FORUMSIGENPper GIOVANIRICERCATORI

3 -4 - 5 marzo2 0 1 1

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GIOVEDÌ 3 MARZO

18.00 Aggiornamento sulle attività delle Aree tematiche della SIGENP:Endoscopia e diagnostica strumentaleG. Lombardi, Pescara

Fegato e pancreasR. Iorio, Napoli

IMIBDA. Ravelli, Brescia

Neurogastroenterologia e malattie acido-correlateL. Pensabene, Catanzaro

Patologia correlata ad alimentiC. Catassi, Ancona

20.30 Cena

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VENERDÌ 4 MARZO

PRIMA SESSIONE

08.00 Arrivo e registrazione dei partecipanti

09.00 Presentazione del ForumA. Staiano, Napoli - A. Ravelli, Brescia

09.10 Come disegnare un trial clinicoA. Staiano, Napoli

09.50 Cos’è e come si fa una meta-analisiR. Francavilla, Bari

10.30 COFFEE BREAK

10.50 Come scrivere una richiesta di grantR. Berni Canani, Napoli

11.30 Come scrivere un articolo… e farlo pubblicareA. Guarino, Napoli

12.10 Come presentare una comunicazioneA. Ravelli, Brescia

12.40 Discussione

13.00 PRANZO

VENERDÌ 4 MARZO

SECONDA SESSIONEModeratori: R. Francavilla, Bari - R. Iorio, Napoli

14.00 Lettura:Probiotici, microbiota e disordini funzionali GIR. Francavilla, Bari

PRESENTAZIONE DEGLI STUDI DA PARTE DEI DELEGATI

14.30 EFFECTS OF PREBIOTIC SUPPLEMENTATION IN NEWBORNS ONWEIGHT GAIN DURING THE FIRST THREE YEARS OF LIFEA. Di Mauro, Bari

14.50 AGE RELATED PATTERN OF INTESTINAL MICROFLORA INCHILDREN WITH CYSTIC FIBROSIS AND ITS RELATIONSHIP WITHINFLAMMATIONE. Ruberto, Napoli

15.10 INFANTILE COLIC AND REGURGITATION AS A EARLY TRAUMATICINSULT IN THE DEVELOPMENT OF FUNCTIONALGASTROINTESTINAL DISORDERSG. Lassandro, Bari

15.30 LACTOBACILLUSGG INCHILDRENWITHCHRONICABDOMINALPAIN:A DOUBLE-BLIND PLACEBO-CONTROLLED CONTROL TRIALC. Fontana, Bari

15.50 HELICOBACTER PYLORI ACTIVE GASTRITIS IN ASYMPTOMATICSIBLINGS OF INFECTED CHILDRENP. Quitadamo, Napoli

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VENERDÌ 4 MARZO

16.10 COFFEE BREAK

16.40 Lettura:NAFLD in età pediatrica: una valutazione critica delle evidenzedisponibili ed implicazioni per ricerche futureR. Iorio, Napoli

PRESENTAZIONE DEGLI STUDI DA PARTE DEI DELEGATI

17.10 RE-EVALUATION OF DIAGNOSTIC CRITERIA AND THERAPEUTICMANAGEMENT IN CHILDREN WITH WILSON’S DISEASEG. Ranucci, Napoli

17.30 RELATIONSHIP BETWEEN ACTIVATED INFLAMMATORY CELLSAND NON-ALCOHOLIC FATTY LIVER DISEASE IN CHILDRENA. Alisi, Roma

17.50 DOCOSAHEXAENOIC ACID SUPPLEMENTATION DECREASESLIVER FAT CONTENT IN CHILDREN WITH NON-ALCOHOLIC FATTYLIVER DISEASE: A DOUBLE-BLIND, RANDOMIZED CLINICAL TRIALA. Alterio, Roma

VENERDÌ 4 MARZO

18.10 INCREASED CAROTID INTIMA-MEDIA THICKNESS AND HEPATICSTEATOSIS IN INSULIN RESISTENT OBESE CHILDRENM. Mancini, Foggia

18.30 OUTCOME OF SEVERELY MALNOURISHED CHILDREN TREATEDACCORDING TO THE WORLD HEALTH ORGANIZATIONGUIDELINES IN AN AREA WITH HIGH PREVALENCE OF CO-MORBIDITY. A ONE-YEAR EXPERIENCE IN RURAL BURKINA FASOM. Paci, Firenze

18.50 EFFICACY AND SAFETY OF A NEW AMINO ACID-BASED FORMULAVERSUS HYDROLYZED FORMULA IN WEANING CHILDREN WITHINTESTINAL FAILURE AND PARENTERAL NUTRITIONM.A. Caiazzo, Napoli

20.30 CENA

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SABATO 5 MARZO

TERZA SESSIONEModeratori: L. Stronati, Roma - A. Muraro, Padova

08.30 LetturaNuovi meccanismi molecolari implicati nella patogenesi delle IBDL. Stronati, Roma

PRESENTAZIONE DEGLI STUDI DA PARTE DEI DELEGATI

09.00 MUCOSA-ASSOCIATED MICROBIOTA IN IBD PEDIATRICPATIENTS, AND METABOLIC PHENOTYPESV. Iebba, Roma

09.20 ROLE OF INTERLEUKIN-23 RECEPTOR IN PEDIATRIC-ONSETINFLAMMATORY BOWEL DISEASE AND GENOTYPE-PHENOTYPEASSOCIATIONC. Friano, Napoli

09.40 NEW INSIGHTS INTO THE PATHOGENESIS OF INFLAMMATORYBOWEL DISEASE: TRANSCRIPTION FACTORS ANALYSIS INBIOPTIC TISSUES FROM PEDIATRIC PATIENTSM. Pierdomenico, Roma

10.00 ATG16L1 AND IRGM CONTRIBUTE TO THE REGULATION OFIMMUNE RESPONSESC. Strisciuglio, Napoli

SABATO 5 MARZO

10.20 CEACAM6: A POSSIBLE ROLE IN PEDIATRIC CROHN’S DISEASE?M. Costanzo, Roma

10.40 USEFULNESS OF SINGLE BALLOON ENTEROSCOPY IN PEDIATRICCROHN’S DISEASES. Oliva, Roma

11.00 COFFEE BREAK

11.20 Lettura:Recenti acquisizioni sulla patogenesi delle allergie alimentariA. Muraro, Padova

PRESENTAZIONE DEGLI STUDI DA PARTE DEI DELEGATI

11.50 PREDICTION OF CELIAC DISEASE RISK IN A COHORT OF 183FAMILIES FROM SOUTHERN ITALYV. Izzo, Napoli

12.10 IS THERE A DIAGNOSTIC TRANSGLUTAMINASE ANTIBODIESCUT-OFF IN A SELECTED PEDIATRIC POPULATION?M. Pescarin, Padova

12.30 USE OF AN AMINO-ACID-BASED FORMULA IN THE TREATMENTOF COW'S MILK PROTEIN ALLERGY PRESENTING AS ALLERGICPROCTOCOLITISM.F. Mastrototaro, Bari

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SABATO 5 MARZO

12.50 MODIFICATIONS OF CYTOKINES (IL-5, IL-13) AND CHEMOKINES(EOTAXIN-3) MUCOSAL PATTERNS IN CHILDREN WITHEOSINOPHILIC ESOPHAHITISA. Chiaro, Messina

13.10 INTESTINAL MICROFLORA MODULATION AS POSSIBLEINNOVATIVE APPROACH FOR CHILDREN WITH COW’S MILKALLERGYL. Cosenza, Napoli

13.30 Chiusura dei lavori e lunch

abstract

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EFFECTS OF PREBIOTIC SUPPLEMENTATION INNEWBORNS ON WEIGHT GAIN DURING THE FIRSTTHREE YEARS OF LIFE

A. Di Mauro, M. Bisceglia, M. Delvecchio, L. Cavallo,R. Francavilla, F. Indrio

Department of Pediatric University of Bari Italy

BACKGROUND: Recent experimental studies in rats suggest that theaddition of oligofructose to the diet reduces energy intake and protectsagainst weight gain and fat mass development.AIM: To establish if the specific modulation of microbiota by thefermentation of prebiotics during early infancy has any influence onweight gain and length growth.SUBJECTS AND METHODS:106 infants were studied prospectively.18were breast fed and 98 randomly assigned to receive gos-fos mixture(0.8 g/dl of a mixture from scGOS and lcFOS, ratio 9:1IMMUNOFORTIS TM), and placebo (maltodextrin) for one month. Theywere re-evaluated 2.2-3.5 years later. Standing height and weight inunderwear were measured and body mass index (BMI) calculated bydividing weight in kilograms by the square of height in metres andexpressed as SDS in keeping with the Italian growth Chart (Cacciari E. etal 2006). The newborns were evaluated for gastric motility and intestinalpermeability and hyperbilirubinemia.RESULTS: There was no difference in chronological age among groups:breast fed newborns 3.2 (2.6–3.5) yrs, prebiotic 2.6 (2.2–3.5) yrs,placebo 3.1 (2.3–3.5) yrs. The mean BMI in the placebo group was 1.3± 2.1 SDS, higher than in the breast fed newborns, -0.1 ± 0.8 SDS (p =0.004), and in the newborns receiving prebiotic supplementation -0.1 ±0.6 SDS (p = 0.005).CONCLUSION: The addition of a prebiotic mixture significantlyameliorates both gastric motility and intestinal permeability and decrease

faster neonatal jaundice during the first month of life. The present dataindicates that these early effects might influence intestinal functions alsolater in life with possible consequences for entire metabolism. These effectsare possibly mediated by gut microbiota manipulation and warrant furtherstudies.

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AGE RELATED PATTERN OF INTESTINAL MICROFLORAIN CHILDREN WITH CYSTIC FIBROSIS AND ITSRELATIONSHIP WITH INFLAMMATION

E. Ruberto*, V. Raia*, E. Bruzzese*, ML. Callegari**,V. Buccigrossi*, M. Arigliani*, R. Merone*, A. Guarino*

*University “Federico II”/Department of Paediatrics/Naples, **University ”Sacrocuore”/Department of microbiology/Piacenza

AIMS: Intestinal microflora includes a heterogeneous population ofmicroorganisms that colonize the human gut shortly after birth, establishinga symbiotic relationship with the host. Intestinal microflora compositionresults from genetic and environmental factors, such as mode of delivery,diet, drugs and infections. Modifications of gut microbiota have beenrecently described in intestinal and extraintestinal chronic inflammatorydiseases, such as IBD and in obesity. We have previously reported thatintestinal inflammation is a frequent feature in children with Cystic Fibrosis(CF) and this improves with lactobacillus GG (LGG) administration. Wehypothesized that in CF children intestinal microflora is different fromhealthy children and this pattern changes with age. Aim of this study wasto comparatively evaluate intestinal microflora in age-matched CF childrenand controls and to explore its relationship with intestinal inflammation inCF patients.MATERIALS AND METHODS: CF children and age-matched controlswere enrolled. Two stool samples for each patient were collected andstored at -20° to evaluate intestinal inflammation and composition ofintestinal microflora. Intestinal inflammation was determined using fecalcalprotectin (ELISA method). DGGE (Denaturing Gradient GelElectrophoresis) analysis and RT-PCR (Real Time Polymerase ChainReaction) were performed to characterize gut microflora composition.RESULTS: Forty-one CF patients were enrolled, distributed as follows: 6infants (median age: 4 months), 19 children younger than 10 years(median age: 7 years) and 16 children older than 10 years of age

(median age: 12,5 years). They were 24 males and 17 females. 35/41were ΔF508homo/heterozygote and 6/41 had other CFTR mutations. Ininfants there was no statistical difference in fecal calprotectin concentrationbetween CF and controls. In contrast significantly higher concentrations offecal calprotectin were found in both CF children younger and older than10 years compared to healthy controls (184±146 µg/g vs 52±46 µg/gand 181±147µg/g vs 41±54 µg/g, respectively).Intestinal microflora in CF was generally characterized by a low richnessand a major interindividual variability compared to healthy controls.However, the composition of intestinal microbiota showed an age-dependent pattern. In infants, DGGE analysis showed a decrease inBacteroides species. In CF children younger than 10 years of age,qualitative and quantitative analysis indicated a significant reduction ifcompared to healthy controls in Eubacterium rectale (1x 1010± 2x 1010vs 2.75x 1011± 2.36x 1011 CFU/g of stools; p<0.01) and Bacteroidesspecies (total Bacteroides: 1.28x 109± 3x 109 vs 8.5x 109± 5.74x 109CFU/g of stools, p<0.01; Bacteroides uniformis: 1.23x 107± 4.7x 107 vs3.32x 108± 3.37x 108 CFU/g of stools; p<0.01) and a non significantreduction in Bifidobacterium pseudocatenulatum. In CF children older than10 years, DGGE profile showed more evident bands corresponding toEscherichia Coli and Dialister invisus, whose relative abundance wasconfirmed by RT-PCR, even if not statistically significant. No correlationbetween the reduction of specific bacterial species and fecal calprotectinconcentration in CF children was observed.CONCLUSIONS: Intestinal microecology has peculiar features in CFchildren with quanti-qualitative differences if compared to age-matchedhealthy children.Intestinal inflammation is frequently observed in CF patients. Modifications ofgut microbiota occur early in life and could well play a role in intestinaland extraintestinal inflammation. In addition, the composition of microbialpopulation varies according to the age of children. These findings are inagreement with the antinflammatory effects observed with LactobacillusGG administration in CF children in the intestinal and respiratory tract.Targeting intestinal microecology early in life may be a novel strategy inthe approach to CF.

This research has been founded by Fondazione per la ricerca sulla fibrosicistica (grant FFC#23/2009) and adopted by Lega Italiana Fibrosi CisticaOnlus - Associazione Toscana Onlus e For me S.r.l.

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INFANTILE COLIC AND REGURGITATION AS A EARLYTRAUMATIC INSULT IN THE DEVELOPMENT OFFUNCTIONAL GASTROINTESTINAL DISORDERS

G. Lassandro, L. Cavallo, R. Francavilla, F. Indrio

Department of Pediatric University of Bari Italy

OBJECTIVE: To investigate the development of functional gastrointestinaldisorders (FGIDs) in children affected in a neonatal age by colic,functional regurgitation and constipationSTUDY DESIGN: A cohort of children 3 to 16 years old referred tooutcome patient of Pediatric Gastroenterology Unit of the UniversityHospital in for recurrent abdominal pain from January 2002 troughDecember 2009 were reviewed. The children were diagnosed with RomeII and III criteria according to the time of evaluation. History of colicregurgitation and constipation during neonatal period, from first to threemonths of age was collected. The population studied was matched withhealthy control of the same age and sex without history of recurrentabdominal pain enrolled among family pediatrician.RESULTS: 3000 children 1456 boys; mean age 7.9; age range 3-16)were recruited. Regurgitation (35% vs 26% control population p<0.001),colic (24% vs 20% control population p<0.002), and constipation (30%vs 22% control population p<0.001).CONCLUSIONS: there is a significant increase in incidence of FIGDs inchildren affected during the neonatal period by colic and functionalregurgitation and constipation. These events can act as a traumatic insultin and influence the development of gastrointestinal tract disturbanceslater in life.

3LACTOBACILLUS GG IN CHILDREN WITH CHRONICABDOMINAL PAIN: A DOUBLE-BLIND PLACEBO-CONTROLLED CONTROL TRIAL

C. Fontana, S. Fico, G. Leone, F. Indrio, R. Francavilla

Ambulatorio di Gastroenterologia Pediatrica, Dipartimento di Biomedicina dell’Età Evolutiva,Policlinico di Bari

OBJECTIVE: Recurrent abdominal pain is a common in school-agedchildren and no reliable treatment is still available. The aim of the presentstudy is to determine if oral administration of Lactobacillus GG wouldimprove frequency (F) and severity (S) of symptoms in children with FAPand if the administration has an effect on the intestinal permeability.METHODS: A total of 136 children [78 F (58%); 6,4 yrs ± 2,1yrs] whofulfilled the Rome II criteria (functional abdominal pain (FAP) or irritablebowel syndrome (IBS) were enrolled in a double-blind, randomizedplacebo controlled trial in which they received LGG (n = 67), or placebo(n = 69) for 8 weeks. F and S were monitored before (week 1-4), during(week 5-12), and after treatment (week 13-20), with Visual AnalogueScale and validated Faces Pain Scale. At entry and at the end of the trialon voluntary basis children underwent double sugar (lactulose andmannitol) intestinal permeability test according using pulsed amperometricdetection (DIONEX DX 600) and result were expressed as ratio betweenthe two sugars (La/Ma).RESULTS: Children receiving placebo or LGG were similar at entry fordemographic characteristics. Compared to baseline, at 12 weeks, therewas a mean reduction of the F of pain of about 63% in the LGG groupcompared to a 26% reduction in the placebo group (p<0,01). Thisdifference was still present at the end of follow-up (mean reduction of 68%in LGG compared to a 24% reduction in placebo group; p<0,01).Similarly, at week 12 there was a mean reduction of the S of pain of 42%in the LGG compared to a 23% reduction in the placebo group (p<0,01).At the end of follow-up there was still a mean reduction of 62% in the

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HELICOBACTER PYLORI ACTIVE GASTRITIS INASYMPTOMATIC SIBLINGS OF INFECTED CHILDREN

P. Quitadamo, G. Boccia, D. Russo, M. Russo, E. Miele, andA. Staiano

Department of Pediatrics, University “Federico II”, Naples, Italy

BACKGROUND: Helicobacter pylori (Hp) has been identified as a classI carcinogen for gastric cancer. The critical time during which Hpproduces its carcinogenic effect may be childhood. The aim of our sudywas to evaluate the presence of Hp gastric active lesions amongasymptomatic siblings of Hp infected children.METHODS: Forty-eight index cases (IC) (M/F: 26/22; mean age ± SD:11.9 ± 3.15 years; range: 3-17 years) were consecutively recruited frompatients complaining of upper gastrointestinal (GI) symptoms whounderwent upper gastrointestinal endoscopy (UGE). Hp infection wasproven by bacterial culture and/or histology and 13C urea breath test(UBT). Fifty-six asymptomatic siblings of IC were invited to undergo UBTand to complete a standardized questionnaire for GI symptoms. UBTpositive siblings underwent UGE in order to evaluate Hp infection andgastric active lesions.RESULTS: Twenty-seven out of 48 (56.25%) IC were Hp infected. Apositive UBT was reported in 17 out of 32 (53.1%) siblings of Hp infectedIC, compared with 4 out of 24 (16.7%) siblings of Hp negative IC(p=0.005). Ten of 13 (77%) UBT positive siblings of Hp infected IC whounderwent UGE reported chronic gastric active histological lesionstogether with histological Hp infection. One out of 4 (25%) UBT positivesiblings of Hp uninfected IC showed gastric active lesions and thepresence of Hp at histology (p=0.011, siblings of infected vs siblings ofuninfected IC).CONCLUSION: Hp gastric active lesions are present in asymptomaticsiblings of Hp infected children. It could be worthwhile to evaluate a testand treat strategy in this subset of children.

5LGG compared to a 28% reduction in the placebo group (p<0,01). Atweek 12 we found that LGG but not placebo determined a significantdecrease in: a) the number of patients with an altered IPT (-40% vs. -21%;p<0,03) and b) La/Ma [(0,026 ± 0,005 (95%CI: 0,012-0,033) vs.0,034 ± 0,01 (95%CI: 0,27-0,041); p<0,02]. All the differences werestatistically significant in IBS but not in FAP.CONCLUSION: LGG, and not the placebo, significantly reduces thefrequency and severity of abdominal pain in children with IBS after acourse of 8 weeks treatment and the effect appears to be sustained longtern after the end of treatment. We speculate that this effect may besecondary to the improvement of the gut barrier induced by theLactobacillus GG in children with IBS..

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RE-EVALUATION OF DIAGNOSTIC CRITERIA ANDTHERAPEUTIC MANAGEMENT IN CHILDREN WITHWILSON’S DISEASE

G. Ranucci, C. Della Corte, E.Nicastro, M. Tufano, F. Di Dato,P. Vajro, R. Iorio

Department of Pediatrics, University of Naples Federico II, Italy

Wilson’s disease (WD) is an autosomal recessive disorder of coppermetabolism (1). The distribution of the metal in diverse organs over timeaccounts for the wide range of clinical manifestations. In the pediatricage bracket, most cases have a hepatic presentation: the percentage ofWD children presenting with isolated elevated serum aminotransferasesranges from 14% to 88% (2,3). If WD is not recognized and adequatelytreated, the progression of hepatic and neurological damage can be veryrapid, and fulminant liver failure can occur. Therefore, the promptdetection of this condition is vital. Unfortunately, the diagnosis of WD isan especially challenging task in children because the conventionalcriteria established for adults are not always appropriate.It is widely accepted that penicillamine is the first-choice therapy for WDchildren with liver disease while zinc is indicated in presymptomaticpatients and as maintenance therapy (1). The optimal medical therapy inpatients presenting with isolated elevated serum aminotransferasesremains unestablished. This probably reflects the absence of anagreement on classification of WD patients with isolatedhypertransaminasemia as presymptomatic cases, requiring zinc, orcases with hepatic onset, requiring chelating agents.The aim of our study was to re-evaluate the conventional diagnosticcriteria and the efficacy of penicillamine and zinc therapy in WD childrenwith isolated hypertransaminasemia.PATIENTS AND METHODS. All WD patients referred to our Departmentof Pediatrics for diagnostic investigation of elevated serum

aminotransferases were analyzed. The diagnosis of WD was initiallyestablished in presence of at least two of the following features: a lowceruloplasmin level (<20 mg/dL), an increased basal urinary copper level(>100 mcg/24 hours), an increased urinary copper level after thepenicillamine challenge test (PCT; >1575 mcg/24 hours), an increasedliver copper level (>250 mcg/g of dry weight). In order to re-evaluate theaccuracy of the diagnostic criteria in WD children, we analyzed onlypatients for whom the diagnosis of WD was supported by the presenceof an abnormal liver copper value and/or the identification of twodisease-causing mutations. Therefore, of the 43 patients (28 males) whofulfilled the aforementioned diagnostic criteria, 40 (26 males, medianage at diagnosis 6.1 years, range 1.1-20.9) were selected for the firstpart of the study.We recruited as control group, 52 patients (30 males, median age atdiagnosis 7.1 years, range 1-20), referred for elevated serumaminotransferases not due to WD. Receiver operating characteristic(ROC) analysis was performed to determine the sensitivity and specificitywith 95% confidence intervals (CIs) for ceruloplasmin, basal 24-hoururinary copper and 24-hour urinary copper after PCT, at differentpreviously proposed cutoff values.In all 43 WD patients, initial and maintenance therapy (drugs used,dosage and duration), response to therapy (especially in terms of serumALT activity), side effects and compliance to therapy were retrospectivelyevaluated. Normalization of serum ALT activity was the main parameterof treatment efficacy in this study. Compliance to therapy was evaluatedon the basis of clinical history and serum and urine copper and zinclevels, as established by AASLD guidelines (1).RESULTS: The serum ceruloplasmin concentration was significantly lowerin children with WD (9.6 + 1.3 mg/dL) versus controls (27.45 + 0.9mg/dL, P < 0.0001). ROC analysis of ceruloplasmin suggested that themost useful cutoff value was 20 mg/dL that showed a sensitivity of 95%[95% confidence interval (CI) 83.1%-99.4%] and a specificity of 84.5%(95% CI 72.6%-92.6%). The optimal basal urinary copper diagnosticcutoff value was found to be 40 mcg/24 hours (sensitivity 78.9%, 95%

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CI 62.7%-90.4%; specificity 87.9%, 95% CI 76.7%-95%). Urinarycopper values after penicillamine challenge did not significantly differbetween WD patients and control subjects, and the ROC analysis showeda sensitivity of only 12.5%.Among WD children, 31 (72%) were treated with penicillamine as firstchoice. Twelve of 31 patients (39%) normalized ALT levels within a medianof 14 months (range, 4 to 48 months). Of the 19 patients with persistenthyper-ALT, 16 (84%) switched to zinc; six of these (38%) normalized ALTon zinc within a median period of 9.5 months (range, 5 to 151). Eleven(92%) of the 12 patients, given zinc alone as first-choice, normalized ALTwithin a median period of 14 months (range, 2 to 46 months). The onlypatient with persistent hyper-ALT on zinc showed a poor compliance totreatment. As maintenance therapy, zinc was used in 34 children (79%).Among penicillamine-treated and zinc-treated children, 16 (37%) had sideeffects, but drug withdrawal was not required in any case.CONCLUSION: In children with mild liver disease, the measurement ofceruloplasmin serum levels may be considered a reliable first-step test forthe diagnosis of WD; as for urinary copper excretion, basal valuesgreater than 40 mcg/24 hours are suggestive of WD, whereas thepenicillamine challenge test does not have a diagnostic role in this subsetof patients.Although penicillamine therapy is generally used for the initial treatmentof Wilson’s disease, the present study has showed that zinc monotherapymay be used, as both first line and maintenance therapy, in WD childrenwith isolated hypertransaminasemia at presentation.

REFERENCES:1. Roberts EA, Schilsky ML. Diagnosis and treatment of Wilson disease:an update. Hepatology 2008;47:2089-21112. Dhawan A, Taylor RM, Cheeseman P, De Silva P, Katsiyiannakis L,Mieli-Vergani G. Wilson's disease in children: 37-year experience andrevised King's score for liver transplantation. Liver Transpl 2005;11:441-4483. Iorio R, D'Ambrosi M, Marcellini M, Barbera C, Maggiore G, ZancanL, Giacchino R, et al. Serum transaminases in children with Wilson'sdisease. J Pediatr Gastroenterol Nutr 2004;39:331-336.

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RELATIONSHIP BETWEEN ACTIVATED INFLAMMATORYCELLS AND NONALCOHOLIC FATTY LIVER DISEASE INCHILDREN

A. Alisi1, R. De Vito2, A. Masotti3, N. Panera1, A. Citti2,L. Valenti4, A.E. Feldstein5,6, V. Nobili6

1Liver Unit, 2Units of Pathology, 3Gene Expression - Microarrays laboratory, Pediatric HospitalBambino Gesù, Rome, 4Department of Internal Medicine, Università degli Studi, OspedaleMaggiore Policlinico “Ca’ Granda” IRCCS, Milan, 5Department of Pediatric Gastroenterologyand 6Cell Biology, Cleveland Clinic, Cleveland, Ohio

BACKGROUND: Paediatric nonalcoholic fatty liver disease (NAFLD)rises in parallel to the obesity. This chronic disease comprises simplesteatosis alone or associated to necro-inflammation (nonalcoholicsteatohepatitis, NASH). The idea that inflammatory cells may participateto the development and progression of NAFLD/NASH is growing.OBJECTIVE: Here we investigate the cellular nature of inflammatoryinfiltrate in liver tissue from children with NAFLD.Patients and Methods Liver expression of specific population of immunecells was firstly assessed in 8 rats with diet-induced NAFLD vs. controlrats (5); then in 34 patients (23 males and 11 females, median age 10.2years) with biopsy proven NAFLD. Anthropometric and biochemicalparameters of patients were obtained by internal database. Here we usedthree different superficial markers to investigate the cellular nature ofinflammatory infiltrate in liver tissue from children with NAFLD: CD45,CD3 and CD163. CD45, known as common leukocyte antigen, is one ofthe most abundant leukocyte cell surface glycoproteins and its expressionis restricted to haematopoietic cells. CD3 antigen represents the mostspecific as well the most sensitive T cells lineage marker, including NKand NKT. CD163 is a member of the cystein scavenger receptorsuperfamily that stains the cells of monocyte/macrophage origin,including activated Kupffer cells.

RESULTS: Diagnosis of NAFLD was histopathologically confirmed in ratsand all subjects. Rats with diet-induced NAFLD presented an increasednumber of CD45 and CD163positive cells, with respect to the control ratstreated with a standard diet. No CD3 positive cells were found in liverfrom NAFLD and control rats.Children with NAFLD/NASH had markedly higher hepatic cell positivityfor CD45 (P<0.0001) and CD163 (P<0.0001) than children with simplefatty liver (No NASH/NAFLD), whereas the number of CD3 positive cellswas significantly decreased (P<0.01) in children with NASH (NAFLDactivity score, NAS 5). Statistical analysis demonstrated a associationbetween C45, CD3 and CD163 positivity and the degree of steatosis,lobular inflammation, ballooning or the stage of fibrosis in all patients.Moreover, at logistic regression analysis there was a significantassociation of all markers with NAS 5 (P<0.01).CONCLUSION:Our results demonstrate that leukocytes markers stronglycorrelate with the severity of disease suggesting that the nature andcomplexity of the inflammatory infiltrate may be used as useful markers notonly to determine the type of disease (No NASH NAFLD orNAFLD/NASH), but also to evaluate its potential progression to fibrosis.

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DOCOSAHEXAENOIC ACID SUPPLEMENTATIONDECREASES LIVER FAT CONTENT IN CHILDREN WITHNONALCOHOLIC FATTY LIVER DISEASE: A DOUBLE-BLIND, RANDOMIZED, CLINICAL TRIAL

A. Alterio1, G. Bedogni2, A. Alisi1, A. Pietrobattista1, P. Risé4,C. Galli4, C. Agostoni3, and V. Nobili1

1Liver Unit, Pediatric Hospital Bambino Gesù, Rome, Italy, 2Clinical Epidemiology Unit, LiverResearch Center, Basovizza, Trieste, 3Department of Maternal and Pediatric Sciences,University of Milan, 4Department of Pharmacological Sciences, University of Milan

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the mostcommon liver disease in children and adolescents living in Westerncountries. Lifestyle changes should be the first step of NAFLD treatment;while effective drugs may be a reasonable second step, but fewrandomized controlled trials are available in children and no definitiveconclusions can be presently reached. In adults, clinical trials have shownthat the supplementations of N-3 LCPUFA, including botheicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA),improves the biochemical and ultrasonographic features of liver steatosis.OBJECTIVE: To investigate whether dietary supplementation with DHAdecreases liver fat content in children with non-alcoholic fatty liver disease(NAFLD).PATIENTS AND METHODS: We performed a randomized controlledtrial of DHA supplementation (250 mg/day and 500 mg/day) vs.placebo in 60 children with biopsy-proven NAFLD (20 children pergroup). Tthe main outcome was the change in liver fat content as detectedby ultrasonography after 6 months of treatment. Secondary outcomeswere the changes in insulin sensitivity index (ISI), alanine transaminase(ALT), triglycerides and body mass index (BMI) after 6 months of treatment.

RESULTS: Blood DHA increased in children supplemented with DHA

(0.65%, 95%CI 0.30 to 1.10% for the DHA 250 mg group and 1.15%,0.87 to 1.43% for the DHA 500 mg group). The odds of more severeversus less severe liver steatosis after treatment was lower in childrentreated with DHA 250 mg/day (OR = 0.01, 0.002 to 0.11, p < 0.001)and DHA 500 mg/day (OR = 0.04, 0.002 to 0.46, p = 0.010) ascompared to placebo but there was no difference between the DHAgroups (p = 0.370). ISI increased and triglycerides decreased to a similardegree in both DHA groups as compared to placebo but there was noeffect on ALT and BMI.CONCLUSION: In conclusion, our randomized clinical trial showsbeneficial effects of DHA on liver steatosis and insulin sensitivity inchildren with NAFLD. Further paediatric RCT are needed to confirm thisvery encouraging finding. Management of children with NAFLD mightconsider incorporating DHA as part of a multifaceted approach.

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INCREASED CAROTID INTIMA-MEDIA THICKNESS ANDHEPATIC STEATOSIS IN INSULIN RESISTENT OBESECHILDREN

M. Mancini, A. Campanozzi, I. Rutigliano, R. Vinci*, M.D’Apolito, M. Falcone, N. D’Altilia, M. Orban, I. Giardino°, M.Pettoello Mantovani.

Clinica Pediatrica, *Radiologia, °Medicina di Laboratorio, Facoltà di Medicina e Chirurgia,Università di Foggia - Italy

Hepatic steatosis and atherosclerosis are conditions often associated withoverfeeding. Although their complications (coronary artery disease, strokeand cirrhosis) usually occur in middle and late age, atheroscleroticprocess in the vascular wall and liver damage might begin very early inobese children.AIM OF THE STUDY: was to investigate, in children attending ouroutpatient clinic for obesity, 1) any early vascular structural change andthe presence of fatty liver; 2) any possible metabolic risk factor.PATIENTS AND METHODS: the study population consisted of 156children (71 girls), whose BMI z-score was 4,5±1,4 and their mean agewas 8,2±2,7 years. They all underwent ultrasonography to measure theintima-media thickness (IMT) of carotids and to study the morphology ofthe liver. Their serum glucose, insulin, HOMA index, lipids, TNF-α weremeasured as well. For those who gave their consent (70 pts), 6-keto PGF1α was also evaluated.RESULTS: according to IMT measurement, patients were divided inGroup A (0,6-0,7 mm) with a BMI z-score of 4,6±1,4* and Group B (0,4-0,5 mm) with a BMI z-score of 4,1±0,9* (*p= 0,2). Their data arereported in the table:

Glucose Insulin HOMA Cholesterol Triglyceride TNF-α 6-keto PG(mg/dl) (µUI/ml) (mg/dl) (mg/dl) (pg/ml) (pg/ml)

GROUP A 89,3±6,6 14,6±7,7 3,2±1,7 161±29 105,4±60 10,1±5,4 185,1±24,1(76 pts) (44 pts)GROUP B 88,0±8,5 11,0±5,8 2,4±1,3 162±32 85,4±50 14±7,5 492,6±91,8(80 pts) (26 pts)p NS 0,001 0,002 NS 0,025 NS 0,002

Liver steatosis was present in 23/76 pts of Group A (30,2%) and in11/80 pts of Group B (13,7%)Conclusions: Increased carotid IMT was documented in 76/156 children(48,7%) and fatty liver in 34/156 (21,8%). Fasting insulin levels, HOMAindex, triglyceride levels and 6-Keto-PGF1α were significantly differentbetween Group A and Group B. These data might suggest that higherinsulin resistance and triglyceride levels, together with low levels of 6-ketoPGF 1α could be considered risk factors for early vascular damage andhepatic steatosis in obese children.

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OUTCOME OF SEVERELY MALNOURISHED CHILDRENTREATED ACCORDING TO THE WORLD HEALTHORGANIZATIONGUIDELINES IN AN AREAWITH HIGHPREVALENCE OF CO-MORBIDITY. A ONE-YEAREXPERIENCE IN RURAL BURKINA FASO

M. Paci, S. Collini, C. Brondello e P. Lionetti.

Dipartimento di Scienze della Salute della Donna e del Bambino, Università degli Studi di Firenze,AOU Meyer, Firenze

OBJECTIVE: To evaluate the implementation of the World HealthOrganization (WHO) guidelines for the treatment of children with severeacute malnutrition and moderate acute malnutrition and associatedcomplications in an area with high prevalence of co-morbidities.PATIENTS AND METHODS: The study was carried out in a districthospital in rural Burkina Faso. Between April 2008 and March 2009,children with severe acute malnutrition and moderate malnutrition plus aconcomitant pathological condition were hospitalized and treated by stafftrained according to the WHO guidelines. After discharge childrenentered the follow-up phase as outpatients until complete rehabilitation.RESULTS: 175 children with severe acute malnutrition (64%) andmoderate malnutrition with illness (36%) were treated. The most frequenttype of acute malnutrition was marasmus (90.1%) followed bykwashiorkor (7.1%) and mixed malnutrition (2.6%). Children withmoderate malnutrition were significantly younger than those with severemalnutrition. Frequency of co-morbidity at admission was very high beingmalaria the most common (74.8%) followed by diarrhea (34.8%) andpneumonia (21.7%). 54.2% of children were discharged reaching -2Standard Deviations (SD) Weight for Height (W/H) reference valueswhereas the others were dismissed gaining weight before attaining -2 SDW/H because of socioeconomic and epidemic reasons.

Mortality rate was 6.8%. Lack of follow-up in the outpatient phase wasvery high, especially during the rainy season.CONCLUSIONS: Implementation of WHO guidelines for inpatienttreatment of children with severe malnutrition and moderate complicatedmalnutrition can lead to a low mortality rate in developing world also inareas with high prevalence of co-morbidity. Lack of attendance ofoutpatient care in the hospital support the concept of a community-basedtherapeutic model.

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EFFICACY AND SAFETY OF A NEW AMINO ACID-BASEDFORMULA VERSUS HYDROLYZED FORMULA INWEANING CHILDREN WITH INTESTINAL FAILURE ANDPARENTERAL NUTRITION

M.A. Caiazzo, G. Ranucci, A. Wudy, M.R. Pirozzi,B. Aceto and M.I. Spagnuolo

Department of Pediatrics, University of Naples Federico II, Italy

INTRODUCTION: Intestinal failure (IF) can be defined as the reduction offunctional gut mass below the minimal amount necessary for digestion andabsorption, adequate to satisfy the nutrient and fluid requirements forgrowth in children. Short bowel syndrome (SBS) was the first commonlyrecognized cause of protracted IF. The normal physiologic process ofintestinal adaptation, after extensive resection, usually allows for recoveryof sufficient intestinal function within weeks to months. During this time,patients can be sustained on parenteral nutrition (PN)1. Complications oflong-term PN include those associated with central venous catheter use andliver failure2. There is no consensus in the recent literature about the idealformula [standard (non-hydrolyzed), semielementary (hydrolyzed), or aminoacid-based)] that might be used to wean these patients from PN. The idealformula should be able to ensure an adequate nutrient intake with minimaladverse effects, such as osmotic diarrhea and allergy3-4. The aim of ourstudy was to assess the impact of a new amino acid-based formula (AF) onnutritional status and its safety in children with IF during four weeks ofobservation, compared with an hydrolyzed formula.PATIENTS AND METHODS: It was enrolled 38 patients referred to theDepartment of Pediatrics (University Federico II, Naples, Italy) for IF withdifferent etiologies: SBS 73%, Motility Disorders 16.2%, Cystic Fibrosis2.7%, Crohn Disease 5.4%, Intestinal Malabsorption 5.4%. Twenty-twopatients (11 males, median age 3.25 years, range 0-10 years) received AF

during weaning from PN, while the remaining 16 patients (9 males, medianage at diagnosis 2.1 years, range 0-7 years) received hydrolyzed formula.Among children that received AF, nutritional status and feeding tolerancewere assessed during four weeks of nutritional rehabilitation from PN usingthe new formula. For each child it was recorded gender, age, body weight,height, body mass index (BMI), etiology of IF and serum levels of nutritionalbiochemical parameters, including albumin, prealbumin, iron, ferritin,calcium, insulin-like growth factor-1 (IGF-1). It was tested xylosemia, urinaryiodine, fecal calprotectin and nitric oxide, as markers of intestinal functionand inflammation. All these parameters were recorded at the time ofenrolment (T0) and after four weeks of dietary management (T1). Allergytests, such as serum total IgE, skin prick test (SPT), atopy patch test (APT),serum specific IgE antibodies (radioallergosorbent test, RAST), wereperformed in every children who had symptoms of cow’s milkallergy/intolerance (CMA/I). Patients were considered affected by CMA/Iif they had positive SPT and/or APT and/or RAST.RESULTS. Among 22 patients that received AF, mean z-scored body weightincreased from -2.01 ± 0.31 to 0.5 ± 0.98 (p<0.0001) at enrolment (T0)and after four weeks of dietary management (T1), respectively. Mean serumalbumin levels increased from 2.38 ± 0.62 g/dL to 4.02 ± 0.46 g/dL(p<0.0001); mean prealbumin values increased from 0.16 ± 0.05 mg/dLto 0.25 ± 0.09 mg/dL (p<0.0006); mean serum iron levels of 18.08 ±9.75 g/dL at T0 increased to 66.62 ± 39.82 g/dL after four weeks(p<0.0001); mean calcium values changed from 8.82 ± 1.16 mg/dL to9.85 ± 1.03 mg/dL (p<0.005); mean IGF-1 increased from 69.75 ±45.02 ng/mL to 109.89 ± 67.46 ng/mL (p<0.05). The children had meanurinary iodine values of 56.23 ± 27.84 g/L at T0 that increased after fourweeks to 119.21 ± 60.8 g/L (p<0.0001). Mean xylosemia values of17.91 ± 10.26 mg% increased to 29.14 ± 11.93 mg% (p<0.005). Fecalcalprotectin and nitric oxide values were within normal range both beforeand after four weeks of administration of AF. No side effects were recorded.None of the patients of the AF branch developed symptoms suggestive ofCMA/I during weaning from PN. All children reached a free diet during the

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follow-up. Among 16 patients that received hydrolyzed formula, alldeveloped symptoms suggestive of CMA/I, during weaning from PN, sothey had undergone allergy tests. Twelve children (75%) had the diagnosisof CMA/I. Only 5 of 16 (31.2%) reached a free diet during the follow-up.CONCLUSION: Significant improvements in anthropometric parameterswere achieved in all patients with IF that received AF, indicating itsnutritional efficacy. Furthermore the good feeding tolerance of AF wasclearly demonstrated by the absence of adverse reactions, allergicmanifestations, or osmotic diarrhea. Our data suggest a possible primaryrole of AF in weaning children with IF from PN, considering its ability toimprove the growth, the nutritional status and the intestinal function, withminimal adverse effects. Furthermore, AF seems to be safer than hydrolyzedformula during transition from PN to EN, because of its lower allergenicity.

REFERENCES1. Goulet O., Ruemmele F. Causes and management of intestinal failure in

children. Gastroenterology. 2006;130:S16-28.2. Goulet O., Ruemmele F, Lacaille F, Colomb V. Irreversible Intestinal

Failure. J Pediatr Gastroenterol Nutr 2004; 38:250-269.3 Ksiazyk J., Piena M, Kierkus J, Lyszkowska M. Hydrolyzed versus

nonhydrolyzed protein diet in short bowel syndrome in children. J PediatrGastroenterol Nutr. 2002;35:615–8.

4. Vanderoof J.A. Hypoallergenicity and effects on growth and tolerance ofa new amino acid-based formula with DHA and ARA. J. PediatrGastroenterol Nutr. 2008;47(2):S60–1.

MUCOSA-ASSOCIATED MICROBIOTA IN IBD PEDIATRICPATIENTS, AND METABOLIC PHENOTYPES

V. Iebba1, M. Proietti Checchi1, G. Di Nardo2, S. Oliva2,M.P. Conte1, A. Macone3, S. Cucchiara2, S. Schippa1

1Microbiology Unit, Dept of Public Health Sciences, Sapienza University of Rome, Italy, 2PediatricGastroenterology and Liver Unit, Dept Pediatrics, Sapienza University of Rome, Italy, 3BiochemistryUnit, Dept of Biochemistry, Sapienza University of Rome, Italy

AIMS: The pathogenesis of Inflammatory Bowel Disease (IBD), includingCrohn’s disease (CD) and ulcerative colitis (UC) as the two major types,is a complex interplay among different interacting elements. IBD patientstypically have higher numbers of mucosa-associated bacteria comparedwith normal subjects. The specific aim of this study is to analyze thecomposition of the dominant mucosa-associated microbiota in IBDpediatric patients, and to analyze human host urine metabolites in orderto cross-correlate and to associate specific bacterial species presence tospecific urinary metabolites induction.METHODS: Temporal temperature gradient gel electrophoresis (TTGE)was used in order to analyze the biodiversity of bacteria, both in IBDpatients and healthy subjects, and to identify bacterial species or groupsthat may be more specifically associated with CD or UC disease. Biopsiesfrom ileum, colon and rectum of 12 CD, 7 UC and 19 controls patientsageing from 8 to 17 years were collected. Total DNA was extracted, andamplification products (by reconditioning PCR) of 16S ribosomal DNAwere compared by TTGE. Solid Phase Micro-Extraction coupled to GasChromatography/Mass Spectrometry (SPME-GC/MS) was used tocharacterize urinary metabolites from the same patients.RESULTS: First results obtained from TTGE profiles revealed a peculiardominant microbiota in relation to the different IBD pathology (UC or CD),evidencing a strong relationship between the dominant intestinal

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microbiota and the type of pathology. Clustering by Factorial DiscriminantAnalysis (FDA) showed a clear separation between TTGE profiles of CDpatients and the other two ones (p<0.0001,Wilks’ Lambda=0.01). Sameresults were obtained with Partial Least Square Discriminant Analysis (PLS-DA). The median bands number in the three patients groups differedsignificantly between CD patients and controls (p=0.00012), and UC andcontrol patients (p=0.012), with a higher bands number in IBD groups.Results obtained showed high intra-individual similarity in all patients,ranging between 80% to 95% (Dice similarity index). The similaritybetween patients within each group is around 50% for CD patients, 60%for UC patients and 30% between control patients. By PLS-DA weextrapolated the TTGE bands discriminating the three groups that needfurther characterization. A major abundance of Proteobacteriaceaebacterial members in IBD was found. PLS-DA analysis on SPME-GC/MSspectra revealed a distinct separation between CD patients and controls(Chi-square=11.827, P=0.0006) with a model predictability of 90.9%(Fisher’s P=0.00014), showing qualitative and quantitative differencesamong peaks.CONCLUSIONS: The presence of a dominant microbiota and a peculiarurinary metabolites profile associable to IBD supports the hypothesis thatintestinal bacteria play a role in the pathogenesis of IBD. These results,even if at a preliminary stage, highlight the peculiarity of the microflora/host interplay in such disorders, and point new functional insights towardsa reformulated ‘probiotic reconstitution’ of dysbiotic IBD microflora.

ROLE OF INTERLEUKIN-23 RECEPTOR INPEDIATRIC-ONSET INFLAMMATORY BOWEL DISEASEAND GENOTYPE-PHENOTYPE ASSOCIATION

C. Friano1, L. Quaglietta1, A.A. te Velde2, S. Meisner2,E. Miele1, A. Staiano1, R. Troncone1

1 Department of Pediatrics, University of Naples “Federico II”, Italy, 2 Centre for Experimentaland Molecular Medicine Academic Medical Centre - Amsterdam, the Netherlands

AIM: To establish the allele frequencies of Interleukine-23 receptor (IL-23R) gene and NOD2/CARD15 gene polymorphisms in a pediatricpopulation affected by Inflammatory Bowel Disease (IBD) and to assessthe impact on susceptibility and disease phenotype.METHODS: The DNA was isolated from peripheral blood of childrenaffected by IBD and screened for polymorphisms of NOD2/CARD15gene (R702W, G908R and 3020insC) and IL-23R gene (s11209026Arg381Gln, rs7517847 intron and rs10889677 exon-3'UTR). Inaddition, the contribution of genetic variants to IBD phenotype wasevaluated. 919 healthy volunteers represented the control group.RESULTS: We investigated the frequencies of the three NOD2/CARD15mutations (Arg702Trp, Gly908Arg, 3020insC) in 71 (M/F: 41/30;mean age 10.8 years, range 4.1-15.1) children with a diagnosis of IBD,22 affected by Crohn’s Disease (CD) and 49 by Ulcerative Colitis (UC).In total 18.2% (4/22) of CD patients and 22.4% (11/49) of patients withUC carried at least one mutant allele within NOD2/CARD15 comparedto 9.2% (85/919) of controls (p<0.05). The frequencies of the three IL-23R gene variants tested showed that 86.4% (19/22) of CD patientscarried at least one mutant allele within IL-23R gene, compared with71.4% (35/49) of patients with UC and 76.2% (700/919) of controls.In particular, Arg381Gln heterozygous polymorphism frequency resultedhigher within the healthy volunteers (48.6%, 280/575), compared to

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14.3% (2/14) of CD patients and 7.7% (3/39) of UC patients carried(p<0.05). None of CD and UC patients carried the Arg381Glnhomozygous variant, confirming the protective role of this polymorphismin our pediatric population too.Genotype/phenotype analysis showed that NOD2/CARD15 mutationswere associated to ileal and ileo-colonic involvement and tomoderate/severe activity in CD affected children, whereas IL-23Rmutations resulted associated to a less extensive ileum involvement. IL-23Rmutations was associated with a more extensive disease in children withUC (pancolitis is reported in 42.8% (15/35) of patients carrying at leastone mutation of IL-23R gene) and to moderate/severe activity disease.CONCLUSIONS: Our data provide additional support regarding thecontribution of the IL-23R gene in CD and UC pathogenesis. Future studieson larger cohorts of pediatric patients are necessary to further explorethe impact on CD and UC phenotypes.

NEW INSIGHTS INTO THE PATHOGENESIS OFINFLAMMATORY BOWEL DISEASE: TRANSCRIPTIONFACTORS ANALYSIS IN BIOPTIC TISSUES FROMPEDIATRIC PATIENTS

M. Pierdomenico, L. Stronati, M. Costanzo, R. Vitali,G. Di Nardo, F. Nuti, S. Oliva, S. Cucchiara, A. Negroni.

Sapienza Università di Roma – Dipartimento di Pediatria, ROMA

OBJECTIVES:Our work is aimed at identifying ex vivo new transcriptionfactors, potentially involved in the pathogenesis of pediatric inflammatorybowel disease (IBD), by using a microarray approach.METHODS: Microarray, including 84 transcription factors, wasperformed in inflamed and uninflamed mucosal tissues of Crohn’s Disease(CD) pediatric patients and in healthy controls. Real time-PCR was usedto confirm microarray results on a larger size of CD and ulcerative colitis(UC) patients. Protein expression was evaluated by western blot assay.RESULTS: Microarray assay showed 40 genes differentially regulated inthe inflamed mucosa and 17 in the uninflamed mucosa of CD patients ascompared to controls. Real-Time PCR analysis revealed 10 transcripts inCD and 4 in UC, selected among those with higher differences ascompared to healthy controls, significantly overexpressed in the inflamedtissues of patients. Moreover, 4 transcripts in CD and 2 in UC were foundsignificantly upregulated in the uninvolved tissue. A further investigationevidenced an increased protein expression of ATF3 and HIF1α in CDpatients as well as in Caco2 cell line stimulated by cytokines and hypoxia.CONCLUSION: This study shows an evident upregulation of severaltranscription factors in the inflamed and uninflamed mucosa of childrenwith IBD, suggesting that the inflammatory process is somehow activatedat molecular levels even in the macroscopically normal mucosa ofpatients. A differential pattern of gene expression between CD and UCindicates distinct molecular mechanisms underlying the pathogenesis oftwo diseases. Finally, ATF3 and HIF1α are proposes as new transcriptionfactors potentially involved in the onset and maintenance of IBD.

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ATG16L1AND IRGMCONTRIBUTE TO THE REGULATIONOF IMMUNE RESPONSES

C. Strisciuglio1,2, C. Friano1, A.C.W. Vos2, M. Duijvestein2,A.P. Verhaar2, G.R. van den Brink2, D.W. Hommes2 and M.E.Wildenberg2

1Dept. of Pediatrics, University Federico II, Naples, Italy, 2Dept. of Gastroenterology andHepatology, Leiden University Medical Center, Leiden, The Netherlands

INTRODUCTION: Various polymorphisms in the autophagy relatedgenes ATG16L1 and IRGM have been associated with the developmentof Crohn’s disease (CD). Autophagy is primarily known to be importantin the survival of cells during starvation as well as the processing ofintracellular bacteria. Although the link between decreased autophagyand an inflammatory disorder like IBD suggests a role for this process inthe regulation of immune responses, no data has been available on thistopic thus far. Therefore, this study focused on the effects of decreasedATG16L1 and IRGM expression on the immunogenicity of dendritic cells.METHODS: Monocytes obtained from healthy volunteers were culturedin the presence of GM-CSF and IL-4 to generate dendritic cells. Geneknockdown was achieved using siRNA technology. Dendritic cellphenotype was studied by flow cytometry, and functionality was tested inmixed lymphocyte reactions, an OVA processing assay and cytometricbead arrays.RESULTS: Knockdown efficiency achieved ranged from 25-85% inindividual experiments. Interestingly, even the lower level knockdownresulted in a clear decrease in functional autophagy, indicating thatrelatively small changes in the levels of autophagy proteins (e.g. such asthose caused by some point mutations) have a strong impact on thepathway as a whole. Decreased levels of autophagy did not decreaseviability of dendritic cells under nutrient-rich conditions. Strinkingly,ATG16L1low and IRGMlow dendritic cells induced significantly more

T-cell proliferation in both an allogeneic mixed lymphocyte reaction andan antigen specific proliferation assay. This finding was consistent in bothhuman and mouse cells, suggesting a conserved role for autophagy inthe regulation of the immune reaction.Flow cytometry showed ATG16L1low and IRGMlow dendritic cells toexpress levels of HLA-DR and co-stimulatory molecules comparable to thatof control cells. Furthermore, decreased levels of autophagy did not resultin an altered cytokine profile of these cells, indicating that mechanismsother than increased maturation underlie the increased immunoreactivity.

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CEACAM6: A POSSIBLE ROLE IN PEDIATRIC CROHN’SDISEASE?

M. Costanzo, M. Pierdomenico, L. Stronati, A. Negroni,R. Vitali, G. Di Nardo, S. Oliva, M. Aloi and S. Cucchiara.

Department of Pediatrics, Pediatric Gastroenterology and Liver Unit, Sapienza UniversityHospital Umberto I, Rome, Italy.

Crohn’s disease (CD) is an inflammatory disorder that is thought toresult from an inappropriated host immune response to the normalgastrointestinal bacteria flora. Recent microbiological research in CD hasmainly been focused on specific Escherichia coli strains, termed AIEC(Adherent and Invasive E.coli), able to adhere and to invade intestinalepithelial cells, surviving within macrophages and inducing secretion ofhigh levels of TNFα. Adhesion of AIEC depends on the expression ofspecific surface receptor CEACAM6 (cell adhesion molecule 6 related tocarcinoembryonic antigen), that was shown to be abnormally expressedby epithelial cells in adults with CD.AIMS OF STUDY: 1) to assess gene/protein expression levels ofCEACAM6 in human CaCo2 (human epithelial colorectal adenocarcinoma)cell line, as well as in bioptic specimens of children with CD, before and afterincubation with TNFα and IFNγ; 2) to evalutate the ability of the knownAIEC strain, LF82, to alter in vitro CEACAM6 expression; 3) to identify newAIEC strains in CD patient biopsies and evaluate the ability to modulateCEACAM6 expression.METHODS: Mucosal biopsy specimens were taken during endoscopyand mRNA and protein expressions were detected by using real-timepolymerase chain reaction and Western blot; Organ Culture of mucosalbiopsies were induced with TFNα and IFNγ and AIEC strains;Immunoistochemistry of mucosal biopsies.

RESULTS:- TNFα and IFNγ are able to increase the gene/protein expression of

CEACAM6 in CaCo2 cell line.- CEACAM6 is up-regulated in intestinal inflamed specimens of children

with CD as compared to healthy controls.- TNFα and IFNγ are able to increase the gene expression of CEACAM6

in cultured healthy ileal tissue of CD patients.- AIEC LF82 is able to induce in CaCo2 CEACAM6 expression.- also new potential AIEC, that we have isolated by CD patients, are able

to induce CEACAM6 gene expression.CONCLUSION: Our work demostrates for the first time that CEACAM6expression:- is up-regulated in intestinal inflamed specimens from CD pediatric

patients as compared to healthy controls;- is strongly influenced in vitro and ex vivo by the pro-inflammatory

cytokines, TNFα and IFNγ;- is in vitro inducible by the presence of the AIEC strain, LF82, and also

by new potential AIEC strains that we have isolated by CD patients.These results might contribute to highlight the pathogenetic mechanismsunderlying CD and identify new therapeutic targets in the management ofpediatric IBD.

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USEFULNESS OF SINGLE BALLOON ENTEROSCOPYIN PEDIATRIC CROHN’S DISEASE

S. Oliva, G. Di Nardo, F. Ferrari, S. Mallardo, P. Rossi, M.Aloi and S. Cucchiara.

Department of Pediatrics, Pediatric Gastroenterology and Liver Unit, Sapienza – Universityof Rome

BACKGROUND: Endoscopic visualization of the small bowel (SB) inCrohn’s disease (CD), either with wireless capsule endoscopy (WCE) orwith balloon assisted techniques is indicated in case of diagnosticuncertainty and when traditional upper gastrointestinal (GI) endoscopydoes not account for the clinical activity of the disease. We describe theuse of single-balloon enteroscopy (SBE) in consecutive pediatric patientswith suspected or established CD. This investigative tool is rarely reportedin children.METHODS: Thirty patients (age range: 7-18 years) were prospectivelyinvestigated: 16 (group A) with suspected CD and unspecific conventionalupper and lower GI endoscopy; 14 (group B) with longstanding CD, 13of which with previous surgery and showing signs unaccountable byconventional endoscopy. All underwent magnetic resonance (MR) andgroup A also WCE.RESULTS: In group A, SBE allowed to diagnose CD in 12, eosinophilicenteropathy in 2, unspecific abnormalities in 2. Of 10 patients, WCEwas diagnostic of CD only in 3, but was suggestive of CD or unspecificin the remaining subjects. In group B, SBE revealed a moderate-to-severedisease activity in most patients, leading to introduction or change inbiological therapy, with subsequent marked decrease in the PCDAI. In 1and 3 patients of group A and group B, respectively, SBE allowedsuccessful dilation of SB strictures. No complications occurred in allinvestigated subjects.

CONCLUSIONS: SBE is a useful and safe endoscopic procedure toevaluate SB in pediatric subjects with suspected or established CD. It canallow to achieve a definite diagnosis of CD when the latter is uncertainand may redirect therapeutic choices in selected CD patients.

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PREDICTION OF CELIAC DISEASE RISK IN A COHORTOF 183 FAMILIES FROM SOUTHERN ITALY

V. Izzo1, M. Pinelli2, F. Tucci1, V. Bruno1, D. Cielo1,M. Morelli1, N.Tinto3, A. Cola3, M.V. Esposito3, S. Cocozza2,L. Greco1, L. Sacchetti3

1Dipartimento di Pediatria Università di Napoli ‘‘Federico II’’, 2Dipartimento di Biologia ePatologia Cellulare e Molecolare "L. Califano" Università di Napoli ‘‘Federico II’’,3Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli ‘‘Federico II’’

AIMS: Predisposition to Celiac Disease (CD) is polygenic trait and ischaracterised by the presence of the HLA-DQ2 heterodimer in more than95% of the patients. But HLA genes explain only 40% of the geneticvariation. Through large Genome Wide Association studies (GWAs) havebeen identified more than 40 non-HLA genes which are likely involved inthe pathogenesis of CD.The aim of the study is to confirm, in 183 CD families, the association ofthe most strongly associated 10 SNPs obtained in the recent GWAs andin their replications and to validate a model of risk prediction for thesiblings in the same cohort.MATERIALS AND METHODS: We recruited 183 families (794 patients)from Southern Italy characterized by a CD-child (proband), both parentsand, at least, one sibling of the proband. HLA genotyping was performedin all the study sample, in order to group the subjects into 5 HLA classesof decreasing CD risk (double DQ2, DQ2 in trans, DQ2 in cis, DQ8, NoDQ2 no DQ8), as we previously reported. Ten Single NucleotidePolymorphisms (SNPs) were genotyped using TaqMan chemistry on anABI Prism 7900HT Instrument (Applied Biosystems): rs6441961(CCR1/CCR3), rs17810546 and rs9811792 (IL12A/SCHIP1 and IL12Arespectively), rs1738074 (TAGAP), rs2816316 (RGS1), rs1464510(LPP), rs2327832 (OLIG3), rs842647 (REL), rs6822844 (IL2/IL21),rs3184504 (SH2B3).

To test whether associated SNPs could have a predictive role, we split thesample in a training set and a testing set. The training set was composedby the proband-parent trios whether the testing set by the sibs of affectedchildren. In the training set we performed a Transmission DisequilibriumTest (TDT) to identify associated SNPs and, by means of a Bayesianapproach, we calculated an arbitrary risk score. Then, in the testing setwe checked if CD recurrence was more frequent in sibs with larger valuesof the arbitrary risk score.RESULTS: By means of a TDT we found an association between threeSNPs, namely those in LPP, REL, and RGS1 genes, and the CD risk. Thebest result regarded the LPP SNP that showed a very strong association(p<0.001, Odds Ratio (OR)=2.38; 95% Confidence Interval (CI)= 1.66-3.33).We assigned to all the sibs the arbitrary risk score from the genotype ofthese 3 SNPs and the HLA haplotype. Then in each HLA risk class, wedivided siblings in two groups (high and low risk) with regards to theirarbitrary risk score. The disease frequency in low risk sibs is 6% while inhigh risk sibs is 16% with a Relative Risk (RR)= 2.62 (95% CI= 1.19 -5.84) and a p value= 0.026.CONCLUSIONS: Three out of ten SNPs were reconfirmed to beassociated to CD in a family study. In addition, our Bayesian resultsshowed that affected siblings line up more frequently in high arbitrary riskscore independently from HLA type. Although more studies on larger anddifferent samples are needed, we believe that present results suggest thatconsidering also 3 non-HLA genes, can improve the prediction of CDrecurrence in high risk siblings.

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IS THERE A DIAGNOSTIC TRANSGLUTAMINASEANTIBODIES CUT-OFF IN A SELECTED PEDIATRICPOPULATION?

M. Pescarin1, D. Basso2, M. Gasparetto1 and G. Guariso1

1Department of Pediatrics, Gastroenterology Unit, University of Padova, Italy, 2Department ofLaboratory Medicine, University of Padova, Italy

BACKGROUND AND SPECIFIC AIMS: To date, serological tests forCeliac Disease (CD) diagnosis available for clinicians have reached ahigh diagnostic accuracy. As yet, in addition to the serologicalexamination, small bowel biopsy remains the gold standard. The purposeof this study is to evaluate a relationship between the serum levels of IgAanti-human transglutaminase (hTTG) and the histological pattern in orderto identify a possible serologic cut-off to be diagnostic amongsymptomatic pediatric patients. In addition, we propose a new diagnosticalgorithm which would allow, in some specific cases with unconfirmeddiagnosis, to eliminate the need for intestinal biopsy.PATIENTS AND METHODS: The analysis employs a retrospective andprospective approach on a sample of 711 symptomatic children, enrolledbetween 2002 and 2010. tTG IgA serum levels (ELISA, Quanta Lite h-tTG IgA – INOVA Diagnostics ) and small bowel mucosal morphology(classified according to Marsh-Oberhuber) were investigated in allchildren. Total IgA were measured using an immunonephelometric assay(Dade Behring GmbH). For tTG IgA measurement, sensitivity was 92.5%,specificity 97.6%, positive predictive value 98% and negative predictivevalue 91.2%. A total IgA deficiency was defined when total IgA valuewas < 0.06 g/L. For the statistic elaborations ROC curves were used,and diagnostic cut-offs with the corresponding specificity, sensitivity levels,positive and negative predictive values were identified on the basis of thebest "delta positive rate” (DPR = (sensitivity-(1-specificity)). All patientswere then divided by age into 5 groups: group I of 81 children aged 2

years; group II of 143 children aged> 2 years and 5 years; group III of146 children aged> 5 years and 8 years; group IV of 200 childrenaged> 8 and 12 years; group V of 142 children aged> 12 years.RESULTS: Among the 711 patients, the histological diagnosis of CD wasmade in 333 patients (229 females), with mean age of 6.7 years.Serological and histological data of both celiac and non-celiac patientswere furtherly compared on the basis of the best DPR, in order to calculatea diagnostic cut-off level. The so obtained value thus is 20 U (withsensitivity 93.9%, specificity 97.3%, PPV 96.8%, NPV 94.7%). Setting thespecificity 100%, the cut-off is equal to 100 U with a sensitivity of 75%,PPV of 100% and NPV of 81%.When patients are grouped according to their age, the following cut-offlevels with a specificity of 100% are found: group I: 60 U; group II: 20.5U; group III: 101.4 U, group IV: 38.5 U, group V: 77 U. Consequently,the range width of cut-off levels among the different groups is remarkable.CD with non-classic symptoms was statistically more frequent among theolder children. Furthermore, children younger than eight years old presentintestinal biopsies with a more severe degree (grade 3 Marsh-Oberhuber)than the older children.CONCLUSIONS: There are differences in clinical presentation relatedto patients’ age at diagnosis: non-classic symptoms are more frequentamong children over 8 years old. The anti- tTG IgA serum levels correlatewith the degree of mucosal lesions and are thus suitable for verifyingpatients’ compliance to dietary treatment.We conclude suggesting a new diagnostic approach whereby symptomaticpatients with anti- tTG IgA antibodies’ level above the value of 100 U donot need to undergo endoscopy, whereas they can start a gluten-free diet.Using this cut-off among symptomatic children, biopsy would be necessaryonly for those cases without response to gluten-free diet.

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USE OF AN AMINO-ACID-BASED FORMULA IN THETREATMENT OF COW'S MILK PROTEIN ALLERGYPRESENTING AS ALLERGIC PROCTOCOLITIS

M.F. Mastrototaro, C. Fontana, S. Fico, G. Leone, F. Indrio,R. Francavilla.

Ambulatorio di Gastroenterologia Pediatrica, Dipartimento di Biomedicina dell’Età Evolutiva,Policlinico di Bari

BACKGROUND: Food allergy to cow's milk proteins (APLV) is frequentlyfound in young infants and may present as proctocolitis. Treatment is bystarting an elimination diet; different substitution products have beenproposed: soya milk, extensive casein/whey protein hydrolysate,hydrolysed soya and pork collagen and amino-acid-based formula.AIM: The aim of our study was to evaluate the efficacy of a new amino-acid-based formula (Nutramigen AA) for the treatment of infantileproctocolitis.PATIENTS AND METHODS: Twelve consecutive infants, 38 to 166days of age, presenting with persistence of bloody stools for an averageof 2 weeks with or without vomiting, failure to gain weight and colic wereenrolled. In all infants the infective/inflammatory causes of rectal bleedingwere excluded. Eight were on extensive hydrolysed formula, three oncow’s milk formula and one was breast fed. At entry all received anaminoacid based formula (Nutramigen AA) for three months and all wereevaluated clinically every 2 weeks. After one and three months faecalcalprotectin, faecal occult test and after three months hemoglobin, serumiron and ferritin were evaluated.RESULTS: After 2 weeks of treatment, 9 of the infants (75%)demonstrated resolution of their symptoms while blood was still present,although reduced, in three; it disappeared after further 2 weeks in oneand 4 weeks in two. Baseline histology, where available, varied fromeosinophilic infiltration to normal findings. Only six infants had failure to

thrive at presentation and this was resolved after a median of six weeks(range: 3-12 weeks). At entry fecal calprotectin/faecal occult test wasabnormal/positive in all while at one and three months these tests werestill abnormal/positive in 5/7 an 1/0 respectively (p<0,001 frombaseline). Hemoglobin, serum iron and ferritin at three months wereimproved compared to baseline (p<0,02). Of the 8 who were challenged,5 tolerated the extensive hydrolysed formula and the remainder hadrecurrence of their symptoms and returned to the amino-acid-basedformula.CONCLUSION: Infants presenting with allergic proctocolitis may haveresolution of their symptoms when fed with the new amino acid-basedinfant formula even if not responsive to extensive hydrolysed formula. Therecovery is followed by improved growth parameters, normalization offaecal calprotectin and amelioration of blood parameters.

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MODIFICATIONS OF CYTOKINES (IL-5, IL-13) ANDCHEMOKINES (EOTAXIN-3) MUCOSAL PATTERNS INCHILDREN WITH EOSINOPHILIC ESOPHAHITIS

A. Chiaro and C. Romano

Pediatric Department, Pediatric Endoscopy Unit, University of Messina

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronicinflammatory disorder, with a persistent or spontaneously fluctuatingcourse, characterized by dense eosinophilic infiltration of the esophagealmucosa. The clinical symptoms are characterized by chronic and/orrecurrent dysphagia, vomiting, epigastric or chest pain until foodimpaction. Treatment goals have been directed toward symptomselimination and reduction/normalization of esophageal inflammation. Therationale later end-point is complete histologic remission. Effectivetreatments include oral gavage with budesonide or fluticasoneproprionate, methylprednisolone and nutritional restrictions. Despite theacute efficacy of steroids, most patients treated with steroids haveclinicopathological relapse when treatment is discontinued. Thecomplications of untreated disease include fibrosis of the esophageallamina propria and strictures formation. The pathogenesis of EoE involvesenvironmental and genetic factors, particularly food antigens, and over-expression levels of cytokines (IL-5) and chemokines (eotaxin-3).Translational studies show the presence of IL-5 in the esophageal mucosawith variable pattern of expression and up-regulation of IL-13 and INF-gamma.OBJECTIVES: evaluate the expression of IL-5 and IL-13, eotaxin-3/CCL26 on esophageal mucosal, their modifications after therapy andcorrelations with severity of clinical symptoms.PATIENTS AND METHODS: this is a multicenter prospective cohortstudy that will provide the recruitment of patients with diagnosis of EoE

(>15 eosinophils/high-power field on esophageal mucosal) agedbetween 3-16 years. We exclude patients with complicated EoE(esophageal stenosis). The patients will be randomized in two group:Group A will start oral steroid treatment for 4 weeks while Group B willbe taken only clinical follow-up. Esophageal biopsies will be obtainedform proximal, medium ad distal esophagus at the moment of recruitmentand after discontinuation of therapy. Cytokines (IL-13, IL-5) and eotaxin-3/CCL26 levels will be performed at T0 (recruitment) and T1 (after 4weeks) on biopsy specimen by enzyme-linked immunosorbent assay(ELISA). Each patient will be evaluated with appropriate clinical score(Pentiulk S, et al. JPGN 2009) at T0 and T1 with correlation betweenclinical response and cytokines pattern.CONCLUSIONS: The primary end point of the study is attempt todemonstrate a significative correlation between clinical score, histologicalmodifications and specific mucosal cytokines expression after steroidtreatment.

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INTESTINAL MICROFLORA MODULATION ASPOSSIBLE INNOVATIVE APPROACH FOR CHILDRENWITH COW’S MILK ALLERGY

L. Cosenza, R. Nocerino, G. Terrin, A. Passariello, A. Coruzzo,L. Leone, M.R. Velardo, M. Di Costanzo, R. Troncone, andR. Berni Canani

Department of Pediatrics and European Laboratory for the Investigation on Food InducedDiseases (ELFID),University of Naples “Federico II”, Italy

BACKGROUND: Intestinal microflora has a crucial role in thedevelopment of atopic disorders. Children with atopic diseases havedifferent commensal bacterial groups in the gut compared to non-atopicchildren, and differences are also found between countries with high andlow incidence of atopic diseases. Lactobacillus GG (LGG) is the moststudied probiotic in the prevention and treatment of atopic disorders. Itelicits potent immunoregulatory effects and influences intestinal microfloratoward a more beneficial composition for prevention and treatment ofatopic disorders. Cow’s milk allergy (CMA) is the most frequent foodallergy in children worldwide. Extensively hydrolysed formula are widelyused for dietotherapy of children with CMA, they are efficacious to resolvethe symptoms but are ineffective to limit disease duration. Recently, LGGhas been added to commercially available extensively hydrolyzed caseinformula (EHCF), and it has been demonstrated effective to promote afaster recovery of intestinal symptoms in infants with CMA-induced allergiccolitis. The effect of this approach on tolerance acquisition in childrenwith CMA is unknown.AIM: The main purpose of our study was to determine whether theaddition of LGG to an extensively hydrolyzed casein formula (EHCF)could be able to influence the time of tolerance acquisition in childrenwith CMA at 6 and 12 months of follow-up, compared with a EHCFwithout LGG.

METHODS: Children (1-24 months of age) affected by IgE-or non-IgE-mediated CMA, confirmed by oral food challenge, were randomlyallocated to two different dietary interventions: 1, EHCF (Nutramigen®,Mead Johnson, Italy); 2, EHCF containing LGG for at least 1x106 CFU/gof formula powder LGG (Nutramigen LGG®, Mead Johnson, Italy). Toinvestigate the possible tolerance acquisition an oral food challenge wasperformed at 6 and 12 months after diagnosis.RESULTS: The presence of LGG in the study formula was checked in twobatches and the results showed a total count of 4.5x107 CFU/gr ofpowder and 8.5x107 CFU/gr of powder, respectively. Sixty-four patientswere enrolled (male 58%; age 5.2 ± 4.8 m; body weight 7.1 ± 2.4 kg;IgE-mediated 41%; gastrointestinal symptoms 61.8%; cutaneoussymptoms 41.8%; respiratory symptoms 16.4%). Kaplan-Meyer methodshowed that the rate of patients acquiring tolerance at 6 (22.2% vs46.4%) and 12 month (37.0% vs 64.3%) was significantly higher ingroup 2 (log-rank test, p<0.05). Linear regression analysis revealed thatthe result was influenced by the presence of gastrointestinal symptoms (B-8.2, p<0.05) but not by age, sex, presence of cutaneous or respiratorysymptoms, or by the mechanism IgE or non-IgE mediated.CONCLUSIONS: To the best of our knowledge this is the firstdemonstration of an effect of LGG on the time of tolerance acquisition.Our data suggest the possibility of an “active dietotherapy” in childrenaffected by CMA. Future studies are necessary to see whether if theimmunoregolatory role elicited by LGG could be also able to be protectiveagainst the atopic march.

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