasone o ciclosporina (Leonardi et al. 1995). Anche il numero di linfociti T è aumentato nella congiuntiva dei pazienti con VKC. L'attivazione di questi linfociti sembra giocare un ruolo vitale nella patogenesi della VKC. I cloni di cellule T derivate da tessuti di VKC sono principalmente di tipo Th2 (Leonardi et al. 1999a). I linfociti Th2, in virtù del loro profilo di citochine, sono responsabili dell’aumento della produzione di IgE, del reclutamento e l'attivazione di mastocic e eosinofili (Umetsu 1997; Bielory et al. 2002a) Un nostro lavoro, ha recentemente dimostrato un aumento delle cellule natural killer nella congiuntiva dei pazienti con VKC, suggerendo un ruolo di queste cellule e dell’ immunità 22 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. innata nella VKC. (Lambiase 2007-lavoro allegato, Bonini 2005) Cellule epiteliali E 'stato dimostrato che le cellule epiteliali congiuntivali non costuiscono solo una barriera meccanica, ma partecipano all’infiammazione esprimendo antigeni di superficie come ad esempio le molecole di adesione (ICAM-1, VCAM-1 e HLADR) e rilasciando citochine (eotassina, IL-8, IL-6, RANTES). È stato ipotizzato che l’istamina rilasciata dai mastociti congiuntivali, potrebbe stimolare la sintesi di IL-6 e IL-8 dalla cellule epiteliali congiuntivali e contribuire ad amplificare la risposta allergica (Irkec 2003). Anche i fibroblasti di cornea e congiuntiva contribuiscono alla induzione e amplificazione dell’ infiammazione allergica oculare così come al rimodellamento del tessuto. TGFb-1, IL-1 e le citochine prodotte dalle cellule Th2 inducono la produzione di VEGF da parte dei fibroblasti congiuntivali, che possono svolgere un ruolo cruciale nella neovascolarizzazione e nella formazione delle papille giganti (Asano-Kato et al. 2005). 23 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 5. Trattamento della cheratocongiuntivite primaverile Poiché la VKC è una malattia cronica, e recidivante la gestione del paziente è piuttosto impegnativa. L'esposizione a stimoli non-specifici come sole, vento e acqua può provocare arrossamento congiuntivale frequenti tra i pazienti VKC, evitare i fattori scatenanti, è complicato e comunque non sufficiente per controllare i sintomi. L’applicazione di impacchi freddi e l'uso di lacrime artificiali hanno dimostrato di essere efficaci nel sollievo dei sintomi (Bielory 2002b). La varietà dei farmaci attualmente disponibili per il trattamento della VKC include l’uso di antistaminici, stabilizzatori mastocitari, agenti multiazione, corticosteroidi e immunomodulatori ma, a tutt’oggi, non ci sono terapie in grado di agire sui molteplici aspetti della fisiopatologia della VKC. Infatti, la maggior parte dei farmaci utilizzati sono palliativi e non specifici volti a trattare i sintomi o a deprimere la risposta immunitaria in toto senza agire sulla complessa risposta immunitaria, per cui quando la terapia viene interrotta vi è spesso una recidiva della malattia. Perché ci sono pochi studi randomizzati e controllati la selezione di un farmaco tra le molte opzioni disponibili è per lo più sulla base dell'esperienza personale e la preferenza del medico curante. Una meta-analisi di studi randomizzati controllati ha infatti evidenziato come ci siano scarse evidenze per raccomandare uno specifico farmaco, e come ci sia anche una mancanza di criteri standardizzati di trattamento basati sulla diagnosi e la stadiazione della severità. (Mantelli 2007) A tale scopo, ci siamo proposti di creare una stadiazione della severità e dell’attività della VKC utilizzando un metodo di analisi dei dati che ci consentisse di ricavare un albero decisionale, di semplice e immediata interpretazione nalla pratica clinica. Per far ciò, uno studio retrospettivo su 207 casi di VKC è stato effettuato, i dati sono stati analizzati con il metodo CART®, al fine di ricavare un algoritmo decisionale, di proporre una stadiazione della severità e 24 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. individuare i fattori di rischio per un peggiore esito visivo (Sacchetti M 2010-IN PRESS-lavoro allegato). I colliri contenenti antiistaminici vengono spesso utilizzati dai pazienti durante a primi stadi della malattia, come trattamento di prima linea. Questi sono in grado di alleviare il prurito. Gli antistaminici H1- selettivi, qauli emedastina e levocabastina, sono efficaci nel controllo dei segni e sintomi della VKC live e moderata. (Bielory et al. 2005). Gli stabilizzatori di membrana inibiscono la degranulazione dei mastociti che è un evento centrale nella VKC. I mediatori rilasciati dai mastociti, infatti, sono responsabili di molti sintomi e segni associati VKC. Tali farmaci sono quindi in grado di alleviare i sintomi acuti della malattia attiva e anche di ridurre lo stimolo per lo sviluppo dell’ infiammazione allergica cronica (Church 2002). L'efficacia del sodio cromoglicato (QID), lodoxamide (qid), Nedocromil (bid) e pemirolast (QID) nel controllo dei sintomi e nella prevenzione dell’esacerbazione è stato dimostrato da molti studi (Tabbara 1977; Bonini et al. 1992b). Questi farmaci dovrebbero essere usati come trattamento di prima scelta al momento della comparsa delle manifestazioni allergiche stagionali e dovrebbe essere usato continuo per tutta la stagione. Gli antistaminici sistemici possono essere una buona scelta quando allergia coinvolge gli occhi, il naso o faringe simultaneamente. Non trovano invece indicazione quando l’allergia è limitata agli occhi, il trattamento con farmaci antistaminici topici è efficace e privo di effetti indesiderati. Recentemente, una nuova generazione di farmaci antiistaminici multiazione,come l’Olopatadina, l’Epinastine, il Ketotifene e l’Azelastina hanno dimostrato la duplice azione di stabilizzatori di membrana e antagonismo del recettore H1. L'azione di questi farmaci non è limitata ai mastociti, al blocco dei recettori H1, essi esercitano anche un’azione antiinfiammatoria attraverso diversi meccanismi quali l’inibizione del rilascio di TNF-alfa e di varie citochine da parte dell’epitelialio congiuntivale e delle cellule infiammatorie, in modo da controllare l’infiammazione in modo più efficace rispetto ai soli antiistaminici (Lambiase 2009). 25 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. L’uso degli anti-infiammatori non steroide (FANS) in collirio, quali il ketorolac e il diclofenac hanno dimostrato di diminuire prurito oculare e congiuntivale e l’iperemia associata alle congiuntiviti allergiche. (D'Angelo et al. 2003, Kosrirukvongs 2004). I corticosteroidi topici sono i farmaci più efficaci farmaci per il controllo dei segni e sintomi della VKC. A causa delle complicazioni associate al loro uso prolungato, questi non dovrebbero essere prescritta come trattamento di prima linea. Infatti, il trattamento prolungato con corticosteroidi può causare effetti collaterali gravi e permanenti quali cataratta e glaucoma e aumentare la suscettibilità alle infezioni virali e da funghi. (Tabbara 1995, Bonini 2000) La Ciclosporina A topica, è stata proposta per il trattamento della VKC per ridurre i segni e i sintomi della VKC in numero studi clinici in aperto. La ciclosporina blocca la proliferazione dei linfociti Th2 e la produzione di IL-2. Inoltre, inibisce il rilascio di istamina attraverso una riduzione della produzione di IL-5 (Ben - Ezra et al. 1988; Secchi et al. 1990). Ben Ezra et al. (1986) ha descritto l’uso di ciclosporina collirio 2% per il trattamento di gravi VKC quasi due decenni fa, riportando efficacia nel sollievo dei sintomi soggettivi nell’ 86% dei pazienti trattati. Un altro studio, il trattamento topico con Ciclosporina (0,5-2%) in emulsione di olio d'oliva o di ricino, instillata quattro volte al giorno, ha dimostrato essere efficace nel trattamento della VKC. (Abu El-Asrar et al. 1996). Alcuni studi in doppio cieco, controllati con placebo hanno dimostrato che la ciclosporina (2%) in collirio è efficace e sicura nel trattamento delle VKC severe (Bleik JH 1991, Pucci et al. 2002; Kilic 2006). In uno studio prospettico in aperto, su 10 casi VKC severa, il trattamento con ciclosporina topica (2%) ha ridotto significativamente gli score dei segni clinici e dei sintomi. Dopo il trattamento è stata inoltre osservata la riduzione della popolazione di cellule CD4 e di CD28 congiuntivali. (Avudunk et al. 2001). La Ciclosporina a concentrazioni inferiori 0,05% e 1% è stata anche utilizzata nel trattamento delle VKC, ma le evidenze disponibili sono ancora scarse per trarre conclusioni sull’efficacia. (Kosrirukvongs 2004, Spadavecchia et al. 2006). 26 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 5.1 Studio multicentrico randomizzato controllato in doppio cieco per la valutazione dell’efficacia del trattamento con ciclosporina A in collrio nella prevenzione delle recidive dell’infiammazione nella VKC Uno studio della durata di 2 anni, in doppio cieco, multicentrico, randomizzato, controllato con cross-over è stato effettuato presso il nostro centro per valutare l’efficacia del trattamento con Ciclosporina nella prevenzione delle recidive della VKC e nel trattamento delle fasi acute della malattia. L’obiettivo primario studio era valutare la differenza nel numero di recidive di infiammazione oculare tra i gruppi di pazienti trattati con ciclosporina e ketotifene. Inoltre, le differenze di sintomi e segni tra i due gruppi sono stati valutati ad ogni visita. Metodi: Trentaquattro pazienti con VKC (30M e 4F, età media 14±7anni)sono stati inclusi nello studio e trattati in doppio cieco con ciclosporina senza conservanti 0,05% in emulsione cationica (N=16) o ketotifene 0,025% soluzione oftalmica (N=18) due volte al giorno per 6 mesi da marzo a ottobre. Nel secondo anno di studio, i pazienti sono stati inclusi per sei mesi (da Marzo a Ottobre) in cross-over: i pazienti precedentemente trattati con ciclosporina hanno ricevuto ketotifene (N=15) e i pazienti precedentemente trattati con ketotifene hanno ricevuto ciclosporina (N=15). I pazienti sono stati valutati con un esame oculare completo, i segni (iperemia, secrezione, del tarso e / o papille limbal, Trantas punti, congiuntivale fibrosi, cheratite puntata superficiale, ulcera corneale e vasi della cornea) e i sintomi (prurito, fotofobia, arrossamento, lacrimazione, secrezione e la riduzione della visione) sono stati valutati e un punteggio da 0 a 3 è stato assegnato (0 = assente, 1 = lieve, 2 = moderata, 3 = grave) al basale, dopo 12 settimane di trattamento, dopo 3 mesi e alla fine del trattamento (6 mesi). Ulteriori visite sono state effettuate in caso di recidive della malattia durante il periodo dello studio. L’Oxford score è stato anche valutato ad ogni visita. 27 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Il test Anova e la regressione lineare sono stati effettuati per valutare la differenza tra il numero di recidive nei gruppi trattati la ciclosporina (CsA) vs ketotifene. La curva di sopravvivenza di Kaplan-Meier e le stime di rischio sono stati eseguiti per valutare le differenze nell’obiettivo primario (numero di recidive VKC). Il modello lineare generalizzato per misure ripetute è stato utilizzato per valutare le differenze di segni e sintomi tra i due gruppi. Risultati: Nel primo anno dello studio, tre pazienti hanno abbandonato lo studio, a causa di una reazione avversa (n=1, trattata con CsA),e di inefficacia della terapia con scarso controllo dell'infiammazione (n=2 trattati con ketotifene). Nel secondo anno di studio, i pazienti sono stati trattati in cross-over. In totale 30 pazienti (un paziente ha abbandonato lo studio prima di iniziare il trattamento per mancanza di compliance) sono stati trattatti CSA-(n = 15) o Ketotifene (n = 15). Durante il secondo anno dello studio, 5 pazienti sono usciti dallo studio per inefficacia del trattamento (4 nel grppo trattato con ketotifenee 1 nel gruppo trattato con CsA). Considerando tutti i pazienti abbiamo osservato in tutto 49 esacerbazioni, 15 in pazienti trattati con CsA e 34 in pazienti trattati con ketotifene. L'analisi statistica ha mostrato una differenza statisticamente significativa nel numero di riacutizzazioni della VKC tra i due gruppi di trattamento, indicando che i pazienti trattati con CsA avevano un numero significativamente inferiore di riacutizzazioni rispetto ai pazienti trattati con ketotifene (p <0,05). Inoltre, di un minor numero di pazienti ha sviluppato recidive (10 nel gruppo ciclosporina vs 18 nel gruppo Ketotitifene) e un minor numero di pazienti con ha sviluppato più di una recidiva (9% nel gruppo ciclosporina vs 33% ketotifene). I pazienti trattati con ketotifene hanno mostrato un rischio di recidiva 2,4 volte superiore rispetto ai pazienti trattati con CsA. Infine, il trattamento con CsA ha garantito un più lungo intervallo libero da malattia. (Figura 6) 28 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Figura 6.Il trattamento con CsA ha consentito di ridurre significativamente il numero di riacutizzazioni rispetto al trattamento con ketotifene (A, B). Il pazienti trattati con ketotifene hanno mostrato un rischio di recidiva 2,4 volte superiore rispetto alla CsA (C). Il trattamento con CsA ha garantito un più lungo intervallo libero da malattia. (D) Inoltre, l'analisi statistica ha evidenziato che il trattamento con ciclosporina A 0,05% è più efficace del trattamento con ketotifene nel controllare tutti i sintomi valutati: prurito (p = 0.003), fotofobia (p = 0.023), secrezione (p = 0.002), lacrimazione (p = 0.045), arrossamento (p = 0,016)e score totalae die sintomi (TSYS) (p = 0,045) e solo uno dei segni valutati (secrezione mucosa; p = 0,041). (Figura 7,8) 29 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Figura 7. Il trattamento con ciclosporina A 0,05% è più efficace del trattamento con ketotifene nel controllare tutti i sintomi valutati: prurito (A), fotofobia (B), secrezione (C), lacrimazione (D), arrossamento (E)e score totalae dei sintomi (F). 30 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Figura 8. La Ciclosporina A in collirio è risultata più efficace nel controllo dei segni come la secrezione mucosa (A) rispetto al Ketotifene in collirio, ma non degli altri segni valutati: iperemia (B), papille limbari e tarsali (C, D), total score dei segni(E) e Oxford score (F). Nessuna differenza significativa è stata osservata in dell'acuità visiva e la pressione intraoculare tra i due gruppi di pazienti 5.2 Studio aggiuntivo per valutare l’efficacia della ciclosporina per il trattamento delle fasi acute della VKC. In caso di recidiva di VKC i pazienti sono stati inclusi in questo studio aggiuntivo per valutare l'efficacia della ciclosporina 1% vs il trattamento standard, con steroide topico (Etacortilen ® 0,15%, Sifi) per il trattamento delle infiammazioni allergiche acute. 31 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. L'obiettivo di questo studio era di confrontare gli effetti clinici della ciclosporina vs trattamento con steroidi. Metodi: Questo studio è stato condotto in doppio cieco, randomizzato e controllato. Quando una ricaduta di VKC è stato confermato dal medico (con un incremento di almeno il 100% della somma di iperemia, prurito e punteggi lesione corneale rispetto al basale dei valori), tutti i segni e sintomi clinici sono stati registrati. A seguito di randomizzazione (1:1), i pazienti hanno ricevuto ciclosporina 1% o steroide topico quattro volte al dì per 7 giorni. Una volta che raggiunta la remissione della fase acuta, il trattamento è stato sospeso e tutti i segni e sintomi clinici sono stati registrati. La remissione è stata definita come riduzione di almeno il 100% della somma di iperemia e prurito e punteggi lesione corneale rispetto ai valori di fase acuta. Se la remissione non è stata ottenuta dopo 7 giorni di trattamento, i paziente sono stati considerati "non responder" e trattati con steroidi topici 4 volte al giorno per 7 giorni (rescue medication). Dopo la remissione della fase acuta, i pazienti, ancora una volta iniziato il trattamento precedente (0,05% ciclosporina o fumarato ketotifene 0,025%). Il Test Chi quadrato è stato eseguito per confrontare il numero di pazienti che hanno ricevuto il trattamento rescue nel gruppo CsA versus gruppo di steroidi. Gli score dei sintomi e dei segni sono stati anche analizzati con modello lineare generalizzato per misure ripetute, per valutare le differenze tra i trattamenti con CsA 0,1% e del 0,15%. Risultat: Venti recidive di infiammazione acuta hanno richiesto il ricorso al trattamento rescue con desametasone 0,15% 4 volte al giorno per 7 giorni, secondo quanto previsto dal protocollo dello studio. In particolare è stato necessario amministrare il trattamento rescue in 16 pazienti che hanno ricevuto il trattamento con CsA e in 7 trattati con steroidi (p <0,029). (Figura 9) 32 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Figura 9 I pazienti con riacutzzazione trattati con CsA 0,1% hanno richiesto un numero maggiore di rescue medication rispetto ai pazienti trattati con desametazone (A,B) Allo stesso modo, i pazienti trattato con desametasone hanno mostrato un miglioramento significativo del prurito (p = 0,037), dell’ iperemia congiuntivale (p = 0,009) e del total score dei segni (p = 0,038) rispetto ai pazienti trattati con CsA 0,1%. (Figura 10) 33 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Figura 10. il trattamento con desametazone è risultato più efficace nel controllo del prurito(A), iperemia(B), total score dei segni (C) rispetto al trattamento con CsA 0.1%, ma non ha raggiunto la significatività nel total score dei sintomi (D), nell’Oxford score (E) e negli altri parametri valutati. Questo studio ha dimostrato con il trattamento con CsA 0,05% consente una riduzione significativa delle recidive stagionali di infiammazione oculare nei pazienti con VKC, rispetto al 34 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. trattamento standard con collirio antiallergico (Ketotifene 0,025%). I risultati di questo studio suggeriscono che la ciclosporina in collirio può essere utilizzata nei pazienti con VKC come trattamento preventivo delle riacutizzazioni che consenta un risparmio nell’uso degli steroidi. Nelle fasi di acuzie, invece, il trattamento più efficace resta quello steroideo topico, usato ad alte dosi per brevi periodi. 5.3 Trattamento chirurgico della cheratocongiuntivite primaverile Sebbene sia stato proposto il trattamento delle papille giganti con l’escissione chirurgica, la sua efficacia non è stata dimostrata, e la rapida ricomparsa della reazione papillare rende inutile tale chirurgia. Anche la crioterapia delle papille giganti è da sconsigliare in quanto promuove l'infiammazione e può causare cicatrici congiuntivali. (Tanaka et al. 2004). Differente è l’approccio ai pazienti che hanno sviluppato una ulcera corneale a placca, in cui la rimozione chirurgica della placca è necessaria per ottenere la riepitelizzazione della ulcera a scudo ulcera. (Solomon et al. 2004). Anche l’innesto di membrana amniotica è stato descritto in pazienti con ulcere persistenti refrattarie alla terapia medica, al fine di favorire la riepitelizzazione (Rouher et al. 2004). 35 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 6. Conclusioni Nonostante lo sviluppo di nuovi farmaci nel corso dell'ultimo decennio, al momento, la situazione dei pazienti con VKC severa rimane preoccupante per la dipendenza da steroidi topici, con tutti i rischi che ne conseguono. Nuovi e selettivi farmaci sono necessari per un miglior controllo della VKC. (Hingorani1995). Finora, i risultati di attualità ciclosporina sono molto incoraggianti, ma a causa della indisponibilità dei preparazioni commerciali, il suo uso in VKC è limitato. Gli sviluppi futuri nella modulazione di prodotti eosinofili, citochine e molecole di adesione potranno essere rilevanti. La dimostrazione di alti livelli di leucotrieni nelle lacrime di pazienti con VKC e del miglioramento nei segni e sintomi della VKC quando viene somministrato per via orale montelukast, un antagonista del recettore dei leucotrieni, suggerisce che gli anti-leucotrieni hanno potenziale terapeutico e la necessità di ulteriori studi (Akman et al. 1998; Lambiase et al. 2003). Anche molecole probiotiche sono state proposte per inibire le allergie oculari. Uno studio preliminare ha dimostrato che il trattamento con Lactobacillus acidophilus diluito in soluzione fisiologica somministrata come collirio migliora i segni ed i sintomi nei pazienti con VKC. (Iovieno 2008-lavoro allegato) 36 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 7. Bibliografia Abelson MB , Schaefer K (1993): Conjunctivitis of allergic origin: immunologic mechanisms and current approaches to therapy.Surv Ophthalmol 38: 115–132. Abelson MB, Baird RS , Allansmith MR (1980): Tear histamine levels in vernal conjunctivitis and other ocular inflammations. Ophthalmology 87: 812–814. Abu El-Asrar AM, Struyf S, Al-Mosallama AA, Missotten L, Van Damme J , Geboes K (2001): Expression of chemokine receptors in vernal keratoconjunctivitis. Br J Ophthalmol 85: 1357– 1361. Abu El-Asrar AM, Tabbara KF, Geboes K, Missotten L , Desmet V (1996): An immunohistochemical study of topical cyclosporine in vernal keratoconjunctivitis. Am J Ophthalmol 121: 156–161. Akman A, Irkec M , Orhan M (1998):Effects of lodoxamide, disodium cromoglycateand fluorometholone on tear leukotrienelevels in vernal keratoconjunctivitis.Eye 12: 291–295. Allansmith MR , Ross RN (1988): Ocular allergy. Clin Allergy 18: 1–136. Asano-Kato N, Fukagawa K, Okada N, Kawakita T, Takano Y, Dogru M, Tsubota K , Fujishima H (2005): TGF-beta1, IL-1beta, and Th2 cytokines stimulate vascular endothelial growth factor production from conjunctival fibroblasts. Exp Eye Res 80: 555–560. Avunduk AM, Avunduk MC, Erdol H, Kapicioglu Z , Akyol N (2001): Cyclosporine effects on clinical findings and impression cytology specimens in severe vernal keratoconjunctivitis. Ophthalmologica 215: 290–293. 37 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. BenEzra D, Pe’er J, Brodsky M , Cohen E (1986): Cyclosporine eye drops for the treatment of severe vernal keratoconjunctivitis. Am J Ophthalmol 101: 278– 282. BenEzra D, Matamoros N , Cohen E (1988): Treatment of severe vernal keratoconjunctivitis with cyclosporine A eyedrops. Transplant Proc 20: 644–649. Bielory L (2000): Allergic and immunologic disorder of the eye. Part 2: ocular allergy. J Allergy Clin Immunol 106: 1019–1032. Bielory L (2002b): Ocular allergy guidelines: a practical treatment algorithm. Drugs 62:1611– 1634. Bielory L , Frohman LP (1992): Allergic and immunologic disorders of the eye. J Allergy Clin Immunol 89: 1–15. Bielory L, Bonini S , Bonini S (2002a): Inflammatory mechanism in allergic diseases. In: Zweiman B , Schwartz LB (eds). Allergic eye disorders. New York: Marcel Dekker, 311– 323. Bielory L, Lien KW , Bigelsen S (2005): Efficacy and tolerability of newer antihistaminesin the treatment of allergic conjunctivitis. Drugs 65: 215–228. Bleik JH, Tabbara KF.Topical cyclosporine in vernal keratoconjunctivitis.(1991) Ophthalmology;98(11):1679-84. Bonini S, Bonini S, Schiavone M, Centofanti M, Allansmith MR , Bucci MG (1992a): Conjunctival hyperresponsiveness to ocular histamine challenge in patients with vernal conjunctivitis. J Allergy Clin Immunol 89: 103–107. 38 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Bonini S, Barney NP, Schiavone M, Centofanti M, Berruto A, Bonini S , Allansmith MR (1992b): Effectiveness of Nedocromil sodium 2% eyedrops on clinical symptoms and tear fluid cytology of patients with vernal conjunctivitis. Eye 6: 648–652. Bonini S, Bonini S, Lambiase A et al. (1995a): Vernal keratoconjunctivitis: a model of 5q cytokine gene cluster disease.Int Arch Allergy Immunol 107: 95–98. Bonini S, Lambiase A, Schiavone M, Centofanti M, Palma LA , Bonini S (1995b): Estrogen and progesterone receptors in vernal keratoconjunctivitis. Ophthalmology 102: 1374–1379. Bonini S, Bonini S, Lambiase A et al. (2000):Vernal keratoconjunctivitis revisited. A case series of 195 patients with long-term followup. Ophthalmology 107: 1157–1163. Bonini S, Lambiase A, Sacchetti M, Bonini S.(2003) Cytokines in ocular allergy. Int Ophthalmol Clin;43(1):27-32. Bonini S, Coassin M, Aronni S , Lambiase A (2004): Vernal keratoconjunctivitis. Eye 18: 345– 351. Bonini S, Micera A, Iovieno A, Lambiase A, Bonini S. (2005) Expression of Toll-like receptors in healthy and allergic conjunctiva. Ophthalmology112(9):1528; Bonini S, Sacchetti M, Mantelli F , Lambiase A (2007): Clinical grading of vernal keratoconjunctivitis. Curr Opin Allergy Clin Immunol 7: 436–441. Buckley RJ (1988): Vernal keratoconjunctivitis.Int Ophthalmol Clin 28: 303–308. Calder VL, Jolly G, Hingorani M, Adamson P, Leonardi A, Secchi AG, Buckley RJ ,Lightman S (1999): Cytokine production and mRNA expression by conjunctival T-cell lines in chronic allergic eye disease. Clin Exp Allergy 29: 1214–1222. Cameron JA (1995): Shield ulcers and plaques of the cornea in vernal keratoconjunctivitis. phthalmology 102: 985–993. 39 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Chen W, Mempel M, Schober W, Behrendt H, Ring J.Gender difference, sex hormones, and immediate type hypersensitivity reactions.Allergy. 2008;63(11):1418-27. Church MK , Levi-Schaffer F (1997): The human mast cell. J Allergy Clin Immunol 99: 155– 160. Church MK , McGill JI (2002): Human ocular mast cells. Curr Opin Allergy Clin Immunol 2: 419–422. Cutolo M, Sulli A, Capellino S, Villaggio B, Montagna P, Seriolo B et al. Sex hormones influence on the immune system: basic and clinical aspects in autoimmunity. Lupus 2004;13:635–638 D’Angelo G, Lambiase A, Cortes M, Sgrulletta R, Pasqualetti R, Lamagna A , Bonini S (2003): Preservative-free diclofenac sodium 0.1% for vernal keratoconjunctivitis. Graefes Arch Clin Exp Ophthalmol 241: 192–195. Fujishima H, Takeyama M, Takeuchi T, Saito I , Tsubota K (1997): Elevated levels of substance P in tears of patients with allergic conjunctivitis and vernal keratoconjunctivitis. Clin Exp Allergy 27:372–378. Fujishima H, Saito I, Takeuchi T , Tsubota K (2002): Immunological characteristics of patients with vernal keratoconjunctivitis. Jpn J Ophthalmol 46: 244–248. Fukagawa K, Nakajima T, Saito H, Tsubota K, Shimmura S, Natori M , Hirai K (2000): IL-4 induces eotaxin production in corneal keratocytes but not in epithelial cells. Int Arch Allergy Immunol 121: 144– 150. Hingorani M , Lightman S (1995): Therapeuticoptions in ocular allergic disease.Drugs 50: 208– 221. 40 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Iovieno A, Lambiase A, Sacchetti M, Stampachiacchiere B, Micera A , Bonini S (2008): Preliminary evidence of the efficacy of probiotic eye-drops treatment in patients with vernal keratoconjunctivitis. Graefes Arch Clin Exp Ophthalmol 246: 435–441. Iqbal A, Jan S, Babar TF , Khan MD (2003): Corneal complications of vernal catarrh. J Coll Physicians Surg Pak 13: 394–397. Irkec M , Bozkurt B (2003): Epithelial cells in ocular allergy. Curr Allergy Asthma Rep : 352– 357. Kilic A , Gurler B (2006): Topical 2% cyclosporine A in preservative-free artificial tears for the treatment of vernal keratoconjunctivitis. Can J Ophthalmol 41: 693–698. Kosrirukvongs P , Luengchaichawange C (2004): Topical cyclosporine 0.5 per cent and reservative-free ketorolac tromethamine 0.5 per cent in vernal keratoconjunctivitis. J Med Assoc Thai 87:190–197. Labrie F, Bélanger A, Simard J, Van Luu-The, Labrie C.DHEA and peripheral androgen and estrogen formation: intracinology.Ann N Y Acad Sci. 1995;774:16-28 Lambiase A, Bonini S, Bonini S, Micera A, Magrini L, Bracci-Laudiero L , Aloe L (1995): Increased plasma levels of nerve growth factor in vernal keratoconjunctivitis and relationship to conjunctival mast cells. Invest Ophthalmol Vis Sci 36: 2127–2132. Lambiase A, Bonini S, Micera A, Tirassa P, Magrini L, Bonini S , Aloe L (1997): Increased plasma levels of substance P in vernal keratoconjunctivitis. Invest Ophthalmol Vis Sci 38: 2161– 2164. Lambiase A, Bonini S, Rasi G, Coassin M,Bruscolini A , Bonini S (2003): Montelukast,a leukotriene receptor antagonist,in vernal keratoconjuctivitis associatedwith asthma. Arch Ophthalmol 121: 615–620. 41 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Lambiase A, Normando EM, Vitiello L et al. (2007): Natural killer cells in vernal keratoconjunctivitis. Mol Vis 13: 1562–1567. Lambiase A, Minchiotti S, Leonardi A, et al. (2009);Prospective, multicenter demographic and epidemiological study on vernal keratoconjunctivitis: a glimpse of ocular surface in Italian population.Ophthalmic Epidemiol. 16(1):38-41. Lambiase A, Micera A, Bonini S.(2009)Multiple action agents and the eye: do they really stabilize mast cells?Curr Opin Allergy Clin Immunol;9(5):454-65.. Leonardi A (2002a): Vernal keratoconjunctivitis: pathogenesis and treatment. Prog Retin Eye Res 21: 319–339. Leonardi A, Papa V, Milazzo G , Secchi AG (2002b): Efficacy and safety of desonide phosphate for the treatment of allergic conjunctivitis. Cornea 21: 476–481. Leonardi A, Borghesan F, Faggian D, Secchi A , Plebani M (1995): Eosinophil cationic protein in tears of normal subjects and patients affected by vernal keratoconjunctivitis. Allergy 50: 610– 613. Leonardi A, Borghesan F, DePaoli M, Plebani M , Secchi AG (1998): Procollagens and inflammatory cytokine concentrations in tarsal and limbal vernal keratoconjunctivitis. Exp Eye Res 67: 105–112. Leonardi A, De Franchis G, Zancanaro F, et al (1999a): Identification of local Th2 and Th0 lymphocytes in vernal conjunctivitisby cytokine flow cytometry.Invest Ophthalmol Vis Sci 40: 3036–3040. Leonardi A, Jose PJ, Zhan H , Calder VL (2003a): Tear and mucus eotaxin-1 and eotaxin-2 in allergic keratoconjunctivitis.Ophthalmology 110: 487–492. 42 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Leonardi A, Cortivo R, Fregona I, Plebani M, Secchi AG , Abatangelo G (2003b): Effects of Th2 cytokines on expression of collagen, MMP-1, and TIMP-1 in conjunctival fibroblasts. Invest Ophthalmol Vis Sci 44: 183–189. Leonardi A, Brun P, Abatangelo G, Plebani M , Secchi AG (2003c): Tear levels and activity of matrix metalloproteinase (MMP)-1 and MMP-9 in vernal keratoconjunctivitis. Invest Ophthalmol Vis Sci 44: 3052–3058. Malkin CJ, Pugh PJ, Jones RD, Jones TH, Channer KS. Testosterone as a protective factor against atherosclerosis-immunomodulation and influence upon plaque development and stability. J Endocrinol 2003;178:373–380. Mantelli F, Santos MS, Petitti T, Sgrulletta R, Cortes M, Lambiase A, Bonini S. (2007)Systematic review and meta-analysis of randomised clinical trials on topical treatments for vernal keratoconjunctivitis.Br J Ophthalmol.;91(12):1656-61. Metz DP, Hingorani M, Calder VL, Buckley RJ , Lightman S (1997): T-cell cytokines in chronic allergic eye disease. J Allergy Clin Immunol 100: 817–824. Mitchell VL, Gershwin LJ. Progesterone and environmental tobacco smoke act synergistically to exacerbate the development of allergic asthma in a mouse model. Clin Exp Allergy 2007;37:276–286. Miyoshi T (2001): Interleukin-8 concentrations in conjunctival epithelium brush cytology samples correlate with neutrophil, eosinophil infiltration, and corneal damage. Cornea 20: 743– 747. Montan PG, Ekstrom K, Hedlin G, van Hage-Hamsten M, Hjern A , Herrmann B (1999): Vernal keratoconjunctivitis in a Stockholm ophthalmic centre – epidemiological, functional, and immunologic investigations. Acta Ophthalmol Scand 77: 559–563. 43 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Narita S, Goldblum RM, Watson CS, Brooks EG, Estes DM, Curran EM etal. Environmental estrogens induce mast cell degranulation and enhance IgE-mediated release of allergic mediators.Environ Health Perspect 2007;115:48–52. Neumann E, Gutmann MJ, Blumenkranz N, Michaelson IC (1959): A review of four hundred cases of vernal conjunctivitis. Am J Ophthalmol 47: 166–172. Pitton C, Michel L, Salem P, Benhamou M, Mencia-Huerta JM, Maclouf J et al. Biochemical and morphological modifications in dexamethasone-treated mouse bone marrow derived mast cells. J Immunol1988;141:2437–2444. Pucci N, Novembre E, Cianferoni A, Lombardi E, Bernardini R, Caputo R, Campa L , Vierucci A (2002): Efficacy and safety of cyclosporine eyedrops in vernal keratoconjunctivitis. Ann Allergy Asthma Immunol 89: 298–303. Rocha EM, Wickham LA, da Silveira LA, Krenzer KL, Yu FS, Toda I, Sullivan BD, Sullivan DA. Identification of androgen receptor protein and 5alpha-reductase mRNA in human ocular tissues. Br J Ophthalmol. 2000 Jan;84(1):76-84. Rouher N, Pilon F, Dalens H, Fauquert JL,Kemeny JL, Rigal D , Chiambaretta F(2004): Implantation of preserved human amniotic membrane for the treatment of shield ulcers and persistent corneal epithelial defects in chronic allergic keratoconjunctivitis. J Fr Ophtalmol 27: 1091–1097. Sacchetti M, Baiardini I, Lambiase A, Aronni S, Fassio O, Gramiccioni C, Bonini S, Bonini S.Development and testing of the quality of life in children with vernal keratoconjunctivitis questionnaire.Am J Ophthalmol. 2007 Oct;144(4):557-63. 44 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Schirra F, Suzuki T, Dickinson DP, Townsend DJ, Gipson IK, Sullivan DA.Identification of steroidogenic enzyme mRNAs in the human lacrimal gland, meibomian gland, cornea, and conjunctiva.Cornea. 2006;25(4):438-42. Secchi AG, Tognon MS , Leonardi A (1990): Topical use of cyclosporine in the treatment of vernal keratoconjunctivitis. Am J Ophthalmol 110: 641–645. Solomon A, Zamir E, Levartovsky S ,Frucht-Pery J (2004): Surgical managementof corneal plaques in vernal keratoconjunctivitis:a clinicopathologic study. Cornea 23: 608–612. Spadavecchia L, Fanelli P, Tesse R et al. (2006): Efficacy of 1.25% and 1% topical cyclosporine in the treatment of severe vernal keratoconjunctivitis in childhood. Pediatr Allergy Immunol 17: 527–532. Sudo N, Yu XN, Kubo C. Dehydroepiandrosterone attenuates the spontaneous elevation of serum IgE level in NC/Nga mice. Immunol Lett 2001;79:177–179. Sullivan DA.Tearful relationships? Sex, hormones, the lacrimal gland, and aqueous-deficient dry eye.Ocul Surf. 2004 Apr;2(2):92-123. Tabbara KF (1999): Ocular complications of vernal keratoconjunctivitis. Can J Ophthalmol34: 88–92. Tabbara KF (2001): Tear tryptase in vernal keratoconjunctivitis. Arch Ophthalmol 119:338–342. Tabbara KF , Arafat NT (1977):Cromolyn effects on vernal keratoconjuctivitis in children. Arch Ophthalmol 95: 2184–2186. Tabata N, Tagami H, Terui T.Dehydroepiandrosterone may be one of the regulators of cytokine production in atopic dermatitis. Arch Dermatol Res 1997;289:410–414. 45 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Tanaka M, Takano Y, Dogru M, Fukagawa K, Asano-Kato N, Tsubota K , Fujishima H (2004): A comparative evaluation of the efficacy of intraoperative mitomycin C use after the excision of cobblestone- like papillae in severe atopic and vernal keratoconjunctivitis. Cornea 23: 326–329. Tomassini M, Magrini L, Bonini S, Lambiase A , Bonini S (1994): Increase levels of eosinophil cationic protein and eosinophil derived neurotoxin (protein X) in vernal keratoconjunctivitis. Ophthalmology 101: 1808–1811. Trocme SD, Kephart GM, Allansmith MR, Borne WM , Gleich GJ (1989): Conjunctival deposition of eosinophil granule major basic protein in vernal keratoconjunctivitis and contact lens-associated giant papillary conjunctivitis. Am J Ophthalmol 108: 57– 63. Trocme SD, Kephart GM, Bourne WM, Buckley RJ , Gleich GJ (1993): Eosinophil granule major basic protein deposition in corneal ulcers associated with vernal keratoconjunctivitis. Am J Ophthalmol 115: 640–643. Tuft SJ, Dart JK , Kemeny M (1989): Limbal vernal keratoconjunctivitis: clinical characteristics and immunoglobulin E expression compared with palpebral vernal.Eye 3: 420–427. Uchio E, Ono SY, Ikezawa Z , Ohno S (2000): Tear levels of interferon-g, interleukin (IL) -2, IL-4 and IL-5 in patients with vernal keratoconjunctivitis, atopic keratoconjunctivitis and allergic conjunctivitis. Clin Exp Allergy 30: 103–109. Uekert SJ, Akan G, Evans MD, Li Z, Roberg K, Tisler C et al. Sex-related differences in immune development and the expression of atopy in early childhood. J Allergy Clin Immunol 2006;118:1375–1381. Vasiadi M, Kempuraj D, Boucher W, Kalogeromitros D, Theoharides TC. Progesterone inhibits mast cell secretion.Int J Immunopathol Pharmacol 2006;19:787–794. 46 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Verin PH, Dicker ID , Mortemousque B (1999): Nedocromil sodium eye drops are more effective than sodium cromoglycate eye drops for the long-term management of vernal keratoconjunctivitis. Clin Exp Allergy 29: 529–536. Yu CK, Liu YH, Chen CL. Dehydroepiandrosterone attenuates allergic airway inflammation in Dermatophagoides farinae-sensitized mice. J Microbiol Immunol Infect 2002;35:199–202. 47 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Development and Testing of the Quality of Life in Children with Vernal Keratoconjunctivitis Questionnaire MARTA SACCHETTI, ILARIA BAIARDINI, ALESSANDRO LAMBIASE, SILVIA ARONNI, OMAR FASSIO, CLAUDIA GRAMICCIONI, SERGIO BONINI, AND STEFANO BONINI ● PURPOSE: To develop and validate a questionnaire that measures health-related quality of life (HRQoL) in children with vernal keratoconjunctivitis (VKC). ● DESIGN: Prospective, observational case series. ● METHODS: An initial list of 42 items was developed and administered to 30 children with active VKC (six girls and 24 boys; mean age, nine ⴞ two years). The 30 most significant items were selected and converted into questions on a three-step scale for validation in 41 children with active VKC (eight girls and 33 boys; mean age, 9.5 ⴞ 2.1 years). Twenty-two children also completed the generic KINDL questionnaire. Clinical signs were evaluated and scored and total sign scores (TSSs) were calculated. Validation was performed by factorial analysis and Pearson correlation. Internal consistency was computed by the Chronbach ␣ on the extracted factors. ● RESULTS: Factorial analysis extracted two factors with good internal consistency: symptoms (12 items; ␣ ⴝ 0.89) and daily activities (four items; ␣ ⴝ 0.77). Correlations of Quality of Life in Children with Vernal Keratoconjunctivitis (QUICK) scores to KINDL scores were in the expected direction. Most patients reported itching (93%), burning (90%), redness (90%), the need to use eye drops (90%), tearing (83%), and photophobia (80%). The children’s greatest concerns were limitations on going to the pool (71%), playing sports (58%), and meeting friends (58%). QUICK symptom scores were correlated significantly to conjunctival hyperemia (P < .001), secretion (P ⴝ .042), chemosis (P ⴝ .012), superficial punctate keratopathy (P < .001), and TSS (P ⴝ .010). ● CONCLUSIONS: The QUICK questionnaire is a new, simple instrument to measure HRQoL in children with severe allergic conjunctivitis. This test is effective for the global evaluation of the impact of VKC on children’s Accepted for publication Jun 19, 2007. From the Department of Ophthalmology, University of Rome Campus Bio-Medico, Rome, Italy (M.S., A.L., S.A., St.B.); the Section for Allergy and Respiratory Diseases, Department of Internal Medicine, University of Genoa, Genoa, Italy (I.B.); the Department of Psychology, Turin University, Turin, Italy (O.F.); the Department of Internal Medicine, Second University of Naples, Naples, Italy (Se.B.); and the Institute of Neurobiology and Molecular Medicine, Italian National Research Council, Rome, Italy (C.G., Se.B.). Inquiries to Stefano Bonini, Via E. Longoni, 83, 00155 Roma, Italy; e-mail: [email protected] 0002-9394/07/$32.00 doi:10.1016/j.ajo.2007.06.028 © 2007 BY daily lives. (Am J Ophthalmol 2007;144:557–563. © 2007 by Elsevier Inc. All rights reserved.) V ERNAL KERATOCONJUNCTIVITIS (VKC) IS A SEVERE allergic conjunctivitis mainly affecting preadolescent boys. It is characterized by recurrent inflammation of the conjunctiva and cornea causing intense ocular symptoms such as redness, itching, photophobia, and mucous discharge with corneal involvement and potential impairment of visual function. These symptoms may last all year with seasonal exacerbations and often require long-term therapy.1,2 Clinical objective evaluation of signs and symptoms commonly are used to evaluate VKC severity. However, clinical evaluations may not necessarily reflect the impact of the disease on the patients’ health-related quality of life (HRQoL). In fact, because the presence of severe signs and symptoms of VKC may significantly influence the well-being and daily functioning of patients, often they or their parents refer problems with daily activities, even in mild and moderate cases, to VKC. The availability of an instrument to assess the impact of symptoms of ocular surface inflammation on children’s daily functioning would aid in the evaluation of VKC severity and treatment outcomes. HRQoL parameters have become important outcome measures in allergic diseases, and many questionnaires have been developed to evaluate the impact of allergy on HRQoL.3–9 In particular, rhinoconjunctivitis HRQoL questionnaires, addressed to adults, adolescents, and children, also include the evaluation of eye symptoms. However, these questionnaires are not specific to ocular allergy, because symptoms of both nasal and ocular allergy influence HRQoL scores.10 –13 Moreover, allergic rhinoconjunctivitis only accounts for a limited percentage of cases of allergic conjunctivitis in children,14 usually the mildest forms without corneal and visual involvement. In fact, studies of quality of life in children with allergic rhinoconjunctivitis, in contrast to studies in adults and adolescents, demonstrated minimal interference with their normal daily activities and emotions,6,13,15 whereas the severity of signs and symptoms in VKC patients significantly influences daily life and social interactions.1 This study describes the development and initial validation of a new instrument, the Quality of Life in Children with Keratoconjunctivitis (QUICK) questionnaire, for the measurement of HRQoL in children between five and 12 ELSEVIER INC. ALL RIGHTS RESERVED. 557 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. years of age with allergic conjunctivitis, keratoconjunctivitis, or both. Our goal was to develop an instrument: 1) able to capture physical, psychosocial, and practical aspects of HRQoL of particular relevance in patients with VKC; 2) that is generated and tested according to well-established procedures applied to other, similar questionnaires; and 3) designed to be used by children as a quick and simple test. METHODS THIS STUDY WAS CARRIED OUT IN TWO SEPARATE PHASES: the development and the validation processes, involving two different groups of patients with VKC. The questionnaire was developed using a standard multistep method. These phases are described in detail below. ● PATIENTS: Children (age, between four and 12 years) with active VKC were recruited consecutively by the Corneal and External Disease Center in Rome, Italy, from March 20, 2003 through July 18, 2005 for the development and validation phases of the new questionnaire. VKC diagnosis was based on clinical history and objective examination (presence of mild to severe cobblestone-like appearance in the upper tarsal conjunctiva) and was confirmed by the presence of eosinophils in a conjunctival scraping. VKC was considered in the active phase when patients reported symptoms of itching, photophobia, and tearing associated with conjunctival hyperemia, mucous discharge, epithelial keratopathy, or a combination thereof at slit-lamp examination. Patients in treatment with topical steroid drugs were not included. Clinical histories were collected and clinical signs (conjunctival tarsal or bulbar papillary reaction, secretion, hyperemia and chemosis, corneal superficial keratopathy) were evaluated and scored from 0 to 3 (0 ⫽ absent, 1 ⫽ mild, 2 ⫽ moderate, 3 ⫽ severe) to correlate questionnaire scores to clinical findings. The total sign score was calculated as the sum of all the sign scores (range, zero to 18). FIGURE 1. Diagram showing the procedure of query selection during the development and validation phases of the Quality of Life in Children with Vernal Keratoconjunctivitis (QUICK) questionnaire in children with vernal keratoconjunctivitis (VKC). the field of ocular allergy was required to indicate symptoms and problems troubling children with VKC.1,2,14 3) Ten children with VKC in the active phase (seven boys and three girls; mean age, 6.5 ⫾ three years) and their parents (six men and four women; mean age, 44 ⫾ six years) were interviewed by a clinician and a psychologist to identify those areas of daily life that were impaired by the presence of VKC. 4) Two psychologists (I.B. and O.F.) with experience in the process of development and validation of qualityof-life questionnaires for allergic diseases were requested to adapt the items to a pediatric population.8 ● DEVELOPMENT PROCESS: Item Generation. The aim of this phase was to identify symptoms and problems perceived as troublesome by patients with VKC. A preliminary list of items was developed on the basis of the following sources: Item Reduction. A qualitative selection was performed from the preliminary list of items by eliminating those that were redundant, ambiguous, difficult to understand, or expressed in the negative form. The resulting list of items was listed randomly and was administered to 30 children with VKC in the active phase (24 boys and six girls; age range, five to 12 years; mean age ⫾ standard deviation [SD], nine ⫾ two years), who had to indicate which of the selected items they had experienced directly in the preceding two weeks on a three-point scale: 1 ⫽ never, 2 ⫽ sometimes, and 3 ⫽ always. The highest scoring items then were adopted to construct the new specific tool. The 1) An international literature review was performed to identify existing questionnaires for HRQoL assessment in children with ocular surface symptoms, allergy, or both by using the key words conjunctivitis, rhinoconjunctivitis, keratoconjunctivitis, allergy, atopy, children, quality of life, questionnaire.7,13,16,17 2) A panel of four ophthalmologists (M.S., A.L., S.A., and St.B.) and two immunologists (St.B. and C.G.) who perform clinical and investigational research in 558 AMERICAN JOURNAL OF OPHTHALMOLOGY OCTOBER 2007 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. TABLE 1. Characteristics of Patients with Vernal Keratoconjuctivitis Included in the Development Phase (n ⫽ 30) and in the Validation Process (n ⫽ 41) of the QUICK Questionnaire Characteristic of Patients Item Reduction Phase Validation Phase No. of patients Age (yrs) Mean (range) Gender (n) Male Female Associated atopic conditions (n) Asthma Rhinitis Atopic dermatitis Urticaria Vernal keratoconjunctivitis course (n) Seasonal Perennial Duration of disease (yrs) Range Mean Topical treatment (n) Anti-allergic eye drop Cyclosporine A eye drop None Vernal keratoconjunctivitis form (n) Limbal Tarsal Mixed Visual acuity (mean) Total signs score (mean) 30 41 9 ⫾ 2 (5 to 12) 9.5 ⫾ 2.1 (5 to 12) 24 6 33 8 1 10 3 0 4 16 5 3 13 17 21 20 1 to 6 3.1 ⫾ 2 1 to 9 4.5 ⫾ 2.3 25 2 3 34 3 4 4 22 4 0.9/1 ⫾ 0.16/1 5 ⫾ 2.2 7 29 5 0.9/1 ⫾ 0.2/1 6.4 ⫾ 3 QUICK ⫽ Quality of Life in Children with Vernal Keratoconjunctivitis. resulting questionnaire then was administered to a different patient population for validation (Figure 1). ● VALIDATION PROCESS: Forty-one children with VKC in the active phase (33 boys and eight girls; age range, five to 12 years; mean age ⫾ SD, 9.5 ⫾ 2.1 years) completed the preliminary QUICK questionnaire, converted into questions on a Likert scale of three steps. Children were asked to point out difficult questions or problems with any item. Twenty-four of the 41 children also completed the KINDL questionnaire, a generic tool designed to obtain an assessment of HRQoL, independent of current health status, in children of a wide age range.18 Nineteen children were administrated the Kid-KINDL version (28 items for children between eight and 12 years), and five children were administered the Kiddy KINDL (12 items for children between four and seven years). Both KINDL versions include six domains (physical well-being, emotional wellbeing, self-esteem, family, friends, and school) and an additional domain of six items for chronically ill children. The subscales of KINDL are calculated such that a higher score corresponds to a higher HRQoL. VOL. 144, NO. 4 QUALITY OF LIFE IN The aim of the validation phase was to evaluate the following psychometric properties of the new instrument: 1) Validity. This parameter assesses whether an instrument actually measures what it was designed to measure. Factorial analysis was performed on the preliminary QUICK questionnaire scores to identify potential subscales according to our hypotheses; the principal component method with Varimax rotation was adopted. By means of Pearson correlation coefficients, we also evaluated the relationship between the new questionnaire and the KINDL questionnaire to evaluate the convergent validity. 2) Internal consistency. This parameter measures the homogeneity of a scale. It was computed using the Cronbach correlation coefficient on the extracted factors. Measurements with reliability of 0.70 or more have been recommended for the purpose of comparing groups. The statistically significant cutoff value was set at P ⬍ .05. The entire statistical analysis was conducted by using SPSS software version 10.0 (SPSS, Inc, Chicago, Illinois, USA). CHILDREN WITH VKC 559 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. TABLE 2. Mean Response Scores (range, zero to three) to the Preliminary List of 42 Questions Asked to 30 Children with Vernal Keratoconjunctivitis in the Development Phase of the QUICK Questionnaire During the Last Two Weeks, Because of Conjunctivitis . . . Score 1 . . . you had to use eye drops 2 . . . you had trouble going to the swimming pool 3 . . . you rubbed your eyes 4 . . . you had red eyes 5 . . . you had tearing 6 . . . you had, in the morning, closed, and sticky eyes 7 . . . you had itchy eyes 8 . . . you felt burning in your eyes 9 . . . you had problems playing with pets 10 . . . you had problems going to the cinema 11 . . . you had problems in the light 12 . . . you had to use tissues 13 . . . you had difficulties in reading 14 . . . you had eye secretion 15 . . . you were nervous and restless 16 . . . you had trouble meeting your friends 17 . . . you had problems playing video games or computer 18 . . . you had trouble practicing sports (football, gym, etc.) 19 . . . you felt embarrassed 20 . . . you had difficulties concentrating on homework or other activities 21 . . . you had blurred vision 22 . . . you had problems at school 23 . . . you had trouble playing outdoors 24 . . . you had puffy eyes 25 . . . you have eaten little 26 . . . you had reduction in eyesight 27 . . . you had difficulties watching TV 28 . . . you had trouble staying in air-conditioned rooms 29 . . . you cried easily 30 . . . you slept badly 31 . . . you had problems going to school 32 . . . you had problems riding your bicycle 33 . . . you were proud of yourself 34 . . . you had problems going to the country 35 . . . you had problems falling asleep 36 . . . you felt at ease with other people 37 . . . you attended school regularly 38 . . . you enjoyed playing with other children 39 . . . you had difficulty opening your eyes completely 40 . . . you had problems participating in social activities (boy scouts, football team, etc.) 41 . . . you were satisfied with yourself 42 . . . you searched for the company of your friends 2.46 TABLE 3. Factorial Analysis Allowed Selection of 16 Items To Be Allocated into Two Domains (Symptoms and Daily Activities) for the Final Version of the QUICK Questionnaire to Assess Quality of Life in Children with Vernal Keratoconjunctivitis Factor II: 2.30 2.24 2.24 2.22 . . . you felt burning in your eyes . . . you had trouble staying in air-conditioned rooms . . . you had to use tissues . . . you had puffy eyes . . . you had problems in the light . . . you had tearing . . . you had itchy eyes . . . you had red eyes . . . you had blurred vision . . . you had eye secretion . . . you had to use eye drops . . . you had, in the morning, closed and sticky eyes . . . you had trouble playing outdoors . . . you had trouble practicing sports (football, gym) . . . you had trouble meeting your friends . . . you had trouble going to the swimming pool % Variance 2.20 2.17 2.15 2.11 2.08 2.05 2.02 1.95 1.95 1.80 1.79 1.78 1.74 1.71 1.69 1.66 1.54 1.54 1.47 1.44 1.43 1.41 ⫺0.143 0.719 0.717 0.715 0.694 0.689 0.689 0.664 0.635 0.596 0.565 0.042 0.070 ⫺0.015 ⫺0.123 ⫺0.109 ⫺0.343 ⫺0.405 ⫺0.105 ⫺0.152 ⫺0.091 0.536 0.217 ⫺0.338 0.734 0.562 0.683 0.549 0.677 0.401 38.71 0.422 13.34 RESULTS ● SUBJECT CHARACTERISTICS: Thirty children (24 boys and six girls) with VKC completed the initial list of 42 items for the development of the new questionnaire, and 41 children with VKC (33 boys and eight girls) completed the preliminary QUICK test of 30 items for the validation phase. All patients were between five and 12 years of age. The characteristics of patients are listed in Table 1. All patients showed signs and symptoms of conjunctival inflammation with conjunctival hyperemia (100%), mucus discharge (58%), and superficial punctuate keratitis (55%). At the time of inclusion in this study, only seven patients (10%) were not receiving treatment. The remaining patients were receiving treatment with antiallergic eye drops (83%), whereas a small percentage (7%) also was treated with topical cyclosporine A. 1.41 1.37 1.35 1.35 1.34 1.34 1.21 1.20 1.10 1.07 1.06 1.04 0.99 0.98 0.78 AMERICAN JOURNAL 0.756 QUICK ⫽ Quality of Life in Children with Vernal Keratoconjunctivitis. Total variance, 52.05%. The 30 final items selected for the validation process are in bold face. 560 Factor I: QUICK QUICK Daily Symptoms Activities QUICK Items ● QUICK DEVELOPMENT PROCESS: Item Generation. The initial list of expressions comprised 51 items. Whenever OF OPHTHALMOLOGY OCTOBER 2007 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. TABLE 4. The QUICK Questionnaire Scores Showed Significant Correlations to KINDL Scores in Children with Vernal Keratoconjunctivitis QUICK Symptoms QUICK Daily Activities KINDL Domains P Value R P Value R Physical well-being Emotional well-being Self-esteem Family Friends School Disease Total score .006 .335 .271 .824 .965 .605 .001 .101 ⫺0.635 ⫺0.239 0.283 0.824 ⫺0.012 ⫺0.135 ⫺0.699 ⫺0.359 .105 .584 .508 .947 .960 .862 .012 .281 ⫺0.407 ⫺0.143 0.172 ⫺0.17 ⫺0.13 ⫺0.46 ⫺0.526 ⫺0.240 QUICK ⫽ Quality of Life in Children with Vernal Keratoconjunctivitis. FIGURE 2. Bar graphs showing the percentage of children referring the presence of (Top) vernal keratoconjunctivitis (VKC)-related symptoms and (Bottom) daily life limitations in the previous two weeks. possible, children’s and parents’ language was maintained. The qualitative selection omitted nine items that were considered redundant, ambiguous, difficult to understand, expressed in negative form, or a combination thereof. The item generation phase resulted in a pool of 42 items (Figure 1). Item Reduction. Thirty patients with active VKC completed the preliminary questionnaire of 42 items (Table 1). Analysis of children’s answers allowed us to select 30 items with higher mean score for the validation process (Table 2). ● QUICK VALIDATION PROCESS: Validation was undertaken in a group of 41 children with active VKC (Table 1). Validity. Factor analysis revealed a bidimensional structure, which explained up to 52% of the total variance. This solution was deemed appropriate on the basis of eigenvalues. Fourteen items were excluded because they were not allocated to the extracted dimensions (Figure 1). The 16 items selected for the final version of the QUICK questionnaire were allocated into two domains: 1) symptoms VOL. 144, NO. 4 QUALITY OF LIFE IN (12 items) and 2) daily activities (four items; Table 3). The percentages of children with VKC referring symptoms and problems in daily life are shown in Figure 2. Item and scale scores were oriented so that higher scores indicated worse HRQoL. To make scores more meaningful and to permit comparisons among different populations of patients, linear transformations were performed on raw scores, indicating the percentage of maximum possible scores. Thus, the minimum possible score is defined as zero and the maximum possible score is defined as 100. Twenty-four of 41 children (mean age, nine ⫾ two years; 19 boys and five girls) also completed the KINDL questionnaire. Correlations between QUICK and KINDL scores were in the expected direction: as shown in Table 4, conceptually similar domains had higher correlations than conceptually unrelated domains. Internal Consistency. The dimensions of QUICK showed satisfactory Cronbach ␣ values: symptoms, ␣ ⫽ 0.889; and daily activities, ␣ ⫽ 0.772. The QUICK symptom factor showed a significant correlation to objective clinical sign scores: conjunctival hyperemia (P ⬍ .001; ⫽ 0.656), secretion (P ⫽ .042; ⫽ 0.409), chemosis (P ⫽ .012; ⫽ 0.469), superficial punctuate keratopathy (P ⬍ .001; ⫽ 0.657), and limbal papillary reaction (P ⫽ .042; ⫽ 0.387), but not with tarsal papillary reaction and duration of disease. The total signs score also was significantly related to the QUICK symptoms score (P ⫽ .010; ⫽ 0.442). The QUICK daily activity factor showed no significant correlation to clinical sign scores. DISCUSSION THIS STUDY DESCRIBES THE DEVELOPMENT AND INITIAL validation of an age- and disease-specific quality-of-life questionnaire for allergic keratoconjunctivitis in a group of children with VKC. VKC is, together with atopic keratoCHILDREN WITH VKC 561 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. conjunctivitis, the most severe form among ocular allergies.1,2,14,19 VKC is a rare disease affecting young patients with severe ocular symptoms that undoubtedly interferes with their quality of life. In addition to their intense ocular discomfort, children with VKC often experience negative consequences because of the limiting of many of their preferred activities in the attempt to avoid worsening of their disease. What children really experience in daily life, and their subjective viewpoint about symptoms and problems resulting from VKC, should not be neglected in the planning of patient management. In contrast to existing quality of life tests that include items for allergic conjunctivitis, the QUICK questionnaire was developed to be used specifically in children with severe forms of ocular allergy with or without corneal involvement. The final version of the QUICK questionnaire contains 16 items. It was designed specifically to be administered easily and quickly to five- to 12-year-old children with chronic keratoconjunctivitis. The validation process showed that the new questionnaire met the standards for internal consistency and validity. Psychometric characteristics make QUICK useful to evaluate the specific burden of VKC on HRQoL. At present, it also seems to be the first validated questionnaire specific for the assessment of HRQoL in VKC. Conceptually similar domains of QUICK showed significant correlations to KINDL, providing good evidence for QUICK validity. In particular, the QUICK symptoms domain was significantly related to the physical well-being and disease domains of KINDL, suggesting that VKCrelated symptoms are associated with a reduction of health status and to a subjective perception of illness. The disease domain of KINDL also was related significantly to the daily activity domain of QUICK, indicating that the children’s functioning in everyday life is influenced negatively by the presence of VKC. The QUICK questionnaire was validated in a population of children with VKC aged between five and 12 years. The item generation phase of the questionnaire included children younger than five years who initially were interviewed with their parents to collect information regarding VKC-related problems. However, because of the lack of collaboration of younger children, only children aged more than five years were included in the subsequent phases of administration of the questionnaire. The differences in age between the groups of patients should not affect the validity of the QUICK questionnaire, because children included in all subsequent phases of development and validation were aged between five and 12 years. In addition, because topical steroids exert a prompt and intense effect by reducing signs and symptoms of VKC, patients in treatment with topical steroids were not included in the study to avoid interferences of such treatments on the children’s perception of the disease. 562 AMERICAN JOURNAL The QUICK questionnaire was developed for its initial validation in the Italian language because the prevalence of VKC is higher in Mediterranean countries than in other regions.1,2,19 An English version of the questionnaire also was produced (© 2007 Barzanò & Zanardo) for which the Italian version was translated into English and back by two independent bilingual translators. The administration of the QUICK questionnaire in other languages and in different geographic and cultural regions may contribute to its further validation. The QUICK symptoms domain showed significant correlation to the clinical, objective parameters of VKC severity, indicating that the QUICK symptom score is related to the disease process. The lack of significant correlation between the QUICK daily activities domain and clinical sign scores may be explained by daily life adjustments related to the perception of the disease by the children, their parents, or both, rather than the severity of the disease at the time of the visit. In addition, the parents’ perception of the disease may have a profound impact on the child’s own perception, influencing the child’s diseaserelated quality of life and capacity for adjustment. Because the aim of the study was to provide a tool capable of assessing VKC-related quality of life, its relationship to the child’s psychological state or to a combination of the child’s and parents’ psychological condition has not been investigated. Further studies should be performed to clarify such an association. In our opinion, the lack of correlation between the QUICK daily activities domain and clinical sign scores should not be interpreted as a sign of reduced validity, but rather suggests the importance of combining clinical, objective evaluations to HRQoL instruments to balance all kinds of health outcome measures. Itching, burning, redness, photophobia, and the need to use eye drops were the most frequent symptoms reported, because these are the expression of the severity of the allergic inflammation.1,14 However, children with VKC also experienced disease-related limitations in their daily life and social interactions such as playing sports and meeting friends. Patients reported limitations in activities that cause exposure to allergens such as playing outdoors20 and to irritant stimuli, such as going to the pool because of the notoriously powerful irritating effects of chlorinated air and water.21,22 These findings suggest that treatment of VKC should improve not only the children’s signs and symptoms, but also their daily life and functioning. In conclusion, the QUICK questionnaire is a new tool for the measurement of HRQOL in children with allergic conjunctivitis, including those with severe symptoms, corneal involvement, or both. This new instrument may allow collection of information on relevant additional clinical variables in subjects with severe ocular allergy, both in clinical practice and in drug trials. OF OPHTHALMOLOGY OCTOBER 2007 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. THIS STUDY WAS SUPPORTED BY AN EU-FRAMEWORK PROGRAMME FOR RESEARCH, CONTRACT NO. FOOD-CT-2004-506378, the GA2LEN project (Global Allergy and Asthma European Network). The authors indicate no financial conflict of interest. Involved in design and conduct of study (M.S., I.B., A.L., S.A., C.G., Se.B., St.B.); collection (M.S., S.A., St.B.), analysis (O.F.), and interpretation of the data (M.S., I.B., A.L., O.F., Se.B., St.B.); and preparation (M.S., I.B., A.L., S.A., O.F., C.G., Se.B., St.B.), review (A.L., S.A., Se.B., C.G., St.B.), and approval of the manuscript (M.S., I.B., A.L., S.A., O.F., C.G., Se.B., St.B.). The study was conducted following the tenets of the Declaration of Helsinki; the use of the QUICK questionnaire was approved by the Institutional Review Board of the University of Rome Campus Bio-Medico, and informed consent was obtained from the participating children’s parents. We would like to thank Annalisa Alesii, San Raffaele La Pisana, Rome, Italy, and Cristina Artesani, Allergy Unit C.I. Columbus, Rome, Italy, for their contribution in the first phase of the development of the questionnaire. The copyright for the Italian and English versions of the QUICK questionnaires has been provided by Barzanò & Zanardo, 2007. REFERENCES 1. Bonini S, Bonini S, Lambiase A, et al. Vernal keratoconjunctivitis revisited: a case series of 195 patients with long-term follow-up. Ophthalmology 2000;107:1157–1163. 2. Bonini S, Coassin M, Aronni S, Lambiase A. Vernal keratoconjunctivitis. Eye 2004;18:345–351. 3. Guyatt GH, Feeny DH, Patrick DL. Measuring healthrelated quality of life. Ann Intern Med 1993;118:622– 629. 4. Felce D, Perry J. Quality of life: its definition and measurement. Res Dev Disabil 1995;16:51–74. 5. Leynaert B, Soussan D. Monitoring the quality-of-life in allergic disorders. Curr Opin Allergy Clin Immunol 2003;3: 177–183. 6. Juniper EF. Measuring health-related quality of life in rhinitis. J Allergy Clin Immunol 1997;99:S742–S749. 7. Rutishauser C, Sawyer SM, Bowes G. Quality-of-life assessment in children and adolescents with asthma. Eur Respir J 1998;12:486 – 494. 8. Baiardini I, Pasquali M, Giardini A, et al. Rhinasthma: a new specific questionnaire for patients with rhinitis and asthma. Allergy 2003;58:289 –294. 9. Cohen BL, Noone S, Munoz-Furlong A, Sicherer SH. Development of a questionnaire to measure quality of life in families with a child with food allergy. J Allergy Clin Immunol 2004;114:1159 –1163. 10. Juniper EF, Guyatt GH. Development and testing of a new measure of health status for clinical trials in rhinoconjunctivitis. Clin Exp Allergy 1991;21:77– 83. 11. Bunnag C, Leurmarnkul W, Jareoncharsri P, et al. Development of a health-related quality of life questionnaire for Thai patients with rhinoconjunctivitis. Asian Pac J Allergy Immunol 2004;22:69 –79. 12. Juniper EF, Guyatt GH, Dolovich J. Assessment of quality of life in adolescents with allergic rhinoconjunctivitis: development and testing of a questionnaire for clinical trials. J Allergy Clin Immunol 1994;93:413– 423. VOL. 144, NO. 4 QUALITY OF LIFE IN 13. Juniper EF, Howland WC, Roberts NB, Thompson AK, King DR. Measuring quality of life in children with rhinoconjunctivitis. J Allergy Clin Immunol 1998;101:163–170. 14. Bielory L, Bonini S, Bonini S. Allergic eye disorders. In: Zweiman B, Schwartz LB, eds. Inflammatory mechanisms in allergic diseases. New York, New York: Marcel Dekker, 2002:311–323. 15. Meltzer EO. Quality of life in adults and children with allergic rhinitis. J Allergy Clin Immunol 2001;108:S45–S53. 16. Juniper EF, Thompson AK, Ferrie PJ, Roberts JN. Development and validation of the mini rhinoconjunctivitis quality of life questionnaire. Clin Exp Allergy 2000;30:132–140. 17. French DJ, Carroll A, Christie MJ. Health-related quality of life in Australian children with asthma: lessons for the cross-cultural use of quality of life instruments. Qual Life Res 1998;7:409 – 419. 18. Ravens-Sieberer U, Bullinger M. Assessing health-related quality of life in chronically ill children with the German KINDL®: first psychometric and content analytical results. Qual Life Res 1998;7:399 – 407. 19. Leonardi A. Vernal keratoconjunctivitis: pathogenesis and treatment. Prog Retin Eye Res 2002;21:319 –339. 20. Halken S. Prevention of allergic disease in childhood: clinical and epidemiological aspects of primary and secondary allergy prevention. Pediatr Allergy Immunol 2004;15: S9 –S32. 21. Bernard A, Carbonelle S, Michel O, et al. Lung hyperpermeability and asthma prevalence in schoolchildren: unexpected associations with the attendance at indoor chlorinated swimming pools. Occup Environ Med 2003; 60:385–394. 22. Massin N, Bohadana AB, Wild P, Hery M, Toamain JP, Hubert G. Respiratory symptoms and bronchial responsiveness in lifeguards exposed to nitrogen trichloride in indoor swimming pools. Occup Environ Med 1998;55: 258 –263. CHILDREN WITH VKC 563 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Clinical grading of vernal keratoconjunctivitis Stefano Bonini, Marta Sacchetti, Flavio Mantelli and Alessandro Lambiase Purpose of review The purpose of the present review is to provide an overview on the clinical features of vernal keratoconjunctivitis on the basis of cases series presented in the literature. Furthermore, a new grading system of vernal keratoconjunctivitis based on the severity of the disease is proposed. Different treatment options are discussed based on the clinical grade of vernal keratoconjunctivitis. Recent findings Recent epidemiological studies on the demographic, clinical and immunologic features of vernal keratoconjunctivitis are presented. The efficacy and complications of treatments are described. Summary Diagnosis and treatment of patients is a challenge for ophthalmologists as no precise diagnostic criteria have been established, the pathogenesis is unclear, and antiallergic treatments are often unsuccessful. This review describes old and new concepts of vernal keratoconjunctivitis diagnosis and treatment: the clinical features, the diagnostic criteria, the common features between this and other ocular allergies and the therapeutic strategies. On the basis of this knowledge, a new grading system is introduced based on clinical signs and symptoms of ocular surface inflammation. This new grading of vernal keratoconjunctivitis may help clinicians and researchers to classify disease activity and to establish a common agreement for treatments. Keywords allergy, antiallergic treatments, cyclosporine A, steroids, vernal keratoconjunctivitis Curr Opin Allergy Clin Immunol 7:436–441. ß 2007 Lippincott Williams & Wilkins. Department of Ophthalmology, University of Rome Campus Bio-Medico, Rome, Italy Correspondence to Stefano Bonini, MD, University of Rome Campus Bio-Medico, Department of Ophthalmology, Via E. Longoni, 83, 00155 Rome, Italy Tel: +39 0622541342; fax: +39 0622541456; e-mail: [email protected] Current Opinion in Allergy and Clinical Immunology 2007, 7:436–441 Abbreviations PAC SAC VKC perennial allergic conjunctivitis seasonal allergic conjunctivitis vernal keratoconjunctivitis ß 2007 Lippincott Williams & Wilkins 1528-4050 Introduction Vernal keratoconjunctivitis (VKC) is a disease usually contained under the classification of allergic conjunctivitis, together with seasonal allergic conjunctivitis (SAC), perennial allergic conjunctivitis (PAC) and atopic keratoconjunctivitis (AKC) [1]. In spite of common clinical and immunological features, however, VKC differs from other ocular allergic diseases for age of onset, clinical findings, low percentage of patients with positive response to standard allergic diagnostic tests and scarce response to antiallergic treatments [2]. The lack of standardized diagnostic criteria and the lack of a common language between physicians regarding the definition and the severity of VKC renders this disease more difficult to diagnose and treat. This article summarizes the authors’ longstanding clinical and research experience in order to propose a new clinical grading according to the clinical phases of VKC. This proposal may help physicians use a common language in the diagnosis and management of patients. Furthermore, this grading may be used to allow a more homogenous selection of patients for clinical trials. Definition and features of vernal keratoconjunctivitis VKC, an orphan disease characterized by severe chronic inflammation of the ocular surface, mainly affects boys in the first decade of life [2,3]. The disease is generally bilateral and is characterized by seasonal or perennial symptoms that exacerbate in spring or early autumn. During exacerbations, patients complain of symptoms of intense itching and photophobia associated with tearing and sticky mucus discharge. The disease may primarily involve the tarsal or limbal conjunctiva leading to different forms of VKC: limbal, tarsal or mixed (both limbal and tarsal) forms. The presence of giant papillae with a cobblestone-like appearance in the upper tarsal conjunctiva or at the limbus is considered the hallmark of the disease. These signs are associated with the presence of Horner–Trantas dots and corneal involvement, ranging from superficial punctuate keratopathy to corneal erosions and shield ulcers [2,3,4–6]. VKC generally has a good prognosis; however, in our case series of 195 patients with VKC, 6% of patients showed a permanent reduction in visual acuity as a result of corneal damage [2,7–9]. Treatments Topical steroids have been shown to be effective in controlling ocular surface inflammation in VKC, but they 436 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Vernal keratoconjunctivitis Bonini et al. 437 should be used with particular caution due to their ocular side effects including glaucoma and cataract formation [2,4,9–13]. As alternative, new topical agents with dual antiallergic activity (mast cell stabilizers and antihistamine) may be used for long-term treatment of allergic inflammation, to alleviate signs and symptoms of the disease [4,12,14–20]. In spite of treatments, symptoms and signs of severe forms are not adequately controlled unless prolonged steroid-based therapies are used [2,3,4]. Recently, topical treatment with cyclosporine A 1% and 2% eye-drops has been proposed for patients with VKC. The efficacy of topical cyclosporine A in VKC has been already reported, but it requires further investigations with long-term randomized clinical studies [21–28]. Common features of vernal keratoconjunctivitis and other ocular allergies Patients with VKC may share several anamnestic, diagnostic and clinical features with other ocular allergic diseases. For instance, a personal or family history of allergy is detectable in approximately 50% of patients with VKC and is a common finding in patients with seasonal or perennial allergic conjunctivitis [1,2,3,29]. In addition, atopic dermatitis is present in 10% of VKC patients and is constantly present in patients with atopic keratoconjunctivitis [1,30]. High levels of specific serum IgEs as well as a positive skin test to common allergens are constant features of almost all ocular allergies. The presence of specific IgEs to seasonal or perennial allergens can be documented in approximately all cases of SAC and PAC, but many patients with VKC show a negative response to skin test or radioallergosorbent test (RAST) for common allergens. In agreement with other studies, our case series of VKC reported positive skin tests in 58% of patients with VKC, and positive RAST tests in 52% of patients [2,3,31]. Signs and symptoms of VKC may not always allow a differential diagnosis with respect to other ocular allergic diseases. Severe forms of VKC typically present with a cobblestone-like appearance of the upper tarsal or limbal conjunctiva, associated with mucus discharge, corneal damage and intense itching. These severe forms are easily distinguishable from other allergic conjunctivitis, while the mildest forms do not always show these typical manifestations. Indeed, the presence of giant papillae may certainly orient towards a diagnosis of VKC. Corneal involvement is present in the clinical picture of AKC and VKC, but not in SAC and PAC. The presence of severe itching is a constant feature of VKC that is detectable in more than 96% of patients. Moderate to intense itching, however, is a common feature of all ocular allergic diseases representing a diagnostic criterion to distinguish allergic conjunctivitis from other diseases of the ocular surface [1,2,3,4,31]. The presence of eosinophils in the conjunctival epithelium may be an additional tool to distinguish VKC from other diseases of the ocular surface. This feature is present in 85% of conjunctival scrapings of VKC patients, but may also be present in patients with other allergic conjunctivitis [1,2,31]. Variability of clinical features of vernal keratoconjunctivitis The term ‘vernal’ does not appropriately describe the course of the disease in a significant percentage of patients. In fact, most patients in our series of 195 patients showed a seasonal presentation of the disease, but 23% of patients showed a perennial form and 60% of patients also had recurrences during winter [2]. In addition, prolonged inflammation (presence of the disease for more than 3 years) leads to a greater chance of developing perennial symptoms all year round [2,7]. Moreover, intermittent daily worsening of symptoms (during seasonal recurrences) is a common finding in most of our patients. These daily changes have not been adequately studied but they seem to be related to unknown environmental factors and are scarcely influenced by antiallergic treatments. Symptom variability could influence the outcome of clinical trials especially if patient enrolment occurs during a prolonged period of time [2,32,33]. Signs of the disease are also extremely variable in severity and presentation: cobblestone-like appearance or mild papillary reaction, corneal keratopathy or absence of corneal involvement, signs of conjunctival fibrosis or conjunctival oedema. These findings are not necessarily all simultaneously present in VKC patients, but may represent different evolutions of the disease. Clinical grading of vernal keratoconjunctivitis Based on the clinical characteristics of VKC, we suggest the following grading system to be adopted to grade the severity of the disease at the moment of the clinical examination (Table 1): (1) Grade 0, quiescent: the disease was present in the past (i.e. 1 year ago during spring season) or in the recent present, but at the time of visit the patient is free of symptoms, the conjunctiva shows no (or mild) hyperemia and the cornea is not involved in the allergic reaction. In contrast to the lack of symptoms and ocular involvement, papillae may be present (þ1 up to þ3 papillary reaction), although they are not inflamed. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Absent Absent/mild (2) Grade 1, mild intermittent: the patient refers to onset of symptoms, such as itching and mild photophobia, during spring season. Occasional symptoms may be present during the day but their occurrence is short and well tolerated. Slight ocular inflammation leads to mild hyperemia (Fig. 1a) but no corneal involvement. Once again, papillary reaction ranges from þ1 to þ3, and giant papillae may be present notwithstanding the mild form of the disease (Fig. 1b). (3)Grade2,moderate,isdividedintotwomoregradesbased on the severity of symptoms and corneal involvement: (a) 2A Moderate intermittent: symptoms occur as in grade 1 but they are more frequent and disturbing during the day. Mild conjunctival secretion and tearing may interfere with daily activity. Mild to severe papillary reaction may be observed as well as conjunctival hyperemia (Fig. 1c), without corneal involvement. (b) 2B Moderate persistent the patient is symptomatic every day during season. Conjunctival hyperemia, secretion and itching occur everyday. The cornea may be occasionally involved with superficial punctuate keratitis. Mild to severe papillary reaction may be observed (Fig. 1d). (4) Grade 3, severe: symptoms are present everyday and the patient’s daily activities are influenced by the intense itching and photophobia. Moderate to severe conjunctival hyperemia (Fig. 1e) and secretion may be associated to the presence of Horner-Trantas dot. The cornea is involved with superficial punctuate keratitis. Mild to severe conjunctival papillae (þ3 to þ4 papillary reaction) are common, and present injection and swelling (Fig. 1f). (5) Grade 4, very severe: severe itching and photophobia are present everyday with mucus discharge on the ocular surface and between papillae. Presence of superficial keratopathy or corneal erosions and ulcerations are common features. Horner-Trantas dots are present (more than 3), and mild to severe papillary reaction (þ3 to þ4), with injection and swelling, is usual (Fig. 1g,h). VKC, vernal keratoconjunctivitis. Severe Moderate to severe Absent or mild and occasional symptoms Grade 5, evolution Grade 4, very severe Few Trantas dots may be present Numerous Trantas dots may be present Absent Moderate to severe Superficial punctuate keratitis may be present Superficial punctuate keratitis Corneal erosion or ulceration. Absent Absent Mild to severe Moderate to severe with injection and swelling Moderate to severe with injection and swelling Mild to severe fibrosis Mild to moderate Moderate to severe Mild to moderate Grade 0, quiescent Grade 1, mild intermittent Grade 2A, moderate intermittent Grade 2B, moderate persistent Grade 3, severe Table 1 Clinical grading (from 0 to 5) of vernal keratoconjunctivitis Corneal involvement Absent Absent Absent Trantas dot Absent Absent Absent Papillary reaction Mild to moderate Mild to moderate Mild to severe Conjunctival secretion Absent Absent/mild Mild Conjunctival hyperemia Absent/mild Mild Mild Symptoms Absent Mild and occasional symptoms Mild to moderate intermittent symptoms Mild to moderate persistent symptoms Moderate to severe persistent symptoms Severe and persistent symptoms VKC grading 438 Eye allergy (6) Grade 5, evolution: the patient has occasional symptoms during seasonal periods. Conjunctival papillary reaction may be present but the cornea is spared. Evidence of conjunctival fibrosis may be seen on the upper tarsal conjunctiva or at the fornix (Fig. 1i,j). Treatment options Although clinicians in the field of ocular allergies agree that there are several features to aid in their differential diagnosis, once they describe therapeutic options, they generally refer to the wide range of topical treatments rather than considering different treatments for the different clinical Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Vernal keratoconjunctivitis Bonini et al. 439 Figure 1 Clinical grading of vernal keratoconjunctivitis The patient with vernal keratoconjunctivitis (VKC) at grade 1 shows mild conjunctival hyperemia (a) and papillary reaction (b). The grade 2 of the disease is characterized by moderate conjunctival hyperemia (c) and papillary reaction (d). More severe ocular surface inflammation (e) with severe papillary reaction (f) are present in grade 3. Severe corneal involvement and ocular surface inflammation with limbal reaction (g) or papillary injection with mucous discharge (h) are considered as grade 4. Conjunctival fibrosis at upper tarsal (i) or fornix (j) are associated with VKC evolution phase (grade 5). presentations. At present, there is a wide range of antiallergic drugs in the market, but it must be highlighted that all these drugs are exclusively symptomatic, and have no effect on disease activity or progression. Although with peculiar features, the mildest grades of VKC may show many similarities to the other forms of allergic conjunctivitis, and thus they may all share a common therapeutic approach. On the other hand, the more severe grades (3 and 4) of the VKC require more effective and prompt therapeutic approaches. Indeed, therapeutic options must be considered on the basis of disease severity and personalized according to the single patient. In line with the proposed grading, we indicate possible therapeutic options as follows (Fig. 2): (1) Grade 0, quiescent: no treatment is needed in this phase of the disease; the usual approach is to wait for symptoms to occur. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 440 Eye allergy Figure 2 Different therapeutic approach proposed for the different vernal keratoconjunctivitis grades Grade 4 Very severe Grade 2 Moderate: A intermittent, B persisent Grade 3 Severe Topical pulsed steroid (high dose) Topical pulsed steroid Cyclosporine A eye drops Grade 1 Mild intermittent Grade 0 Quiescent No treatments Occasional Antiallergic eye drops Daily administration Antiallergic eye drops (Mast cell stabilizers/ anti-histamine/ Dual action) Grade 5 Evolution Occasional Antiallergic eye drops Lubricant eye drops/Vasoconstrictors eye drops when necessary (2) Grade 1, mild intermittent: topical vasoconstrictors or antihistamines may be used in this phase to control the mild symptoms of the disease. (3) Grade 2, moderate, in grades 2A and 2B the therapeutic approach is based on the use of topical antiallergic eye drops such as antihistamines or mast cell stabilizers, as well as dual action antiallergic drugs: (a) 2A Moderate intermittent: brief courses of antiallergic eye drops may be used to control symptoms of the disease. Topical steroid are not required in this grade of the disease. (b) 2B Moderate persistent: daily administration of topical antiallergic drugs are generally required to maintain low levels of ocular inflammation and to control daily manifestations of the disease. (4) Grade 3, severe: daily administration of topical antihistamines or mast cell stabilizers, as well as dual action antiallergic drugs, is recommended. As a possible alternative, topical cyclosporine A from 1% to 2%, has been proposed [21–28]. In addition, a brief pulse of high dose topical steroids may be considered in the recurrences of ocular inflammation. Moreover, to avoid possible abuses, steroids should be prescribed in a controlled regimen, in order to reach a maximum of 10 drops per month, during season. (5) Grade 4, very severe: in general, treatment recommendations are the same as for grade 3, although topical steroids are more frequently required. Clinicians should strongly suggest patients to record the use of steroid eye drops in order to limit possible abuses. The use of topical cyclosporine should be considered on a daily basis. When present, corneal shield ulcers should be surgically removed. No treatment is necessary for the presence of giant papillae, including cryotherapy. (6) Grade 5, evolution: brief courses of topical antihistamines or mast cell stabilizers, as well as multiple active antiallergic drugs, may be used to control symptoms of the disease during recurrences. No further therapies are required during periods of disease quiescence. Conclusion In conclusion, we have proposed a new grading for clinical signs and symptoms of VKC patients. We believe that grading of the disease may allow a clearer evaluation of disease severity and progression, and may aid in the choice of therapeutic options and analysis of treatment efficacy. Grading of the disease may also help create a common language among researchers and clinicians, allowing a more careful and homogenous selection of patients for enrolment in clinical trials. Further clinical trials are necessary to validate the proposed grading system. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 456). 1 Bielory L. Allergic and immunologic disorders of the eye. Part II: Ocular allergy. J Allergy Clin Immunol 2000; 106:1019–1032. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Vernal keratoconjunctivitis Bonini et al. 441 2 Bonini S, Bonini S, Lambiase A, et al. Vernal keratoconjunctivitis revisited: a case series of 195 patients with long-term follow-up. Ophthalmology 2000; 107:1157–1163. Leonardi A, Busca F, Motterle L, et al. Case series of 406 vernal keratoconjunctivitis patients: a demographic and epidemiological study. Acta Ophthalmol Scand 2006; 84:406–410. This study evaluated the specific allergic sensitization and epidemiological characteristics based on data collected in 406 cases of vernal keratoconjunctivitis (VKC). 3 17 Lee S, Allard TR. Lodoxamide in vernal keratoconjunctivitis. Ann Pharmacother 1996; 30:535–537. 18 Foster CS. Evaluation of topical cromolyn sodium in the treatment of vernal keratoconjunctivitis. Ophthalmology 1988; 95:194–201. 19 Avunduk AM, Tekelioglu Y, Turk A, Akyol N. Comparison of the effects of ketotifen fumarate 0.025% and olopatadine HCl 0.1% ophthalmic solutions in seasonal allergic conjunctivities: a 30-day, randomized, double-masked, artificial tear substitute-controlled trial. Clin Ther 2005; 27:1392– 1402. 4 Leonardi A. Vernal keratoconjunctivitis: pathogenesis and treatment. Prog Retin Eye Res 2002; 21:319–339. 5 Pucci N, Novembre E, Lombardi E, et al. Atopy and serum eosinophil cationic protein in 110 white children with vernal keratoconjunctivitis: differences between tarsal and limbal forms. Clin Exp Allergy 2003; 33:325–330. 6 Tuft SJ, Dart JK, Kemeny M. Limbal vernal keratoconjunctivitis: clinical characteristics and immunoglobulin E expression compared with palpebral vernal. Eye 1989; 3 (Pt 4):420–427. 22 Mendicute J, Aranzasti C, Eder F, et al. Topical cyclosporin A 2% in the treatment of vernal keratoconjunctivitis. Eye 1997; 11:75–78. 7 Bonini S, Coassin M, Aronni S, Lambiase A. Vernal keratoconjunctivitis. Eye 2004; 18:345–351. 23 Secchi AG, Tognon MS, Leonardi A. Topical use of cyclosporine in the treatment of vernal keratoconjunctivitis. Am J Ophthalmol 1990; 110:641– 645. 8 Iqbal A, Jan S, Babar TF, Khan MD. Corneal complications of vernal catarrh. J Coll Physicians Surg Pak 2003; 13:394–397. 9 Tabbara KF. Ocular complications of vernal keratoconjunctivitis. Can J Ophthalmol 1999; 34:88–92. 10 Tabbara KF, al-Kharashi SA. Efficacy of nedocromil 2% versus fluorometholone 0.1%: a randomised, double masked trial comparing the effects on severe vernal keratoconjunctivitis. Br J Ophthalmol 1999; 83:180–184. 11 Akman A, Irkec M, Orhan M. Effects of lodoxamide, disodium cromoglycate and fluorometholone on tear leukotriene levels in vernal keratoconjunctivitis. Eye 1998; 12 (Pt 2):291–295. 12 Coutu RB. Treatment of vernal keratoconjunctivitis: a retrospective clinical case study. Wallace F. Molinari Ocular Pharmacology Award. Optom Vis Sci 1991; 68:561–564. 13 Dada T, Konkal V, Tandon R, et al. Corneal topographic response to intraocular pressure reduction in patients with vernal keratoconjunctivitis and steroid-induced glaucoma. Eye 2007; 21:158–163. 14 Kosrirukvongs P, Vichyanond P, Wongsawad W. Vernal keratoconjunctivitis in Thailand. Asian Pac J Allergy Immunol 2003; 21:25–30. 15 Verin P, Allewaert R, Joyaux JC, et al., Lodoxamide Study Group. Comparison of lodoxamide 0.1% ophthalmic solution and levocabastine 0.05% ophthalmic suspension in vernal keratoconjunctivitis. Eur J Ophthalmol 2001; 11:120– 125. 16 Verin PH, Dicker ID, Mortemousque B. Nedocromil sodium eye drops are more effective than sodium cromoglycate eye drops for the long-term management of vernal keratoconjunctivitis. Clin Exp Allergy 1999; 29:529–536. 20 Abelson MB. A review of olopatadine for the treatment of ocular allergy. Expert Opin Pharmacother 2004; 5:1979–1994. 21 BenEzra D, Matamoros N, Cohen E. Treatment of severe vernal keratoconjunctivitis with cyclosporine A eyedrops. Transplant Proc 1988; 20 (Suppl 2): 644–649. 24 Gupta V, Sahu PK. Topical cyclosporin A in the management of vernal keratoconjunctivitis. Eye 2001; 15:39–41. 25 Bleik JH, Tabbara KF. Topical cyclosporine in vernal keratoconjunctivitis. Ophthalmology 1991; 98:1679–1684. 26 Spadavecchia L, Fanelli P, Tesse R, et al. Efficacy of 1.25% and 1% topical cyclosporine in the treatment of severe vernal keratoconjunctivitis in childhood. Pediatr Allergy Immunol 2006; 17:527–532. 27 Pucci N, Novembre E, Cianferoni A, et al. Efficacy and safety of cyclosporine eyedrops in vernal keratoconjunctivitis. Ann Allergy Asthma Immunol 2002; 89:298–303. 28 Kilic A, Gurler B. Topical 2% cyclosporine A in preservative-free artificial tears for the treatment of vernal keratoconjunctivitis. Can J Ophthalmol 2006; 41:693–698. 29 Friedlaender MH. Current concepts in ocular allergy. Ann Allergy 1991; 67:5–13. 30 Bonini S. Atopic keratoconjunctivitis. Allergy 2004; 59 (Suppl 78):71– 73. 31 Bonini S, Bonini S. Studies of allergic conjunctivitis. Chibret Int J 1987; 5:12– 22. 32 Bonini S, Bonini S. IgE and non-IgE mechanisms in ocular allergy. Ann Allergy 1993; 71:296–299. 33 Bonini S, Bonini S, Schiavone M, et al. Conjunctival hyperresponsiveness to ocular histamine challenge in subjects with vernal conjunctivitis. J Allergy Clin Immunol 1992; 89:103–107. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Molecular Vision 2007; 13:1562-7 <http://www.molvis.org/molvis/v13/a173/> Received 17 March 2007 | Accepted 25 August 2007 | Published 30 August 2007 ©2007 Molecular Vision Natural killer cells in vernal keratoconjunctivitis Alessandro Lambiase,1 Eduardo Maria Normando,1 Laura Vitiello,3 Alessandra Micera,1,2 Marta Sacchetti,1 Eleonora Perrella,6 Luigi Racioppi,3 Sergio Bonini,4,5 Stefano Bonini1 1 Interdisciplinary Center for Biomedical Research (CIR), Laboratory of Ophthalmology, University Campus Bio-Medico of Rome, Rome Italy; 2IRCCS-G.B. Bietti Eye Foundation, Rome, Italy; 3Department of Biology and Molecular and Cellular Pathology, Second University of Naples, Naples, Italy; 4Internal Medicine, Second University of Naples; Naples, Italy; 5Institute of Neurobiology and Molecular Medicine, National Research Council (INMM-CNR) of Rome, Italy; 6Pathology, University Campus Bio-Medico of Rome, Italy Purpose: Recent studies suggest that natural killer (NK) cells exert effector/regulatory properties on both innate and adaptive responses via release of different cytokines. While some information indicates NK cells in allergic asthma and atopic dermatitis, no data are available for allergic conjunctivitis. The aim of this study was to evaluate NK in the blood and the conjunctiva of patients with vernal keratoconjunctivitis (VKC). Methods: Six patients with active VKC and six healthy subjects were included in the study. Blood samples and conjunctival biopsies were taken from each patient. NK cells in blood and conjunctiva were quantified by flow cytometry and immunohistochemistry, respectively. Clinical findings of the patients were recorded, conjunctival immune infiltrates were characterized, and both parameters were correlated to NK cell number. Results: Compared to healthy subjects, NK cells were significantly decreased in the blood and increased in the conjunctiva of patients with VKC. Conclusions: Together with lymphocytes, eosinophils, and mast cells, NK cells constitute a significant proportion of the immune cells infiltrating VKC conjunctiva. This finding indicates a potential role of NK and innate immunity in the regulation of allergic reactions and in diseases such as VKC. New therapeutic alternatives for modulating allergic inflammation might target NK cells. Vernal keratoconjunctivitis (VKC) is a chronic bilateral inflammation of the conjunctiva characterized by hyperemia, chemosis, photophobia, and mucous discharge. The hallmark of the disease is the presence of giant papillae on the upper tarsal conjunctiva or limbus [1]. Originally classified as an allergic conjunctivitis-type disease, VKC was considered for many years to be the expression of a classical type I immunoglobulin-E (IgE)-mediated hypersensitivity reaction of the conjunctiva, according to the Coombs and Gell definition [2]. Recent studies have demonstrated that only 50% of patients with VKC are sensitive to the skin/Radio-Allergo-Sorbent Test (skin/RAST) [3], and that a more complex IgE-independent pathogenic mechanism is involved with the contribution of many cells and mediators detected in the serum, tears, and conjunctiva of patients with VKC [4]. Conjunctival allergen challenge and biochemical/functional studies indicate a Th2driven mechanism [5,6] for VKC, and a definition, similar to that of asthma, of “an allergic inflammatory disease with mast cells, eosinophils and lymphocytes” [7]. This traditional view of the allergic reaction has been recently revisited, and an adjuvant role of innate immunity in allergic inflammation has been proposed. Epithelial cells, dendritic cells (prototypical antigen presenting cells), natural killer (NK) cells, as well as mast cells/basophils, through expression of various membrane receptors, particularly toll-like receptors (TLR), might also participate in the innate immunity response by playing a regulatory role in allergy [8-10]. In line with this hypothesis, TLRs have been shown to be differentially expressed in the conjunctiva of patients with VKC when compared to healthy subjects [11]. NK cells are granular lymphocytes that play a crucial role in innate immunity due to their cytotoxic ability to destroy virus-infected cells and certain tumor cell targets without prior stimulation [12]. Recent data suggest that a subpopulation of NK cells (NK-T) are important regulators of both innate and acquired response through the release of several immunoregulatory mediators (including TNFα, TNFβ, TGFβ, GM-CSF, MIP-1α, IL-1, IL-2, IL-3, IL4, IL5, IL-8, IL10 and IL-13) [13-16]. Indeed, NK cells can be triggered by specific T-cell receptor activation to secrete cytokines that promote Th2 responses instead of Th1 responses, suggesting that NK cells might play a role in regulating the balance between Th1/ Th2 lymphocytes [15,16]. In the present study, we sought to compare NK cells in the peripheral blood and in the conjunctiva of VKC patients with those of healthy subjects. Correspondence to: Professor Stefano Bonini, M.D., Chair of Ophthalmology, University Campus Bio-Medico, Via Emilio Longoni 83, 00155 Rome Italy; Phone: +39 06-22541342; FAX: +39 0622541456; email: [email protected] METHODS Patients and tissue handling: The study, performed in spring, involved six patients with active VKC (males between 7-35 years old) and six healthy subjects with no history of atopic 1562 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Molecular Vision 2007; 13:1562-7 <http://www.molvis.org/molvis/v13/a173/> ©2007 Molecular Vision ocular/systemic diseases (males between 24-26 years old). VKC diagnosis was based on the presence of recurrent symptoms of itching, photophobia, and tearing in early spring. The symptoms were associated with a mild to severe cobblestonelike appearance in the upper tarsal conjunctiva, mucous discharge, epithelial keratopathy, and at least two eosinophils per optic field (x100/oil immersion) in the conjunctival scraping. Two out of six patients showed sensitization to Dermatophagoides pteronissinus with a skin prick test. Symptoms (itching, tearing, and photophobia) and signs (tarsal and bulbar papillae, hyperemia, secretion, and corneal epithelial involvement) were recorded and graded on a scale of 0-3 (0, absent; 1, weak; 2, mild; and 3, severe). A total symptom score (from 0-9) and a total sign score (from 0-15) were calculated. Table 1 summarizes the characteristics of the patients included in the study. One physician (A.L.) carried out all ocular examinations by slit lamp biomicroscopy. All patients were without treatment for at least one week prior to the study. Blood samples and conjunctival biopsies were collected from both VKC and healthy subjects and flow cytometry and immunohistochemistry were performed to identify circulating and conjunctival NK cells. Conjunctival biopsy from healthy subjects was performed at the time of surgery, for strabismus. The study adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from both VKC and healthy subjects, and institutional human experimentation committee approval was granted. Flow cytometry: For specific staining, 150 µl of peripheral blood were pretreated with NH4CL-EDTA for 10 min at room temperature to lyse erythrocytes It was then incubated for 30 min at 4 °C with CD3-FITC, CD16-PE, or CD56-PerCP conjugated monoclonal antibodies, according to the manufacturer’s instructions (Simultest CD3/CD16+CD56; Code 340042; Becton Dickinson Biosciences, San Jose, CA). All samples were analyzed through a FACScan flow cytometer (Becton Dickinson) equipped with an argon laser emitting at 488 nm. Forward and side scatter signals were collected as linear signals, and all emissions were collected on a four-decade logarithmic scale. FITC, PE, and PerCP signals were measured, respectively at 530 nm, 575 nm and 670 nm. Spectral overlap was minimized by electronic compensation with Calibrite beads (Becton Dickinson) before each series. CellQuest® software (Becton Dickinson) was used to acquire and evaluate 5,000 events with preserved side scatter signals and high membrane staining for specific antibodies. Histological and immunohistochemical evaluations: Conjunctival specimens were post-fixed in 4% ρ-formaldehyde-100 mM phosphate-buffered saline (PBS) and cut in 3 µm sections (HM 325 Microm; BioOptica, Milan, Italy). Sections were either processed for basic histology (hematoxylin and eosin; BioOptica), immunohistochemistry, or immunofluorescence. Briefly, sections were stained with monoclonal antihuman CD4 (1/50; Dako Corp, Carpenteria, CA), EG2 (1/ 50; Peninsula, Upsala, Sweden), AA1 (1/200; R&D Systems, Minneapolis, MN), and CD56 (undiluted clone T199; BD) antibodies and developed using ABC-peroxidase (Vectastain Elite ABC, Vector Laboratories, Inc., Burlingame, CA) or with the appropriate fluorescent technique. As a negative control, sections were stained with a nonspecific isotype antibody. Thelper lymphocytes (CD4+), activated eosinophils (EG2+), mast cells (AA1+), and NK cells (CD56+) were counted under light microscopy, in a blind-fashion, in three optic fields (40X magnification) for each of three randomly selected areas. Arkon32 software (version 10.06.06) was used for image analysis. Data are shown as mean±SEM. For fluorescent analysis, specific binding of the primary antibody was detected using a cy2-conjugated donkey antimouse-antirabbit-antigoat antibody (Jackson Laboratory, West Grove, PA), and evaluated at 60X magnification in an oil immersion (E2000U confocal microscope; Nikon, Tokyo, Japan). Brightness and contrast level were evaluated using C1 software (Nikon), and the images were transferred to the Adobe Photoshop 7.0 program (Adobe Systems Inc., San Jose, CA). To detect nonspecific binding, we performed control immunofluorescent staining by substituting the primary antibody with control isotype IgG (data not shown). Statistical evaluation: The nonparametric Mann-Whitney test (StatView II for PC; Abacus Concepts Inc., Barkley, CA) was used for statistical comparison of the number of circulating and conjunctival immune cells in patients with VKC versus healthy subjects. The Spearman rho test was employed to correlate the number of circulating and conjunctival NK cells to signs, symptoms, and IgE serum levels and to the number TABLE 1. CHARACTERISTICS OF PATIENTS WITH VERNAL KERATOCONJUNCTIVITIS This table summarizes the characteristics of the patients included in the study. 1563 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Molecular Vision 2007; 13:1562-7 <http://www.molvis.org/molvis/v13/a173/> ©2007 Molecular Vision of circulating and conjunctival eosinophils. Data are represented in graphs as mean±SEM, and p values less than 0.05 were considered statistically significant. RESULTS Circulating NK cells (CD56+ and CD16+) were decreased in patients with VKC compared to healthy subjects. The decrease in NK cells was significant when considering NK cell number (Figure 1A; VKC: 744±401 versus healthy subjects: 1554±649; p<0.05) as well as percent NK cells (CD56+ and CD16+ cells/ CD3+ cells; VKC: 5.6%±1.9% versus healthy subjects: 10.9%±5%; Figure 1B; p<0.05). Immunohistochemistry of the conjunctiva showed a significant increase of CD4+ T-helper lymphocytes (VKC: 56.1±17.5/optic field versus control: 5.6±2.6/optic field; p<0.01), activated eosinophils (VKC: 30.9±8.3/optic field versus control: 0±0/optic field; p<0.01) and AA1+ mast cells (VKC: 41.3±11.6/optic field versus control: 2.2±2.5/optic field; p<0.01) in patients with VKC compared to healthy subjects (Figure 2). CD56+-bearing NK cells were detected in all conjunctiva of patients with VKC and in 75% (4/6) of healthy subjects. Statistical analysis showed a significant (p<0.01) increase in the number of CD56+ cells infiltrating the conjunctiva of patients with VKC (24±10.5 cells/optic field) when compared to healthy subjects (5.9±2.6 cells/optic field; Figure 3). No significant correlations were observed between the number of circulating or conjunctival NK cells and the signs and symptoms scores or plasma IgE levels. DISCUSSION This report demonstrates that, relative to healthy normal subjects, patients with VKC have a decreased number of circulating NK cells and an increased number of NK cells together with Th lymphocytes, activated eosinophils, and mast cells in the conjunctiva. NK cells play a crucial role in innate immunity, having the ability to directly lyse virus-infected cells [12]. Aside from their cytotoxic activity, NK cells can produce and release high amounts of both Th1 (IFNγ) and Th2 (IL4, IL5, and IL13) cytokines, enabling these cells to also regulate acquired immune response [13-16]. A new concept proposes classifying NK cells by their predominant Th1 (NK-1) or Th2 (NK-2) cytokine pattern [15]. Both subsets might inhibit or stimulate aspects of the allergic and normal immune response by specific patterns of cytokine release [17]. Recently, it was proposed that NK cells play a crucial role in the pathogenesis of allergic diseases by altering the balance between Th1/Th2 lymphocytes. NK cells might be triggered to secrete cytokines (IL-4, IL-5, and IL-13) that promote Th2 rather than Th1 responses [15,16]. In an animal model of allergic asthma, Schuster and coworkers detected increased numbers of NK cells along with CD4 and CD8 expressing T cells in both bronchoalveolar lavage fluid (BALF) and lung parenchyma [18]. In a murine model of allergic peritonitis, NK-derived IL-5 contributed to the observed eosinophil infiltration [19]. Furthermore, NK-specific depletion inhibited allergen-induced pulmonary eosinophil and CD3-T cell infiltration, and blunted the increase of IL-4, IL-5, and IL-12 in BALF [20]. Allergenspecific immunotherapy in humans was also shown to decrease NK cell activity [21]. All these data indicate a key role of NK cells in the control of allergic disease. The present findings of decreased circulating NK in VKC patients appear at first to Figure 1. Circulating natural killer cells in patients with vernal keratoconjunctivitis compared to healthy subjects. A: Flow cytometry demonstrated a significant decrease of circulating natural killer cells (cells/µl) in vernal keratoconjunctivitis (VKC) patients compared to healthy subjects (p<0.05). B: The percent natural killer cell decrease was significant (CD56+ and CD16+ cells/CD3+ cells; p<0.05). 1564 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Molecular Vision 2007; 13:1562-7 <http://www.molvis.org/molvis/v13/a173/> ©2007 Molecular Vision diverge from the hypothesis that NK is an important regulator of the allergic reaction. However, similar decreases in circulating NK cells have been described in atopic diseases such as asthma and atopic dermatitis [22-24]. In these prior studies, the decrease of circulating NK cells was shown to not be due to any intrinsic defect in the ability of NK cells to proliferate, but merely reflected accumulation of these cells in the site of inflammation [22]. In line with this hypothesis, we demonstrated a significant increase of NK cells infiltrating the conjunctiva in inflamed VKC tissues. The number of NK cells in VKC conjunctiva was similar to that of eosinophils—cells long considered to be crucial players in the pathogenesis of VKC. This finding indicates that NK cells may be involved in the pathophysiology of VKC, modulating allergic inflammation through the release of cytokines that influence the balance between Th1 and Th2 responses and the resulting conjunctival eosinophil infiltration. Surprisingly, no significant correlation was observed between the number of circulating or conjunctival NK cells and the clinical signs and symptoms of VKC. This lack of correlation might indicate an all or none relationship with VKC that is unaffected by changes in the severity signs and symptoms. Conversely, it might simply reflect the low sample size of VKC Figure 2. Evaluation of inflammatory cells in the conjunctiva of patients with vernal keratoconjunctivitis. The graph (A) illustrates a significant increase of T-helper lymphocytes (CD4+/green; B), activated eosinophils (EG2+/green; C), and mast cells (AA1+/green; D) in vernal keratoconjunctivitis (VKC) conjunctiva compared to healthy subjects (E, F, G, respectively). No specific binding was detected when sections were incubated with isotype IgG (data not shown; 40X magnification). 1565 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Molecular Vision 2007; 13:1562-7 <http://www.molvis.org/molvis/v13/a173/> ©2007 Molecular Vision Figure 3. Immunohistochemical evaluation of natural killer cells in vernal keratoconjunctivitis conjunctiva. Graph A illustrates the significant increase of natural killer cells in the conjunctiva of patients with vernal keratoconjunctivitis (VKC) compared to healthy subjects. Natural killer cells were identified by CD56 expression quantified by immunohistochemistry (B=VKC and C=healthy subjects) and confocal analysis (D=VKC and E=healthy subjects). No specific immune staining was observed in the presence of isotype IgG (F=control staining; 40X magnification). 1566 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Molecular Vision 2007; 13:1562-7 <http://www.molvis.org/molvis/v13/a173/> patients. A more extensive study including a larger population is required to confirm these initial findings. Nonetheless, this study indicates a potential role of NK cells in VKC pathophysiology as well as a link between innate and specific immunity in allergic diseases. These novel findings appear of clinical relevance since a deeper understanding of the role of NK in VKC might lead to new pharmacological strategies focused on selective modulation of Th1 and Th2 polarization of NK cells, using factors such as IL-7, IL-15, and αgalactosylceramide [25,26]. ACKNOWLEDGEMENTS This study was presented in part at the 2006 Association for Research in Vision and Ophthalmology meeting (ARVO, Fort Lauderdale, FL). REFERENCES 1. Bonini S, Coassin M, Aronni S, Lambiase A. Vernal keratoconjunctivitis. Eye 2004; 18:345-51. 2. Coombs RRA, Gell PGH. The classification of allergic reactions underlying disease. In: Gell PGH, Coombs RRA, editors. Clinical Aspects of Immunology. London: Blackwell; 1963. p. 317320. 3. Bonini S, Bonini S, Lambiase A, Marchi S, Pasqualetti P, Zuccaro O, Rama P, Magrini L, Juhas T, Bucci MG. Vernal keratoconjunctivitis revisited: a case series of 195 patients with long-term followup. Ophthalmology 2000; 107:1157-63. 4. Leonardi A. Vernal keratoconjunctivitis: pathogenesis and treatment. Prog Retin Eye Res 2002; 21:319-39. 5. Maggi E, Biswas P, Del Prete G, Parronchi P, Macchia D, Simonelli C, Emmi L, De Carli M, Tiri A, Ricci M, Romagnani S. Accumulation of Th-2-like helper T cells in the conjunctiva of patients with vernal conjunctivitis. J Immunol 1991; 146:116974. 6. Metz DP, Bacon AS, Holgate S, Lightman SL. Phenotypic characterization of T cells infiltrating the conjunctiva in chronic allergic eye disease. J Allergy Clin Immunol 1996; 98:686-96. 7. Bonini S, Bonini S. IgE and non-IgE mechanisms in ocular allergy. Ann Allergy 1993; 71:296-9. 8. Dombrowicz D. Exploiting the innate immune system to control allergic asthma. Eur J Immunol 2005; 35:2786-8. 9. Lambrecht BN, Hammad H. Taking our breath away: dendritic cells in the pathogenesis of asthma. Nat Rev Immunol 2003; 3:994-1003. 10. Marone G, Triggiani M, de Paulis A. Mast cells and basophils: friends as well as foes in bronchial asthma? Trends Immunol 2005; 26:25-31. 11. Bonini S, Micera A, Iovieno A, Lambiase A, Bonini S. Expression of Toll-like receptors in healthy and allergic conjunctiva. Ophthalmology 2005; 112:1528; discussion1548-9. 12. Kiessling R, Klein E, Wigzell H. “Natural” killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to geno- ©2007 Molecular Vision type. Eur J Immunol 1975; 5:112-7. 13. Biron CA. Activation and function of natural killer cell responses during viral infections. Curr Opin Immunol 1997; 9:24-34. 14. Scharton-Kersten TM, Sher A. Role of natural killer cells in innate resistance to protozoan infections. Curr Opin Immunol 1997; 9:44-51. 15. Stuhlsatz HW, Eberhard A, Kristin H, Vojtisek O, Greiling H. [Glycosaminoglycans in the synovial fluid of chronic joint diseases (proceedings)]. Z Rheumatol 1976; 35:suppl444-54. 16. Hoshino T, Winkler-Pickett RT, Mason AT, Ortaldo JR, Young HA. IL-13 production by NK cells: IL-13-producing NK and T cells are present in vivo in the absence of IFN-gamma. J Immunol 1999; 162:51-9. 17. Korsgren M. NK cells and asthma. Curr Pharm Des 2002; 8:18716. 18. Schuster M, Tschernig T, Krug N, Pabst R. Lymphocytes migrate from the blood into the bronchoalveolar lavage and lung parenchyma in the asthma model of the brown Norway rat. Am J Respir Crit Care Med 2000; 161:558-66. 19. Walker C, Checkel J, Cammisuli S, Leibson PJ, Gleich GJ. IL-5 production by NK cells contributes to eosinophil infiltration in a mouse model of allergic inflammation. J Immunol 1998; 161:1962-9. 20. Korsgren M, Persson CG, Sundler F, Bjerke T, Hansson T, Chambers BJ, Hong S, Van Kaer L, Ljunggren HG, Korsgren O. Natural killer cells determine development of allergen-induced eosinophilic airway inflammation in mice. J Exp Med 1999; 189:55362. 21. Sin B, Misirligil Z, Aybay C, Gurbuz L, Imir T. Effect of allergen specific immunotherapy (IT) on natural killer cell activity (NK), IgE, IFN-gamma levels and clinical response in patients with allergic rhinitis and asthma. J Investig Allergol Clin Immunol 1996; 6:341-7. 22. Ikegami Y, Yokoyama A, Haruta Y, Hiyama K, Kohno N. Circulating natural killer T cells in patients with asthma. J Asthma 2004; 41:877-82. 23. Oishi Y, Sakamoto A, Kurasawa K, Nakajima H, Nakao A, Nakagawa N, Tanabe E, Saito Y, Iwamoto I. CD4-CD8- T cells bearing invariant Valpha24JalphaQ TCR alpha-chain are decreased in patients with atopic diseases. Clin Exp Immunol 2000; 119:404-11. 24. Takahashi T, Nakamura K, Chiba S, Kanda Y, Tamaki K, Hirai H. V alpha 24+ natural killer T cells are markedly decreased in atopic dermatitis patients. Hum Immunol 2003; 64:586-92. 25. Hong S, Wilson MT, Serizawa I, Wu L, Singh N, Naidenko OV, Miura T, Haba T, Scherer DC, Wei J, Kronenberg M, Koezuka Y, Van Kaer L. The natural killer T-cell ligand alphagalactosylceramide prevents autoimmune diabetes in non-obese diabetic mice. Nat Med 2001; 7:1052-6. 26. van der Vliet HJ, Nishi N, Koezuka Y, von Blomberg BM, van den Eertwegh AJ, Porcelli SA, Pinedo HM, Scheper RJ, Giaccone G. Potent expansion of human natural killer T cells using alpha-galactosylceramide (KRN7000)-loaded monocytederived dendritic cells, cultured in the presence of IL-7 and IL15. J Immunol Methods 2001; 247:61-72. The print version of this article was created on 30 Aug 2007. This reflects all typographical corrections and errata to the article through that date. Details of any changes may be found in the online version of the article. α 1567 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Manuscript . 1 2 3 Tailored approach to the treatment of vernal keratoconjunctivitis 4 5 Marta Sacchetti MD1 , Alessandro Lambiase MD PhD1, Flavio Mantelli MD1, Velika 6 Deligianni MD2, Andrea Leonardi MD2Stefano Bonini MD1 7 8 1 9 2 Department of Ophthalmology, University of Rome, Campus Bio-Medico, Rome, Italy Dept. of Neuroscience, Ophthalmology Unit, University of Padua, Padua, Italy 10 11 Meeting presentation: None 12 Financial Support: None 13 Conflict of Interest: No authors have any financial/conflicting interests to disclose 14 Running head: clinical grading of vernal keratoconjunctivitis 15 16 17 18 Address for reprints: 19 Prof. Stefano Bonini 20 Department of Ophthalmology 21 University of Rome, Campus Bio-Medico 22 Via Alvaro del Portillo, 21 23 00128 Rome, Italy 24 Phone number. +3906225419185 25 Fax. +390622541456 26 E-mail. [email protected] 27 28 1 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 29 Abstract 30 Purpose: To develop a standardized clinical grading system for the management of vernal 31 keratoconjunctivitis (VKC) patients and to identify the risk factors associated with a worsened 32 outcome of the disease. 33 Design: Retrospective cohort study 34 Participants: Two-hundred seven consecutive patients with VKC, referred to our center from 35 1997 to 2007, were included in the study. One-hundred ten of those patients were included in 36 the follow-up study (range 1-10 years). 37 Methods: Classification tree analysis (CART®) was performed to separate the patients into 5 38 subgroups by therapeutic approach. Regression tree analysis and multivariate logistic 39 regression analysis were performed during follow-up to identify predictors of worse visual 40 outcome. 41 Main Outcome Measures: Age, gender, duration and course of disease, signs, symptoms, 42 overall symptoms score, history of atopy, markers of allergy, best corrected visual acuity and 43 therapy were collected at baseline and during follow up. Number of relapses and the final best 44 corrected visual acuity were also evaluated in follow-up. 45 Results: A decision tree for VKC treatment was developed by CART analysis and a new 46 clinical grading system was proposed accordingly. Sixteen patients were classified as grade 0 47 (absence of symptoms and no therapy); 59 patients were grade 1 (presence of symptoms 48 without photophobia, occasional use of anti-allergic eye drop); 74 patients were graded as 2 49 (presence of symptoms including photophobia, daily anti-allergic treatment); 22 patients with 50 superficial punctuate keratopathy were graded as 3 (daily anti-allergic treatment associated 51 with occasional topical steroid); and 36 were graded as 4 (diffuse SPK and/or corneal ulcer; 52 pulsed high dose topical steroid). A higher number of relapses and a higher baseline grade of 53 VKC were the main predictor factors for worse visual outcome. 2 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 54 Conclusions: This grading system allows for identifying the more severe forms of VKC that 55 are at higher risk of recurrences, corneal ulcer, and worse final visual outcome. 56 3 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 57 Introduction 58 Vernal keratoconjunctivitis (VKC) is a chronic allergic disease with seasonal recurrences of 59 ocular surface inflammation characterised by intense itching, tearing, photophobia and 60 mucous discharge, associated with conjunctival hyperemia and chemosis.1-3 Most severe cases 61 show corneal signs of inflammation ranging from superficial punctate keratitis to corneal 62 ulcers that can lead to visual impairment.1,4, 5 Signs and symptoms of VKC are variable and 63 may present with daily fluctuations in individual patients. Episodes of acute itching and 64 redness lasting for several hours are often referred by the patients’ parents following short 65 symptom-free periods. 66 are variable among patients. As a consequence, the management of VKC is a challenge to 67 ophthalmologists, since the therapy needs frequent adjustments according to the specific 68 needs of each patient.1,2,6,8,9 69 Anti-allergic eye drops have been shown to control ocular signs and symptoms when used on 70 a routine basis while acute episodes of the disease require more potent agents such as ocular 71 topical steroids.8 These compounds are very effective but their use is limited due to possible 72 complications associated with chronic use, such as cataract development and glaucoma.1-5, 9-10 73 A standardized clinical grading system based on VKC severity has been recently proposed in 74 order to provide a common description of the disease and to avoid selection bias in the 75 recruitment of patients for clinical trials.11 However, a standardized therapeutic approach 76 corresponding to these different grades has not yet been defined. 77 In this study, we retrospectively review the clinical records of 207 patients with VKC 78 observed in our cornea and external disease Unit over the last ten years. On the basis of the 79 treatments prescribed in this patient population, we developed a decision tree for VKC 80 treatment that accurately reflects the clinical grading of the disease. We also identified the 4 1,3,6,7 Intervals between acute symptomatic and symptom-free periods Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 81 occurrence of relapses, variability of clinical grading during follow-up and the predictive 82 factors for worse clinical outcome, including final visual acuity. 83 5 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 84 Methods 85 Patients 86 We retrospectively reviewed the clinical records of 207 patients with VKC referred to our 87 Cornea and External Disease Unit from 1997 to 2007. Institutional Review Board 88 (IRB)/Ethics Committee approval was obtained. The diagnosis was based on a history of 89 ocular surface inflammation characterised by itching, photophobia, tearing and mucous 90 discharge, the presence of a papillary reaction on the upper tarsal conjunctiva and/or at the 91 limbus associated with the presence of eosinophils in conjunctival scrapings.1,10 Patients had a 92 complete examination by the same physician (SB) at least once a year. Additional visits were 93 planned by the physician according to the clinical severity of the disease or if required by 94 patients/parents in the case of worsening ocular symptoms. 95 The following clinical and demographic data were collected for each patient: age, gender, 96 familial history of atopy, associated ocular diseases, presence of associated atopic diseases, 97 positive reaction to skin PRICK tests, age of onset of VKC, seasonal/perennial course of 98 VKC, history of corneal shield ulcer and any previous topical treatments used. Each clinical 99 sign (conjunctival hyperemia, chemosis and secretion, upper tarsal and limbal papillary 100 reaction, subconjunctival fibrosis, corneal ulcer) and symptom (itching, tearing, burning and 101 photophobia) observed at the first clinical examination was assessed and scored: 0=absent, 102 1=mild, 2=moderate, and 3=severe. Corneal involvement was scored as 0=spared, 103 1=superficial punctate keratopathy in less than half the cornea, 2= superficial punctate 104 keratopathy in more than half the cornea, and 3= corneal ulcer. In addition, patients and/or 105 their parents were asked to score the overall symptoms according to the following scale: 106 0=absence of symptoms, 1=presence of mild intermittent symptoms, 2=presence of mild 107 persistent or moderate symptoms, 3= presence of severe symptoms. The best corrected visual 6 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 108 acuity (BCVA) and the topical treatments prescribed were also recorded and evaluated at each 109 visit. 110 111 Vernal keratoconjunctivitis decision tree 112 Classification tree (CT) analysis (CART® V6.0; Salford system, CA, USA) was performed on 113 the entire population (n=207) of patients with VKC in order to develop a decision tree for 114 treatment. 115 analysis: 12 Five therapeutic approaches were considered as dependent variables for the CT 116 - Score 0 = no treatment; 117 - Score 1 = anti-allergic eye-drop treatment used occasionally; 118 - Score 2 = anti-allergic eye-drop treatment used daily; 119 - Score 3 = anti-allergic eye-drop treatment used daily or associated with 120 low 121 dose topical steroid pulsed treatment (i.e. no more than 10-15 drops for 122 a month) 123 - Score 4 = high dose pulsed treatment with topical steroids (4-6 times 124 daily for 125 - 3 days, tapered in one week). 126 The clinical sign (conjunctival hyperemia, chemosis and secretion, upper tarsal and limbal 127 papillary reaction, subconjunctival fibrosis, superficial punctate keratitis and corneal ulcer) 128 and symptom (itching, tearing, burning and photophobia) scores and the overall symptoms 129 score (OSS) collected at the first clinical examination were included in CT analysis as 130 independent variables. 131 The CART® method generates a classification procedure by partitioning the entire patient 132 population into subdivisions according to the clinical variable that produces the best split in 7 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 133 the population. All possible separations or cut-off points (for continuous variables) are 134 examined to determine which variable produces the best population split. In each of the 135 resulting strata, the process is repeated until none of the variables shows significant influence. 136 The resulting decision tree is composed of a root node (including all of the data), a set of 137 internal nodes (splits), and a set of terminal nodes. A class label is assigned to each terminal 138 node.12 139 140 141 Follow-up study 142 Patients with at least 1 year of follow-up (n=110) were included in this phase of the study. 143 Vernal keratoconjunctivitis severity at the time of each follow-up visit was retrospectively 144 graded according to the new decision tree. All patients with incomplete follow-up visits and 145 patients included in clinical trials requiring pre-established control visits and treatments were 146 excluded. 147 At the end of the follow-up period, the mean number of recurrences of ocular inflammation 148 per year, the development of corneal shield ulcer, the mean number of visits per year and the 149 mean number of changes in clinical grading were evaluated as outcome parameters. The 150 BCVA at the last examination in the quiescent phase was also recorded. Lastly, any 151 development of long term ocular complications (i.e. glaucoma and cataract) was noted. 152 Bivariate and multivariate logistic regression analyses were performed to obtain a relative risk 153 evaluation of worse visual outcome associated with the potential predictor variables. A 154 regression tree of predictors of worse visual outcome was constructed using the CART® 155 method. 12,13 The following clinical and demographic features were used as predictor variables 156 for a worse BCVA at the final examination: 1) gender (male/female); 2) course of VKC 157 (seasonal/perennial); 3) presence of associated atopic diseases (yes/no); 4) familial history of 8 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 158 atopy (yes/no); 5) history of corneal ulcer (yes/no); 6) upper tarsal papillary reaction score (0- 159 3); 7) limbal papillary reaction score (0-3); 8) recurrence rate of inflammation during the 160 follow-up period; 9) total duration of disease (years); 10) VKC grade at presentation (VKC 161 grade 0-4); 11) age of onset of VKC (years). 162 To perform logistic regression analyses, the final BCVA was used as a dependent variable: 163 patients with a final BCVA of 1 were classified as having “excellent vision“, patients with a 164 final BCVA<0.9 were classified as having “fair/poor vision”. 165 166 167 Statistics 168 The classification regression tree analysis (CART® V6.0; Salford system, CA, USA) was 169 performed to develop a decision tree for treatment of VKC and to identify predictive factors 170 for a worse visual outcome. 171 Bivariate logistic regression analyses were conducted to examine the relationship between 172 each demographic and clinical potential predictive factor and final BCVA (outcome variable). 173 Lastly, all significant predictors resulting from the bivariate analysis were included in a 174 multivariate logistic regression analysis for predicting fair/poor final BCVA. The clinical 175 grading of patients at the first visit was correlated to the mean number of recurrences of 176 inflammation per year, to the mean number of visits and to BCVA at the final examination, 177 using the Spearman rho test (SPSS V15 software, Illinois, CA, USA). 178 179 9 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 180 Results 181 VKC decision tree 182 Two-hundred seven patients with VKC were included in the tree analysis. The demographic 183 and clinical characteristics of the population are described in Table 1. 184 The VKC decision tree is shown in Figure 1. The most optimal tree was generated by splitting 185 the population by overall symptoms score (OSS): patients without symptoms (OSS=0) were 186 graded as 0-quiescent (n=16). The presence of ocular symptoms (OSS>1) but not photophobia 187 identified patients with grade 1-mild VKC (n=59), while patients graded as 2-moderate 188 (n=74) also complained of photophobia. Patients with corneal involvement were graded as 3- 189 severe (mild to moderate SPK; n=22) and 4-very severe (diffuse SPK and/or corneal ulcer; 190 n=36) based on the severity of corneal involvement. This new VKC grading system and 191 related treatments are summarized in Table 2. 192 Follow-up study 193 One-hundred and ten patients were included in the follow-up analysis (range 1-10 years; 194 mean ± SD: 4 ± 2 years). Twenty-four were females and 86 were males, aged from 4 to 31 195 years (mean, 10 ± 6 years). 196 During the entire follow-up period, patients underwent 2.3 ± 1.9 (mean ± SD) visits per year, 197 showing changes in VKC grade from 0 to 8 times per year (mean ± SD: 2 ± 1.6 changes per 198 year). Patients had from 0 to 3.5 recurrences per year (mean ± SD: 0.4 ± 0.6 recurrences for 199 year). Moreover, 14% of patients developed a corneal shield ulcer and 30% of patients 200 showed complete recovery of the disease after a mean duration of 10 ± 6 years. The BCVA at 201 final examination ranged from 0.2 to 1 (mean ± SD: 0.9 ± 0.1). No patients developed 202 glaucoma and/or cataract during the follow-up period. 10 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 203 A higher VKC grade at the first examination was significantly related to a higher number of 204 recurrences per year (p<0.001; rho=0.36), a higher number of visits (p=0.002; rho=0,29) and 205 a worse final BCVA (p=0.001; rho=-0.36). 206 All 11 potential predictive factors were included in the regression tree model performed with 207 CART® to predict a worse BCVA. The CART® decision tree for VKC treatment is shown in 208 Figure 2. The mean recurrence rate of inflammation appeared at the root node: patients (n=9) 209 with a higher mean recurrence rate (> 1.1 per year) showed a higher risk of worse visual 210 outcome (mean ± SD BCVA: 0.6±0.3). The remaining patients were then split based on VKC 211 grade at presentation and then by age of onset of VKC: a higher baseline VKC grade and 212 lower age of onset resulted in a mean BCVA of 0.7±0.2 (i.e., medium risk). Conversely, a 213 lower baseline VKC grade and a higher age of onset were associated with a lower risk of 214 visual impairment (mean ± SD BCVA: 0.9 ± 0.1). 215 Bivariate logistic regression analysis revealed a significant risk for worse BCVA with the 216 following factors: a perennial VKC course (p=0.013), the limbal VKC form (p=0.036), a 217 history of corneal ulcer (p=0.004), the severity of baseline VKC grade (p<0.05) and mean 218 recurrences of ocular inflammation per year (p=0.001). Multivariate logistic regression, 219 conducted with all significant risk items included as predictors, showed that the number of 220 recurrences per year (p=0.003) and a more severe baseline VKC grade at presentation 221 (p=0.048) were significantly associated with worse final visual outcome. 222 223 11 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 224 Discussion 225 We retrospectively analysed data from 207 patients with VKC treated in our Cornea and 226 External Disease Unit over a 10-year period. Results showed that these patients underwent 227 from 1 to 9 examinations per year and showed a great variability of clinical manifestations 228 with improvement and/or worsening of clinical signs and symptoms up to 8 times per year. 229 Such high clinical variability forces the physician to almost constantly check and modify the 230 patient’s treatment .1,4,8 A simple clinical decision tree and a grading system that allows 231 ophthalmologists to share a common language in the assessment and treatment of VKC and in 232 the development of clinical trials on potential new therapeutic compounds, would greatly 233 improve the management of this challenging disease.1,6,8 234 Starting from the retrospective evaluation of therapies used in our case series of 207 VKC 235 patients, we developed a standardized grading system using CART®, a statistical approach to 236 generating a decision tree.14,15,16 Classification tree analysis has the advantage that the results 237 are easier to interpret than in conventional statistical analyses. In fact, decision trees have 238 intuitive appeal because the classification structure is explicit. In our study, the decision tree 239 obtained allowed us to identify five classes of VKC severity, characterised by different signs 240 and symptoms and requiring different therapeutic approaches. Our analysis identified the 241 presence of ocular symptoms and corneal involvement as the two most important features of 242 VKC that influence therapy. The overall presence and severity of ocular symptoms should be 243 considered the foremost parameter when prescribing topical anti-allergic therapy1,4,6,8-10 The 244 frequency of instillation of ocular therapy may be decided by the course of symptomatology: 245 anti-allergic eye drops may be used as needed in patients with mild intermittent symptoms, or 246 daily in patients complaining of persistent symptoms. 17-19 Of ocular surface symptoms, the 247 presence of photophobia appears to discriminate the most severe stages, which are mostly 248 characterized by corneal involvement. In these cases, topical steroids are successfully used to 12 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 1,4,6,8 249 control ocular inflammation. 250 stages of disease, these parameters were not appropriate splitting criteria for the decision tree, 251 although they still are considered the main diagnostic criteria of VKC. 252 The new VKC grading system proposed in this study is based on a retrospective analysis and, 253 therefore, its validity should be confirmed by a prospective study. Nevertheless, it must be 254 noted that the VKC grading system developed using CART® alone correctly identified 255 patients with more severe forms of VKC, since severity grade was significantly related to a 256 higher number of recurrences per year, a higher number of visits and a lower final BCVA. 257 Similarly, the logistic regression analysis showed that a higher recurrence rate and higher 258 grade of VKC at presentation were associated with a higher risk of poor visual outcome. 259 These same predictive factors were identified by the regression tree analysis performed with 260 CART. Furthermore, CART identified a lower age of VKC onset as an additional risk factor 261 for a worse final visual outcome. 262 Our results indicate that VKC patients with frequent recurrences of inflammation and/or those 263 with a severity grade of 3 or 4 (i.e., with corneal involvement), and a lower age of onset of the 264 disease, need to be more closely followed and treated with a more aggressive approach to 265 control the disease and to prevent a reduction in BCVA. The therapeutic approach described 266 in this study allowed us to reduce long-term complications due to prolonged or self- 267 administered topical steroids, which was shown in our previous case series of 195 patients to 268 be responsible for glaucoma in 2.1% of patients and cataract in one patient.1 Furthermore, 269 this new approach to clinical grading of VKC might be incorporated into clinical trials to 270 improve evaluation of new treatment options that better control signs, symptoms and 271 recurrences of VKC. 272 In conclusion, in this study, we propose a new clinical grading system for VKC based on a 273 simple decision tree that can be easily applied to patients by any ophthalmologist. Our aim is 13 Since itching and conjunctival papillae are present in all Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 274 to achieve a standardized approach to VKC treatment based on clinical severity of the disease 275 as defined by the decision tree. This new grading system will also allow us to identify severe 276 forms of VKC, patients who are at higher risk of developing recurrences, corneal ulcer, and a 277 worse final visual outcome, and should therefore be followed by ophthalmologists on a more 278 frequent basis or addressed to referral centers for corneal diseases. 279 14 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 280 References 281 1. Bonini S, Bonini S, Lambiase A, et al. Vernal keratoconjunctivitis 282 revisited: a case series of 195 patients with long-term follow-up. Ophthalmology. 283 2000;107:1157–63 284 2. 285 Leonardi A. Vernal keratoconjunctivitis: pathogenesis and treatment. Prog Retin Eye Res 2002;21:319–39. 286 3. Bonini S, Coassin M, Aronni S, et al. Vernal Keratoconjunctivitis. Eye. 287 2004;18:345-51. 288 4. Leonardi A, Busca F, Motterle L et al. Case series of 406 vernal 289 keratoconjunctivitis patients: a demographic and epidemiological study. Acta 290 Ophthalmol Scand. 2006;84:406-10. 291 5. 292 Ophthalmol. 1999;34:88-92. 293 6. 294 demographic and epidemiological study on vernal keratoconjunctivitis: a glimpse of 295 ocular surface in Italian population. Ophthalmic Epidemiol. 2009;16:38-41 296 7. 297 quality of life in children with vernal keratoconjunctivitis questionnaire. Am J 298 Ophthalmol. 2007;144:557-63. 299 8. 300 randomised clinical trials on topical treatments for vernal keratoconjunctivitis. Br J 301 Ophthalmol. 2007;91:1656-61. 302 9. 303 2003;43:41–58. 15 Tabbara KF. Ocular complications of vernal keratoconjunctivitis. Can J Lambiase A, Minchiotti S, Leonardi A et al. Prospective, multicenter Sacchetti M, Baiardini I, Lambiase A et al. Development and testing of the Mantelli F, Santos MS, Petitti T, et al. Systematic review and meta-analysis of Leonardi A, Secchi AG. Vernal keratoconjunctivitis. Int Ophthalmol Clin Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 304 10. 305 allergy. J Allergy Clin Immunol. 2000;106:1019-32. 306 Bielory L. Allergic and immunologic disorders of the eye. Part II: ocular 11. 307 Bonini S, Sacchetti M, Mantelli F, Lambiase A. Clinical grading of vernal keratoconjunctivitis. Curr Opin Allergy Clin Immunol. 2007;7:436-41. 308 12. Breiman L, Friedman JH, Olshen RA, Stone CJ. Classification and 309 Regression Trees. Wadsworth & Books/Cole Advanced Books & Software, Monterey, 310 CA, 1984. 311 13. Worth AP, Cronin MTD The use of discriminant analysis, logistic 312 regression and classification tree analysis in the development of classification models 313 for human health effects. J Mol Structures (Theochem). 2003; 622:97-111 314 14. Muller R, Möckel M. Logistic regression and CART in the analysis of 315 multimarker studies. Clin Chim Acta. 2008;394:1-6. 316 15. 317 Podgorelec V, Kokol P, Stiglic B, Rozman I. Decision trees: an overview and their use in medicine. J Med Syst. 2002;26:445-63. 318 16. Costanza MC, Paccaud F. Binary classification of dyslipidemia from the 319 waist-to-hip ratio and body mass index: a comparison of linear, logistic, and CART 320 models. BMC Med Res Methodol. 2004;4:7 321 17. Verin P, Allewaert R, Joyaux JC et al. Comparison of lodoxamide 0.1% 322 ophthalmic solution and levocabastine 0.05% ophthalmic suspension in vernal 323 keratoconjunctivitis. Eur J Ophthalmol. 2001;11:120-5. 324 18. Avunduk AM, Avunduk MC, Kapicioglu Z et al. Mechanisms and 325 comparison of anti-allergic efficacy of topical lodoxamide and cromolyn sodium 326 treatment in vernal keratoconjunctivitis. Ophthalmology. 2000;107:1333-7. 16 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. . 327 19. Verin PH, Dicker ID, Mortemousque B. Nedocromil sodium eye drops are 328 more effective than sodium cromoglycate eye drops for the long-term management of 329 vernal keratoconjunctivitis. Clin Exp Allergy. 1999;29:529-36. 17 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. * Precis Precis (35 words) This study presents a decision tree for vernal keratoconjunctivitis based on a classification regression analysis that provides an intuitive means of optimizing treatment and identifying patients at higher risk and in need of closer follow-up. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Table 1 Table 1 Characteristics of patients included in this retrospective study of VKC patients followed over a ten year period in our referral center. Patients (N) 207 (%) Sex Male 162 (78%) Female 45 (22%) Age (years) Range 3-32 Mean ± SD 10±6 Age of onset of VKC (years) Range 0.5-26 Mean ± SD 6.2±4.5 VKC type (N) Tarsal 151 (73%) Limbal 20 (10%) Mixed 36 (17%) VKC course (N) Seasonal 143 (69%) Perennial 64 (31%) Patients with atopic 91 (44%) associated diseases (N) Skin PRICK tests (N) Positive 98 (47.3%) Negative 69 (33.3%) Not done 40 (19.3%) Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Table 2 Table 2. Clinical grading of VKC obtained from the classification tree analysis (CART®) Grade Clinical findings Treatment 0 quiescent Absence of symptoms No treatments 1 mild Presence of symptoms with no corneal Anti-allergic eye drops occasionally involvement 2 moderate Presence of symptoms associated with Anti-allergic eye drops daily photophobia with no corneal involvement 3 severe Presence of symptoms associated with Anti-allergic eye drops daily photophobia and mild to moderate SPK associated with pulsed low dose topical steroid 4 very severe Presence of symptoms associated with Pulsed high dose topical steroid photophobia and diffuse SPK and/or corneal ulcer eventually associated with surgical removal of corneal plaque Figure 1 Click here to download high resolution image Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Figure 2 Click here to download high resolution image Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Legends Figure Legends Figure 1. Classification tree analysis (CART®) stratified the VKC population into 5 subsets defined by severity grade of signs and symptoms. Figure 2. Regression tree analysis (CART®) identified that a higher frequency of recurrences, a more severe VKC grade and a younger age of onset of VKC were risk factors for a worse visual outcome. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Graefes Arch Clin Exp Ophthalmol (2008) 246:435–441 DOI 10.1007/s00417-007-0682-6 INFLAMMATORY DISORDERS Preliminary evidence of the efficacy of probiotic eye-drop treatment in patients with vernal keratoconjunctivitis Alfonso Iovieno & Alessandro Lambiase & Marta Sacchetti & Barbara Stampachiacchiere & Alessandra Micera & Stefano Bonini Received: 20 February 2007 / Revised: 11 July 2007 / Accepted: 25 August 2007 / Published online: 27 November 2007 # Springer-Verlag 2007 Abstract Background Probiotics have been shown to improve allergic inflammation. The aim of this study was to evaluate the efficacy of Lactobacillus Acidophilus eye-drops in controlling signs and symptoms of vernal keratoconjunctivitis (VKC). Methods Seven patients (mean age 11.8±4.3; five M, two F) with mild to moderate VKC were included in the study. Lactobacillus Acidophilus diluted in saline solution (2×108 CFU/ml) was administrated as eye-drops four times daily for 4 weeks in both eyes. Clinical signs (conjunctival hyperemia, chemosis, secretion, Trantas dots, superficial punctuate keratitis) and symptoms (itching, photophobia, burning, tearing) were evaluated and scored from 0 to 3 at baseline, after 2 and 4 weeks of treatment. Total sign (TSS) and symptom (TSyS) scores were calculated. Conjunctival impression cytology was performed in three patients at baseline and after 4 weeks of treatment, in order to evaluate the expression of ICAM-1 and TLR-4. Results In the six out of seven patients who completed the study, symptoms were significantly improved after both 2 weeks (TSyS: baseline 6.7±0.9 vs 4.1±1.2; p=0.017) and 4 weeks (TSyS: baseline 6.7±0.9 vs 3.6±1.2, p=0.011) of treatment. A significant improvement of clinical signs was observed after 4 weeks of treatment (TSS: baseline 7.5±1.6 vs 3.9±1.7, p=0.034) but not after 2 weeks of treatment (TSS: baseline 7.5±1.6 vs 5.3±1.5; NS). In particular, photophobia was significantly reduced (2±0.6 vs 1±0.3; p=0.023) at 2 weeks, while at 4 weeks the scores for itching (1.8±0.3 vs 1±0.3), tearing (1.6±0.4 vs 0.8±0.2), conjunctival hyperemia (2.3±0.2 vs 1.4±0.5) and chemosis (1.2±0.4 vs 0.4±0.4) were significantly lower compared to baseline. A down-regulation of ICAM-1 and TLR-4 was observed in two patients showing clinical improvement after 4 weeks of treatment. Conclusion Our open pilot study showed that 1-month treatment with probiotic eye-drops improves signs and symptoms in patients with VKC. Additional double-blind controlled clinical trials with a larger sample of patients are needed to confirm the effects of topical Lactobacilli on VKC patients. Keywords Vernal keratoconjunctivitis . Allergy . Conjunctiva . Lactobacillus Acidophilus . Probiotic Introduction Presented at the annual meeting of the Association for Research in Vision and Ophthalmology (ARVO), Fort Lauderdale, Florida, May 2006. A. Iovieno : A. Lambiase : M. Sacchetti : B. Stampachiacchiere : A. Micera : S. Bonini (*) Interdisciplinary Center for Biomedical Research (CIR), Laboratory of Ophthalmology, University of Rome “Campus Bio-Medico”, Via E. Longoni, 83, 00155 Rome, Italy e-mail: [email protected] B. Stampachiacchiere : A. Micera IRCCS-G.B. Bietti Eye Foundation, Rome, Italy Vernal keratoconjunctivitis (VKC) is a chronic allergic eye disease affecting children in pre-puberal age. However, VKC is not associated with a positive skin test or RAST in 42–47% of patients, confirming that it is not solely an IgEmediated disease, but that other mechanisms are involved in VKC pathogenesis [2, 3]. It is characterized by recurrent ocular surface inflammation leading to conjunctival hyperemia, mucus discharge and tarsal and/or bulbar papillary reaction. Patients complain of intense ocular symptoms such as itching, photophobia, burning and tearing, lasting all year with seasonal recurrences [2, 3]. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 436 Graefes Arch Clin Exp Ophthalmol (2008) 246:435–441 Topical antiallergic treatment is commonly used to reduce signs and symptoms of the disease but is not always successful. Topical cyclosporine has shown good effectiveness in long-term maintenance of clinical remission in VKC patients, but it is not commercially available for the treatment of VKC [2, 3]. Topical steroids represent the most effective therapy for moderate to severe forms of VKC; however, their long-term use should be strictly limited as it exposes patients to potential ocular side effects. If untreated, VKC leads to severe corneal complications and visual impairment [2, 3]. A novel therapeutic approach to allergic diseases based on the oral administration of probiotic bacteria has been recently proposed [5, 18, 27, 30]. Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” [11]. The best-known and commonly used probiotics are strains of Bifidobacterium and Lactobacillus species. Lactobacilli are nonpathogenic, gram-positive bacteria, and represent the major components of the commensal microbial flora of the small and large intestinal tract of both humans and animals. Previous clinical trials demonstrated the efficacy of Lactobacilli therapy for the prevention and treatment of atopic dermatitis, food allergy and allergic rhinitis [14–16, 23, 28, 31]. The health-promoting properties attributed to Lactobacilli strains are multiple, and include their capacity to interact with the host immune system. The ingestion of Lactobacilli has been shown to enhance IL-10 serum concentrations in patients with atopic dermatitis, therefore displaying anti-inflammatory activities [24]. The chronic use of Lactobacilli may strengthen the mucosal immunological barrier of the gut through the promotion of local IgA production and the reduction of epithelial permeability to allergens [1, 9, 10, 17]. In vitro studies have demonstrated that Lactobacilli are able to induce Th1-type cytokines such as IL-12 and IFN-γ; the stimulation of the Th1 phenotype may normalize the Th1/Th2 imbalance present in allergic conditions [19, 26]. Since VKC is mainly characterized by the increase of Th2type cytokines and by conjunctival immune cell infiltration, the aim of this study was to investigate the effects of the topical administration of Lactobacillus Acidophilus on signs and symptoms of patients with active VKC. Materials and methods Lactobacillus Acidophilus eye drops were prepared as follows: freeze-dried inactivated Lactobacillus Acidophilus (Lacteol forte, Bruschettini srl, Genova, Italy) was diluted in saline solution (NaCl 0.9% solution) to obtain a final concentration of 200 millions CFU/ml. The preparation was then aliquoted in eye-drop bottles at PH 7–7.4 and stored at −70°C until use. The eye-drop solution was prepared once for the 4 weeks of treatment under the hood in the hospital pharmacy using sterile eye-drop bottles, therefore preserving a sterile condition, aliquoted in eye-drop bottles at PH 7–7.4 and stored at −70°C. One bottle of eye-drop solution (7 ml) was given to the patient once every week, for 1 month. The patients were instructed to keep the eye-drops bottles at 4°C. This study followed the guidelines of the Declaration of Helsinki for research involving human subjects, and approval was received from The Intramural Committee, in accordance with the ethical principles for medical research involving human subjects. Informed consent was obtained from each patient or his/her parents. Seven patients with mild to moderate VKC in the active phase (mean age 11.8±4.3 years; range: 7–17 years; sex: five M, two F; Table 1) were included in the study during Table 1 Characteristics of patients enrolled in the study Patient Age/ gender VKC form Duration (ys) Previous therapies *TSyS (baseline) *TSyS (postLactobacilli treatment) **TSS (baseline) **TSS (postLactobacilli treatment) 1 13/M 4 Lodoxamide 12 8 10 8 2 13/M 4 Ketotifen 16 14 20 19 3 9/F 4 Lodoxamide 16 12 25 19 4 7/M 6 Ketotifen 16 8 18 6 5 7/M 2 Ketotifen 6 6 6 6 6 17/M 2 None 16 12 14 12 7 17/F Tarsal/ perennial Mixed/ perennial Tarsal/ perennial Mixed/ perennial Tarsal/ seasonal Limbal/ seasonal Tarsal/ seasonal 9 Ketotifen 12 0 12 0 *TSyS = total symptoms score; **TSS = total sign score. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Graefes Arch Clin Exp Ophthalmol (2008) 246:435–441 springtime. Diagnosis of VKC was based on history, clinical presentation (presence of mild to severe cobblestone-like appearance in the upper tarsal and/or limbal papillary reaction) and the presence of eosinophils in upper tarsal conjunctival scrapings. None of the patients were using any form of systemic therapy or contact lenses, had corneal ulceration, had a history of infectious conjunctivitis, or had undergone eye-surgery in past three months. After 3 days of wash-out, in which patients were only treated with artificial tears and cold compresses, patients received probiotic eye-drops four times daily in both eyes for 1 month. Clinical symptoms (itching, photophobia, burning and tearing) and signs (conjunctival hyperemia, chemosis, secretion, Trantas dots and superficial punctuate keratitis) were evaluated according to slit lamp examination and fluorescein staining, and scored from 0 to 3 (0=absent, 1= mild, 2=moderate, 3=severe) at baseline, after 2 weeks and after 4 weeks of treatment with probiotic eye-drops. Total sign (TSS; range: 0–24) and symptom (TSyS; range: 0–30) scores were calculated as the sum of each symptom and sign score. At each time point, patients were asked about the tolerability of Lactobacillus eye-drops. Tolerability was expressed as a score from 0 to 3 (0=no discomfort, 1=mild discomfort, 2=moderate discomfort, 3=severe discomfort, cannot use study drug). Conjunctival impression cytology (Millicell 0.22 μm filters, Millipore, Milan, Italy) was performed at baseline and after 1 month of Lactobacillus Acidophilus eye drop treatment in the three patients who had given their written consent to sampling (patients n° 4-5-6; see Table 1). Millicell metilcellulose membranes were removed from plastic supports with a surgical blade and placed in an autoclaved microtube containing 300 μl cell lysis solution (Puregene RNA purification kit; Gentra Systems, MI, USA). Membranes were immediately processed to obtain maximum recovery of total RNA from each sample. Total RNA was reverse transcribed, and amplification reaction 437 was carried out in a programmable PTC100 thermocycler and an Opticon2 machine respectively (MJ Research, Watertown, MA, USA) as described elsewhere [4] (Table 2). Samples were amplified in duplicate and from these replicates, averages were calculated and differences in C(t) data were evaluated by the REST software package [25]. Target gene expressions were shown in the graph as percentages of increase/decrease values that were obtained after calculating differences between real pre and post Ct values. All sterile RNase-Free plasticware and analytical reagents were purchased from NUNC (Roskilde, Denmark), Euroclone (Milan, Italy) and GIBCO-Invitrogen (Carlsbad, CA, USA), unless otherwise specified in the text. The non-parametric Mann-Whitney U-test was used to compare signs and symptoms at different time-points. Analysis was performed by using the statistical package StatView II for PC (Abacus Concepts Inc., Berkley, CA, USA) [32]. A probability <0.05 was considered statistically significant. All data is reported as mean ± SEM. Results Six out of seven patients (86 %) completed the entire study. Patient 3 discontinued treatment because there was no improvement in his clinical condition, and dropped out from the study at day 14. No side-effects were observed during clinical examination, and all patients tolerated probiotic eyedrop administration (0 score at each time point). All six patients (100%) reported improvement of clinical conditions after 2 weeks of treatment, and five out of six patients (83%) reported improvement of clinical conditions after 4 weeks of treatment. Accordingly, total symptom score (TSyS) was significantly lower after both 2 weeks (TSyS 6.7±0.9 vs 4.1±1.2; p=0.017) and 4 weeks (TSyS: 6.7±0.9 vs 3.6±1.2, p=0.011) of treatment when compared to baseline (Fig. 1a). In particular, photophobia was Table 2 Sequence of primers used for gene amplification in real-time PCR Gene (amplicon size) Sequence Reference ICAM-1 (107 bp) Sense: 5′-ATGAGTGCCCAGGGAATATG-3′ Antisense: 5′-CCCTGATCACTGCAGGAAA -3′ Sense: 5′-AAT CCC CTG AGG CAT TTA GG-3′ Antisense: 5′- CAG GGC TAA ACT CTG GAT GG-3′ Sense: 5′-GAA GGG GTC ATT GAT GGC AAC-3′ Antisense: 5′- GGG AAG GTG AAG GTC GGA GTC-3′ J03132 TLR-4 (102 bp) GAPDH (100 bp) U88880 BC013310 cDNAs were run for amplification with primers (10 pM, prepared by MWG, Biotech, Ebersberg, Germany) specific for ICAM-1 and TRL4 designed by the primer3 software available online at http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi [29]. GenBank software was used to select the mRNA complete sequence of each gene investigated (http://www.ncbi.nlm.nih.gov/ Genbank; provided to the public domain by the National Center for Biotechnology Information, Bethesda, MD, USA). The PCR amplification profile included: 1 cycle of activation 95°C/15 min followed by 43 cycles of denaturation at 94°C/30 sec, annealing at 60°C/25 sec and extension at 75°C/30 sec, completed by a further extension at 75°C/5 min. Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 438 Graefes Arch Clin Exp Ophthalmol (2008) 246:435–441 Fig. 1 Effect of Lactobacillus Acidophilus eye-drops on symptoms of VKC patients. a Total symptom score at different time-points. A significant improvement (*) in TSyS was observed after 2 and 4 weeks of treatment. b Symptom scores at different time-points. Photophobia was significantly improved (*) after 2 weeks of treatment while itching and tearing were significantly in improved after 4 weeks of treatment significantly improved (2±0.6 vs 1±0.3; p=0.023) at two weeks time-point, while at 4 weeks time-point itching (1.8±0.3 vs 1±0.3) and tearing (1.6±0.4 vs 0.8±0.2) were significantly improved (Fig. 1b). A significant improvement in total sign score (TSS) was observed after 4 weeks of treatment with respect to baseline (TSS: 7.5±1.6 vs 3.9±1.7, p=0.034) but not after 2 weeks (TSS: 7.5±1.6 vs 5.3±1.5; NS) (Fig. 2a). In particular, conjunctival hyperemia (2.3±0.2 vs 1.4±0.5) and chemosis (1.2±0.4 vs 0.4±0.4) scores were significantly lower at the 4-week time-point compared to baseline (Figs. 2b, 3). Figure 4 shows modifications of conjunctival epithelial expression of ICAM-1 and TLR-4 after 4 weeks of treatment with Lactobacillus Acidophilus eye-drops. Interestingly, a down-regulation of both ICAM-1 and TLR-4 was observed in patient 4 and patient 6, and both these patients showed clinical improvement after 4 weeks of treatment. On the other hand, up-regulation of both conjunctival markers was observed in patient 5, who showed no improvement after treatment. Discussion Fig. 2 Effect of Lactobacillus Acidophilus eye-drops on clinical signs of VKC patients. a Total sign score at different time-points. A statistically significant improvement (*) of TSS was observed after 4 weeks of treatment. b Symptom scores at different time-points The improvement in hyperemia and chemosis reached statistical significance (*) at the 4-week time-point This pilot open study demonstrated that 4-week treatment with Lactobacillus Acidophilus eye-drops was effective in reducing signs and symptoms of patients with mild to moderate vernal keratoconjunctivitis in the active phase. Lactobacilli eye-drops did not cause any side effects and were well-tolerated by patients, as reported after systemic administration. All patients reported improvement of symptoms, except one who dropped out of the study because his clinical condition did not improve after 2 weeks of treatment. This patient had the most severe form of VKC included in the study, with the highest total symptom score, total sign score and punctuate keratopathy scores at baseline. Thus, total symptom score of the remaining patients was significantly improved after 2 and 4 weeks of treatment, demonstrating that Lactobacillus Acidophilus eye-drops can improve symptoms in patients with VKC in the active phase. A statistically significant improvement of clinical signs was also observed after 1 month of treatment; in Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Graefes Arch Clin Exp Ophthalmol (2008) 246:435–441 439 Fig. 3 Patient characteristics at slit-lamp examination. An improvement of conjunctival hyperemia (A, B; p<0.05), Trantas dots (C, D; p>0.05) and secretion (E, F; p>0.05) was observed after 4 weeks of Lactobacilli eye-drop treatment particular, hyperemia and chemosis scores were significantly lower with respect to baseline. Little is known regarding the precise mechanisms by which lactobacilli may improve the clinical conditions of allergic patients. The therapeutic effects of Lactobacillus Acidophilus on VKC patients after 1 month of therapy could be related to its antiinflammatory properties. Lactobacilli act by inducing antinflammatory cytokines such as IL-10 e TGF-β, that play a crucial role in down-regulating inflammatory cascades. Pessi et al. showed that serum IL10 concentrations were significantly elevated after giving oral Lactobacillus rhamnosus GG at a daily dose of 2× 1010 CFU for 4 weeks to nine children affected by atopic dermatitis [24]. Peripheral blood mononuclear cells, monocytes and lymphocytes from healthy donors, if stimulated with lactobacilli and with polyclonal T cell stimulator Fig. 4 Characteristics of the patient that underwent TLR4/ICAM-1 analysis. A significant down-regulation in ICAM-1 and TLR-4 mRNA was observed in patients (Pt4 and Pt6) showing a reduction in total symptoms score (TSyS) and total signs score (TSS) after 4 weeks of treatment with Lactobacilli eye-drops. A slightly increase of both ICAM-1 and TLR-4 mRNA was detected in the patient (Pt5) that did not show an improvement of TSyS and TSS following Lactobacilli treatment phytohaemagglutinin (PHA), produce high levels of IL-10 and low levels of Th2-type cytokines [20]. In line with this hypothesis, the reduction of conjunctival epithelial ICAM-1 expression observed in two patients showing clinical improvement supports the hypothesis of an antiinflammatory effect of topical Lactobacillus on the ocular surface. Another hypothesis is based on a direct effect of Lactobacilli eye-drops in modulating the allergic reaction. The concept of probiotics as possible means of antiallergic therapy emerged from substantial but indirect evidence linking low incidence rates of allergies in poor countries with a certain composition of intestinal flora. These finding are in line with the “hygiene hypothesis”, reporting that the exposure to microbial products early in life could be essential for the proper development of the immune system [30]. The greatest microbial exposure throughout life, and the newborn’s first major challenge, is the intestinal microbiota. Bjorkstein et al. observed that Lactobacilli are more commonly found in the intestinal microflora of populations (eg. Estonian) with low incidence of allergic diseases [6]. Besides, Kalliomaki et al. showed that children who developed allergic diseases were less often colonized by lactic acid producing bacteria, but had higher counts of Clostridia when compared with healthy children [13]. There is evidence that probiotic microorganisms preferentially elicit T-helper 1 cells (Th1) and T-helper3/T regulatory cells (Treg) that counterbalance the predominance of T-helper 2 (Th2) direct immune responses in allergic disease. The ability of probiotic bacteria to induce Th1 and Treg responses is based on the recognition of bacterial cell components by cell receptors such as Toll-like receptors (TLR), a family of glycoproteins expressed by epithelial cells, dendritic cells, monocytes and mast cells/ basophils [8, 12]. TLRs are involved in the production of inflammatory cytokines, cell stimulation and activation of the specific immune response [12]. The expression of TLR4 has been correlated with the allergic phenotype. Dabbagh Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. 440 et al. showed that TLR4-defective mice subjected to sensitization and pulmonary challenge with a protein allergen had reductions in airway inflammation by eosinophils, allergen-specific IgE levels, and Th2 cytokine production, compared with wild-type mice [7]. In addition, in vitro studies observed that TLR-4 activation on mast cells resulted in the induction of a subset of genes including Th2 cytokines and chemokines [21, 22]. In a recent study, VKC conjunctiva displayed upregulation of TLR-4, when compared to healthy conjunctiva [4]. Thus, the reduction of conjunctival expression of TLR-4 observed in patients after 4 weeks of therapy could be indirect evidence of the immunomodulating effects of Lactobacillus Acidophilus. Our preliminary data could suggest that topical treatment with Lactobacillus Acidophilus may offer a safe and welltolerated support to standard antiallergic therapy for the treatment of mild to moderate cases of vernal keratoconjunctivitis. However, it is not possible to exclude that the beneficial effects referred by patients after 2 weeks of treatment might be due to washing out of allergens from the ocular surface rather than the effect of Lactobacillus administration. The topical administration of Lactobacillus Acidophilus may require at least 4 weeks of treatment to exert a clinically significant effect. Additional double-blind controlled clinical trials with a larger sample of patients are needed to confirm the therapeutic effects of topical Lactobacilli on VKC patients. In conclusion, the administration of Lactobacilli eyedrops in VKC patients represents the first description of a topical use of probiotic bacteria in humans. References 1. Ait-Belgnaoui A, Han W, Lamine F, Eutamene H, Fioramonti J, Bueno L, Theodorou V (2006) Lactobacillus farciminis treatment suppresses stress induced visceral hypersensitivity: a possible action through interaction with epithelial cell cytoskeleton contraction. Gut 55:1090–1094 2. Bonini S, Bonini S, Lambiase A, Marchi S, Pasqualetti P, Zuccaro O, Rama P, Magrini L, Juhas T, Bucci MG (2000) Vernal keratoconjunctivitis revisited: a case series of 195 patients with long-term followup. Ophthalmology 107:1157–1163 3. Bonini S, Coassin M, Aronni S, Lambiase A (2004) Vernal keratoconjunctivitis. Eye 18:345–351 4. Bonini S, Micera A, Iovieno A, Lambiase A, Bonini S (2005) Expression of Toll-like receptors in healthy and allergic conjunctiva. Ophthalmology 112:1528; discussion 1548–1549 5. Boyle RJ, Tang ML (2006) The role of probiotics in the management of allergic disease. Clin Exp Allergy 36:568–576 6. Bjorksten B, Sepp E, Julge K, Voor T, Mikelsaar M (2001) Allergy development and the intestinal microflora during the first year of life. J Allergy Clin Immunol 108:516–520 7. Dabbagh K, Dahl ME, Stepick-Biek P, Lewis DB (2002) Toll-like receptor 4 is required for optimal development of Th2 immune responses: role of dendritic cells. J Immunol 168:4524–4530 Graefes Arch Clin Exp Ophthalmol (2008) 246:435–441 8. Feleszko W, Jaworska J, Hamelmann E (2006) Toll-like receptorsnovel targets in allergic airway disease (probiotics, friends and relatives). Eur J Pharmacol 533:308–318 9. Galdeano CM, Perdigon G (2006) The probiotic bacterium Lactobacillus casei induces activation of the gut mucosal immune system through innate immunity. Clin Vaccine Immunol 13: 219–226 10. Heyman M, Terpend K, Menard S (2005) Effects of specific lactic acid bacteria on the intestinal permeability to macromolecules and the inflammatory condition. Acta Paediatr Suppl 94:34–36 11. Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food London, Ontario, Canada, April 30 and May 1, 2002 12. Kaisho T, Akira S (2006) Toll-like receptor function and signaling. J Allergy Clin Immunol 117:979–987 13. Kalliomaki M, Kirjavainen P, Eerola E, Kero P, Salminen S, Isolauri E (2001) Distinct patterns of neonatal gut microflora in infants in whom atopy was and was not developing. J Allergy Clin Immunol 107:129–134 14. Kalliomaki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E (2001) Probiotics in primary prevention of atopic disease: a randomised placebo- controlled trial. Lancet 357:1076– 1079 15. Kalliomaki M, Salminen S, Poussa T, Arvilommi H, Isolauri E (2003) Probiotic and prevention of atopic disease: 4-year followup of a randomised placebo-controlled trial. Lancet 361:1869– 1871 16. Majamaa H, Isolauri E (1997) Probiotics: a novel approach in the management of food allergy. J Allergy Clin Immunol 99:179–185 17. Malin M, Suomalainen H, Saxelin M, Isolauri E (1996) Promotion of IgA immune response in patients with Crohn’s disease by oral bacteriotherapy with Lactobacillus GG. Ann Nutr Metab 40: 137–145 18. Matricardi PM, Bjorksten B, Bonini S, Bousquet J, Djukanovic R, Dreborg S, Gereda J, Malling HJ, Popov T, Raz E, Renz H, Wold A; EAACI Task Force 7 (2003) Microbial products in allergy prevention and therapy. Allergy 58:461–471 19. Miettinen M, Matikainen S, Vuopio-Varkila J, Pirhonen J, Varkila K, Kurimoto M, Julkunen I (1998) Lactobacilli and streptococci induce interleukin-12 (IL-12), IL-18, and gamma interferon production in human peripheral blood mononuclear cells. Infect Immun 66:6058–6062 20. Niers LE, Timmerman HM, Rijkers GT, van Bleek GM, van Uden NO, Knol EF, Kapsenberg ML, Kimpen JL, Hoekstra MO (2005) Identification of strong interleukin-10 inducing lactic acid bacteria which down-regulate T helper type 2 cytokines. Clin Exp Allergy 35:1481–1489 21. Nigo YI, Yamashita M, Hirahara K, Shinnakasu R, Inami M, Kimura M, Hasegawa A, Kohno Y, Nakayama T (2006) Regulation of allergic airway inflammation through Toll-like receptor 4-mediated modification of mast cell function. Proc Natl Acad Sci USA 103:2286–2291 22. Okumura S, Kashiwakura J, Tomita H, Matsumoto K, Nakajima T, Saito H, Okayama Y (2003) Identification of specific gene expression profiles in human mast cells mediated by Toll-like receptor 4 and FcepsilonRI. Blood 102:2547–2554 23. Peng GC, Hsu CH (2005) The efficacy and safety of heat-killed Lactobacillus paracasei for treatment of perennial allergic rhinitis induced by house-dust mite. Pediatr Allergy Immunol 16:433–438 24. Pessi T, Sutas Y, Hurme M, Isolauri E (2000) Interleukin-10 generation in atopic children following oral Lactobacillus rhamnosus GG. Clin Exp Allergy 30:1804–1808 25. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30(9):e36 Tesi di dottorato in Immunologia Oculare, di Marta Sacchetti, discussa presso l’Università Campus Bio-Medico di Roma in data 15/03/2010. La disseminazione e la riproduzione di questo documento sono consentite per scopi di didattica e ricerca, a condizione che ne venga citata la fonte. Graefes Arch Clin Exp Ophthalmol (2008) 246:435–441 26. Pohjavuori E, Viljanen M, Korpela R, Kuitunen M, Tiittanen M, Vaarala O, Savilahti E (2004) Lactobacillus GG effect in increasing IFN-gamma production in infants with cow’s milk allergy. J Allergy Clin Immunol 114:131–136 27. Rautava S, Kalliomaki M, Isolauri E (2005) New therapeutic strategy for combating the increasing burden of allergic disease: Probiotics-A Nutrition, Allergy, Mucosal Immunology and Intestinal Microbiota (NAMI) Research Group report. J Allergy Clin Immunol 116:31–37 28. Rosenfeldt V, Benfeldt E, Nielsen SD, Michaelsen KF, Jeppesen DL, Valerius NH, Paerregaard A (2003) Effect of probiotic 441 29. 30. 31. 32. Lactobacillus strains in children with atopic dermatitis. J Allergy Clin Immunol 111:389–395 Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386 Strachan DP (1989) Hay fever, hygiene, and household size. BMJ 299:1259–1260 Vanderhoof JA, Young RJ (2004) Current and potential uses of probiotics. Ann Allergy Asthma Immunol 93:S33–S37 Wilcox RR (2007) New statistical procedures for the social sciences: modern solution to basic problems. Lawrence Erlbaum Associates, Hillsdale (NJ)