Journal of Pediatric Gastroenterology and Nutrition
40:616706
May 2005 Lippincott Williams & Wilkins
Abstracts
38th Annual Meeting of the European Society for Pediatric
Gastroentrology, Hepatology and Nutrition
Porto, Portugal, June 14, 2005
OP1-01 FINE-MAPPING OF THE COELIAC DISEASE LINKAGE
REGION ON CHROMOSOME 19P13 REVEALS A NEW
PLAYER IN THE FIELD AJ Monsuur1, I Lavrijsen1,
A Zhernakova1, L Franke1, C Wijmenga1. 1Complex genetics
section, DBG-Department of Medical Genetics, UMCU,
Utrecht, The Netherlands
Introduction: Coeliac disease (CD) is a complex genetic
disorder. Besides the environmental factor gluten and the
HLA-DQ2 and 8 proteins, other unknown genetic factors are
involved. Several genome-wide screens have been performed
to locate the regions with genes involved in CD. In the Dutch
population this has led to the discovery of two susceptibility
regions, 6q21-22 and 19p13 (CELIAC4). The region on
19p13 is only limited to 3.5 Mb, but since it is a region with
a high density of genes, it still contains 92 candidate genes.
Aim: We set out to fine-map this region with microsatellite
markers and single nucleotide polymorphisms (SNPs) to
search for association between genes and CD.
Methods: We started with a cohort of 216 cases and 216
controls and expanded this to 311 cases and 540 controls.
Microsatellites and SNPs were used.
Results: Association testing using microsatellite markers has
revealed a small region of interest of around 450 kb. Further
fine-mapping with SNP shows association in a 150 kb region,
encompassing a limited number of genes. Adding more SNPs
led to the discovery of MYO9B as the gene on 19p13 most
strongly associated to CD. This gene, which is a single-
headed motor myosin, shows association in its 3 part. This
part of the gene contains the most interesting domains, which
also differentiate the role of this myosin from the other family
members. The Rho-Gap and Dag-Pe domains indicate that
this gene is involved in signal transduction. We are now
elucidating the specific role of this gene in signal transduction
and trying to incorporate it in our models for CD.
Conclusion: Finemapping the Dutch CD linkage region on
chromosome 19p13 has led us to the gene MYO9b which is
associated with the disease.
OP1-02 GLUTEN ATAXIA: INTESTINAL AND BRAIN TARGET-
ING BY TISSUE TRANSGLUTAMINASE ANTIBODIES
IR Korponay-Szabo1, M Hadjivassiliou2, CA Williamson2,
K Laurila1, K Haimila3, N Woodroofe2, DS Sanders2,
M Maki1. 1Paediatric Research Centre, Univ. of Tampere,
Tampere, Finland. 2The Royal Hallamshire Hospital and
Univ. of Sheffield, Sheffield, United-Kingdom. 3Finnish Red
Cross, Helsinki, Finland
616
Aims: The pathophysiology of gluten-sensitivity related
ataxia (gluten ataxia), which can also complicate classical
coeliac disease, is unclear. Some gluten ataxia patients do
not have small intestinal villous atrophy and it is not
clear whether they have the same disease entity as those
with coeliac disease. In this study, we investigated whether
gut and brain tissue from gluten ataxia patients show evidence
of in vivo antibody targeting of tissue transglutaminase
(TG2).
Methods: Unfixed, frozen jejunal biopsy samples from
patients with gluten ataxia (n = 11) and from disease control
patients with other known causes of ataxia (n = 8) were
blindly evaluated for the presence of TG2-related immuno-
globulin deposits using double-colour immunofluorescence.
Results were compared with HLA-DQ haplotypes and with
the presence of jejunal villous atrophy and circulating
antibodies against endomysium, TG2 and gliadin. In addition,
frozen autopsy brain samples from one patient who died of
gluten ataxia were also studied.
Results: IgA deposited on jejunal TG2 was found in ten of
the 11 gut samples of gluten ataxia patients and in none of
the disease controls. The intestinal IgA deposition pattern
was similar to that seen in overt coeliac disease or, in
those cases with normal villous structure, to that seen in
patients with latent coeliac disease. Only four of the ten
gluten ataxia patients who had these IgA deposits were
positive for serum anti-TG2 and endomysial antibodies and
five had partial villous atrophy, whereas all but one carried
HLA-DQ2 or DQ8. A widespread IgA deposition in vessels
was also found in the brain of a patient with gluten ataxia,
which was most pronounced in the cerebellum, pons and
medulla, sparse or absent elsewhere. The in vivo IgA
deposition in the brain co-localised with TG2 and was
confined to intracerebral small vessels as shown by the
co-localisation with the endothelial marker von Willebrand
factor. IgA deposited in the wall of medium sized brain
arteries appeared as in vivo endomysial positivity. There
was no IgM or IgG deposition in either jejunal or brain
sections.
Summary: IgA-class anti-TG2 antibodies are present in the
gut and brain in patients with gluten ataxia with or without an
enteropathy.
Conclusions: Humoral autoimmunity against TG2 seems to
be involved in gluten ataxia. Our findings suggest that
cerebellar and spinal lesions result from immune-mediated
mechanisms perhaps through vascular involvement as the
initial insult.
 ESPGHAN 38TH ANNUAL MEETING
OP1-03 TRAIL A NEW MEDIATOR OF THE INNATE IMMUNE
SYSTEM IN THE INTESTINAL MUCOSA B Begue1,
J Schmitz1, O Goulet1, N Cerf-Bensussan1, F Ruemmele1.
1
INSERM EMI 0212, Necker-Enfants Malades Hospital,
Paediatric Gastroenterology, Paris, France.
TNF-related apoptosis inducing agent (TRAIL) potently
induces apoptosis in tumoral transformed cells. Recent
reports indicate that TRAIL is constitutively expressed in
the intestinal mucosa by intestinal epithelial cells (IEC).
However, no pro-apoptotic effects of TRAIL on normal IEC
were observed and the physiological role of TRAIL in the gut
remains unclear. In the present study, we aimed to analyze
the biological effects of TRAIL under normal and in-
flammatory conditions in the intestinal mucosa.
Methods: TRAIL expression in IEC was analyzed by
quantitative real-time PCR and western blot analysis in vitro
and ex vivo. The effect of TRAIL on IEC was analyzed in
vitro using an NFkB assay (electro mobility shift assay), gene
expression assays for IL-8, TNFa, CARD15. Apoptosis was
quantified using the AnnexinV-PI assay.
Results: Normal IEC express low levels of TRAIL, which are
unregulated under pro-inflammatory conditions. Thus, in
patients with inflammatory bowel disease, TRAIL levels were
markedly higher in zones of acute inflammation compared to
non-inflammatory tissue. Similarly, TRAIL levels were
tremendously upregulated in TNBS-induced colitis in B6
mice. Under non-inflammatory conditions, IEC responded to
TRAIL with NFkB activation, as analyzed in vitro using the
HIEC cell model. In parallel, a marked upregulation of the
pro-inflammatory chemokine IL-8 and the intracellular
pathogen recognition receptor CARD15 was observed after
TRAIL stimulation. But no cytotoxic effect of TRAIL on
HIEC or normal enterocytes in organotypic cultures ex vivo
was observed. However, under inflammatory conditions,
mimicked by pre- or co-stimulation with TNFa, TRAIL
potently induced apoptosis in IEC (in vitro and ex vivo).
Summary and Conclusions: TRAIL is a new inflammatory
mediator implicated in the homeostasis of intestinal epithelial
barrier functions. TRAIL is highly upregulated in IEC in
inflammatory CD ileum and may augment in an auto/para-
crine fashion antibacterial defence of the epithelial barrier
and clearance of infected or damaged IEC via apoptosis.
OP1-04 TIGHT JUNCTIONS FORMATION IN EXPERIMENTAL
MODEL OF NECROTIZING ENTEROCOLITIS - EFFECT
OF EGF TREATMENT J Clark1, T Saunders1, S Doelle1,
K Dvorak2, M Halpern1, B Dvorak1. 1University of Arizona,
Dept. Pediatrics, Tucson, USA. 2University of Arizona, Dept.
Cell Biology & Anatomy, Tucson, USA.
Background: Necrotizing enterocolitis (NEC) is the most
common intestinal disease predominately of formula fed,
premature babies. Epidermal growth factor (EGF) reduces the
incidence of disease in a neonatal rat model of NEC. Claudin
and occludin are the major structural and functional
components of the tight junction barrier. However, the role
of tight junctions in NEC pathogenesis and EGF treatment is
currently unknown.
Aim: The aim of this study was to determine if EGF affects
expression of tight junction genes and proteins in the ileum
during the development of NEC.
Methods: Neonatal rats, either dam fed (DF), milk formula
fed (NEC), or fed with formula plus 500 ng/ml EGF (NEC +
EGF) were exposed to asphyxia and cold stress to develop
NEC. After 96 hours, ileal expression of claudin-3 and
occludin were evaluated using Realtime-PCR, Western blot
and immunohistochemistry.
617
Results: Claudin-3 and occludin mRNA levels in the ileum
were significantly increased 24 fold in NEC animals
compared to DF animals (p , 0.0001). Supplementation
with EGF normalized mRNA expression to DF levels.
Histological localization of claudin-3 and occludin proteins
was disturbed in animals with NEC compared to DF animals.
NEC animals had upregulated expression and disorganization
of the tight junction proteins. EGF treatment resulted in
localization of the proteins at the tight junction complex.
Conclusion: The ability of EGF to normalize expression and
localization of tight junction proteins may be one mechanism
by which integrity of the mucosal barrier is maintained,
thereby protecting the intestinal mucosa against NEC. The
increased expression of mRNA and disorganization of protein
in animals with NEC may represent a compensation for the
inability to form functional tight junctions at the mucosal
barrier.
Supported by the APS Porter Physiology Fellowship (to J.C.)
and NIH Grant HD-39657 (to B.D.)
OP1-05 PRODUCTION OF ANTITRANSGLUTAMINASE AUTO-
ANTIBODIES IN THE JEJUNUM OF PATIENTS WITH
TYPE1 DIABETES F Florian1, M Vecchiet1, S Gazzin1,
F Ziberna1, D Sblattero1, A Franzese2, S Auricchio2,
R Troncone1. 1Department of Biology, University of Trieste,
Trieste, Italy. 2Department of Paediatrics and European
Laboratory for the Investigation of Food-Induced Diseases,
University FedericoII, Naples, Italy.
Summary: An association between type 1 diabetes mellitus
(T1DM) and celiac disease (CD) has been reported since
a long time. The prevalence of CD in T1DM is as high as
1.3%16.4%, and nearly 20% of T1DM children has been
shown to react to rectal instillation of gliadin. A hallmark of
CD are the autoantibodies to tissue transglutaminase (tTG).
By the Phage Display technique we previously demonstrated
that the site of production of tTGautoantibodies is the
intestine.
Aim: The aim of this study was to investigate the presence of
IgA anti-tTG antibodies in jejunal biopsies from patients with
T1DM patients who did not show serological markers of CD.
Materials and Methods: We made antibody libraries from
the jejuna of 7 patients with T1DM (4 EMA/tTG+, 3
EMA/tTG2). We first adopted the One Step Cloning
Strategy: only the genes belonging to the VH5 family, which
has been previously associated with the anti-tTG antibody
response, were cloned. In a second stage the entire antibody
repertoire from 2 EMA/tTG + and 2 negative was cloned.
Results: The VH5 libraries from EMA/tTG + patients were
highly positive for the presence of scFv anti-tTG, whereas
those from EMA/tTG-patients were not. Surprisingly, the
htTG selection of the complete antibody repertoire libraries
revealed to be strongly positive also inpatients EMA/tTG2.
The positive clones were subjected to BstnI restriction
analysis (fingerprinting) and sequencing. The fingerprinting
was highly variable and the sequencing showed that the scFv
anti-tTG, in EMA/tTG-patients, belong prevalently to the
VH3 gene family. A possible gluten-related condition was
confirmed by the isolation of anti-gliadin scFv in EMA/tTG
patients.
Conclusion: Phage Display libraries from T1DMpatients
revealed the intestinal expression of anti-tTG antibodies
also in absence of peripheral antibodies. T1DM patients
EMA/tTG-produce anti-tTG antibodies prevalently with the
VH3 gene family. It will be interesting to evaluate the
percentage of T1DM patients EMA/tTG-producing intestinal
anti-tTG antibodies and their possible progression toward
an overt CD.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
618 ESPGHAN 38TH ANNUAL MEETING
OP1-06 THE ROLE OF FECAL TNF ALPHA AND CALPROTEC-
TION IN THE DIAGNOSIS OF NEONATAL DIARRHOEA
N Kapel1, C Roman2, D Caldari2, F Sieprath2, Y Khalfoun1,
O Goulet2, F Ruemmele2. 1Coprology, Pitie Salpetrie`re
Hospital, Paris, France. 2Paediatric Gastroenterology, Necker-
Enfants Malades Hospital, Paris, France.
The aetiology of severe early-onset neonatal diarrhoea which
persists despite bowel rest is heterogeneous and diagnosis is
often difficult. Two different categories of disorders present-
ing with intractable diarrhoea in the first weeksof life can be
distinguished: constitutive epithelial disorders, such as micro-
villous atrophy (MVA) or epithelial dysplasia (ED) and
immune-inflammatory disorders, such as autoimmune enter-
opathy (AE) or inflammatory colitis (IC). The aim of the
present analysis was to evaluate the use of fecal inflamma-
tory markers in the diagnostic work-up of intractable
diarrhoea.
Material and patients: A total of 18 patients (11 males)
were enrolled in this study with a mean age of 10 months
(range 218 months). Stool samples were collected at the
time of diagnosis and stored at 280 until assay. TNFa and
calprotectin were measured by ELISA (TNF easia, Bio-
source, Belgium; Calprest, Eurospital, Italy). 9 children had
a constitutive enterocyte disorder (group1: MVA = 4, ED = 5)
and 9 patients had an immuno-inflammatory disease (group 2:
AE = 4, IC = 5).
Results: All patients presented with intractable diarrhoea
which persisted despite bowel rest. In group 1 nofecal TNFa
was detectable (,30 pg/g), whereas group 2 showed
dramatically elevated TNFa levels (mean 5568, range 237
18078 pg/g), p , 0.01. Under successful treatment these
inflammatory parameters normalized completely. Similarly,
calprotectin levels were normal (,15 mg/g) in all patients in
group 1and highly raised in group 2 (mean:1025, range 375
3095 mg/g), p , 0.01.
Summary/Conclusions: Determination of inflammatory
markers in the fecesis a simple method helping to distinguish
between constitutive and immuno-inflammatory aetiologies
of severe persistent diarrhoea. These data also suggest that
constitutive enterocyte disorders are not accompanied by an
inflammatory mucosal reaction. This point has to be further
followed-up since in the past an inflammatory mucosal
reaction was discussed for ED, probably secondary to an
altered epithelial barrier function.
OP2-01 SMALL BOWEL MUCOSAL TRANSGLUTAMINASE
AUTOANTIBODIES DURING OATS AND GLUTEN
CHALLENGE IN COELIAC DISEASE K Kaukinen1,
K Holm2, M Villanen2, I Korponay-Szabo2, M Maki1.
1
Tampere University Hospital, Tampere, Finland. 2Paediatric
Research Centre, University of Tampere, Tampere, Finland.
Aim: Small bowel mucosal extracellular transglutaminase 2
(TG2)-specific IgA-deposits are found in early developing
coeliac disease (CD) before TG2-autoantibodies are measur-
able in serum. We studied whether such IgA-deposits might
be of value in monitoring CD during oats or gluten challenge.
Method: 23 CD children in remission were randomized to
start either oats (50 g/day) or gluten (wheat, rye, barley and
oats, altogether 20 g/day); when small bowel histological
relapse was evident after gluten challenge, patients continued
with oats but excluded wheat, barley and rye from the diet.
Unfixed, frozen jejunal biopsies (at the baseline, after 6 and 24
months challenges) were blindly evaluated for the presence of
TG2-specific IgA-deposits using double-colour immunofluo-
rescence, and their intensity was graded 03. The results were
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
compared to serum TG2-antibodies and jejunal mucosal
morphology and inflammation.
Results: At the baseline serumTG2-antibodies were negative
in all 23 patients, but 4 of them had minimal (grade 1/22
1 1/2;) small bowel mucosal TG2-specific IgA-deposits,
mainly around mucosal vessels. In gluten challenge group,
jejunal histological relapse was evident and the densities of
IgA-deposits clearly increased (grade 23) within 6 months in
all 10 patients; afterwards while consuming gluten-free diet
with oats jejunal mucosa recovered fully and densities of
TG2-specific IgA-deposits decreased being negative in all
after two years withoats. In oats challenge group in children
having CD in remission two patients withdrew in a month
from the study because of abdominal symptoms; they had no
inflammation or IgA-depositions in jejunal mucosa after
consumingoats. The rest 11 in oats group remained in clinical
and histological remission; after 2-years with oats mucosal
IgA-deposits were negative in 9, two had minimal depositions
as was seen at the baseline of the study.
Summary: Non-toxicity of oats in CD was indicated by
small bowel mucosalTG2-specific IgA-deposits; they corre-
lated well with mucosal villousmorphology and inflammation
and serum TG2-antibodies.
Conclusions: Small bowel mucosal TG2-antibody responses
can be used in monitoring CD.
OP2-02 MANNAN PREVENTS GLIADIN PEPTIDES TOXICITY
ON CACO-2 CELLS M Silano1, O Vincentini1,
R Iannuccelli2, I De Angelis2, AL Stammati2, M De
Vincenzi1. 1Division of Human Health and Nutrition, Istituto
Superiore di Sanita`, Roma, Italy. 2Division of Toxicity
Mechanisms, Istituto Superiore di Sanita`, Roma, Italy.
Aim: Mannan and oligomers of N-acetyl-D-glucosamine are
demonstrated toexert a protective effect on intestinal mucosa
specimens of celiac patients, when exposed to gliadin pepsin-
tryptic digest (GL-PT). Aim of this work is toevaluate the
mechanisms of toxicity which mannan, N,NDiacetylchito-
biose and N,N,N Triacetylchitotriose interfere with on
epithelial intestinal cells.
Methods: Caco-2 cells were exposed to GL-PT (1 mg/ml)
and mannan (1,5 mg/ml), N,NDiacetylchitobiose (up to
1,5 mg/ml) and N,N,NTriacetylchitotriose (up to
0,65 mg/ml) both separately and simultaneously. The
following determinations were carried out as indexes of
toxicity: cell viability measured by uptake of the vital dye
Neutral Red, NO release in the cell culture medium,
transepithelial electrical resistence (TEER) and caspase-3
activity.
Results: The incubation with GL-PT diminuished the cell
viability, the concomitant icubation with mannan didnt.
Caco-2 monolayer exposed to GL-PT showed a significant
decrease in TEER after 4 and 24 hours of treatment, but not
in the concomitant presence of mannan. Nitric oxiderelease
in the culture medium by the cells increased after exposure
to GL-PT. The simoultaneous treatment of the cells with
mannan and GL-PT resulted in a significant reduction
of NO production and caspase-3 activity. The incubation
of the cells with N,NDiacetylchitobiose or N,N,N
Triacetylchitotriose alone, at concentration of 1,5 mg/ml
and 0,65 mg/ml respectevely, resulted in a significative
increase of NO release and caspase-3activity and decrease
of TEER.
Summary: Mannan protects Caco-2 cells from the noxius
effects of gliadin.The two oligomers of N-acetilglucosamine
at high conncentrations showed toxic effects on the cellular
model, while at low concentration no protective effect could
be noticed.
 ESPGHAN 38TH ANNUAL MEETING
Conclusion: These results are consistent with the hypothesis
that mannan could compete with gliadin peptides for the same
cellular pathways.
OP2-03 RAPID DETECTION OF COELIAC AUTOANTIBODIES
IN THE OFFICE IR Korponay-Szabo1, TM Raivio2,
K Laurila2, J B Kovacs3, E Nemes4, K Kaukinen5,
M Maki2. 1Paediatric Research Centre, Univ. of Tampere,
Finland and Dept. of Paediatrics, Univ. of Debrecen,
Debrecen, Hungary. 2Paediatric Research Centre, Univ. of
Tampere, Tampere, Finland. 3Heim Pal Childrens Hospital,
Budapest, Hungary. 4Dept. of Paediatrics, Univ. of Debre-
cen, Debrecen, Hungary. 5Dept. of Internal Medicine, Univ.
of Tampere, Tampere, Finland.
Aims: Coeliac disease can present with a wide variety of
clinical symptoms ranging from asymptomatic patients to
severely sick ones requiring quick intervention or in whom it
is challenging to exclude other causes. Detection of anti-
bodies against transglutaminase 2 (TG2) or endomysium
(EMA) are very helpful in making a positive diagnosis. We
evaluated whether rapid coeliac antibody detection at the
point of care can reliably sort out coeliac patients and help in
planning diagnostic procedures.
Methods: Antibody testing was performed by a commercial
immunochromatographic rapid test (Biocard Celiac Disease,
AniBiotech, Vantaa, Finland) which detects anti-TG2 anti-
bodies from whole blood in five minutes by their binding to
self TG2 antigen contained in the red blood cells of the
sample. For the initial evaluation of the test kit, stored whole
blood samples from 106 patients with untreated coeliac
disease and from 99 patients with normal mucosa were
blindly tested. The test was then carried out in the outpatient
department office on 80 prospectively enrolled subjects aged
159 years (including 47 first-degree relatives of coeliac
patients) with the suspicion of coeliac disease using fingertip
or venous EDTA blood. The results were read onsite. Serum
endomysial and anti-TG2 antibodies were separately de-
termined. Patients with positive antibody results were sent to
jejunal biopsy.
Results: The Biocard test had 97% sensitivity and 91%
specificity for biopsy-proved coeliac disease on stored
samples. From the onsite-tested subjects 11 clinical patients
and 3 family members were positive with the Biocard test. Up
to now, ten of them underwent a jejunal biopsy which showed
severe villous atrophy in all, whereas six other patients with
negative Biocard results who had endoscopy on independent
indications had normal villous structure. In the last three
Biocard-positive and sick patients, biopsy was performed
within 36 hours after initial admission. The Biocard test was
able to show positivity at haemoglobin levels as low as 68 g/l,
and results were highly concordant with EMA and laboratory
anti-TG2 antibody results (kappa value 0.89 and 0.88,
respectively).
Summary: Rapid coeliac antibody testing was convenient
and highly efficient in detecting coeliac patients.
Conclusions: Onsite detection of anti-TG2 antibodies is
useful for rapid case finding and for the appropriate and fast
use of invasive tools while sparing vulnerable patients
unnecessary other diagnostic procedures.
OP2-04 THE C-13910 ALLELE OF LACTASE NON-PERSIS-
TENCE IS ASSOCIATED WITH THE DOWNREGULA-
TION OF LACTASE GENE EXPRESSION DURING
CHILDHOOD AT THE LEVEL OF TRANSCRIPTION
H Rasinpera1, M Kuokkanen2, NS Enattah2, KL Kolho3,
E Savilahti3, A Orpana4, I Jarvela4. 1Department of Medical
Genetics, University of Helsinki, Helsinki, Finland. 2Depart-
619
ment of Molecular Medicine, National Public Health Institute,
Helsinki, Finland. 3Hospital for Children and Adolescents,
University of Helsinki, Helsinki, Finland. 4HUCH-Laboratory
Diagnostics, Laboratory of Molecular Genetics, Helsinki,
Finland.
Aim: To study whether the C213910 allele of the C/T213910
genotype associated with lactase persistence/non-persistence
is associated with the down regulation of the lactase activity
during childhood.
Methods: Intestinal biopsy specimens of 11 Finnish children,
aged 10 months to 17 years, heterozygous for the C/T213910
polymorphism were studied for the lactase enzyme activity
using Dahlqvist method, lactase/sucrase ratio, and lactase
mRNA levels using quantitative minisequencing. Informative
SNP A/G + 593 on exon 1 of the LPH gene was used as marker
of the relative expression level.
Results: All children had high lactase activity (.20 U/g
protein). An equally high level of lactase mRNA was
transcribed from both the C-13910- and T213910 allele in
children , 4 years. In those aged between 4 and 5, a reduction
in the lactase mRNA from the C213910 allele started to appear,
and became evident in children .6 years of age when the
expression was reduced to 1324 % of the expression from
that of the T213910-allele.
Summary: Relative expression levels of lactase mRNA in
intestinal biopsies of children heterozygous for the lactase
persistence genotype C/T213910 were analyzed using quanti-
tative solid-phase minisequencing. The lactase non-persis-
tence-associated C213910 allele was shown to associate with
the reduced transcription of lactase mRNA in children
beginning from the age of 4.
Conclusion: Expression of the C213910 allele of lactase non-
persistence declines with increasing age and accordingly,
associates with the developmental down regulation of lactase
activity in subjects with the C/C-13910 genotype.
OP2-05 NOD2 MUTATION: A RISK FACTOR FOR GUT GRAFT
VERSUS HOST DISEASE IN CHILDREN POST HAE-
MATOPOETIC STEM CELL TRANSPLANT A Hassan1,
S Adams1, K Lindley2, N Shah2, P Milla2, G Davies1, P Veys1,
M Elawad1. 1Department of Immunology and Bone Marrow
Transplant, Great Ormond Street Hospital, London, United-
Kingdom. 2Department of Paediatric Gastroenterology, Great
Ormond Street Hospital, London, United-Kingdom.
Aim: We previously demonstrated that pre-Haematopoetic
Stem Cell Transplant (HSCT) gut infection was an in-
dependent risk factors for the development of gut-Graft
Versus Host Disease (g-GVHD). Mutations of the NOD2
gene leads an altered immune response by activation of the
pro-inflammatory transcription factor NF-kb. This is proved
to be a key factor in inflammatory bowel disease such as
Crohns disease. Our aim in this study was to look at NOD2
mutation in both donor and recipients of HSCT.
Methods: We prospectively studied DNA samples form
donors and patients who underwent HSCT between January
2000 to June 2004. We tested for the 3 types of NOD2
mutation (at SNPs 8,12 & 13), the presence of g-GVHD and
the incidence of infection pre and post transplant.
Results: 17/59 children had NOD2 gene mutations and 42/59
had no mutations. 10/17 (60%) children with NOD2 mutation
developed g-GVHD compared to 6/42 (14%) children with no
mutations (P , 0.001). SNPs 13 occurred in 6/10 of the
patients who had g-GVHD. There was no significant
difference between the 2 groups in term of donor recipient
HLA matching, intensity of conditioning or GVHD pro-
phylaxis.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
620 ESPGHAN 38TH ANNUAL MEETING
Conclusion: This is the first paediatric study to indicate
significant association between NOD2 mutation and the
development of g-GVHD. SNPs 13 appears to carry higher
risk of g-GVHD compared to 8 and 12. This finding is crucial
in defining patients at high risk for g-GVHD and may be used
in donor selection. Improvement of standard hygiene and
prevention of gut infection might be the way forward in
prevention of g-GVHD.
OP2-06 DEFECTIVE ENTERIC NERVOUS SYSTEM STEM
CELL FUNCTION IN THE PATHOGENESIS OF
HIRSCHSPRUNGS DISEASE? N Thapar1, D Natarajan2,
V Pachnis2. 1Institute of Child Health, London, United-
Kingdom. 2National Institute for Medical Research, London,
United-Kingdom.
Aim: To isolate and characterise Enteric Nervous System
(ENS) stem cells from the gut of post-natal miRet51/51
Hirschsprungs mouse.
Methods: Cultures of dissociated myenteric plexus from the
ganglionic portion of miRet51/51 mouse gut were established
and maintained under specific conditions. After 710 days
they form ENS stem cell-containing neurosphere like bodies
(NLBs). Stem cells were selectively labelled by infecting
dissociated NLBs using a green fluorescent protein tagged
retroviral vector and isolated by flow cytometry. The isolated
stem cells were cultured and characterised using a panel of
ENS lineage and differentiation markers.
Results: ENS stem cells were isolated from the ganglionic
portion of post-natal miRet51/51 Hirschsprungs mouse gut.
In clonogenic cultures (15 days) individual stem cells were
able to differentiate into glia and neurons, including mature
neuronal phenotypes. However, there was a clear delay in
the onto geny of differentiation between miRet51/51 stem cells
compared to their wild-type counterparts. At day 5, dif-
ferentiation into neurons and glia was minimal. By day 10,
6.1 + /23.4% of cells in miRet51/51 colonies expressed markers
of neuronal differentiation compared to 12.8 + /25.1% in wild
type colonies (p , 0.01). Glial markers were expressed by
1.5 + /20.68% and 7.0 + /22.3% of colony cells in miRet51/51
and wild-type respectively (p = 0.014).
Summary of Results: ENS stem cells can be isolated from
Hirschsprungs-like gut, and although bipotential, show
a delayed differentiation profile.
Conclusions: These findings suggest that defective ENS
stem cell function may contribute to the pathogenesis of
Hirschsprungs disease. In miRet51/51 mice there is delayed
rostro-caudal migration of ENS stem cells in the developing
gut during embryogenesis. The results suggest that there may
be a critical requirement for differentiation in completing
ENS formation. Differentiated neurons may be essential for
the directed and chronologically ordered migration of
colonising stem cells. In Hirschsprungs disease the late
arrival of stem cells at critical points along the migration
pathway e.g. the caecum, may disrupt key molecular events
and lead to terminal aganglionosis.
OP3-01 LIVER DISEASE FOLLOWING FONTAN OPERATION:
LONG-TERM RESULTS S Camposilvan1, L Zancan1,
E Talenti2, G Stellin3, O Milanesi1, L DAntiga1. 1Dpt od
Paediatrics, Padova, Italy. 2Dpt of Radiology, Padova, Italy.
3
Dpt of Paediatric Cardiac Surgery, Padova, Italy.
Aim: Patients with congenital cardiac malformations cor-
rected by a Fontan operation (FO) lack a pumping chamber in
the pulmonary arterial circulation, and consequently have
chronically increased central venous pressures and liver
dysfunction. Aim of our study was to assess the features of
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
liver disease in children who underwent a FO and its
relationship to cardiac function.
Methods: Between December 2003 and September 2004 we
contacted all patients with a FO performed at our centre to
carry out a prospective study comprising of: physical
examination, biochemical tests (AST, ALT, GGT, Bilirubin,
INR, factor 5, protein profile, faecal alpha1antitrypsin),
echocardiogram, liver ultrasound. A liver disease score,
comprising of clinical, biochemical and ultrasonografic
parameters was used to compare the degree of liver
involvement with cardiac features.
Results: 34 patients, 20 males, median age at assessment
14,7 years (range 4,126,7),26 with a residual left ventricle, 8
with a residual right ventricle, affected by tricuspid atresia
(17), pulmonary atresia (4), hypoplastic left heart syndrome
(5), double outlet right ventricle (2), single left ventricle (2),
miscellaneous (4), with a median follow up after FO of 11,5
years (1,723,3) accepted to be enrolled. 54% had hepato-
megaly, 9% had splenomegaly. 22% had abnormal trans-
aminases, 40% had abnormal gGT, 50% had elevated
bilirubin, 76% had abnormal clotting profile, 21% had
protein losing enteropathy, 18% had abnormal hepatic vein
flow, 6% had dilated portal vein, 41% had an abnormally
fluctuating portal flow, 63% had increased hepatic artery
resistance index, 23% had increased hepatic artery pulsatility
index. 61% had an heart rate ,1 and 38% ,2 Standard
Deviations of normal. Alteration of liver disease score was
correlated to length of follow up after surgery (r2 = 0.29, p =
0.009). Hepatic dysfunction was strictly correlated to low
cardiac index (r2 = 0.20, p = 0.02) and to reduced heart rate
(r2 = 0.12, p = 0.03).
Conclusions: Liver disease is highly prevalent in children
who underwent FO. Hepatic injury is caused by low cardiac
index, due to reduced heart rate, likely as a consequence of
surgical repair. Therapeutic strategies increasing heart rate
and surgical procedures sparing the sinoatrial node could
improve cardiac performance in these patients, and prevent or
reduce hepatic dysfunction in the long-term.
OP3-02 FIBROSCAN* IS A NEW NON-INVASIVE METHOD
FOR THE DIAGNOSIS OF LIVER FIBROSIS IN CHIL-
DREN: A PROSPECTIVE COMPARISON WITH FIBROT-
EST* AND LIVER BIOPSY. T Lamireau1, B Le Bail2,
L Rebouissoux1, L Castera3, J Foucher3, M Darriet4, V De
Ledinghen4. 1Unite de Gastroenterologie Pediatrique,
opital des Enfants, Bordeaux, France. 2Service dAnatomie
H^
Pathologique, H^opital Pellegrin, Bordeaux, France. 3Service
de Gastroenterologie, H^ opital Haut-Leve`que, Bordeaux,
France. 4Laboratoire de Biochimie, H^ opital Pellegrin,
Bordeaux, France.
Aim: Transient elastography (FibroScan) is a novel, non-
invasive and rapid (, 5 mn) bedside method to assess liver
fibrosis by measuring liver stiffness in adult patients, but its
usefulness in children is unknown. The aim of this prospective
study was to evaluate the feasibility and the accuracy of
FibroScan for the detection of hepatic fibrosis in children with
chronic liver disease, in comparison to Fibrotest and liver
biopsy.
Methods: 67 children (29 boys and 38 girls; median age: 11
years, 4 months18 years) with chronic liver disease were
included: Wilsons disease (n = 8), biliary atresia (n = 14),
cystic fibrosis (n = 16), autoimmune hepatitis (n = 6), chronic
viral hepatitis (n = 11), other (n = 12). In 34 cases (51%),
a liver biopsy was performed and fibrosis was evaluated using
METAVIR score. Performance of FibroScan and Fibrot-
est for the detection of fibrosis was assessed using areas
under the receiving operating characteristic curve (AUROC).
 ESPGHAN 38TH ANNUAL MEETING
Correlation of stiffness values with clinical, biological,
morphological and histological parameters were analyzed
using the Spearman coefficient of correlation.
Results: Liver stiffness measurement using FibroScan was
possible in 100% of children. FibroScan and Fibrotest
values ranged from 2.6 to 57.9 kPa, and 0.004 to 0.97,
respectively. According to METAVIR, fibrosis was F1 in 5
cases, F2 in 5 cases, F3 in 8 cases, and F4 in 16 cases.
Correlation coefficients (r) and associated probability (p) were:
r (p) FibroScan Fibrotest
METAVIR fibrosis 0.74 (0.001) 0.48 (NS)
SQS fibrosis 0.71 (,0.0001) 0.54 (0.004)
US splenomegaly 0.63 (, 0.0001) 0.38 (NS)
US hepatic dysmorphy 0.37 (0.05) 0.23 (NS)
Platelet count 20.57 (0.0004) 20.14 (NS)
Conclusion: Liver stiffness measurement by FibroScan is
a feasible, noninvasive and accurate method for the
assessment of liver fibrosis in children. FibroScan shoud
be useful to the follow-up of children with chronic liver
disease in order to avoid repeated liver biopsies.
OP3-03 FEASIBILITY OF NEONATAL WILSON DISEASE
SCREENING BY MUTATION ANALYSIS G Loudianos1,
A Zappu1, O Magli2, S Diana1, E Kanavakis3, P Nicolaidou4,
A Cao5, S De. Virgiliis1. 1Dipartimento di Scienze
Biomediche e Biotecnologie, USC, Cagliari, Italy. 2General
Hospital of Kalymnos, Kalymnos, Greece. 3Department of
Medical Genetics, Athens University, Athens, Greece. 43rd
Pediatric Department, Athens University, Athens, Greece.
5
Istituto di Neurogenetica e Neurofarmacologia,CNR-
Cagliari, Cagliari, Italy.
Wilson disease (WD) is an autosomal recessive disorder caused
by a defective function of the copper transporting ATP7B
protein. This results in progressive copper overload and
consequent liver, brain and kidney damage. The development
of an accurate, cost-effective diagnostic test may pave the way
to preventing the devastating tissue damage caused by WD. In
some isolated populations, such as the Sardinian population
and the Greek inhabitants of the island of Kalymnos, WD
results from a limited spectrum of mutations. Herein we report
an estimation of the incidence of WD in these two populations
by molecular analysis. In the past few years mutation analysis
of the WD gene in Sardinians has led us to characterize the
mutation in 94.3% of the WD chromosomes and to identify
21 disease-causing mutations. The most common of these is
the -441/-427 del promoter mutation, which accounts for 60%
of the total. Presence of the -441/-427 del was screened for in
a random sample of 5290 subjects mainly newborns. The
results revealed 122 positive heterozygous samples with
a carrier rate of 3.8%. Based on these data and according to
the Hardy-Weinberg law we calculated the expected new cases
of the disease, which are 5 new cases per 150000 live births per
year with an incidence of approximately 1/3000 live births per
year. In the island of Kaymnos (Greece), with a population of
20000 inhabitants, mutation analysis of 10 WD families
revealed the presence of the compound heterozygous or
homozygous state for mutations H1069Q or R969Q. By
screening for these mutations in 400 guthrie cards (correspond-
ing to 50% of the newborns in 4 years) we detected one
compound heterozygous state for mutations R969Q/H1069Q,
18 heterozygous for mutation H1069Q, and 9 heterozygous for
mutation R969Q. According to these results, the carrier
frequency was 4.4% for H1069Q and 2.2% for R969Q, while
the expected new cases of the disease are 23.1/20000
621
inhabitants. These preliminary data are important because they
suggest that an efficient screening program based on mutations
analysis for the early identification and treatment of Wilson
disease in a preclinic stage is not only needed, but also feasible
in these populations, as it would prevent the severe toxic effect
of copper in presymptomatic patients.
OP3-04 CHIMERIC ENGRAFTMENT AND TISSUE ENZYME
ACTIVITY AFTER LIVER CELL TRANSPLANTATION
FOR ARGININOSUCCINATE-LYASE DEFICIENCY
X Stephenne1, M Najimi1, C Sibille2, MC Nassogne3,
R Reding4, J De Ville De Goyet4, E Sokal1. 1UCL-St Luc
Departement de pediatrie, Brussels, Belgium. 2UCL-St Luc
Departement de cytogenetique, Brussels, Belgium. 3UCL-St
Luc Departement de neuropediatrie, Brussels, Belgium.
4
UCL-St Luc-Departement de chirurgie, Brussels, Belgium.
Successful engraftment parallel to appearance of tissue
enzyme activity have so far not been shown after human
liver cell transplantation (LCT).
One 42-months old girl with argininosuccinate-lyase (ASL)
deficiency was treated with protein restriction, phenylbuty-
rate and arginine. She however still presented several
episodes of hyperammonemia up to 472 mg/dl with pro-
gressive psychomotor retardation. LCT was performed, and
she received 0.36 billion/kg male and female, fresh and
cryopreserved liver cells from 3 different donors, given in 11
successive infusions over 5 months.
Engraftment was followed in recipient liver biopsies by
fluorescence in situ hybridisation (FISH), real-time PCR for
the Y chromosome and measurement of tissue ASL activity.
FISH technique applied 2 , 5 months post-LCT showed
engraftment with a XX/XXYY chimerism of 4.7%, 5.5%
respectively (blank = 2%). Further hybridization of the
biopsies with centromeric probes specific for autosomes
confirmed the tetraploidy of those cells. The analysis of the
ploidy in donor liver cell suspensions demonstrated that
a significant proportion of tetraploid cells is only present in
the first donor liver. Presence of donor cells was further
confirmed by real- time PCR at the 5 months biopsy. At the 8
months biopsy two others subpopulations of diploid XXY and
diploid XXYY cells were found in significant proportion
(14%, blank = 2.5% and 5%, blank = 0% respectively). ASL-
activity, originally nul, appeared in the 8 months biopsy in
two different samples (0.15 & 0.34 nmoles min21 mg21
respectively, reaching 3 % of normal activity) showing in situ
metabolic effect of donor origin In parallel, metabolic control
improved together with psychomotor progress, including speech
acquisition, changing clinical phenotype from severe neonatal to
moderate late onset type.
Our findings suggest that tetraploid cells may have an
engraftment advantage on other hepatocyte sub-populations.
The diploid XXY and XXYY cells found at the last biopsy
would result from the division of tetraploid infused cells. The
8-months presence of Y-positive cells and appearance of en-
zyme activity are indicative of repopulation of the host liver.
In this patient, we have demonstrated, ongoing liver cell
engraftment of the host liver 8 months post-LCT correlated
with the recovery of enzyme activity.
This report encourages further attempts to correct some inborn
errors of liver metabolism by liver cell transplantation.
OP3-05 IN A HOMOGENEOUS COHORT OF CAUCASIAN
CHILDREN THE PROGNOSIS OF CHRONIC HEPATITIS
B REMAINS FAVOURABLE DURING A 20-YEAR
SURVEY F Bortolotti1, M Guido2, M Resti3, F Pighin1,
A Gatta1. 1Dept. of Clinical Experimental Medicine, Padua,
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
622 ESPGHAN 38TH ANNUAL MEETING
Italy. 2Dept of Pathology, Padua, Italy. 33rd Pediatric Clinic,
Meyer Hospital, Florence, Italy.
Aim: To update the history of biopsy-proven chronic
hepatitis B after anti- HBe seroconversion in a cohort of 91
consecutive Caucasian children.
Methods: Periodical visits included biochemistry, virologi-
cal screening, ultrasonography and counseling regarding
alcohol intake in older subjects.
Results: The outcome in 89 children (21 treated long ago
with immuno- modulators or IFN), after an average 20-year
period, is reported in the table:
Outcome at Last Visit
(average age 26 y) 68 untr. 21 treated
Inactive HBsAg carriers = normal ALT, 41 (60%) 17 (81%)
HBV DNA , 105 cp/ml
HBsAg negative, normal ALT, 9 (13%) 4 (19%)
undetectable viremia by PCR
(residual occult infection?)
HBsAg carrier with mild fluctuating 10 (15%) 0 should be confined to those without neurological involvement.
ALT alterations, viremia ,105 cp/ml
in 9 (13%) cases
Early cirrhosis regressed to normal liver 2 (3%) 0 OP4-01 ORAL INSULIN ENHANCES INTESTINAL ADAPTA-
- Anti-HBe + hepatitis 4 (6%) 0 TION IN A RAT MODEL OF SHORT BOWEL SYN-
- Early cirrhosis progressed to HCC 2 (3%) 0 DROME R Shamir1, I Sukhotnik2, N Shehadeh3. 1Pediatric
Mild to moderate alcohol intake was recorded in 40% of
HBsAg carriers with abnormal ALT but in 4.8% of untreated
inactive carriers (p = 001); drug abuse was recorded only in 2 of
4 cases with anti-HBe positive hepatitis. Cirrhosis was an early
complication in 4 males: complete regression was histological-
ly documented in 2 at the age of 21 and 28 years, respectively.
Summary: Following anti-HBe seroconversion 75% of
untreated and all treated children became adults with normal
liver function and low or undetectable viremia. In addition
13% of untreated cases had a mild disease with low viremia,
associated in part with alcohol intake.
Conclusions: In a homogeneous cohort of Caucasian
children on regular follow-up the prognosis of chronic
hepatitis B remains favorable during the 3rd-4th decade of
life, especially if exposure to cofactors of liver damage is
prevented by counseling.
OP3-06 OUTCOME OF MITOCHONDRIAL CYTOPATHIES PRE-
SENTING WITH LIVER INVOLVEMENT J Raiman2,
M Champion2, AJ Baker1, A Dhawan1, G Mieli-Vergani1,
N Hadzic1. 1Kings College Hospital, London, United-
Kingdom. 2Guys Hospital, London, United-Kingdom.
Aim: In the absence of a readily available diagnostic test for
mitochondrial cytopathies (MC) we investigated clinical
features of children with liver dysfunction eventually di-
agnosed with MC.
Methods: A retrospective review of cases referred to a tertiary
centre between 1991 and 2004 was made. Diagnostic criteria
included: a) reduced muscle respiratory enzyme complex I-V
activity (,20% of age adjusted mean value), b) presence of mt
DNA mutations, c) clinical evidence of a recognised mitochon-
drial syndrome, or d) combination of suggestive biochemical
and radiological features (elevated serum and/or CSF lactate,
abnormal brain MRI), family history and reduced enzyme
activity (2040%).
Results: 24 children (17 male, 71%) were identified. Median
age at presentation was 0.33y (range,05).12 (50%) children
were from consanguineous families. Presenting features
were acute liver failure (ALF)(10), biochemical liver
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
dysfunction (12), and hepatosplenomegaly (2). Extrahepatic
involvement included neurological (18), haematological (3),
renal (5), pancreatic (2) and failure to thrive (8). Median
serum lactate was 3.45 mmol/L (range,0.917.0) and CSF
lactate 1.5 mmol/L (range,16.45). Brain MRI was abnormal
in 11/15 children with either atrophy or changes in the brain
stem or basal ganglia. Diagnosis was confirmed on muscle or
liver enzymology in 20 [deficiency of complexes IV (9), I
(5), I & IV (2) and II & III (4)] and mt DNA analysis in 1.
Alpers (2), Leigh (1) and Pearson sy. (1) were diagnosed in 4
patients. 14 children died at a median age of 1.8y (range,
05.4). 2 boys underwent transplantation due to deepening
liver failure. One of them died from progressive respiratory
failure 10 months later. The other is well with normal
neurodevelopment and graft function 3y post-transplant. Of
the 10 survivors, 2 have cirrhosis, while in 8 the liver
function stabilised after a median follow up of 3.9y (range,
0.513.8).
Conclusions: MC should be sought in all children with
cryptogenic liver disease, including ALF. Of the non-invasive
clinical tests only brain MRI appears to be helpful Overall
prognosis is poor and consideration of liver transplantation
Gastroenterology and Nutrition, Meyer Childrens Hospital,
Haifa, Israel. 2Pediatric Surgery, Bnai Zion Medical center,
Haifa, Israel. 3Pediatric Diabetes, Meyer Childrens Hospital,
Haifa, Israel.
Background: Experimental studies have suggested that
insulin plays an important role in small intestinal growth
and development. In the present study we investigated the
effect of oral insulin on a rat model of short bowel syndrome
(SBS).
Methods: Male Sprague-Dawley rats were divided into three
experimental groups: sham rats (n = 14) underwent bowel
transection, SBS-rats (n = 13) underwent 75% small bowel re-
section, and SBS-INS rats (n = 13) underwent bowel
resection and were treated with oral insulin given in drinking
water from the third to 15th postoperative day. Parameters of
intestinal adaptation (bowel and mucosal weight, mucosal
DNA and protein, villus height and crypt depth), enterocyte
proliferation and apoptosis were determined on day 15.
Results: SBS-INS rats demonstrated a significant increase
(vs. SBS) in jejunal (84 6 5 vs. 71 6 5 mg/cm/100 g, p ,
0.05) and ileal (60 6 3 vs. 43 6 2 mg/cm/100g, p , 0.05)
bowel weight, jejunal (33 6 2 vs. 24 6 2 mg/cm/100g, p ,
0.05) and ileal (22 6 1 vs. 14 6 1 mg/cm/100g, p , 0.05)
mucosal weight, ileal mucosal DNA (32 6 5 vs. 18 6
2 mg/cm/100g, p , 0.05) and protein (49 6 4 vs 24 6
2 mg/cm/100g, p , 0.05), ileal villus height (776 6 44 vs.
672 6 24mm, p , 0.05) and crypt depth (241 6 8 vs 172 6
11 mm, p , 0.05). SBS-INS rats also showed an increased
cell proliferation index in jejunum and ileum and decreased
apoptotic index in jejunum compared to SBS-animals.
Conclusions: In a rat model of SBS, oral insulin strongly
enhances intestinal adaptation. Possible mechanisms may
include increased cell proliferation and decreased enterocyte
loss via apoptosis.
OP4-02 THE INFLUENCE OF HABITUAL DIET AND IN-
CREASED DOCOSAHEXAENOIC ACID INTAKE DUR-
ING PREGNANCY ON THE FATTY ACID COMPOSITION
OF INDIVIDUAL PLACENTAL LIPIDS H Demmelmair1,
M Klingler1, C Campoy2, J Diaz2, T Decsi3, B Veszpremi4,
 ESPGHAN 38TH ANNUAL MEETING 623

B Koletzko1. 1Dr von Hauner Childrens Hospital-Ludwig- Methods: DHA nutritional status in 29 Spanish healthy
Maximilians-University, Muenchen, Germany. 2School of pregnant women was analyzed in relation with parity; they
Medicine-University of Granada, Granada, Spain. 3Depart- were participants in the NUHEAL study; aged between 18
ment of Pediatrics- University of Pecs, Pecs, Hungary. and 40 years, and were recruited at 20 6 1 wks of gestation.
4
Department of Obstetrics and Gynecology-University of Pecs, Plasma phospholipid fatty acids were measured by gas
Pecs, Hungary. chromatography. An individual food frequency questionnaire
was performed at 20th & 30th wks; Spanish tables of food
Aim: To study the effects of dietary n-3 fatty acids on the composition were used (Novartis Dietsource Version 1.7.01).
composition of individual placenta lipid fractions and ANOVA, General lineal model with repeated measures &
elucidate the involvement of various lipid fraction in correlation study were performed using SPPS Versions 11.5.
placental fatty acid transfer. Results: Table 1.
Methods: We analysed the fatty acid composition of Conclusions: After the first delivery, the women in the
placental phospholipids (PL), triglycerides (TG), cholesterol second pregnancy become to show a significant increase in
esters (CE) and non-esterified fatty acids (NEFA) in pregnant the n6/n3 and AA/DHA ratios, indicating a lower DHA
women receiving placebo or n-3 long-chain polyunsaturated nutritional status. The consequences of a DHA deficiency
fatty acids. Placental samples were available from 202 because a drop in mothers stores due to successive
women participating in a randomised clinical trial in pregnancies may be well evaluated. These results suggest
Germany, Spain and Hungary. During the second half of the need of supplementing pregnant women with DHA,
pregnancy, the women received milk based supplements specially if they are multiparous. This work is a part of the
containing either modified fish oil [500 mg docosahexaenoic 5thEU Framework Program (Research grant Nutrition and
acid (DHA), 150 mg eicosapentaenoic acid daily] or placebo. Healthy Lifestyle [NUHEAL], grant no. CLK1-CT-1999-
Placental samples were collected immediately after delivery, 00888).
lipids extracted, lipid fractions isolated by thin layer chroma-
tography and fatty acids analysed by gas chromatography.
Results: DHA values (M 6 SE) of placebo groups tended to 0 deliveries 1 delivery 2 or more
differ between countries (table). Relative to placebo, (n:12) (n:14) deliveries (n:3)
supplementation increased DHA content (M 6 SE) of all X 6 SEM X 6 SEM X 6 SEM
placental lipid fractions in all study populations. 20th
Total saturated and monounsaturated fatty acids were not pregnancy DHA (mg/dl) 10,35 6 0,89 9,34 6 0,67 10,22 6 2,02
affected by the supplementation, but n-6 long chain poly- n-6/n-3 4,38 6 0,48 4,90 6 0,36 4,38 6 1,37
unsaturated fatty acids tended to be lower after supplemen- AA/DHA 0,95 6 0,08 1,02 6 0,07 1,15 6 0,31
tation (n.s.). There was a significant correlation of DHA 30th
content in TG (r = 0.55) and PL (r = 0.71) to DHA content in pregnancy DHA (mg/dl) 10,87 6 0,71 8,83 6 0,53 7,33 6 1,73
NEFA. n-6/n-3 4,34 6 0,32a 4,99 6 0,19ab 6,06 6 1,59b
Conclusions: Dietary n-3 fatty acids modifies the composi- AA/DHA 0,95 6 0,07a 1,04 6 0,05a 1,44 6 0,09b
tion of all major lipid fractions in placental tissue. The Delivery DHA (mg/dl) 11,44 6 1,48 9,27 6 0,59 6,80 6 0,20
correlation of DHA contents in NEFA with those of TG and n-6/n-3 4,00 6 0,35a 5,42 6 0,33b 6,70 6 0,28b
PL, respectively, are in line with a placental NEFA uptake and AA/DHA 0,89 6 0,08a 1,10 6 0,07ab 1,45 6 0,11b
subsequent incorporation into tissue lipid fractions. Neonates DHA (mg/dl) 5,95 6 0,63 4,85 6 0,47 3,48 6 0,41
n-6/n-3 2,06 6 0,21a 2,62 6 0,11b 2,91 6 0,03b
AA/DHA 1,00 6 0,11a 1,27 6 0,05b 1,51 6 0,11b
Spain (n = 110) Germany (n = 48) Hungary (n = 53)

Placebo vs. Suppl. Placebo vs. Suppl. Placebo vs. Suppl.

OP4-04 HOMOCYSTEINE, FOLATE & METHYLENETETRAHY-
PL 4.9 6 0.1 vs 5.7 6 0.2 5.1 6 0.2 vs 6.2 6 0.2 4.2 6 0.2 vs 6.0 6 0.2 DROFOLATE REDUCTASE (MTHFR) 677C/T POLY-
NEFA 4.3 6 0.2 vs 5.6 6 0.2 4.1 6 0.3 vs 4.9 6 0.3 3.9 6 0.3 vs 5.0 6 0.3 MORPHISM IN SPANISH PREGNANT WOMAN AND IN
TG 4.6 6 0.3 vs 6.6 6 0.5 4.9 6 0.5 vs 7.1 6 0.8 4.5 6 0.4 vs 6.8 6 0.5 THEIR OFFSPRING. V Dolz1, C Campoy1, A Molloy2,
CE 2.9 6 0.2 vs 3.9 6 0.2 2.6 6 0.2 vs 3.7 6 0.3 3.5 6 0.2 vs 4.3 6 0.3 J Scott2, G Marchal3, T Decsi4, E Szabo4, B Koletzko5.
1
Dept. of Paediatrics. School of Medicine. University of
Granada, Granada, Spain. 2Dept. of Biochemistry. Trinity
College, Dublin, Ireland. 3Dept. of Obstetric and Gynecol-
OP4-03 PLASMA PHOSPHOLIPID DHA NUTRITIONAL STA- ogy. Granadas University Hospital San Cecilio, Granada,
TUS IN PREGNANT WOMEN AND THEIR NEONATES Spain. 4Dept. of Paediatrics. University of Pecs, Pecs,
IN RELATION WITH PARITY C Campoy1, A Cano2, Hungary. 5Dept. of Paediatrics. Dr. v. Haunersches Kinder-
S Pardillo2, T Decsi3, H Demmelmair4, M Rodrguez5, spital, Munchen, Germany.
MC Ramrez6, B Koletzko4. 1Dept. of Paediatrics. School of
Medicine. University of Granada, Granada, Spain. 2Obstet- Hyperhomocystinemia is considered a cardiovascular risk
ric & Gynecology. University Hospital San Cecilio de factor and has been related to foetal toxicity. The enzyme
Granada. University of Granada, Granada, Spain. 3Dept. of methylenetetrahydrofolate reductase (MTHFR) catalyzes the
Paediatrics. University of Pe`cs, Pecs, Hungary. 4Dr. v. conversion of 5,10-methylenetetrahydrofolate (5,10-MTHF)
Haunersches Kinderspital, LMU, Munchen, Germany. 5Sci- to 5-methyltetrahydrofolate (5-MTHF) and has a direct
entific Department. Ordesa Laboratories. Barcelona. Spain, influence on the remethylation of homocysteine to methio-
Barcelona, Spain. 6Institute of Human Nutrition. University nine. The common 677C/T mutation of the MTHFR gene
of Granada, Granada, Spain. causes lower MTHFR activity and is a risk factor for neural
Multiple pregnancies could affect the docosahexaenoic acid tube defects and hyperhomocysteinemia. As part of the
(DHA) nutritional status in mother and fetus. Some studies NUHEAL study, 144 Spanish healthy pregnant women, aged
demonstrate that maternal DHA stores will be dangerously between 18 and 40 years, were recruited at 20 6 1 wks of
diminished after successive pregnancies. gestation into a randomized double-blind trial where they

J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005

624 ESPGHAN 38TH ANNUAL MEETING
received one of four daily treatments until delivery (docosa-
hexaneoic acid (DHA): 0.5 g/day; 5-MTHF: 0.4 mg/day; both
or placebo) The effects on plasma folate, whole blood folate
(WBF), plasma total homocysteine (tHcy) and the 677C/T
polymorphism of MTHFR were studied.
Biochemical Analysis: MTHFR genotype analysis was
carried out by PCR and RFLP. tHcy was measured using an
automated IMX assay Plasma and red cell folates were
measured by microbiological assay. Data was analyzed using
General Linear Models for repeated measures & correlation
analysis. *Significance was set at: p , 0.05.
Results: tHcy values were ,15 mmol/L in mothers and their
neonates. At delivery, tHcy was higher in mothers who did
not receive MTHF (placebo: 6.37 6 0.33 mmol/L and DHA:
6.39 6 0.32 mmol/L) compared with those who received 5-
MTHF (5-MTHF: 5.61 6 0.28 mmol/L*; DHA + 5-MTHF:
5.67 6 0.22 mmol/L*). Folate levels were ,9 mg/L in 39% of
the mothers at 20 wks and in 56% at delivery. Whole Blood
Folate (WBF) concentrations were ,120 mg/L in 11.8% at 20
wks and in 15.1% at delivery. Some 10.5% of neonates were
deficient in WBF. The MTHFR 677C T polymorphism was
present in 70% mothers and 63% of neonates with 19.4%
mothers and 15.2% infants being MTHFR 677TT homozy-
gotes. The MTHFR 677/T polymorphism group supple-
mented with 5-MTHF showed lower levels of tHcy than the
MTHFR 677C/T or MTHFR 677CC genotypes.
Conclusions: Maternal folate levels decreased during preg-
nancy, and were correlated with the folate in their offspring.
677TT genotype determined a higher risk of folate deficiency
in mothers and neonates. The MTHFR 677TT genotype was
more sensitive to 5-MTHF supplementation, diminishing the
tHcy levels. This work is a part of the 5th EU Framework
Program (Research grant Nutrition and Healthy Lifestyle
[NUHEAL], grant no. CLK1-CT-1999-00888).
OP4-05 THE EFFECT OF ORAL SUPPLEMENTATION OF
LACTOBACILLUS REUTERI, ON THE IMMUNOLOGIC
COMPOSITION OF BREAST MILK. T Jakobsson1,
T Abrahamsson2, B Bjorksten3, M Fredrikson4, M Bottcher
F5. 1Department of Paediatrics, University Hospital of
Linkoping, Linkoping, Sweden. 2Department of Paediatrics,
University Hospital of Linkoping, Linkoping, Sweden.
3
Centre for Allergy Research, Karolinska Institute, Stock-
holm, Sweden. 4Division of Occupational and Environmental
Medicine, University Hospital of Linkoping, Linkoping,
Sweden. 5Clinical Research Centre, University Hospital of
Linkoping, Linkoping, Sweden.
Background: Human milk contains numerous components,
which may affect the infants immune system, but the role of
breast feeding in allergy prevention is controversial. Oral
supplementation of lactobacilli has been suggested to protect
against atopic disease in breast-fed children.
Aim: To identify a potentially effect of the probiotic
lactobacillus strain Lactobacillus reuteri on the immunolog-
ical composition of human milk as part of a prospective study
of allergy prevention during the first years of life.
Methods: Pregnant women (n = 109) received LB reuteri (108
colony forming units; ATCC 55730) or placebo daily during
the last 4 weeks of pregnancy. Samples of breast milk were
obtained within the first three days after delivery (colostrum)
and after one month. The levels of IL-10, TGF-B1 and -2,
TNF-a, soluble CD14, total IgA and secretory IgA were deter-
mined by ELISA. The Na/K-ratio in the samples was deter-
mined as a marker of membrane leakage suggesting subclinical
mastitis.
Results: The levels of the anti-inflammatory cytokine IL-10
were higher in colostrum of mothers receiving LB reuteri
(median 6.6 pg/mL [10th90th percentiles 1.280]), than in
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
samples from the mothers in the placebo group (4.8 pg/mL
[1.225]); p = 0.046.
In contrast, the levels of TGF-B2 were lower in colostrum
from mothers in the LB reuteri group, (median 674 pg/mL
[2791485] vs. 965 pg/mL [4122410]) than in the placebo
group; p = 0.020. The levels of the other parameters were
similar in the two groups. The Na + /K + -ratio was correlated
with the TGF-B2 level, (r = 0.57; p , 0.001), but did not
differ significantly between the two groups. One month after
termination of the treatment, the composition of the breast
milk was similar in the two groups of mothers.
Conclusion: Administration of LB reuteri to mothers
increases the levels of the anti-inflammatory cytokine IL-10
and reduces TGF-B2 in the breast milk. However TGF-B2 is
produced by many cell types. Thus, the origin of TGF-B2 in
breast milk is uncertain. The correlation between TGF-B2
and the Na + /K + ratio may suggest an association with
subclinical mastitits.
OP4-06 PROGRAMMING OF INFANTS SYSTOLIC BLOOD
PRESSURE BY ACCELERATED FOETAL GROWTH
DURING EARLY PREGNANCY J Aaltonen1, T Ojala2,
K Laitinen2, E Isolauri1. 1University of Turku, Department
of Paediatrics, Turku, Fnland. 2Turku University Central
Hospital, Department of Paediatrics, Turku, Finland.
Restricted growth during the foetal period (Barkers hypoth-
esis), followed by vigorous postnatal catch up growth, has
been related to later risk of cardiovascular disease.
Aim: To evaluate the association between early foetal growth
and blood pressure at six months of age in a prospective
study.
Methods: 126 healthy mothers were randomly assigned into
control and dietary intervention groups. The intervention
consisted of dietary counselling by a nutritionist and was
supported by foods with favourable fat composition. Foetal
growth was assessed at 20 gestational weeks by ultrasound
examination and infants blood pressure at 6 months of age
was measured (Dinamap R).
Result: The systolic blood pressure at six months correlated
with the foetal growth at the 20th gestational week (r = 0.5,
p = 0.003), not with birth weight. Thus, the accelerated
growth during the early foetal period followed by the
restricted growth during the later pregnancy was related to
elevated systolic blood pressure in early infancy. Further-
more, the systolic blood pressure tended to be lower in the
intervention group (Table).
Intervention group Control group
mean (95% CI) mean (95% CI) p
Systolic blood
pressure 93 (91-96) mmHg 97 (94-101) mmHg 0.05
Summary: Systolic blood pressure early in life was regulated
by foetal growth during a vulnerable developmental phase.
Conclusion: This study substantiates the Barkes hypothesis
and specifies the pattern of growth during organogenesis as
the major determinant of systolic blood pressure. The later
risk of cardiovascular disease may hence be reduced by
maternal nutrition counselling during pregnancy.
G1-01 MMP-12 IS UPREGULATED IN THE INTESTINE OF
CHILDREN WITH POTENTIAL COELIAC DISEASE
AND IN TYPE 1 DIABETES V Bister1, KL Kolho2,
R Karikoski3, M Westerholm-Ormio2, E Savilahti2,
 ESPGHAN 38TH ANNUAL MEETING
U Saarialho-Kere1. 1Dept. of Dermatology, Helsinki University
Central Hospital, Helsinki, Finland. 2Hospital for Children
and Adolescents, Helsinki, Finland. 3Dept. of Pathology,
Helsinki University Central Hospital, Helsinki, Finland.
Aim: To find markers predictive for the development of
celiac disease (CD) in paediatric patients with positive
screening tests for CD in serum but inadequate criteria for
diagnosis of CD in biopsy.
Methods: Children with positive screening tests (increase in
autoantibodies to TG2 in serum) but with no changes in
villous structure comprised the study group (n = 28). The
clinical course of these children was followed-up for 2 to 3
years. The MMP profile, proliferative and apoptotic charac-
teristics of jejunal biopsies at the beginning of the follow-up
were studied. MMP-1,-3 and -12 were analyzed by in situ
hybridization and MMP-9, -19 and -26 were immunostained.
Apoptotic cells were identified by TUNEL technique and
proliferating cells by immunostaining for Ki-67.
Results: MMP-12 was detected in macrophages in 16/28
samples and its expression was associated with increased
autoantibodies for TG2 and densities of CD3 and gammadelta
positive T-cells in the epithelium. The number of stromal
MMP-26 positive cells was high in patients with high TG2
titers. Expression of MMP-12, MMP-1 and -3 clustered in
children with type 1 diabetes. The proportion of apoptotic
mucosal cells was increased in patients with type 1 diabetes
compared to the other children. When children with CD were
compared to those who did not develop it, the numbers of IEL,
cryptal Ki-67, CD-3 and MMP-12 positive cells were higher
and showed the most significant differences.
Summary: An increase in autoantibodies to TG2 but normal
villous structure poses a challenge for clinicians. We
followed-up such children and found that high titers of TG2
in serum and positive staining for MMP-12 in the gut was G1-03
associated with overt CD.
Conclusions: In paediatric patients, increased numbers of
MMP-12 positive macrophages in lamina propria associate
with high titers of antibodies to TG2 and proness to CD. It is
possible that the upregulation of several MMPs in the gut of
patients with type 1 diabetes reflects a stage of mild
inflammation in their intestine.
G1-02 SHORT TERM EFFECTS OF GLUTEN DIGESTED WITH
LACTOBACILLI ENDOPEPTIDASE ON COELIAC PA-
TIENTS F Landolfo1, M Gobbetti2, R DI Cagno3,
R Lanzetta4, L Viglione5, S Scognamiglio6, ME Camarca7,
L Greco1. 1European Laboratory for Food Induced Disease
(ELFID), Department of Gastroenterology, University of Naples
Federico II, Naples, Italy. 2Department of Plant Protection and
Applied Microbiology, University of Bari, Bari, Italy. 3De-
partment of Plant Protection and Applied Microbiology,
University of Bari, Bari, Italy. 4Department of Chemistry and
Biochemistry, University Federico II., Naples, Italy. 5Department
of Chemistry and Biochemistry, University Federico II., Naples,
Italy. 6European Laboratory for Food Induced Disease (ELFID),
Department of Gastroenterology, University of Naples Federico
II, Naples, Italy. 7European Laboratory for Food Induced
Disease (ELFID), Department of Gastroenterology, University
of Naples Federico II, Naples, Italy. 8European Laboratory
for Food Induced Disease (ELFID), Department of Gastroen-
terology, University of Naples Federico II, Naples, Italy.
Aims: Gliadine contains a superpeptide, 33-mer, not di-
gestible by human intestinal enzymes. This peptide activates
a specific T-cells toxic response which produces the
characteristic intestinal mucosal lesions in celiac patients.
The 33-mer is hydrolyzed by a prolil-endopeptidase from
625
Flavobacterium meningosepticum. We isolated an endopep-
tidase from selected Lactobacilli, which is capable to
hydrolyze such peptides. The aim of the study is to evaluate
the short term toxicity of gluten pre-digested with this
endopeptidase in celiac subjects.
Methods: Twenty Celiac patients, negative for Anti-
Transglutaminase antibodies on a strict gluten-free diet were
admitted. After a baseline permeability test, patients were
allocated randomly to the ingestion of one single biscuit
containing either 2 g of raw gluten or 2 g of gluten digested
for 24 hours with the Lactobacilli peptidases. Fasting patients
ingested one single biscuit and the permeability test was
started after 5 hours. Intestinal permeability was evaluated by
a 5 hour urine collection after a load of Rhamnose 1 gr,
Lactulose 7,5 g and Sucrose 40 g. Trisilyl derivatives were
prepared to assay the sugars by Gas Chromatography
according to Dutton.
Results: Of the 20 patients: 3 were excluded because they
had ingested gluten after the recruitment; 13 showed a marked
increase of the gastrointestinal permeability after the in-
gestion of the raw gluten biscuit and a much lower value (not
significantly different from the basal value) after the ingestion
of a biscuit with the same dose of gluten pre-digested with
Lattobacilli; three patients showed pathological values of
intestinal permeability with both kind of biscuits, while one
patient showed no alteration after the raw gluten ingestion.
Conclusions: We suggest that it is possible to reduce short
term gluten toxicity by prolonged hydrolysis of raw gluten
with Endopeptidase from bacteria normally used in food
production, for the super-dough. Not all patients show the
same sensitivity to gluten and individual variations cannot be
excluded. We are now running a long-term tolerance study.
COELIAC DISEASE AND RISK OF ADVERSE FETAL
OUTCOME A POPULATION-BASED COHORT STUDY
J Ludvigsson1, S Montgomery2, A Ekbom2. 1Dept. of
Paediatrics, Orebro, Sweden. 2Clinical Epidemiology Unit,
Karolinska Institute, Stockholm, Sweden.
Objectives: Studies of maternal coeliac disease (CD) and
fetal outcome are inconsistent and low statistical power is
likely to have contributed to this inconsistency. We in-
vestigated the risk of adverse outcomes in women with CD
diagnosed prior to pregnancy and in women who did not
receive a diagnosis of CD until after the delivery.
Design: A national register-based cohort study restricted to
women aged 1544 years with singleton live born infants.
Linkage between the Swedish national birth register (1973
2001) and the national inpatient register.
Participants: 2078 offspring to women who had received
a diagnosis of CD (19642001): 1149 offspring to women
diagnosed prior to birth and 929 offspring to women
diagnosed after infant birth. Main outcome measures:
Intrauterine growth retardation, low birth weight (,2500 g),
very low birth weight (,1500 g), preterm birth (,37
gestational weeks), very preterm birth (,30 gestational
weeks), and caesarean section.
Results: Undiagnosed CD was associated with an increased
risk of intrauterine growth retardation (OR = 1.62; 95% CI =
1.222.15), low birth weight (OR = 2.13; 95% CI = 1.662.75),
very low birth weight (OR = 2.45;95% CI = 1.354.43), preterm
birth (OR = 1.71; 95% CI = 1.352.17), and caesarean section
(OR = 1.82; 95% CI = 1.272.60). In contrast, a diagnosis of CD
made before the birth was not associated with these adverse fetal
outcomes.
Conclusions: Undiagnosed maternal CD is a risk factor for
unfavourable fetal outcomes, but the risks are reduced where
CD has been diagnosed.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
626 ESPGHAN 38TH ANNUAL MEETING
G1-04 CREATION OF A MOUSE MODEL OF CELIAC DISEASE
BY IN VIVO LONG TERM EXPRESSION OF DISEASE
SPECIFIC ANTITRANSGLUTAMINASE ANTIBODY
D Sblattero1, R Di Niro1, F Ziller1, M Stebel1, T Not2,
L Zentilin3, A Ventura2, R Marzari3. 1Dep of Biology,
TRIESTE, Italy. 2Dep of Sciences of Reproduction and
Development, TRIESTE, Italy. 3ICGEB, TRIESTE, Italy.
Aim: The appearance of autoantibodies targeted against
tissue transglutaminase (tTG) is a typical feature for Celiac
Disease (CD). Several preliminary reports suggest a role of
the antibody response in the disease but to establish whether
and how the tTG antibodies play a role in CD pathogenesis an
animal model in still lacking. We propose to achieve long-
term expression of CD specific anti tTG antibodies in an
animal model inorder to evaluate their biological role.
Methods: Monoclonal antibody recognizing and inhibiting
human and mouse tTG were isolated from an antibody library
made from CD patient. These antibodies were engineered in
a novel form called miniantibody produced by genetic
fusion of human anti-tTG scFv to mouse IgG CH2-CH3
domains. This confers both effectors functions to the antibody
and prevents from a rapid clearance. The gene was then
cloned into an Adeno Associated Virus (AAV) expression
vector for in vivo secretion of the protein.
Results: Preliminary experiments in eukaryotic cell line
show that the miniantibody is efficiently secreted as dimeric
molecule, is correctly glicosylated and retains binding speci-
ficity and affinity of the original antibody. The AAV vector
was used for in vivo experiments on C57/bl mice. 3 weeks
after a single intramuscularly administration miniantibodies
were present in the serum. The titer remains stable and sus-
tained for more than 1 year. Biological parameters such as
body weight, blood sugar level, lymphocyte activation, motor
dysfunction, and in situ deposits were monitored. Preliminary
data analysis indicates modification on some parameters like
lymphocyte activation markers.
Conclusion: High titre and long time expression of antibody
are typical of the disease. We are now able to mimic this
situation in an animal model providing long-term sustained
production of anti tTG antibodies.
G2-01 SACCHAROMYCES BOULARDII IN THE PREVENTION
OF ANTIBIOTIC-ASSOCIATED DIARRHEA IN CHIL-
DREN: A RANDOMIZED DOUBLE-BLIND PLACEBO-
CONTROLLED TRIAL M Kotowska1, P Albrecht1,
H Szajewska1. 1Dept of Paediatric Gastroenterology &
Nutrition, The Medical Univ of Warsaw, Warsaw, Poland.
Background: Co-treatment with Saccharomyces boulardii
appears to lower the risk of antibiotic-associated diarrhea
(AAD) in adults receiving broad- spectrum antibiotics.
However, it is unclear whether similar benefits occur in
children, as the only published trial involving children was
neither randomized nor blinded. Furthermore, conflicting data
about the effects of another probiotic, Lactobacillus rhamnosus
GG, suggest a different response in adult and pediatric
populations, and that results observed in one population cannot
be simply extrapolated to the other.
Aim: To determine if Saccharomyces boulardii prevents
AAD in children.
Methods: 269 children (age: 6 mo to 14 years) with otitis
media and/or respiratory tract infections were enrolled in
a double-blind, randomized placebo-controlled trial in which
they received standard antibiotic treatment plus 250 mg of
S boulardii (experimental group, n = 132) or a placebo
(control group, n = 137) orally twice daily for the duration of
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
antibiotic treatment. Analyses were based on allocated
treatment and included data from 246 children.
Results: Patients receiving S boulardii had a lower preva-
lence of diarrhea ($3 loose or watery stools/day for $48
hours occurring during or up to 2 weeks after the antibiotic
therapy) than those receiving placebo (9/119 [7.5%] vs.
29/127 [23%], relative risk, RR: 0.3, 95% confidence interval,
CI: 0.20.7). S boulardii also reduced the risk of AAD (diarrhea
caused by C difficile or otherwise unexplained diarrhea)
compared with placebo (4/119 [3.4%] vs. 22/127 [17.3%],
RR: 0.2; 95% CI: 0.070.5). No adverse events were observed.
Conclusion: This is the first RCT evidence that S boulardii
effectively reduces the risk of AAD in children.
G2-02 NSP4 PLAYS A MAJOR ROLE IN THE EARLY SECRE-
TORY DIARRHEA INDUCED BY ROTAVIRUS IN
A CACO-2 EXPERIMENTAL MODEL G De Marco1,
V Buccigrossi1, I Bracale1, S Di Caro1, MT Russo1,
A Russo Raucci1, LR Assante1, A Guarino1. 1Department of
Pediatrics, University Federico II, Naples, Italy.
Aim: Among Rotavirus (RV) multiple mechanisms of
diarrhea a major role is played by viral enterotoxin NSP4.
RV infection induces an early anion secretion and subsequent
cell damage in a Caco-2 cell model. Aim of the study was to
address the role of NSP4 in a model of RV infection in
human- derived intestinal cell line.
Methods: Caco-2 cells were infected with RV strain SA-11
(5PFU/cell) and mounted in Ussing chambers at different
times after infection. Production of NSP4 was assessed by
western blotting in immunoprecipitates of supernatants and
cell lysates. Short circuit current (Isc) and trans-epithelial
resistance (TEER), reflecting transepithelial ion transport and
tissue integrity were recorded. In parallel experiments, non-
specific human immunoglobulins (Ig) preparations for
parenteral use and specific anti-NSP4 Ig were added to
enterocyte monolayers.
Results: RV induced a biphasic response with a significant
increase in Isc after 13 hours of infection, consistent with
active anion secretion. TEER started to fall progressively 24
hours after infection, indicating monolayer loss of integrity.
Starting from 1 hour after infection, NSP4 was detected in the
supernatant of infected cells at increasing concentrations up
to 48 hours post-infection. Addition of anti-NSP4 Ig, as well
as no-specific human Ig, blocked the RV-induced secretion
but not TEER modifications.
Discussion: RV induces a dual, chronologically distinct,
pathogenic effect in human enterocytes: 1) an early anion
secretion which is NSP4-dependent and inhibited by specific Ig
and by non-specific human Ig; 2) a subsequent cytopathic
damage which is not prevented by anti-NSP4 Ig but blocked by
human Ig.
G2-03 TIR PHOSPHORYLATION INDEPENDENT PATHWAY
OF ENTEROPATHOGENIC ESCHERICHIA COLI (EPEC)
COLONISATION OF HUMAN INTESTINAL MUCOSA
WITH RECRUITMENT OF NCK AND N-WASP EX VIVO
A Phillips1, Y Chong1, S Schuller1, B Kenny2, G Frankel3.
1
Centre for Paediatric Gastroenterology, Royal Free Hospi-
tal, London, United-Kingdom. 2Institute for Cell and
Molecular Biosciences, University of Newcastle, Newcastle-
upon- Tyne, United-Kingdom. 3Centre for Molecular Micro-
biology and Infection, Imperial College, London, United-
Kingdom.
Tir, the EPEC translocated intimin receptor, is of bacterial
origin and is translocated into the host cell by a type III
secretion system. It is focused in the host cell membrane by
 ESPGHAN 38TH ANNUAL MEETING
binding to the bacterial outer membrane protein, intimin,
resulting in intimate bacterial adhesion. Phosphorylation of
tyrosine 474 in Tir is necessary to recruit Nck, N-WASP &
Arp2,3 to induce actin accumulation beneath attached
bacteria, producing the typical attaching-effacing (A/E)
lesion. These findings are based on cell culture studies but
recent in vivo work on C. rodentium, a natural A/E mouse
pathogen, found that Tir phosphorylation was absent and was
not needed for colonisation. Our aim was to determine the
involvement of Tir tyrosine phosphorylation in EPEC human
intestinal colonisation using human in vitro organ culture
(IVOC). Small bowel biopsy samples were obtained with
ethical approval and fully informed consent from cases
undergoing routine endoscopic investigation. Biopsy speci-
mens were cultured for 8 hours in IVOC. Bacterial strains used
included EPEC 2348/69 (E69), E69deltir, E69deltir (ptir), and
E69deltir (ptirY474S).
Results: EPEC Tir was translocated into human enterocytes
and was phosphorylated. E69deltir did not colonise samples; H-01
the wild type phenotype was restored with tir complementa-
tion (E69deltir (ptir)). E69deltir (ptirY474S), the Tir tyrosine
474 phosphorylation deficient mutant, colonised intestinal
explants, and immunostaining showed recruitment of Nck
and N-WASP indicating a tyrosine phosphorylation-indepen-
dent mechanism of A/E lesion formation ex vivo. A similar
phenomenon was observed on C. rodentium infected mouse
colon where Nck and N-WASP mediated actin recruitment
occurred despite absent Tir phosphorylation. Thus, Tir is
necessary for human intestinal colonisation ex vivo. Actin
assembly under EPEC and C. rodentium associates with Nck
and N-WASP recruitment and occurs both with and without
Tir tyrosine phosphorylation showing marked differences in the
biological activity of Tir between in vitro cell culture and ex
vivo/in vivo models. The relevance of observations made using
cultured epithelial cells must be confirmed in more biologically
relevant models.
G2-04 CHEMOKINE PRODUCTION BY BUCCAL EPITHELI-
UM IS EXCLUSIVELY ENHANCED IN CHILDREN
WITH CROHNS DISEASE G Damen1, J Hol1, L De
Ruiter1, J Bouquet1, M Sinaasappel1, J Van Der Woude2,
J Escher1, E Nieuwenhuis1. 1ErasmusMC - Sophia Children
Hospital, University Medical Centre, Dpt. Pediatric Gastro-
enterology and Laboratory of Pediatrics, Rotterdam, The
Netherlands. 2ErasmusMC - University Medical Centre, Dpt.
of Gastroenterology, Rotterdam, The Netherlands.
Aim: Inflammatory Bowel Diseases (IBD) represent an
aberrant immune response by the mucosal immune system
to luminal bacteria. Since the oral mucosa harbors the first
epithelial cells that encounter and interact with micro-
organisms, we assessed the immunological activity of buccal
epithelium in children with IBD and adults with Crohns
disease (CD).
Methods: Buccal epithelial cells were obtained by a non-
invasive brush-swab in 17 children with CD, 18 children with
ulcerative colitis (UC), 14 adults with CD and in 40 controls
(adults and children). The cells were cultured with and
without microbial stimulation. Chemokine levels were de-
termined in the culture-supernatants of the cells by cyto-
metric bead array and ELISA. In addition, CXCL-8 (IL-8)
production was studied by immunohistochemical analysis of
the buccal epithelial cells. Finally, CXCL-8 production by
lipopolysaccharide (LPS) stimulated monocyte-derived den-
dritic cells (moDCs) from these patients was determined. H-02
Results: In comparison to controls, pediatric UC patients,
and adult CD patients, only in children with CD buccal
epithelial cells showed enhanced production of CXCL-8,
CXCL-9 (Mig), and CXCL-10 (IP-10). In vitro microbial
627
stimulation (with LPS or zymosan) resulted in a further
increase of chemokine levels only in case these cells were
derived from pediatric CD patients. CXCL-8 production by
LPS stimulated moDCs from children with CD was equal to
that of children with UC.
Summary/Conclusions: Buccal epithelium of children with
CD is immunologically active, even in the absence of oral
lesions. The enhanced production of chemokines, spontane-
ous and upon stimulation, appears to be specifically
associated with pediatric CD. The enhanced chemokine
response appears to be restricted to cells derived from the
epithelial barrier, since the moDCs produce equal amounts of
CXCL-8. Assessment of the chemokine production by the
buccal epithelial cells shows promise as a new, rapid, and
non-invasive test for screening and classification of in-
flammatory bowel disease in children.
PATHOGENESIS OF ROTAVIRUS-INDUCED BILIARY
ATRESIA IN NEWBORN MICE A Matsui1, T Momoya2,
T Kudo1. 1Department of Child Health, Institute of Clinical
Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.
2
Department of Paediatrics, Jichi Medical School, Tochigi,
Japan.
Background: It is shown that Group A rotavirus, adminis-
tered orally or intraperitoneally, produced extrahepatic
obstruction in newborn mice with intrahepatic histology
similar to human biliary atresia.
Aim: In order to elucidate its pathogenesis in this animal
model, we underwent virological and immunohistochemical
studies of extrahepatic bile ducts and livers of neonatal mice
infected with rhesus rotavirus (RRV).
Methods: Two-day-old BALB/c mice were inoculated with
a 50 ul MEM containing 1.0 3 107 pfu of the RRV, or cell
culture medium as a control. 29 mice with RRV were
inoculated orally, 32 intraperitoneally. The en-bloc removal
of the liver, extrahepatic bile ducts, and duodenum as well as
splenectomy were carried out for both paraffin-embedded and
freshly frozen sections. Livers and bile ducts were homog-
enized, centrifugated and the supernatant virus was titered by
a standard plaque assay. Tissue sections from fresh-frozen
samples were for immunohistochemical study using poAb
raised against RRV.
Results: The tissue sections at 15 revealed, i) the common
bile ducts with multiple cross sections tortuous ducts of
varying diameter, ii) severe stricture with fibrosis and
inflammatory cells in the surrounding stroma with mild
epithelial damage, iii) destruction of interlobular bile ducts,
iv) mixed infiltration of polymorphonuclear and mononuclear
cells around the portal area, v) ductular proliferation with bile
plugs, and vi) periportal fibrosis. Plaque assays on homog-
enized hepatobiliary tissues demonstrated the presence of
infectious virus by day 7 but not thereafter. Immunoperox-
idase staining demonstrated RRV antigens mainly in the
surrounding stroma of the common ducts and portal area. The
viral antigens were positive from day 3 to 19.
Summary: Severe stricture due to fibrosis was shown
surrounding the degenerated common ducts before RRV-
associated proteins disappeared.
Conclusion: RRV might target either stromal or bille duct
epithelial cells in the hepatobiliary system and trigger
immune-mediated injury in murine biliary atresia.
DOUBLE-BLIND RANDOMIZED TRIAL OF COLCHI-
CINE IN BILIARY ATRESIA: LONG-TERM CLINICAL
OUTCOME N Hadzic1, M Davenport1, S Tizzard1,
ER Howard1, AP Mowat1, G Mieli-Vergani1. 1Kings
College Hospital, London, United-Kingdom.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
628 ESPGHAN 38TH ANNUAL MEETING
Aim: Biliary atresia (BA) is an obliterative cholangiopathy of
unknown etiology, leading to progressive fibrosis and
cirrhosis. The primary treatment is surgical excision of the
extrahepatic obliterated ducts and portoenterostomy (Kasai
operation) in an attempt to restore bile flow and abate the
fibrotic process. Colchicine is a collagen synthesis inhibitor
tested in various types of chronic liver disease in adults due to
presumed anti-inflammatory and anti-fibrotic properties.
Methods: Between 19841992, infants with histologically
proven BA were randomized to receive either colchicine
(0.5 mg/kg/day) or placebo in addition to a standard post-
Kasai regimen (including fat vitamin supplements, cholestyr-
amine and phenobarbitone, but not corticosteroids). Routine
clinical, ultrasound and laboratory monitoring was under-
taken on a four-monthly basis or sooner, if clinically indicated.
The medication was continued for 3 years. The randomization
code was then broken and the placebo stopped. Colchicine was
continued until the age of 6. The parents would then decide
whether to continue the drug or not. Native liver survival was
illustrated by Kaplan-Meier methods and compared using log-
rank tests. Current liver biochemistry was assessed in the
survivors by non-parametric tests.
Results: 94 infants were randomized to receive either
colchicine (n = 48) or placebo (n = 46). Mean follow-up
was 8.1 (range 0.418.5) years. Overall, there are 36 (21
colchicine) survivors with native liver, 40 (17 colchicine)
have been transplanted and 18 (10 colchicine) have died.
There was no significant difference in native liver survival
(P = 0.38). In the long-term survivors [colchicine, n = 15
(mean age: 13.9 y) and placebo, n = 14 (mean age: 14.3 y)],
where recent biochemistry was available, median bilirubin
was lower in the colchicine arm [9 (range: 115) vs. 12
(range: 447) mmol/L; P = 0.04], with no differences in
aspartate aminotransferase (P = 0.1), g-glutamyl-transferase
(P = 0.21), alkaline phosphatase (P = 0.31), and platelet count
(P = 0.79) values.
Conclusion: Long-term colchicine therapy is safe and well
tolerated in children after Kasai portoenterostomy for BA.
It does not, however, appear to give any long-term survival
advantage.
H-03 NASOBILIARY DRAINAGE INDUCES REMISSION IN
BENIGN RECURRENT INTRAHEPATIC CHOLESTASIS
JM Stapelbroek1, KJ Van Erpecum2, LWJ Klomp3,
MP Schwartz2, AS Knisely4, RHJ Houwen1. 1Dept of
Pediatric Gastroenterology, UMCU, Utrecht, The Nether-
lands. 2Dept of Gastroenterology, UMCU, Utrecht, The
Netherlands. 3Laboratory for Metabolic Diseases, UMCU,
Utrecht, The Netherlands. 4Institute of Liver Studies, Kings
College Hospital, London, United-Kingdom.
Background and Aims: Benign Recurrent Intrahepatic
Cholestasis (BRIC) is characterized by attacks of cholestasis
without anatomical obstruction. Although these episodes do
not result in permanent liver damage, the resulting pruritus is
severely disabling. Sofar no medical treatment can consis-
tently stop a cholestatic episode. In BRIC type 1, caused by
mutations in ATP8B1, cholestasis seems associated with
enhanced intestinal uptake of bile salts. We hypothesized that
a temporary interruption of the enterohepatic circulation
could abort a cholestatic episode in these patients.
Methods: Bile diversion was established by endoscopically
introducing a nasobiliary drain during a cholestatic episode in
three patients with BRIC. Two had documented ATP8B1
mutations, while in the third a rare variant, 1286 A . C, was
found. Following diversion bile was collected for one hour to
analyse its composition.
Results: In all three patients pruritus totally disappeared
within 24 hours and serum bile acid levels returned to normal
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
or near normal levels. After removal of the drain two patients
experienced a short biochemical relapse, which required no
further intervention. Relative amounts of biliary phospholi-
pids versus bile salts were within the normal range in all three
patients: phospholipids/(bile salts + phospholipids) ratio:
0.23, 0.13 and 0.17.
Summary: Temporary nasobiliary drainage (NBD) was able
to abort a cholestatic episode in three patients with BRIC.
Conclusions: NBD is a valuable addition to the present
therapeutic options in BRIC. In BRIC type 1 patients no
evidence for a primary disorder in the hepatic transport of bile
acids could be found by direct analysis of bile obtained at
diversion.
H-04 LONG TERM FOLLOW-UP OF CHILDREN WITH
CHRONIC HEPATITIS B INFECTION TREATED WITH
INTERFERON ALPHA M Woynarowski1, K Piwczynska1,
A Szaflarska-Poplawska2, D Lebensztejn3, E Grudzien4,
W Reyman4, Z Michalska5, J Soch1. 1Childs Health
Memorial Institute, Warsaw, Poland. 2Dept.Gastroenterol.
Medical Academy, Bydgoszcz, Poland. 3Dept Pediatr. Medical
Academy, Bialystok, Poland. 4Regional Hospital, Bielsko-
Biala, Poland. 5Dept.Infect.Dis. Medical Academy, Gdansk,
Poland.
Background: Interferon alpha is an approved treatment for
chronic hepatitis B in children, but majority of clinical studies
describe the effects of the therapy observed at one year after
the last dose of the medication. Long term follow-up is rarely
reported in the literature.
Aim of the Study: The aim of the study was to evaluate
clinical status, ALT activity, HBs and HBe presence in
children observed longer than one year after interferon
discontinuation.
Material & Methods: We reviewed the medical records of
432 children (M/F 282/150) with chronic hepatitis B treated
with interferon in open label study in 5 regional centers
between 1990 and 1999. All children received 3 MU of
interferon TIW for 20 weeks. The mean age of patients at the
beginning of interferon therapy was 5,9 6 3,6 years. 46
children received lamniwudine and in these children the
observation was stopped at the beginning of lamiwudine
treatment.
Results: The mean observation period of all children was
5,8 6 2,8 years (15 mo13,5 yrs). Children who received
lamiwudine treatment had shorter observation period (2,3 6
9,4 yrs p , 0,001). One year after interferon discontinuation
190 (43,9%) were HBeAg negative. At the end of follow-up
HBeAg was negative in 339 (78,5%) and HBs was negative in
54 (11%). HBV-DNA was tested in 16 HBe negative patients
and positive result was found in 7 (44%). 346 (80%) children
had normal ALT activity. 6% of children had increased liver
echogenicity on USG examination and 3 children presented
with splenomegaly. Children did not present any symptoms of
liver disease.
Conclusions: During approximately 6 years of post in-
terferon treatment follow-up almost 80% of children cleared
antigen HBe and normalized ALT activity however some of
them remained HBV-DNA positive.
N-01 RESTING ENERGY EXPENDITURE IN PEDIATRIC
PATIENTS WITH CHRONIC LIVER DISEASE COM-
PARED WITH HEALTHY CONTROLS AND THE
IMPACT OF LIVER TRANSPLANTATION: A THREE
YEARS FOLLOW UP STUDY. Y Gonzalez-Lama1,
A Morais1, R Lama1, S Tabernero1, MC Diaz2, L Hierro2,
C Camarena2, P Jara2. 1Nutrition Unit. La Paz Universitary
Pediatric Hospital, Madrid, Spain. 2Hepatology and Liver
 ESPGHAN 38TH ANNUAL MEETING
Transplantation Department.La Paz Universitary Pediatric
Hospital, Madrid, Spain.
Introduction: Prevalence of malnutrition among patients
with chronic liver disease (CLD) is thought to be high,
although efforts to improve their nutritional status are very
seldom done. Increase of energy expenditure could be cause
of malnourishment. We aimed to assess the influence of CLD
in nutritional status, the role of energy expenditure and the
impact of liver transplantation (OLT) on this clinical issue.
Patients and Methods: We included all non-transplanted
pediatric patients with CLD due to neonatal cholestatic
syndrome followed by both Hepatology and Nutrition
Departments, and a healthy control group. Patients who
underwent OLT followed periodical nutritional evaluation
during 3 years after OLT. Nutritional evaluation included
evaluation of body composition by anthropometry and
bioimpedance (RJL system), resting energy expenditure
(REE) by indirect calorimetry (Pediatric Deltatrac).
Results: 92 children evaluated: 37 healthy controls (21
males, 16 females; 5.1 + 4.6 years), 55 patients with CLD (28
males, 27 females; 3.6 6 4.3 years; 42% with biliary atresia,
26% with Alagille syndrome, 32% with progressive familial
intrahepatic cholestasis). REE was greater in patients than in N-03
healthy controls with statistical significance. REE was
lineally correlated with body composition (lean body mass;
p = 0.0001, r = 20.7). 35 patients underwent OLT (17 males,
18 females; 3.3 6 3 years). Nutritional follow up after OLT
lasted 1, 2 or even 3 years (100%, 88.5% and 80% of the
patients respectively): REE per Kg of body weigh, lean body
mass or cellular body mass descent during the first year after
OLT and achieved normality at the first year after OLT;
Metabolic rate (real REE related to REE calculated by
Shoefields equation) also achieved normality by the first year
after OLT.
Conclusions: REE is enhanced in pediatric patients with
CLD, compared with healthy controls. REE plays a major
role in the development of malnutrition in these patients. OLT
have a clear impact on impaired REE, allowing its
normalization by the first year after OLT.
N-02 MARKED CHANGES IN THE MRNA EXPRESSION OF
PPARS IN THE LIVER OF MICE WITH PERINATAL
ESSENTIAL FATTY ACID DEFICIENCY V Palsdottir1,
B Gabrielsson2, B Holmberg1, B Strandvik1. 1Dept of
Paediatrics, Inst of the Health of Women and Children,
Goteborg University, Goteborg, Sweden. 2Dept of Medicine,
Inst of Internal Medicine, Goteborg University, Goteborg,
Sweden.
Peroxisome proliferators-activated receptors (PPAR) are
nuclear receptors which by transcription are regulating genes
involved in lipid and glucose metabolism as well as in cell
growth and inflammation. Perinatal changes in essential fatty
acid (EFA) metabolism can program for long-term effects
related to the metabolic syndrome in the rat. The aim of the
present study was to investigate if similar changes would be
induced in the mice and to study gene expression in order to
clarify involved metabolic disturbances.
Methods: Mice of C57BL/6 strain were given EFA deficient
or control diet from day 15 of pregnancy and during lactation.
The pups were killed at weaning (3 w) and different organs
dissected and frozen in 270 until analysis. Lipids (HPLC
and GLC) and PPAR mRNA (real-time PCR) were analysed
in liver.
Results: The pups of EFA deficient mothers were sign
smaller and had lower liver weight, also related to bw than
controls. Serum FA showed marked EFA deficiency,
629
mean(SD) linoleic acid (LA) being 1.60(0.07) vs
29.9(2.9) mol% and EFA deficiency index being 3.7(0.5) vs
0.00(0.00) (p , 0.000001). Liver phospholipid (PL) pattern
(mg/g liver weight) showed only differences between
phosphatidylserine (PS), sphingomyelins (SMs) and phos-
phatidylglycerol but the FA pattern was markedly changed in
most PL. LA was decreased in all PL (p , 0.00001) as was
arachidonic acid (AA). The concentration of docosahexae-
noic acid (DHA) was decreased in all PL but phosphatidyl-
ethanolamine (PE) and SMs and increased in
diphosphoglycerol (DPG) (p , 0.0001). The AA/DHA ratio
was significantly decreased compared to controls except in
SMs, PS and PE. The mRNA expression in the liver of
PPARdelta was decreased 0.8 (0.08) vs 1.67 (0.43) (p , 0.005)
and PPARgamma increased 1.67 (0.21) vs 0.61 (0.23) (p ,
0.0001) but PPARalpha was unchanged.
Summary: Selective marked changes in FA composition of
PL was associated with different expression of PPAR delta and
PPAR gammabut relative preservation of PE and PPAR alpha.
Conclusion: Long-term follow up after FA restitution will
reveal if the PPAR changes are permanent and related to the
long-term effects in metabolism previously found in rats.
N-3 PUFA DURING PREGNANCY IMPROVES HYDRO-
LYSIS CAPACITY WITHOUT ALTERING BARRIER
FUNCTION OF NEWBORNS PIGLETS INTESTINE
AND LIMITS THE SMALL INTESTINE IMMATURITY
OF VERY LOW BIRTH WEIGHT INDIVIDUALS.
G Boudry1, V Douard1, JP Lalle`s1, J Mourot1, I Le
Huerou-Luron1. 1UMR-SENAH INRA/Agrocampus, Saint
Gilles, France.
Aim: At birth, the small intestine of neonates has to fulfil
many roles: to provide nutrients for growth, to allow passage
of immunoglobulins from mothers milk but at the same time
to avoid entrance of harmful antigens and bacteria. The
immaturity of the neonate small intestine physiology
enhances risks of developing intestinal diseases, especially
in very low birth weight individuals. Beneficial role of n-3
polyunsaturated fatty acids (PUFA) on small intestine has
been established in adult animal models. However, the effect
of n-3 PUFA via the maternal diet on neonates intestinal
physiology has not been investigated yet. We examined the
effects of n-3 PUFA supplementation during gestation on
hydrolysis capacity and barrier properties of the small
intestine in normal birth weight (NBW) and on immaturity
signs of the gut in very low birth weight (VLBW) newborn
piglets.
Methods: Sixteen pregnant sows were allocated to two
different diets during their gestation: a diet containing either
5% of lard (LAR) or 2% of lard and 3% of linseed oil, rich in
n-3 PUFA (LSO). At birth, piglets (NBW and VLBW, i.e.
weight , 50% of the average litter BW) from each litter were
sacrificed. Digestive tissue mass and jejunal lactase specific
activity were measured in NBW and VLBW piglets whereas
permeability to horseradish peroxidase (HRP) of the NBW
ileum was determined in Ussing chambers.
Results: There was no difference in the gestation character-
istics of the two groups of sows (duration, number of
piglets.). Lactase activity was increased in the LSO group
(LSO 23 6 2 vs LAR 18 6 2 IU, P , 0.05). Permeability to
HRP tended to be enhanced in the LSO group (LSO 13 6 4 vs
LAR 5 6 2 ng.cm22.h21, P = 0.08) but not after degranulation
of mastocytes. In addition, signs of immaturity measured in
VLBW piglets of LAR group (pancreas weight, intestinal
mucosa/muscularis ratio, lactase activity) were suppressed in
VLBW LSO piglets.
Conclusion: Supplementation with n-3 PUFA during preg-
nancy could be beneficial to newborns by increasing
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
630 ESPGHAN 38TH ANNUAL MEETING

hydrolysis capacity of the intestine without altering its barrier Methods: 10 children diagnosed with SDS (30% of all UK
function and by limiting immaturity of the gut of VLBW cases) had their clinical phenotypes carefully assessed by;
individuals. Stool elastase, growth, regular haematology/bone marrow
parameters, skeletal survey, expert dental review, behaviour
N-04 INCREASED ALPHA-LACTALBUMIN INFANT FOR- questionnaire and blood biochemistry. 10/10 had mutation
MULA FED TO HEALTHY TERM INFANTS IN A screening by restriction enzyme digest or direct sequencing.
MULTI-CENTER PLASMA ESSENTIAL AMINO ACID Results: Mutations of the SBDS gene were identified in 7/10
STUDY AM Davis1, BJ Harris1, USA Multi-center Group2. children (Predicted value is 74%). 6/10 had the common
1
Wyeth Pharmaceuticals, Collegeville, USA. 2Multi-center compound heterozygote 183-184TA.CT and 258+2T.C
sites, 12 cities, USA. mutation, 1/10 had a unique inframe stop codon258+2T.
C/R218X predicting a truncated protein. 3/10 had no
mutations in SBDS gene.
Aim: The purpose of this study was to evaluate the plasma
essential amino acid levels after feeding an experimental
formula (EF) (enriched alpha-lactalbumin dominant whey 183184TA.CT/ 258+2T.C Mutation
protein source, 14.4 g/L) in comparison to a control formula 258+2T.C and R218X negative
(CF) (standard formula, 15.1 g protein/L) and human milk Bony problems 5/6 1/1 2/3
(HM). The EF essential amino acid composition is closer to Dental 3/6 1/1 0/3
that of HM than CF. Stool Elastase 40 (20-.200) ,10 60 (20-100)
Methods: This prospective, randomized, masked, parallel, Neutropenia 5/6 1/1 1/3
multi-center, ambulatory study enrolled healthy, term infants BM Transplant 1/6 1/3
at , 14 days post-natal age, between the 10th and 90th Growth 4/6 ,0.4 1/1 ,0.4 3/3,0.4
percentiles in weight-for-length (U.S. CDC) after ethics centile centile centile
approval. Timed post-prandial plasma essential amino acids Feeding problem 3/6 1/1 0/3
levels were analyzed after 8 weeks of ad libitum study Behaviour problem 4/6 1/1 1/3
feeding. Formula intake was assessed at 4 and 8 weeks and Raised Liver ALT 4/6 0/1 3/3
adverse events (AEs) were assessed every 2 weeks. Serum Facial rash 0/3 0/3 3/3
protein markers were measured at baseline and 8 weeks and
anthropometric measurements were determined at baseline Summary: Greatest varaibility was in the compound
and every 4 weeks. hetrozygotes. A novel inframe stop codon lead to severe
Results: 216 infants were enrolled and 166 (76.9%) features. The no mutaion group had least varaibility and
completed the study. The mean plasma essential amino acid a facial rash responsive to treatment with essential fatty acids.
levels in the EF group were statistically equivalent to the HM Conclusion: SDS has genetic heterogeneity with phenotyp-
group for arginine and cysteine, and higher in the EF group ical variability. The homogeneous mutation negative group
than the HM group for other amino acids. The levels of should help identify a new SDS locus.
arginine, cysteine, tryptophan, and tyrosine were lower for
the CF group than the HM group and all essential amino acid
levels were lower in the CF group than the EF group. The SY1-02 HEREDITARY PANCREATITIS IN CHILDREN
incidence of feeding-related gastrointestinal (GI) AEs was B Oralewska1, G Oracz1, A Sobczynska-Tomaszewska2,
signifantly different (p = 0.025) across groups (EF 11/64, CF M Teisseyre1, J Socha1, D Bak2, J Bal2. 1The Childrens
20/64, and HM 12/88) and statistically different between CF Memorial Health Institute, Warsaw, Poland. 2National
and HM (p = 0.015). Study withdrawals due to feeding- Research Institute of Mother and Child, Warsaw, Poland.
related GI AEs were statistically different between all 3
groups (EF 3/64, CF 8/64, HM 0/88, p = 0.001). The
cumulative incidence of total GI AEs was significantly higher The etiology of chronic pancreatitis (CP) in children is varied
in CF than for either EF or HM (p = 0.013). and includes gene mutations, i.e. cationic trypsinogen gene
Summary: Plasma essential amino acid levels from EF were mutations (PRSS1).
generally higher than those measured in CF or HM. EF had Aim: The aim of this study was to assess prevalence of
fewer feeding-related withdrawals and improved GI tolerance cationic trypsinogen gene mutations in children with chronic
compared to CF. or acute recurrent pancreatitis (ARP) and its influence on
Conclusion: This study demonstrates that feeding a higher clinical course of pancreatitis.
quality (alphalactalbumin) lower protein infant formula Methods and Results: We evaluated 94 children with CP or
provides sufficient protein nutrition and has superior toler- ARP aged 3-18 yrs. PRSS1 gene mutations were found in
ance compared to CF due to its similar GI AE profile to HM. 12 children (12,8%) with CP; 11 girls and 1 boy (aged 3,6-16,
3 years); 6 children had R122H/- mutation, 5 children had
R122C/- and one child N29I/-. 11 children had positive
SY1-01 GENOTYPE-PHENOTYPE CORRELATION IN family history of pancreatitis. In two patients with PRSS1
SHWACHMAN-DIAMOND SYNDROME SE Kirkham1, mutations, pancreas divisum was diagnosed. Two additional
J Hinks , M Schwarz , Z Ahmad , C Mason , KJ Lindley1,
4 4 1 3
mutations were found in 2 patients (one patient had SPINK1
P Ancliff2, N Shah1. 1Great Ormond Street Hospital mutation N34S/-, and second one 5T variant in CFTR gene).
Department of Gastroenetrology, London, United-Kingdom. There was no difference in age of the disease onset between
2
Great Ormond Street Hospital Department of Haematology, patients with hereditary pancreatitis and patients without
London, United-Kingdom. 3Great Ormond Street Hospital PRSS1 mutations (7.86 years vs. 9.01 years, NS). Pancreatitis
Department of Dentistry, London, United-Kingdom. 4Royal grading, according to the Cambridge Classification System
Manchester Childrens Hospital Regional Molecular Genet- was 2,83 grade in PRSS1 group vs.1,45 in the group
ics Lab, Manchester, United-Kingdom. without mutations (p , 0,05).
Conclusions: 1. Cationic trypsinogen gene mutations
Aim: The aim of this study was to establish phenotype- (PRSS1) are common in children with chronic pancreatitis.
genotype correlation in Shwachman-Diamond Syndrome 2. Children with CP associated with PRSS1 have worst
(SDS). clinical course of the disease.

J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005

 ESPGHAN 38TH ANNUAL MEETING
SY2-01 MECHANISMS OF IMMUNOLOGICAL RESPONSE IN
DUODENAL MUCOSA IN ACTIVE CELIAC DISEASE.
J Carnicer1, J Rodriguez2, M Lopez2, C Juarez2, S Sainz3,
J Cubells1. 1Pediatrics. Hospital Sta Creu i S. Pau,
Barcelona, Spain. 2Immunology. Hospital Sta. Creu i S.
Pau, Barcelona, Spain. 3Gastroenterolgy. Hospital Sta. Creu
i S. Pau, Barcelona, Spain.
In celiac disease (CD) gluten reactive CD4+ T helper cells
become activated upon recognition of gluten peptides (in the
context of DQ2 or DQ8) and produce many cytokines of
which interferon-gamma (IFN- d) is dominating. The bi-
ological effects of IFN- d rely mainly on the activity of the
transcription factor signal transducer and activator of
transcription (STAT-1). In addition, in active CD there is an
extensive infiltration of intestinal epithelium by CD8+ T
lymphocytes.
Objectives: To evaluate the lamina propia infiltration by
CD4+ T-cells, the presence of CD8+ T lymphocytes in the
intestinal epithelium, and the nuclear content of STAT-1 in
the enterocytes and lymphocytes of lamina propia in patients
with active CD.
Patients and Methods: Biopsy specimens from distal
duodenum of 52 patients with suspected and untreated CD
(1-18 years of age) were obtained during gastrointestinal
endoscopy. One of the biopsy specimens was used for
histologic examination (Marsh modified clasification). Im-
munofluorescence and immunohistochemistry techniques
were used to analyze the lamina propia infiltration by CD4+
T-cells, the presence of CD8+ T lymphocytes in the intestinal
epithelium (. 40%) and the nuclear content of STAT-1.
Mucosal duodenal samples were also studied in 2 patients
with treated CD and in 5 controls.
Results: Histologic evaluation: 41 cases Marsh-3 (78.8%), 5
cases Marsh-1 (9.6%), and 6 cases (11.5%) with minimal
changes or limited sample for evaluation. Infiltration of the
lamina propia by CD4+ T-cells was observed in 34/38 cases
(89.4%) and CD8+ T lymphocytes in the intestinal epithelium
in 30/38 (78.9%). In patients with active CD, STAT-1 was
increased in the nucleus of enterocytes and in lymphocytes of
lamina propia respect of normal controls.
Conclusion: The data suggest that persistent STAT-1
activation may contribute to maintaining the local inflama-
tory response in CD.
SY2-02 EXPRESSION PROFILES OF COELIAC DISEASE BI-
OPSIES DURING MUCOSA RECOVERY B Diosdado1,
H Van Bakel1, MC Wapenaar1, L Franke1, JWR Meijer2,
ML Mearin3, CJJ Mulder4, C Wijmenga1. 1Complex Genetics
Section, DBG-Department of Medical Genetics, University
Medical Centre, Utrecht, Utrecht, The Netherlands. 2De-
partment of Pathology, Rijnstate Hospital, Arnhem, The
Netherlands. 3Department of Paediatrics, Paediatrician Unit
of Paediatric Gastroenterology, Leiden University Medical
Centre, Leiden, The Netherlands. 4Department of Gastroen-
terology, VUmc, Amsterdam, The Netherlands.
Coeliac disease (CD) is a multifactorial, gluten-sensitive
enteropathy of unknown pathogenesis that comprises in three
stages of development: Marsh I (lymphocytosis), Marsh II
(lymphocytosis and crypt hyperplasia), and Marsh III
(lymphocytosis and crypt hyperplasia and villous atrophy).
Treatment with a gluten-free diet (GFD) normalizes the
mucosa of the small intestine of these patients.
To define the molecular pathways of CD pathogenesis and the
tissue remodeling mechanisms upon treatment with a GFD,
we analyzed the expression profiles of 48 CD biopsies taken
631
at different stages of the disease following a GFD and 21
controls.
The samples were hybridized in duplo on microarray slides
containing .21,000 genes. Data analysis was performed by
MANOVA and GeneSpring software.
The analysis revealed 158 differentially expressed genes
involved in the tissue recovery of CD. The genes could be
divided into two groups based on their pattern of expression.
The first group comprises 102 genes that show a gradual up-
regulated pattern of expression towards mucosa normaliza-
tion. These genes are implicated in enterocyte differentiation
processes and represent the functionality of the duodenum.
The second group of genes consists of 56 genes that are
mainly related to immunological pathways. Many of these
genes are regulated by interferon-gamma and their pattern of
expression shows a gradual down-regulation of the immune
response after gluten withdrawal.
Interestingly, our data do not show changes in expression of
genes of previously proposed mechanisms in CD, such as
apoptosis, oxidative stress or metalloproteinases pathways.
Our results reveal that during the mucosa recovery in CD,
there are two opposite processes taking place. There is
a gradual release of the immune response and a complemen-
tarily up-regulation of genes re-stabilising the normal
homeostasis of the mucosa of the small intestine.
SY5-01 LONG CHAIN POLYUNSATURATED FATTY ACIDS
AND LIVERBIOCHEMISTRY IN BREAST-FED IN-
FANTS MH Jrgensen1, K F. Michaelsen2, L Lauritzen2.
1
Department of Pediatrics, Rigshospitalet, Copenhagen,
Denmark. 2Institut of Human Nutrition, Royal Veterinary
and Agricultural University, Frederiksberg, Denmark.
Background: In a previous study we found higher levels of
aspartate aminotransferase (AST) among breast-fed (BF)
infants compared with formula-fed (FF) infants (1). Albumin
was also higher and AST was found to be positively
associated with plasma IGF-1. Thus, the elevated level of
AST in BF-infants is unlikely to reflect liver injury. Rather it
could be explained by induction of hepatocytes by factors in
human milk (HM). In the sinusoidal membrane of the
hepatocyte AST facilitates uptake of fatty acids into the liver.
HM contains docosahexaenoic acid (DHA), whereas formu-
las supply only the precursor. We investigate if DHA intake is
associated with elevated AST in BF infants.
Methods: Within two weeks after delivery 60 mothers with
an average fish intake below the population median were
randomized to daily supplementation during the first 4 mo of
lactation with either 4.5 g fish oil (FO) or 4.5 g olive oil (OO),
a third group of 41 women with a fish intake within the
highest quartile was used as a reference group (HF). DHA in
HM was measured at 4 mo as was albumin, AST, alanin
aminotransferase (ALT) and gammaglutamyl transferase
(GGT).
Results: All 101 infants received HM, 77 infants were
exclusively breast-fed (BF), 24 infants were supplemented
with formula (FS). DHA in human milk was 0.4 wt% (OO),
0.7 wt% (HF) and 1.4 wt% (FO) (2). BF infants had
significant higher AST (p = 0.012), ALT (p = 0.012) and
GGT (p = 0.013) compared with the FS-infants. 83% BF
infants had one or more transaminases above reference level,
compared with 60% of the FS-infants (Chi-Square, p = 0.04).
Albumin tended to be higher among BF-infants (p = 0.09). No
differences were found in transaminases among the 3 feeding
groups or between the two randomized groups (FO and OO),
neither when all infants were included in the analysis or when
only exclusively BF-infants were included. Also no associ-
ation was observed between transaminases and the content of
DHA in HM.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
632 ESPGHAN 38TH ANNUAL MEETING
Conclusion: BF-infants have an altered liver biochemistry.
With the higher albumin levels in BF infants it is more likely
with a stimulatory effect on the liver rather than injury. The
intake of DHA does not account for the variation in
transaminases in exclusively or partially BF infants. However
the difference found between total BF and total FF infants has
not been investigated in this study.
1) Jrgensen, M. H. et al. (2003). J Pediatr Gastroenterol
Nutr 37(5): 559-65.
2) Lauritzen, L. et al. (2003). Lipids 39:195-206, 2004.
SY5-02 INTESTINAL PERMEABILITY IN HEALTHY BREAST-
FED INFANTS, DURING THE POSTNATAL PERIOD
SP Castellaneta1, A Masciale1, A Zaccaro1, S Straziuso2,
V Miniello2, F Gatti3, L Polimeno3, R Francavilla3. 1PO
Pediatria - Ospedale San Paolo, Bari, Italy. 2Clinica
Pediatrica - University of Bari, Bari, Italy. 3CIRGEEE
(Centro Interdipartimentale di Ricerca di Gastroenterologia
ed Epatologia Pediatrica dellEta` Evolutiva) - University of
Bari, Bari, Italy.
Background and Aim: The maturation of the intestinal
epithelial barrier play an important role during the postnatal
period and few data are available on the exact timing of gut
closure in neonates. The aim of our study was to assess the
timing of maturation of the gut barrier by the measure of the
intestinal permeability (IP) in full term infants.
Patients and methods: Twenty-nine full term infants born
by vaginal delivery and exclusively breast fed entered the
study. Caesarian section, therapies with antibiotics or pro-
biotics, partial formula feeding, febrile or chronic diseases
and a positive family history for atopy were considered
exclusion criteria. All underwent double sugar (lactulose and
mannitol) intestinal permeability test according to Generoso
et al. 1 using pulsed amperometric detection (DIONEX DX
600) at birth and at day 20, 40 and 70 of life. The lactulose-to-
mannitol ratio (L/M) was determined as a marker of IP.
Results: 19 infants (65,5%) completed the study, 8 (27,5%)
disattended one or more IP determination and two (7%) were
excluded for antibiotic treatment. Mannitol excretion signif-
icantly increased (Day 0: 3,8%; D10: 5,9%; D20: 7,5%; D40:
8,1%; D70: 7,9%; p , 0.01) and lactulose decreased (Day 0:
0,6%; D10: 0,4%; D20: 0,3%; D40: 0,3%; D70: 0,1%; p ,
0.01) respectively during the study period. L/M progressively
decreased reaching the normal value at day 20 (Day 0: 0.16;
D10: 0,07; D20: 0,04; D40: 0,03; D70: 0,02; p , 0.001). The
test was well tolerated by all infants.
Summary and conclusion: Our study show that the IP to
sugar probes in healthy breast fed infants is high at birth and
progressively decrease during the first two months of life:
however yet at 20 days the maturation of the IP and the gut
closure seems to be completed.
SY6-01 A ZONULIN-OCCLUDENS TOXIN SYNTHETIC ESA-
PEPTIDE MODIFIES THE INTESTINAL PERMEABIL-
ITY IN IN-VIVO ANIMAL MODEL. F Katouzian1, T Not1,
A Tommasini1, A Fasano2, M Stebel3, D Sblattero4,
R Marzari4, A Ventura1. 1Pediatric Department, University
of Trieste, IRCCS Burlo Garofolo, Trieste, Italy. 2Mucosal
Biology Research Centre University of Maryland School of
Medicine, Baltimore, USA. 3CSPA University of Trieste,
Trieste, Italy. 4Department of Biology University of Trieste,
Trieste, Italy.
Background: We have recently demonstrated that the
permeating effect of Zot on intercellular tight junctions is
due to a protease activating receptor(PAR) active pepti-
de(AP)-like motif.This motif is generated by Vibrio cholerae-
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
specific cleavage and secretion of an active Zot derivative
within the intestinal milieu. Aim of this study was to establish
the effect of the Zot-derived PAR-AP synthetic esapeptide
AT1002 in in vivo intestinal permeability in mice.
Methods: Lactulose (L) and rhamnose (R) sugars were orally
administered with or without AT1002 at 25,100,250 mg/dose
to 9 Balb/c mice (three animals for dose). Nine mice were
treated with non-active synthetic octapeptide at the some
dosages of AT1002. Serum sugars concentrations were
measured by HPLC on blood drop. Intestinal permeability
was expressed as L/R ratio.
Results: Only the 250 mg/dose ofAT1002 (continuous line)
induced an increased intestinal permeability compared to
peptide control (dotted line). Permeability was statistically-
significant starting one hour post-AT1002 administration
(p , 0.001). The intestinal permeating effect remained
detectable for 18 hours after a single dose of AT1002
(250 mg) and returned to baseline after 25 hours.
Conclusions: The Zot PAr-AP esapeptide retained the
intestinal permeating effect on intercellular tight junctions
previously described for the native Zot protein.This effect
was immediate, sustained over time for the dose tested, and
reversible within 25 hours, suggesting a modulating rather
than damaging effect on tight junction structures.
SY6-02 IN VITRO ZINC EFFECTS ON DIFFERENT INTES-
TINAL ION SECRETION SIGNAL PATHWAYS
ACTIVATION: IMPLICATIONS FOR TREATMENT
OF DIARRHEA IN CHILDREN R Berni Canani1,
V Buccigrossi1, A Passariello1, S Ruotolo1, F Porcaro1,
MC Siani1, P De Luca1, A Guarino1. 1Dept. of Pediatrics,
University Federico II, Naples, Italy.
We have demonstrated that, through a direct interaction with
the enterocyte, Zn2+ inhibits V. cholerae toxin (cAMP
agonist) but not E. coli heat-stable enterotoxin (cGMP
agonist) secretive action (J Infect Dis, 2005 in press). This
raises the hypothesis that Zn2+ may be effective in selected
rather than all infectious diarrhea etiologies. Enteric patho-
gens determine diarrhea through four established intracellular
signal transduction pathways stimulating fluid fluxes across
intestinal mucosa: cyclic adenosine monophosphate (cAMP);
cyclic guanosine monophosphate (cGMP), Ca2+ and nitric
oxide (NO).
Aim: To investigate whether Zn2+ could reduce intestinal ion
secretion mediated by Ca2+ or by NO in an in vitro model of
secretory diarrhea.
Methods: Ion transport was investigated by monitoring
electrical parameters of a human intestinal epithelial cells
monolayer (Caco2 cell line) mounted in Ussing chambers
and exposed to increasing doses of Zn2+ (ZnCl2) in the
absence on in the presence of an agonist of Ca2+: carbachol
(10-9 M), or of a stimulator of inducible NO synthase activity:
gammaInterferon (gamma-IFN, 50.000 U/mL). Intracellular
 ESPGHAN 38TH ANNUAL MEETING
concentration of Ca2+, as well as the end stable degradation
products of NO (NO-2/NO-3) were also measured.
Results: The Zn2+ addition to mucosal or to serosal side of
the enterocyte resulted in a decrease in basal short-circuit
current (Isc) with a peak after 25 min of incubation (DIsc
1.73 + 0.2 mA/cm2). This effect was entirely due to
transepithelial Cl- absorption, and was dose-dependent with
a maximal effective dose of 35 mM and saturable.
Preincubation with Zn2+ (35 uM) resulted in a significant
reduction of ion secretion elicited by carbachol (-68%) or by
gamma-IFN (-100%). Zinc was also able to significantly
inhibit intracellular increases of Ca2+ and of NO, in response
to carbachol and gamma-IFN, respectively.
Conclusions: Through a direct enterocyte interaction Zn2+
significantly reduces ion secretion elicited by activation of
three out of the four main intracellular signal transduction
pathways of secretory diarrhea. These data support the Zn2+
use as adjunctive therapy for diarrhea induced by the vast
majority of enteric pathogens, including Rotavirus.
PG1-01 INCREASED TOLL LIKE RECEPTOR (TLR2 AND
TLR4) EXPRESSION IN THE SMALL INTESTINAL
MUCOSA OF CHILDREN WITH COELIAC DISEASE
B Szebeni1, G Veres2, A Dezsofi2, A Vannay1, K Rusai1,
L Szonyi2, B Vasarhelyi1, A Arato2. 1Research Group for
Paediatrics and Nephrology, Hungarian Academy of Scien-
ces, Budapest, Hungary. 2First Department of Paediatrics,
Semmelweis University, Budapest, Hungary.
Background: Besides the central role of the adaptive
immunity, innate immune responses also play an important
role in the development of coeliac disease. Recently it has
been found that rod-shaped bacteria are frequently associated
with the mucosa of coeliac disease patients but not with
controls. Bacterial products are recognized by pattern
recognition receptors (PRRs) like Toll-like receptors (TLRs),
which are key regulators of the innate immune system. After
ligand binding these receptors trigger the release of proin-
flammatory or chemotactic cytokines and chemokines, thus
lowering the thereshold for the activation of gluten reactive
CD4 + T cells in the lamina propria.
Aim: The aim of this study was to characterise the expression
of TLR2, TLR3 and TLR4 in intestinal biopsy samples from
children with coeliac disease and controls.
Methods: Duodenal biopsy samples were collected from 16
children with untreated coeliac disease [meadian age (range):
9 (415) yr] and 10 controls [10 (415) yr]. One sample from
each patient was used for routine diagnostic purposes. The
TLR2, TLR3 and TLR4 gene expression were evaluated by
semi-quantitative reverse transcription polymerase chain
reaction (RT-PCR).
Results: All coeliac patients had endomysium IgA positivity
and subtotal villous atrophy in the small intestinal mucosa.
All controls had normal histological finding. An eightfold
increase of mRNA for TLR2 and fourfold for TLR4 were
detected in the coeliac biopsy samples compared to controls
(p , 0.05 for both comparisions). The expression of TLR3
gene was not significantly different.
Summary: Increased TLR2 and TLR4 genes expression was
found in children with coeliac disease.
Conclusions: These data suggest that coeliac disease is
associated with increased TLR expression in the intestinal
mucosa, implying that alterations of innate immune system
may contribute to the pathogenesis of this disorder.
PG1-02 MAGNESIUM AND CITRATE METABOLISM DISOR-
DERS IN CHILDREN WITH COELIAC DISEASE
633
J Zawadzki1, M Syczewska2, A Alsharif3, J Rujner3,
J Socha3. 1The Childrens Mem. Hlth Inst, Dept. Nephrology,
Kidney Transpl., Warsaw, Poland. 2The Childrens Mem. Hlth
Inst, Dept. Paediatric Rehabil., Warsaw, Poland. 3The
Childrens Mem. Hlth Inst, Dept. Gastroenterology, Hep-
atology, Immunol., Warsaw, Poland.
Aim: Low urine citrate excretion may occur in coeliac
patients with symptoms of Mg depletion, despite of normal
plasma HCO3 and K levels. Aim was to evaluate the effect of
Mg load dose on urine citrate excretion in 31 children, with
coeliac disease without malabsorption syndrome.
Methods: Mg loading test, serum Mg and K levels, urinary
Mg and citrate excretion.
Results: In 9 patients with Mg depletion confirmed with Mg
loading test (Mg retention 66.9%) Mg infusion increased
citraturia from 365.8 to 674.1 mg/1g creatinine (p = 0.0013).
In 2 hypocitraturic patients the urine citrate excretion was
increased from 23.3 to 504.1, and from 72.4 to 153.6 mg/1 g
creatinine respectively. In the second patient with severe
hypomagnesemia and hypokaliemia the test dose of Mg did
not normalize citraturia. After 4 months of MgO administra-
tion the results normalized. In remaining 25 patients with
normal results of Mg loading test (Mg retention 23.9%) i.v.
administration of Mg increased urine citrate excretion from
448.9 to 706.8 mg/1g creatinine (p = 0.0008). Among 5
hypocitraturic patients the Mg depletion could be suspected
in 3 children. Administration of Mg test dose increased
citraturia from 219.4 to 515.9 mg/1g creatinine.
Summary: Decreased citraturia was found in 7 out of 31
patients with coeliac disease. Five of them had biochemical
symptoms of Mg depletion, and in their case the i.v. Mg
loading test increased citraturia of more than 120% of the
basic value. In remaining patients with normal citraturia, the
i.v. Mg load led to marked increase of the urinary citrate
excretion.
Conclusions: Considerable increase of urine citrate excretion
after Mg loading test suggests the link between Mg
homeostasis and metabolism of citrate. Mg depletion may
decrease citraturia directly, or secondarily, due to lower K
stores, resulting in intracellular metabolic acidosis. De-
creased diet Mg supply and/or defect of intestinal Mg
absorption may lead to hypomagnesuria and secondary
hypocitraturia, increasing the risk of calcium nephrolithiasis.
PG1-03 DISTURBED PHOSPHOLIPID FATTY ACID PATTERN
IN INTESTINAL BIOPSIES FROM PATIENTS WITH
COELIAC DISEASE D Steel1, W Ryd2, H Ascher1,
B Strandvik1. 1Dept of Pediatrics, Inst of the Health of
Women and Children, Goteborg University, Goteborg, Sweden.
2
Dept of Pathology, Inst of Laboratory Medicine, Goteborg
University, Goteborg, Sweden.
Phospholipid fatty acids (PLFA) in the intestinal mucosa
mainly reflect cell membrane compositions having an impact
on permeability and function of transport proteins, receptors
and membrane bound enzymes. In intestinal diseases these
functions are often disturbed and inflammation may influence
the lipid homeostasis by increase of arachidonic acid (AA)
release from phospholipids for the eicosanoid synthesis. The
aim of this study was to investigate PLFA composition in
paediatric intestinal mucosal biopsies in coeliac disease (CD).
Methods: Thirteen patients with CD were investigated, 7
(median age 75 m) in acute phase with mucosal atrophy and
6 (median age 98.5 m) in remission with normal mucosal
morphology after gluten-free diet for more than one yr.
Intestinal biopsies were performed with a Watson capsule
under fluoroscopic control before the ligamentum of Treitz.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
634 ESPGHAN 38TH ANNUAL MEETING
Control biopsies (n = 11) were from patients investigated for
unspecific intestinal symptoms with normal morphology,
subsequently found healthy. PLFA were analysed in mucosal
biopsies and serum by capillary GLC.
Results: Intestinal mucosa from patients with active CD
differed markedly from controls with low linoleic acid (LA)
11.8 vs 25.5 mol% (p , 0.001) and increased essential fatty
acid ratio index of 0.2 vs 0.05 (p , 0.01). Long chain
polyunsaturated FA were increased, dihomo-gamma-linolenic
acid 2.1 vs 1.1 mol% (p , 0.01) and AA 13.0 vs 9.25 mol%
(ns). The ratio of AA to docosahexaenoic acid (DHA) was
decreased, 5.2 vs 8.6 (p , 0.001). FA pattern in biopsies from
patients with CD in remission were not sign different from
controls. Mucosal FA and morphological changes were not
related to serum PLFA pattern.
Summary: Marked changes of intestinal PLFA were found in
CD patients not related to serum concentrations. The changed
PLFA pattern in the mucosa might contribute to known
physiological changes of transports and permeability in CD.
Conclusion: Prospective studies would be preferable, in-
cluding healthy controls without GI symptoms, in order to
evaluate if the changes are only secondary to inflammation or
if there are basic changes in the structure of the intestinal
membranes in CD, contributing to the susceptibility to gluten.
PG1-04 INTRACELLULAR TRANSPORT STUDIES OF GLIADIN
PEPTIDES IN HT29 CELLS. I Fischer1, Y Reinke2,
H Wieser3, T Mothes4, HY Naim2, KP Zimmer1. 1Klinik
und Poliklinik fur allgemeine Padiatrie der WWU, Munster,
Germany. 2Inst f Physiol Chemie, Tierarztliche Hochschule
Hannover, Hannover, Germany. 3Deutsche Forschungsanstalt f
Lebensmittelchemie, Garching, Germany. 4Inst f Laborator-
iumsmedizin, klin Chem u Mol Diagnostik, Leipzig, Germany.
Aim: The current study examines the role of mucosal
epithelial cells in the antigen processing and in inducing of
immune reactions. For this, the intracellular transport and
localization of different gliadin peptides was assessed in
human epithelial cell lines.
Methods: HT29 cells were incubated with different gliadin
peptides or Frazers Fraction and gliadin and marker proteins
were detected by immunofluorescence or immunoelectron
microscopy. Cellular organelles were fractionated by dense
gradient centrifugation and examined by Western blot and
immunoprecipitation.
Results: HT29 cells express constitutively HLA class I and
class II complexes after stimulation with gamma Interferon.
The toxic peptide 3149 was found in early endosomes as
assessed by its colocalization with EEA-1, a marker of these
vesicles. By contrast, late endosomes in HT29 cells were
devoid of this peptide. The immunodominant peptide 5668
was also found in early endosomes and to a lesser extent in
late endosomes that were stained with LAMP2. On the other
hand, non-toxic peptides were found in both, early and late
endosomes.
Gliadin colocalized with HLA class I but not with class II
complexes. These two classes of antigens revealed different
endosomal localizations, while HLA class I localized in
EEA-1 positive vesicles, HLA II was found in Cathepsin D-or
LAMP-positive late endosomal vesicles.
Summary: The exogenous toxic peptides were not trans-
ported to HLA class II consisting compartments in HT29
cells, which is an important step for the presentation of
antigens to lymphocytes and the induction of oral tolerance.
On the other hand gliadin peptides colocalize with HLA I
which is involved in the development of inflammation.
Conclusion: It is known that enterocytes can act as an antigen
presenting cell. The epithelium is the barrier for exogenous
antigens and is therefore the first site of contact. If the peptides
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
analysed here are targeted into the lysosomal compartment an
association of gliadin peptides and HLA class II molecules
would be expected. Under therapeutical aspects the enter-
ocytes could act as antigen presenting cell for the induction of
oral tolerance.
PG1-05 IMMUNODOMINANT ALPHA-GLIADIN EPITOPES IN
DQ8 MICE FOLLOWING MUCOSAL IMMUNISATION.
S Senger1, F Maurano1, MF Mazzeo1, M Gaita1,
S Auricchio2, RA Siciliano1, R Troncone2, M Rossi1.
1
Istituto di Scienze dellAlimentazione, CNR, Avellino, Italy.
2
Department of Pediatrics and European Laboratory for the
Investigation of Food-Induced Diseases (ELFID), University
Federico II, Naples, Italy.
Aim and Background: Celiac disease (CD) is characterised
by a strong association with HLA-DQ2 and HLA-DQ8
haplotypes. Our aim was to identify new gliadin epitopes by
using DQ8 transgenic mice as an in vivo model and a strategy
of mucosal immunisation.
Methods: DQ8 mice, from a gluten-free diet colony, were
immunised with a chymotryptic digest of a recombinant a-
gliadin (ct-ra-gliadin; EMBL accession n. AJ130948) plus
cholera toxin (CT) by intragastric challenge at weekly
intervals; one week later spleen and mesenteric lymph node
were analysed for in vitro T cell responsiveness and cytokine
expression by using a panel of synthetic peptides encompass-
ing the entire sequence of ra-gliadin.
Results: Only two previously unidentified peptides, p13
(aa.120139) and p23 (aa. 220239), induced significant in
vitro proliferations of spleen and mesenteric lymphnodes
(MLN) T cells following oral immunisation of DQ8 mice.
The response was restricted by DQ and mediated by CD4 + T
cells; tTG-deamidation of p13 and p23 peptides did not
induce further increase of the specific response. The analysis
of the cytokine pathway showed that both (deamidated or
native) peptides induced a IFN-g2dominated response both in
the spleen and MLN.
Conclusions: We have identified two new gliadin peptides
that are able to elicit potent Th1 immune responses both at the
systemic and intestinal levels. Studies are ongoing to confirm
their immuniogenicity in HLA-DQ8 + coeliac patients.
PG1-06 ASSOCIATION OF POLYMORFISMS IN GENOMIC
REGIONS IMPLICATED IN IMMUNE RESPONSE
WITH COELIAC DISEASE A Capilla1, E Donat2,
C Espinos1, F Palau1, C Ribes-Koninckx2. 1Instituto de
Biomedicina, CSIC, Valencia, Spain. 2Gastroenterology Unit.
La Fe Childrens Hospital, Valencia, Spain.
Coeliac disease (CD) genetic susceptibility is strongly
associated with the HLA class II alleles, but other genetic
factors are involved as well. We found a weak association of
CTLA4 locus in our population. Knocked out (K.O) mice of
the FOXP3 gene shows a similar phenotype than CTLA4 K.O
mice. Moreover, dysregulation of FOXP3 promotes the
development of autoimmune diseases. The TNF
(2308
SNP) and the LT
loci, situated in the HLA region, play a role
in immune response and are considered as potential CD markers
in addition to the presence of DQ.
Aim: To investigate the distribution of the TNF
*2 and LT
*1
alleles in those individuals who carry the risk alleles DQ
B1*0201 A1*0501, and the possible association of a (GT)n
microsatellite polymorphism situated on the promoter region
of FOXP3 locus and CD, with no DQ restriction.
Patients: 113 CD paediatric trios and 69 isolated proband
patients (ESPGHAN criteria) and 104 healthy controls.
Methods: Marker alleles were analyzed by PCR amplification
 ESPGHAN 38TH ANNUAL MEETING
of genomic DNA. HLA DQ alleles were typed by high
resolution test. TNF
and LT
alleles were genotyped by
restriction analysis. FOXP3 STR alleles were studied in
PAGE 12% non-denaturing polyacrilamide gels and silver
stain. Association in trios was determined by using the
Transmission/Disequilibrium Test (TDT).
Results: Allele frequencies were estimated and the results
showed that our population is in Hardy-Weinberg equilibrium
for all markers.
n -308*2 and LT
*1 alleles: considering only families with
at least one informative parent, we applied TDT in 44
and 57 nuclear families respectively. The statistic values,
not considering the DQ genotype, were, for 2308*2,
z = 2.135 (p , 0,05) and for LT
*1, z = 0.00 (n.s). When
analysing individuals with DQ B1*0201 A1*0501, we
observe a high increase of z value for the 2308*2 marker:
z = 3.258. Moreover if we considered those individuals
without DQ B1*0201 A1*0501 we found no statistic
significance (z = 0.762). Besides no differences were
observed in the LT
*1.
n (GT)n microsatellite of FOXP3: we applied TDT in
43 nuclear families. No significant association was found
(z = 0.052; n.s).
Conclusion: We found association of 2308*2 allele of TNF

with CD. This association is even stronger in CD patients
DQB1*0201 A1*0501. Thus, the TNF
2308*2 allele is
possibilily in linkage disequilibrium with this DQ alleles, and
not primary associated with CD.
PG1-07 CLINICAL VALUE OF IGA ANTITRANSGLUTAMI-
NASE TITRES TO PREDICT THE MUCOSAL INJURY
IN CELIAC DISEASE. A Diamanti1, F Colistro2,
F Ferretti1, R De. Vito3, A Calce1, F Bracci1, M Castro1.
1
Gastroenterology Unit, Children Hospital Bambino Gesu`,
Rome, Italy. 2Biochemistry Laboratory, Bambino Gesu`
Children Hospital, Rome, Italy. 3Pathology Service, Bambino
Gesu` Children Hospital, Rome, Italy.
Background: Antiendomysial antibodies of IgA class (EMA
A) and antitissue transglutaminase of IgA class (TGA)were
considered sensitive and specific markers of celiac disease
(CD). Its not still known if a specific pattern of screening test
could be consistent with mucosal damage in celiac pts;
notably an eventual association betweeen TGA titres and
outcome of histological evaluation is not still reported.
Aim: To evaluate if a concordance between IgA antitrans-
glutaminase levels and severity of mucosal injury could exist
in celiac disease (CD).
Methods: In the last year 1886 consecutive subjects with
clinical symptoms and familial history suggestive of CD, 305
healthy children and 92 gastrointestinal disease controls
underwent total serum IgA,EMA and TGA.Subjects with IgA
deficiency were excluded from our study. An intestinal biopsy
was performed if positive EMA A and TGA were found.
Histological features of CD were expressed according to
Marsh classification, modified by Oberhuber.
Results: One hundred eighty two pts (70 M/112 F,ranged for
age 117 yrs) showed positive screening tests. According to
intestinal biopsy results two groups were selected: group A
(25 pts,class 0) and group B (157 pts, class 2,3a,3b and 3c).
Group B showed significantly higher levels of TGA than
group A. In suspected CD pts a concordance between TGA
levels and mucosal injury is shown; each value of TGA
seesms to be able to predict the outcome of histological
evaluation.
PG1-08 A CELL SURFACE TISSUE TRANSGLUTAMINASE IS
INVOLVED IN RECOGNITION OF GLIADIN PEPTIDES
635
BY K 562 CELLS M Silano1, O Vincentini1, A Trecca2,
E Mancini1, M De. Vincenzi1. 1Division of Human Health
and Nutrition, Istituto Superiore di Sanita`, Roma, Italy.
2
Division of Endoscopy and Operative Gastroenterology
Fabio Di Giovanbattista, Roma, Italy.
Aim: Several studies have been demonstated that the
agglutination activity of human chronic myelogenous leuke-
mia K562 cells by prolamines peptictryptic digest (PT) is
higly correlated with the toxicity of these prolamines in
coeliac disease. The mechanism of the agglutination activity
is unknown, but the presence of a binding site on cell surface
of K562 cells has been supposed. In the latest years, a cell
surface tissue transglutaminase has been identified in CD8 +
T cell, in monocytes and in K562 cells. Tissue trans-
glutaminase is known to play a pivotal role in pathogenesis
of coeliac disease, binding the gliadin peptides in the small
bowel mucosa of these patients. We hypothesize that the cell
surface transglutaminase could have a role in the agglutina-
tion activity of K562 cells.
Methods: K562 cells were grown in RPMI medium. For the
agglutination test, cells were harvested, washed and resus-
pended in PBS at the final concentration of 108cells/ml. The
agglutination test were carried out as follow: in each well of
a 96 well plate 25 ml of PBS and 25 ml of cells were added.
Then, mAb anti tTG CUB 7402 (Neomarker, USA) 1 ml of
a solution at the concentration of 2 mg/ml and 25 ml of
a 0,5 mg/ml PT solution were added. Finally, an incubation
for 30 min occoured before evaluating the agglutination by
optical microscope. Controls were performed with PT only and
with mAB anti rabbit IgG.
Results: The incubation of K562 cells with PT digest
resulted in a massive agglutinationof the cells. No agglutina-
tion occoured when the mAb anti tTG was added prior to the
PT digest.
Summary: The ability of the mAb anti tTG of preventing the
agglutination of K562 by gliadin peptides indicates that a cell
surface tissue transglutaminase acts as binding site for the
gliadin peptides.
Conclusion: The results of these study suggest that tissue
transglutaminase could play a role in recognition of gliadin
peptides by the cells in coeliac disease and it could be involved
in starting other responses than the activation of the adaptive
immune system in the mucosa of the coeliac disease patients.
PG1-09 INDUCTION OF TISSUE TRANSGLUTAMINASE EX-
PRESSION BY GLIADIN PEPTIDES IN A HUMAN
INTESTINAL EPITHELIAL CELL LINE. M Silano1,
O Vincentini1, F Maialetti1, M De. Vincenzi1. 1Division of
Human Health and Nutrition, Istituto Superiore di Sanita`,
Roma, Italy.
Aim: Coeliac disease (CD) is an autoimmune entheropathy
triggered by the ingestion of gluten and the related alcohol-
soluble protein fraction of rye and barley. It is well known that
tissue transglutaminase (TGAse) has a pivotal role in the
pathogenesis of coeliac desease, but it is yet to be elucidated
the mechanism causing the increasing of tGAse expression in the
intestinal mucosa. Caco-2 cell line provides a suitable model to
study the enterocyte involvement in the small bowel mucosa
damage caused by gliadin peptides. The aim of this study is to
evaluate if transglutaminase expression by Caco-2 cells increases
after gliadin peptides exposure.
Methods: Human intestinal epithelial cells Caco-2 were used
for the experiments in preconfluence (undifferentiated) and in
longterm culture (differentiated) exposed to 1 mg/ml pepsin-
trypsin digest. After 6 and 24 hours incubation transglutaminase
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
636 ESPGHAN 38TH ANNUAL MEETING
expression was evaluated by western blotting and immuno-
fluorescence.
Results: Tissue transglutaminase expression by Caco-2 cells
is increased 1.5 fold after 24 hours incubation with PT digest,
and the simoultaneus stimulation of the cells with gamma
interferon results in a sinergic effects. It has been also noticed
that tissue transglutaminase expression is increased after
Caco-2 cells got differentiated.
Summary: In a human enterocytes in vitro system, gliadin
peptides exposure increases transglutaminase expression.
Conclusion: The results of this study may provide a new
insight in the interaction between gliadin peptides and
enterocytes.
PG1-10 VILLOUS ATROPHY OF THE DUODENAL BULB IN
COELIAC DISEASE OF CHILDREN: A MULTICENTER
STUDY E Thanasi1, R Nenna1, FM Magliocca2, C Barbera3,
G Guariso4, S Martelossi5, AM Staiano6, M Bonamico1.
1
Department of Paediatrics, La Sapienza University,
Rome, Italy. 2Department of Experimental Medicine and
Pathology, La Sapienza University, Rome, Italy. 3De-
partment of Pediatrics, Torino, Italy. 4Department of
Pediatrics, Padova, Italy. 5Department of Pediatrics, Trieste,
Italy. 6Department of Pediatrics, Napoli, Italy.
Aim: In a recent study we described that hyperplastic villous
atrophy, characteristic of coeliac disease (CD), could be
patchily distributed; the bulbar area was always involved,
sometimes being the only one involved (JPGN 2004;38:204).
The aim of this study was to enlarge the paediatric series in
order to evaluate the presence of duodenal bulb lesions in
a statistically significant number of cases.
Methods: We studied 379 coeliac children and adolescents
(144 males, range 10 mos-18 yrs) at the diagnosis, on
a gluten-containing diet, and 350 gastroenterological controls
aged and sex matched, affected by gastroesophageal reflux
disease or abdominal pain These patients have been enrolled
from five Italian paediatric departments. Upper endoscopy
with multiple biopsies (one bulb sample and four distal
duodenum samples) was performed. We measured anti-human
tissue transglutaminase autoantibodies (anti-tTG Abs) by
ELISA and anti-endomysium antibodies (EMA) by immuno-
fluorescence method.
Results: In all coeliac children various degree of villous
atrophy of the bulbar sample was present; in 9 of them (2.4%)
these lesions were limited to the bulb. Patchy villous atrophy
was found in 24 cases (6.3%). All coeliac patients were anti-
tTG Abs and/or EMA positive. Control subjects showed
neither mucosal changes compatible with CD in all the
duodenal samples, nor autoantibodies positivity.
Conclusions: The demonstration in a large series of CD
children that villous atrophy is always present in the bulbar
area, sometimes being the only one to be affected, suggests
a new reccomandation: for the diagnosis of CD intestinal
biopsy should be taken on bulbar area.
PG1-11 EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)
DEPENDENT PROLIFERATION IN CULTURED BIOP-
SIES FROM CELIAC DISEASE (CD) PATIENTS CHAL-
LENGED WITH GLIADIN. MV Barone1, F Paparo1,
M Maglio1, A Gimigliano1, T Ribecco1, E Miele1,
R Troncone1, S Auricchio1. 1University Federico II.
Pediatric Department (ELFID), Neaples, Italy.
Aim: Caracterization of the role of EGFR signalling on the
early effects of gliadin peptides on jejunal colture biopsies
from CD untreated patients.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
Method: Biopsies from untreated CD patients were cultured
over-night in an organ culture system in the presence of peptic-
triptic digest of gliadin (PTG) or of ovalbumin (PTO). In other
experiments gliadin peptides P3143 or P5668 were used. To
test the EGFR involvement in PTG and P3143 effects on cell
morphology and proliferation two inhibitors were used. ZD .
and the bloking antibody anti EGFR 528. Bromodeoxiuridine
was added to the medium to detect cells in to the S-fase of the
cell cycle. Actin cytoskeleton rearrangements (by phalloidin),
number of cells in S-fase (by bromodeoxiuridine) and in
apoptosis (Tunel staining) were examined; EGFR was stained
by specific antibody (SC 1005).
Results: Incubation of untreated celiac biopsies in presence of
PTG or P3143 induces an alteration of the epitelial
morphology and the actin cytoskeleton and a diffused, more
intense EGFR staining. An increase of both apoptotic and
proliferating epitelial cells is detectable with BrdU incorpora-
tion rising to 38% (2/ + 8%) versus 15% (2/ + 5%) of the
control, and Tunel positive cells rising to 28% (2/ + 10%)
versus 8% (2/ + 4%) of the control. The EGFR inhibitors were
able to prevent the morphology alteration, the actin cytoskel-
eton modifications and the increase of the BrdU incorporation
obtained in presence of PTG and P3143. In contrast PTG and
P31-43 induced apoptosis was not greatly altered in presence of
the EGFR inhibitors.
Summary: Peptic-tryptic digests of gliadin (PTG) and P3143
gliadin peptide mimic EGFR (Epidermal Growth Factor
Receptor) signalling and induce actin rearrangements and
proliferation in several cell lines (nota bibliografica). As EGFR
signalling is known to act on cell proliferation and morphology,
PTG ability to exert EGF effects on biopsies from florid CD
patients in colture was tested. Gliadin induced alteration of
epitelial cells morphology, actin cytoskeleton and proliferation
are prevented by EGFR inhibitors; strongly suggesting that this
pathway is ingaged by gliadin peptides at least for these read
outs. Probably, other receptors may be involved in gliadin
induced apoptosis since this effect is not prevented by EGFR
inhibitors.
Conclusion: Gliadin and gliadin peptide P3143 induce
EGFR dependent proliferation but not apoptosis in cultured
biopsies from CD patients.
PG1-12 PREVALENCE OF CELIAC DISEASE IN CHILDREN
AND ADOLESCENTS WITH TYPE I DIABETES MEL-
LITUS IN SOUTHERN POLAND. Z Grzenda-Adamek1,
M Ciechanowska2, J Nazim2, A Gorska2, K Fyderek1,
J Starzyk1. 1Dept. of Pediatrics Gastroenterology and
Nutrition, Polish-American Childrens Hospital, Jagiellonian
Univ. Medical College, Krakow, Poland. 2Dept. of Endocri-
nology of Children and Adolescents, Polish-American Child-
rens Hospital, Jagiellonian Univ. Medical College, Krakow,
Poland.
Prevalence of celiac disease (CD) in patients with diabetes
mellitus type I (DM) has been estimated from 1.7 to 16.4% in
different populations.
The aim of the study was to evaluate the frequency of CD in
patients with DM and to assess whether the coexistence of
CD has an influence on physical development of the patient
and clinical course of DM.
Patients and Methods: 459 patient with DM: 214 girls, 245
boys, mean age 13.7 yr, range 1.625 yr. All patients, except 4
in which CD was diagnosed before development of DM, were
screened for endomysial antibodies (EMA). EMA were
detected by immunofuorescence. In 139 patients EMA was
performed at least twice. The following parameters were
evaluated: height (expressed as standard deviation score-
SDS), body mass index (BMI) in SDS, hemoglobin (Hb)
 ESPGHAN 38TH ANNUAL MEETING
concentration and clinical course of diabetes i.e.: HbA1c,
total insulin requirement and occurrence of severe hypogly-
cemia. Results: EMA were positive in 38 patients (in 6 they
appeared in second assay). In 26 patients total or subtotal
villous atrophy was found confirming the diagnosis of CD, in
4 small bowel mucosa was normal. The 6 patients tested
initially positive became negative on follow up. Two children
are waiting for biopsy. The prevalence of CD in DM was
estimated for 6.5% (in 4 patients CD was recognized before,
in 26 after DM onset). Diabetics with CD had an earlier onset
of diabetes (6.45 vs 8.6 y. p = 0.02) and their Hb concentration
was lower in comparison with patients with DM without CD
(12.9g% vs. 13.8g%, p = 0.0004). No differences in height-
SDS, BMI-SDS, HbA1c, total insulin requirement and
frequency of severe hypoglycemia were found. In patients
with CD and DM after at least 6 months (median = 1.7y) of
gluten free diet, height-SDS (p = 0.04) and BMI-SDS (p =
0.02) increased. Hb concentration, HbA1c, insulin require-
ment have not changed.
Conclusions: Routine serological screening for CD should be
performed in patients with type I DM because of high
prevalence and the oligosmptomatic course of CD. Test should
be repeated because of the possibility of seroconversion.
PG1-13 A SEQUENTIAL STUDY IN THE SCREENING OF
COELIAC DISEASE AMONG FIRST DEGREE RELA-
TIVES? R Nenna1, M Ferri1, P Mariani1, E Thanasi1,
R Luparia1, B Mora2, C Tiberti3, M Bonamico1. 1De-
partment of Paediatrics, La Sapienza University, Rome,
Italy. 2Department of Experimental Medicine and Pathology,
La Sapienza University, Rome, Italy. 3Department of
Clinical Sciences, La Sapienza University, Rome, Italy.
Aim: The prevalence of coeliac disease (CD) among the
relatives (from 5.5% to 8.5%) and the complications of an
undiagnosed CD prompted us to identify the best strategy to
screen these subjects.
Methods: We studied 441 first degree relatives of 208 CD
patients by IgA EMA and RIA immunoglobulin A anti-
transglutaminase autoantibodies (TGAA). 364 subjects were
typed for HLA-DRB1, -DQA1 and -DQB1 genes by PCR-
SSP method using commercial kits. Endoscopic intestinal
biopsies were proposed to the serology positive subjects.
Results: Two immunodeficient fathers (one IgA deficient, the
other on steroid therapy) both with duodenal villous atrophy,
were excluded from serological evaluation. The TGAA
revealed a higher sensitivity (100%) than IgA EMA (90%)
(table). On the whole, CD prevalence in our series resulted
9.5%. 231/364 relatives (63.4%) and 38/40 biopsy proved
coeliac subjects (95%) carried the DQ2/DQ8 haplotypes.
3 DQ2 positive parents, previously negative, became positive
to the serology during the follow-up.
Intestinal biopsy
Villous Normal Not
N atrophy mucosa done
IgA EMA + TGAA + 38 36 1 1
IgA EMA-TGAA + 8 4 2 2
IgA EMA - TGAA - 393 - 21 372
Total 439 40 24 375
Conclusion: CD prevalence in our series resulted very high.
These data suggest an accurate algorithm to select candidates
for intestinal biopsy among CD high risk subjects: as a first
step an evaluation of the sensitive and non-expensive RIA
TGAA and of total IgA (if IgA deficiency, RIA IgG anti-tTG
637
assay); a second evaluation of the TGAA and the genetic
study should be performed 23 yrs later to the negative
subjects. The relatives carrying the DQ2/DQ8 haplotipes
should continue the serological follow-up, while the others
need only a clinical follow-up.
PG1-14 IS CELIAC DISEASE SCREENING IN RISK GROUPS
JUSTIFIED? A LONG-TERM FOLLOW-UP STUDY
K Kaukinen1, M Viljamaa1, H Huhtala2, M Maki1,
P Collin1. 1Tampere University Hospital, Tampere, Finland.
2
University of Tampere, Tampere, Finland.
Aim: The benefits of celiac disease (CD) screening are
debatable and the compliance to gluten-free diet (GFD) in
screen-detected patients is thought to be poor; we investigat-
ed the dietary compliance, quality of life (QoL), physical
well-being and bone mineral density in screen-detected CD
patients after long-term treatment.
Methods: 53 consecutive screen-detected CD patients (both
children and adults at the diagnosis of CD) were enrolled to
follow-up study after median of 14 (range 521) years of
commencement to GFD; screening was carried out because of
family history of CD or associated autoimmune diseases.
Dietary compliance was assessed by an interview, a 4-day
food record and CD serology. QoL was evaluated by
Psychological General Well-Being (PGWB), SF-36 and
gastrointestinal symptoms by Gastrointestinal Symptom
Rating Scale
questionnaires. The results were compared to
those in 44 symptom-detected treated CD patients, non-CD
controls and general population.
Results: The long-term compliance to GFD did not differ
significantly between screen-detected and symptom-detected
CD patients, being good in 83% and 77% respectively. QoL
was as good in screen-detected treated CD patients as in
symptom-detected CD and nonceliac controls when mea-
sured by PGWB. Mean value of mental health in SF-36 was
significantly better in screen-detected treated CD than in the
general population; in other items there was a similar
tendency. Screen-detected long-term treated CD patients
had slightly less abdominal symptoms and better bone
mineral density than symptom-detected treated CD patients
(not significant).
Summary: Long-term compliance to GFD in screen-
detected CD patients was good. Despite evident dietary
restrictions, the QoL was comparable to that of symptom-
detected patients, and even to non-celiac controls or general
population.
Conclusions: In terms of QoL and compliance CD screening
and early treatment seems to be conceivable in CD risk
groups. These data can be applied when decisions about
mass-screening of CD in general population are rationalized.
PG1-15 COELIAC DISEASE AND GENETIC FACTORS: STUDY
ON HLA AND NON-HLA GENES F Pereira1, A Ferreira1,
M Tavares2, P Canedo1, E Trindade2, F Carneiro3,
J Machado1, J Amil-Dias2. 1IPATIMUP, Porto, Portugal.
2
Hospital S. Joao, Porto, Portugal. 3School of Medicine,
Porto, Portugal.
Background: Coeliac disease (CD) has multifactorial
etiology, with genetic and environmental factors. Little is
known about non-HLA-linked genes. Polymorphisms in
genes that are potentially involved in the regulation of
inflammatory and immune responses may play an important
role in the susceptibility to the development of CD.
Aim: To investigate the association between HLA-DQ2 and
HLA-DQ8 heterodimers, polymorphisms in the TNFA,
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
638 ESPGHAN 38TH ANNUAL MEETING
IFNGR1, IL1B, IL1RN, CTLA4 and MIF genes and the risk
of developing CD.
Methods: A case-control study including 64 CD patients
and 930 controls (313 adults; 617 children) was performed.
The following polymorphisms were genotyped: TNFA
(2308G/A; 2857C/T), IFNGR1 (256C/T), IL1B
(2511C/T), IL1RN (86bp intron 2 VNTR), MIF (2794
VNTR), and CTLA4 (+49A/G) genes. DQ2 and DQ8 hetero-
dimers were identified through DQA1*0501, DQB1*0201,
DQA1*03 and DQB1*0302 allele identification.
Results: We observed a significant association between: (a)
CD and carriers of the HLA-DQ2 heterodimer, with an OR of
25.7 (95% CI = 11.756.6); (b) CD and carriers of TNFA-
308*A allele, with an OR of 3.8 (95% CI = 2.07.0) and 7.0
(95% CI = 1.727.9), for heterozygotes and homozygotes,
respectively. However, after sample stratification in DQ2-
positive and negative carriers, the TNFA association with the
disease was not reproduced. In a multivariate regression
model, only DQ2-positivity was shown to be independently
associated with risk of CD (p , 0.0001 for DQ2 and p = 0.14
for TNFA). Among DQ2-negative CD patients only one case
was shown to be DQ8-positive. No relevant associations were
found with the TNFA-857, IFNGR1-56, IL1B-511, IL1RN
VNTR, CTLA4 + 49 and MIF-704 VNTR polymorphisms.
Conclusion: We confirmed the association of the DQ2
heterodimer with CD. In contrast to what has been dem-
onstrated in other populations, the DQ8 heterodimer may not
be significantly prevalent among DQ2-negative CD patients.
Our findings also suggest that the association of the TNFA-
308A polymorphism with CD is dependent, perhaps due to
linkage disequilibrium, on the association with DQ2.
PG1-16 HIGH PREVALENCE OF CELIAC DISEASE IN SWED-
ISH CHILDREN WITH DIABETES MELLITUS S Lantz1,
EO rtqvist2, L Grahnquist1. 1Unit of Pediatric Gastroenter-
ology and Nutrition, Karolinska University Hospital, Stock-
holm, Sweden. 2Unit of Pediatric Diabetology, Karolinska
University Hospital, Stockholm, Sweden.
Aim: To investigate the prevalence of celiac disease (CD) in
a large group of children with type 1 diabetes (T1DM).
Methods: All children with T1DM, screened for CD, at
Astrid Lindgren Childrens hospital from 1995 to 2004 (n =
847), were retrospectively studied through three patient file
systems (BMS, Diabase and paper charts). Patients where
screened with different combinations of antibody tests over this
period. Children with CD before the T1DM diagnose or CD
diagnosed only with small intestinal biopsy were also included.
Results: 123 out of the 847 children had at least one positive
antibody test. The prevalence of biopsy-confirmed celiac
disease among the diabetic children was 8.8 % (75 of 847
patients). 67/75 (89%) were diagnosed after the T1DM onset.
Three were diagnosed as having CD only with biopsy. Eight
children had CD verified by biopsy before onset of diabetes.
The majority of children with CD (58 %) had no symptoms.
33 % were diagnosed at the first screening, 57% within the
first 2 years and 87 % within 4 years of T1DM. 39% (48/123)
of the T1DM patients with a positive antibody test had not
been referred for a biopsy.
Summary: In this group of 847 children with T1DM, 123
children were positive at least one time in screening test for
CD. Despite that not all 123 children had been followed up
with small bowel biopsy we found an extremely high
prevalence of 8.8 %. 58 % of biopsy verified CD cases, had
no GI symptoms. 57% of the children with CD were
diagnosed within 2 years of the T1DM diagnosis.
Conclusions: The prevalence of 8 % is among the highest
reported. A large group were not further investigated with
biopsy, maybe due to the lack of overt symptoms of CD.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
Using the positive predicted value for each of the three
antibodies in the biopsy group to calculate the expected
frequency of silent CD in the non-biopsy group could
eventually result in a prevalence of 14%.
PG1-17 IS COELIAC DISEASE A DOMINANTLY INHERITED
DISORDER? IR Korponay-Szabo1, A Kapitany2,
JB Kovacs3, M Lorincz3, J Opre1, E Nemes1, J Tumpek2,
S Sipka1. 1Dept. of Paediatrics, Univ. of Debrecen,
Debrecen, Hungary. 23rd Dept. of Internal Medicine, Univ.
of Debrecen, Debrecen, Hungary. 3Heim Pal Childrens
Hospital, Budapest, Hungary.
Aims: Coeliac disease (CD) occurs in approx. 1015 % of
first-degree relatives. Manifestation of the disease is re-
stricted to HLA-DQ2 or DQ8-positive individuals, but the
exact mode of inheritance is unclear and certain pedigrees
show that CD is not co-inherited with HLA-DQ allels. In this
study, we investigated the occurrence of CD in the offsprings
of the patients.
Methods: Patients diagnosed with CD in childhood by
Interlaken or by current ESPGHAN criteria were prospec-
tively followed up and their children were screened using IgA
and IgG endomysial (EMA) and transglutaminase antibodies.
Seropositive subjects were referred for jejunal biopsy.
Offsprings positive for EMA and/or with biopsy-proven CD
were regarded as affected. The results were compared with
the evaluation of children of CD patients diagnosed as adults.
Children first evaluated below the age of six years or on
a gluten-restricted diet were yearly re- screened.
Results: Altogether 481 offsprings (228 males) of 270 CD
parents were studied with a median number of 2 children per
parent and at a median age of 7.3 years. Percentage of
affected offsprings varied according to the mode the coeliac
parent was initially diagnosed (table), and was significantly
higher already at an earlier median age (4.0 years) for
childhood-diagnosed CD parents than for adult-diagnosed
CD parents at a median child age of 7.7 years. HLA-DQ
typing was performed in 85 offsprings. All affected and 76%
of not affected offsprings carried DQ2 or DQ8.
Offsprings
Coeliac parent N (N) Affected %
Childhood-diagnosed 32 40 16 40
Adult-diagnosed, EMA-positive 75 138 28 20
Adult-diagnosed, EMA unknown 72 108 9 8
Screening-diagnosed adult 91 195 (107*) 128 (40*) 66 (38*)
Total 270 481 181 38%
*without index children
Summary: Occurrence rate of CD was 38% in the second
generation which seems to be much higher than for first-
degree relatives in general.
Conclusions: Our data suggest that nearly half of HLA-DQ2
or DQ8-positive offsprings were affected raising the possi-
bility that dominantly inherited gene(s) might be operative at
least in certain subsets of CD patients. These data also
warrant the careful screening of offsprings.
PG1-18 EFFECT OF BREAST-FEEDING ON RISK OF COELIAC
DISEASE: A SYSTEMATIC REVIEW AND META-
ANALYSIS OF OBSERVATIONAL STUDIES AK Ako-
beng1, AV Ramanan2, D Basude1. 1Department of Paediatric
Gastroenterolgy, Booth Hall Childrens Hospital, Man-
chester, United-Kingdom. 2Department of Paediatrics, Booth
Hall Childrens Hospital, Manchester, United-Kingdom.
 ESPGHAN 38TH ANNUAL MEETING
Background: Coeliac disease (CD) is a multifactorial
disorder that may depend on genetic, immunological, and
environmental factors. Recent observational studies suggest
that breast-feeding may prevent the development of CD.
Aim: To evaluate articles that compared effects of breast-
feeding on risk of CD.
Design: Systematic review and meta-analysis of observational
studies published between 1966 and June 2004 that examined
the association between breast-feeding and the development of
CD.
Results: Six case-control studies met the inclusion criteria.
With the exception of one small study, all the included studies
found an association between increasing duration of breast-
feeding and decreased risk of developing CD. Meta-analysis
showed that the risk of CD was significantly reduced in
infants who were being breastfed at the time of gluten
introduction (OR 0.48, 95% CI 0.40 to 0.59) compared with
infants who were not being breast-fed during this period.
Conclusions: Breast-feeding appears to offer some pro-
tection against the development of CD. Being breast-fed
during the introduction of dietary gluten, and increasing
duration of breast-feeding were associated with decreased
risk of developing CD. It is, however, not clear from the
primary studies whether breast-feeding only delays the onset
of symptoms or provides a permanent protection against the
disease.
PG1-19 SEROLOGICAL SCREENING OF CELIAC DISEASE:
CHOOSING THE BEST TESTS UNDER TWO YEARS.
SIGENP Working Group1 V Baldas2, S Martellossi1. 1Dott.
Accomando-Palermo, Dott.ssa Barbato-Roma, Prof.ssa
Bonamico-Roma, Dott. Fontana-Milano, Dott. Gasperrini-
Ancona, Dott. Lambertini-Bologna, Dott. Lionetti-Firenze,
Dott. Romano-Reggio Calabria, Prof. Troncone-Napoli.,
Italy. 2IRCCS Burlo Garofolo, Dip. di Scienze della
Riproduzione e dello Sviluppo-Univ degli Studi di Trieste,
Trieste, Italy.
Background: Anti-transglutaminase antibodies (anti.h-tTG)
have been proposed as a reliable marker of celiac disease
(CD) and a useful means of selecting subjects for intestinal
biopsy. However, no data are available regarding the
diagnostic efficacy of these auto-antibodies during the first
years of life in the diagnosis of CD.
Aims: The present collaborative, retrospective study was
undertaken in order to compare the sensitivity and specificity
of the anti-h-tTG and anti gliadin antibodies (AGA) among
children aged ,2 years in the diagnosis of CD.
Methods: A total of 222 patients aged, 2 years with
symptoms of CD (diarrhea 73%, failure to thrive 40%,
dystrophy 36%, abdominal distention 33%, anemia 13%,
vomiting 7%) underwent intestinal biopsy for the diagnosis of
CD. In these patients we compare the serological marker for
identifying the best test to use in patients ,2 years.
Results: 207/222 (93.2%) patients resulted positive both
anti-h-tTG and AGA test. 15/222 (6.8%) patients resulted
positive only for AGA tests. All of these patients underwent
intestinal biopsy and 218/222 resulted CD patients. In 4/222
the diagnosis of CD were not confirmed: 2 of these had
a score 1 of Marsh 2 have a score II and the follow-up
demonstrated no efficacy of gluten free diet. All these 4
patients were tested positive for only AGA antibodies.
Conclusion: In our experience the AGA test identified 11
(4.9%) patients loose by anti-h-tTG. In the class of age
considered the AGA assay seems more sensitive than anti-h-
tTG even if it demonstrated a low specificity. Based on these
preliminary data we suggest to use both AGA and anti-h-tTG
639
for the serological diagnosis of CD among children aged
,2 years.
PG1-20 SCREENING FOR CELIAC DISEASE IN A FRENCH
ADULT POPULATION: LOWER THAN EXPECTED
PREVALENCE D Caldari1, AM Yamamoto2, N Brousse3,
L Chatenoud2, P Galan4, S Hercberg4, J Schmitz1,
MC Boutron-Ruault4. 1Division of Gastroenterology, Hep-
atology, Nutrition, Department of Pediatrics, Necker Enfants
Malades, Paris, France. 2Department of Immunology, Necker
Enfants Malades, Paris, France. 3Department of Pathology,
Necker Enfants Malades, Paris, France. 4French Institute of
Health and Medical Research (INSERM) Unit 557, Paris,
France.
In Europe, prevalence of silent celiac disease (CD) ranged
between 1/300 and 1/100. The aim of this study was to
estimate the prevalence of CD in French adult population and
to validate the screening tests.
Methods: IgG and IgA antigliadin antibodies (AGA) were
performed in 8465 subjects among voluntary subjects (mean
age: 54.8; range: 4070) enrolled in the SU.VI.MAX study
(Hercberg et al. Arch Intern Med. 2004 Nov) between
September 1999 and June 2000. Those positive or in-
termediate for IgG and/or IgA AGA were tested for anti-
endomysium antibodies (AEA). To improve the screening of
CD IgG and IgA anti-transglutaminase antibodies (ATA) by
radioligand assay (RLA) were performed in respectively
1557 and 1619 subjects, and IgA ATA by ELISA were
performed in 1590 subjects. Those positive were also tested
for AEA. All positive subjects for AEA had an jejunal biopsy.
Results: There were 40 men (1.1%) and 43 women (0.9%)
with at least one positivity for AGA. Among them, 5 women
and 2 men had AEA. One patient negative for all AGA but
positive for all ATA was tested positive for AEA. All had
intestinal biopsies which demonstrated total villous atrophy.
In addition, 1 woman had a known celiac disease, and had
been on a gluten-free diet for over one year. All antibodies
were negative. Therefore, prevalence of CD in that population
was 1/940. Sensitivity and specificity of IgA AGA were
respectively 75 and 90% and for IgG AGA 50 and 97.4%.
Sensitivity was higher for ATA by ELISA (100 %) whereas
specificity was better with RLA (99.1% for IgA and 97.8%
for IgG).
Discussion: Our study is the first french large scale screening
study. The prevalence was lower in our population than in
others, but the mean age was higher than in most other
European studies. An interesting finding is the case of CD
screened through ATA positivity, although all tests were
negative for AGA. It could be advocated to use ATA instead
of AGA as the first screening test, to be confirmed by AEA
before proposing an intestinal biopsy.
Conclusion: There seems to be a low rate of celiac disease in
French adults. The reason for this finding is unknown. Our
data suggest that a screening strategy for celiac disease would
be inappropriate in France.
PG2-01 CTLA4/CT60 POLYMORPHISM IS NOT RELEVANT IN
SUSCEPTIBILITY TO AUTOIMMUNE INFLAMMATO-
RY INTESTINAL DISORDERS B Rueda1, A Zhernakova2,
M Gomez-Garcia3, E Ortega4, A Pinero5, A Nieto6,
B Koeleman2, J Martin1. 1Instituto de Parasitologa y
Biomedicina Lopez-Neyra, CSIC, Granada, Spain. 2Complex
Genetics Group, Department of Biomedical Genetics, Univer-
sity Medical Center, Utrecht, The Netherlands. 3Servicio de
Digestivo, Hospital Virgen de las Nieves, Granada, Spain.
4
Servicio de Pediatra, Hospital Clnico San Cecilio, Granada,
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
640 ESPGHAN 38TH ANNUAL MEETING
Spain. 5Servicio de Digestivo, Hospital Puerta del Mar, Cadiz,
Spain. 6Servicio de Inmunologa, Hospital Puerta del Mar,
Cadiz, Spain.
Aim: The aim of this work was to investigate the possible
influence of the recently described CT60 A/G (SNP 3087243)
dimorphism of the CTLA4 gene, which interestingly has been
recently associated with functional relevance and with
susceptibility to a variety of autoimmune diseases, in the
susceptibility to IBD and celiac disease.
Methods: We analysed a case-control cohort composed of
a total of 528 Spanish IBD patients (284 with Crohns disease
and 244 with ulcerative colitis) and 454 unrelated healthy
individuals, and additionally a group of 90 celiac disease
families. CT60 genotyping was performed using a Taqman 5
allelic discrimination assay.
Results: After comparing IBD patients with the control
population we found no significant deviation in the distribu-
tion of the alleles or genotypes of CTLA4/CT60 dimorphism.
In addition, using familial and case-control analysis, no
evidence for a statistically significant association was
observed between CTLA4/CT60 and celiac disease suscep-
tibility.
Summary: In this work we investigated for the first time the
role of CTLA4/CT60 polymorphism in IBD susceptibility
and no association was observed. In addition we have
observed that CTLA4/CT60 polymorphisms is not relevant
for celiac disease predisposition in our population, accord-
ingly with recent studies that observed no association or
borderline significance between this genetic marker and
celiac disease susceptibility.
Conclusion: Our results suggest that the CTLA4/CT60
polymorphism does not play a major role in autoimmune
inflammatory intestinal disorders.
PG2-02 STUDY ON CARD15 AND TLR4 POLYMORPHISMS
IN CHILDREN WITH EARLY DIAGNOSED IBD.
M Bueno De Mesquita1, M Ferrante2, M Joossens2, G De
Assche2, S Vermeire2, P Rutgeerts2, V Janssens1,
I Hoffman1. 1Paediatric Gastroenterology, Leuven, Belgium.
2
Gastroenterology, Leuven, Belgium.
Aims: Genetic studies in adult IBD patients have highlighted
associations with CARD15 and Toll Like Receptor (TLR)
polymorphisms. These studies further suggest that variants
influence disease location, behaviour and natural history.
However, only few paediatric studies are available and only
on CARD15. Therefore, we examined a large pediatric cohort
of IBD patients for CARD15 and TLR-4 mutations as well as
for ASCA and pANCA.
Methods: We collected 100 children followed at a tertiary
referral center with a diagnosis of IBD before the age of 17
(CD = 79, UC = 18, IC = 3). We also included 45 children
without IBD (control group). All individuals were analysed
for CARD15 R702W, G908R and L1007fs, for TLR4
Asp299Gly and for ASCA and pANCA. Clinical character-
istics were examined at onset as well as at follow up.
Results: Mean age at diagnosis was 11.9 6 3.2 yr. Familial
IBD (defined as at least one first or second degree family
member with proven IBD) was present in 34/100 (34%) of
patients. Besides traditional symptoms, 34% of children
presented with failure to thrive and 21% with anemia. Most
children (.90%) presented with an inflammatory behaviour
at diagnosis, however, 35% evolved towards stricturing and
32% towards fistulising disease after a mean of 7.6 yr of
follow-up. We observed a high frequency of CARD15
variants in CD (43/79 or 54%), as compared to UC (23%,
p = 0.01) and controls (21.9%, p , 0.001). Even more
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
striking was the finding that 15/43 CARD15 + patients (35%)
were homozygous or compound heterozygote. The presence
of CARD15 mutations was negatively associated with an
inflammatory CD behaviour (p = 0.007, OR 0.33 95%CI
0.1450.746) and positively associated with ASCA (p =
0.024 OR 1.634 95%CI 1.0632.510) and need for surgery
(p = 0.01, OR 2.528 95%CI 1.2185.247). The TLR4 mutated
allele frequency was 10.6% in CD, compared to 0% in UC
and 5.3% in controls (p = 0.053 and p = 0.016 resp) and was
associated with ileitis (p = 0.027, OR 7.1 95%CI 1.3537.31).
ASCA was present in 32% of CD, compared to 7.1% in UC
(p = 0.08) and 7.1% in controls (p = 0.001) and was
associated with a need for surgery (p = 0.015, OR 3.40 95%
CI 1.249.33). For pANCA, prevalence was 40% in UC
compared to 6.8% in CD and 2% in controls (all p , 0.01).
pANCA positivity was seen in 4/5 PSC patients.
A number of concomitant diseases were observed: 7 patients
(all CD) had ankylosing spondylitis, 4 psoriasis, 5 PSC (2 CD
and 3 UC), 1 thyroiditis, 1 diabetes, 10 asthma, 2 celiac
disease and 2 patients had Turners Syndrome (both CD).
Summary & Conclusions: In this paediatric IBD cohort,
frequency of CARD15 variants was as high as 54% among
CD patients and was associated with a more complicated
disease phenotype. We also identified a higher frequency of
TLR4 Asp299Gly polymorphisms among CD patients, and
this was associated with an ileal disease location. The
immune marker ASCA was positive in 32% of CD patients
and was also indicative of a more complicated disease
phenotype. The most intriguing was probably the high
frequency of concomitant immune-mediated diseases, in-
cluding 2 cases of Turners Syndrome, suggesting a common
genetic background.
PG2-03 OCTN 1/2 VARIANTS ARE ASSOCIATED WITH DIS-
EASE SUSCEPTIBILITY AND PHENOTYPE IN EARLY
ONSET INFLAMMATORY BOWEL DISEASE (IBD)
RK Russell1, HE Drummond1, ER Nimmo1, DC Wilson2,
PM Gillett3, LT Weaver4, M Bisset5, J Satsangi1. 1GI Lab,
Edinburgh University, Edinburgh, United-Kingdom. 2Dept
Child Life and health, RHSC, Edinburgh, United-Kingdom.
3
Dept Paediatric Gastoenterology, RHSC, Edinburgh, United-
Kingdom. 4Dept Child Life and health, Glasgow University,
Glasgow, United-Kingdom. 5Dept Paediatric Gastoenterology,
Aberdeen, United-Kingdom.
Aims: The IBD5 locus was strongly linked to early-onset
IBD in the index population studied; most recently mutations
of OCTN1/OCTN2 and the combined OCTN1/2 (TC
haplotype) within the IBD5 locus have been implicated in
adult susceptibility to CD. We aimed to study these variants in
an early-onset population.
Methods: Patients diagnosed with IBD ,16 years were
recruited and fully phenotyped. Parental DNA was also
collected to allow Transmission Disequilibrium Testing
(TDT). OCTN variants were genotyped using the Taqman
system.
Results: 299 (200 CD, 74 UC, 25 IC patients) were studied.
TDT for the OCTN1 variant demonstrated association with
IBD (p = 0.01), CD (p = 0.04) but not UC (p = 0.06). OCTN2
did not reach significance. The OCTN1/2 haplotype was
associated with IBD (p = 0.01). TC haplotype contribution
was analysed by case-control analysis (see table).
In CD univariate analysis demonstrated OCTN1/2 mutations
and TC haplotype carriage were associated with decreased
weight (,9th centile) at presentation (p = 0.01, 0.04 and 0.007
respectively) and lower Body mass index (BMI) (,9th
Centile) for OCTN2 and TC haplotype (p = 0.03, 0.01
respectively). This association was also present at 2 years
after diagnosis for the TC haplotype: weight ,9th centile
 ESPGHAN 38TH ANNUAL MEETING
(p = 0.001), BMI ,25th centile (p = 0.008). OCTN1 also
showed association with erythema nodosum (p = 0.04). The
TC haplotype and OCTN2 mutations were protective for ileo-
colonic disease at presentation (p = 0.02 for both). No
associations were found for UC.
Summary: OCTN1/2 and the TC haplotype are linked to
susceptbility to IBD, UC and CD. They are associated with
lower weight/BMI centile at CD diagnosis and follow up.
Conclusion: These data implicate variants of the OCTN1/2
gene as predictors of disease susceptibility and phenotype in
early-onset IBD.
Subjects TC TC TC
(number Haplotype Heterozygosity Homozygosity
studied) carriage rates rates
Controls (248) 170 (68.5%) 117 (47.2%) 40 (16.1%)
CD (185) 153 (82.7%) 112 (60.5%) 41 (22.1%)
P value 0.0008 0.006 0.11
UC (72) 56 (78.9%) 36 (50.7%) 20 (28.2%)
P value 0.09 0.59 0.02
PG2-05 THE DLG 5-113A MUTATION IS ASSOCIATED WITH
PG2-04 CARD15, TLR4, ASCA AND PANCA IN PAEDIATRIC
IBD: A FAMILY STUDY M Bueno De Mesquita1,
M Ferrante2, L Henckaerts2, G Van. Assche2, S Vermeire2,
P Rutgeerts2, V Janssens1, I Hoffman1. 1Paediatric Gastro-
enterology, Leuven, Belgium. 2Gastroenterology, Leuven,
Belgium.
Background & Aims: Around 1015% of IBD patients have
another family member with the diagnosis of IBD and this
familial aggregation is reported to be even higher (2540%)
in paediatric IBD patients, suggesting that genetic factors
may be of more importance in early onset of disease. NOD2
(CARD15) is the first gene identified underlying susceptibil-
ity to Crohns disease and recently also associations with Toll
Like Receptor (TLR) 4, DLG5 and OCTN have been
reported. IBD is also characterized by the presence of
immunologic responses to microbial antigens. Of these,
ASCA and pANCA have been studied the most. We
investigated 100 paediatric IBD patients (age at diagnosis
,17 years) and found a high prevalence of CARD15 (54%)
but also of TLR4 variants (17.7%) in CD patients compared
to controls (21.9% and 9.4% respectively). Our aim was to
investigate if these genetic and serological markers could
help in predicting who is at risk for developing the disease.
Therefore, we studied their prevalence and distribution in
a large cohort of unaffected family members of our patient
group.
Methods: All families of the previously studied patient group
(n = 100) agreed to participate and blood samples were
collected from 339 first degree family members (190 parents
and 149 siblings). Individuals were genotyped for CARD15
variants R702W, G908R and L1007fs, and for the TLR4-
Asp299Gly mutation. Serologic markers ASCA and pANCA
were also determined. A group of 45 unaffected children that
consulted the outpatient clinic served as a control group.
Results: Familial IBD was present in 34/100 (34%) patients.
Of those 34 familial IBD cases, 25 had one other relative
affected, 7 had 2 and 2 patients had 3 other family members
affected. These affected relatives were not included in our
further analysis. Amongst unaffected relatives, 38.6%
(110/285) carried at least one CARD15 variant (38.4% in
parents and 38.8% in siblings) and this was higher than the
prevalence seen in controls (21.8% p , 0.01) but significantly
lower than the prevalence seen in the affected CD patients
(43/79 or 54%, p = 0.012). For TLR4, 17.3% (50/289) of
unaffected relatives carried the 299Gly variant (15.6% in
641
parents and 19.7% in siblings) as compared with 17.7%
(14/79) in affected CD patients (p = NS) and 9.6% in controls
(p = 0.007). The prevalence of ASCA in unaffected relatives
(76/280 or 27%) was as high as in the affected CD patients
(25/79 or 32%). Interestingly, this was due to a high
prevalence only observed in the parents (52/163 or 32.1%)
and not in the unaffected siblings (24/117 or 20.5%) (p =
0.03). In contrast to ASCA, we did not find a higher
prevalence of pANCA in healthy family members of UC
patients (5/285; 1.8% compared to 7/18, 38.9% for relatives
and UC respectively).
Summary & Conclusions: CARD15 variants are more
frequently observed in unaffected family members of CD
patients than in healthy controls. However, their frequency is
still lower than the frequency in patients with CD. In this
large cohort of paediatric IBD and their unaffected family
members, we further found a high prevalence of ASCA but
not of pANCA in healthy relatives. This suggests that ASCA
in particular might be a genetic marker of disease suscepti-
bility. Follow up of all family members is ongoing to see if
any of these relatives eventually will develop the disease.
SUSCEPTIBILITY TO EARLY ONSET INFLAMMATO-
RY BOWEL DISEASE AND DEMONSTRATES A COM-
PLEX GENOTYPE PHENOTYPE RELATIONSHIP
RK Russell1, HE Drummond1, ER Nimmo1, DC Wilson2,
P Mcgroigan3, K Hassan3, G Mahdi4, J Satsangi1. 1GI Lab,
Edinburgh University, Edinburgh, United-Kingdom. 2Dept
Child Life and health, RHSC, Edinburgh, United-Kingdom.
3
Dept Paediatric Gastoenterology, RHSC, Glasgow, United-
Kingdom. 4Dept Paediatric Gastoenterology, Aberdeen, United-
Kingdom.
Aims: Genome wide scanning in a cohort of European IBD
patients identified a potential IBD locus on chromosome 10.
Recently the 113 mutation on the DLG5 gene (Drosophila
Discs Large Homolog 5) at 10q23 has been linked to IBD
susceptibility in adults. We aimed to study the role of this
mutation in a national early-onset population.
Methods: Patients diagnosed with IBD ,16 years were
recruited. Full clinical phenotype including growth data were
obtained. Parental DNA was also collected to allow Trans-
mission Disequilibrium Testing (TDT) to be performed (Both
parents collected in 69% of cases). The DLG5-113A G
A
missense mutation was genotyped using the Taqman system.
A validated scoring system for deprivation category (DEP-
CAT score) was used to judge social class (scored 17, 1 = no
deprivation 7 = maximum deprivation).
Results: A total of 296 patients (197 CD, 73 UC, 26 IC) were
analysed. TDT demonstrated an association with IBD (p =
0.045) but not CD(p = 0.18) or UC(p = 0.10). The allele
frequency for IBD patients was 12.9%, with no significant
differences between CD, UC and IC. 24.9% of patients were
carriers of the mutant allele. Carrier status was associated
with higher weight and height centile (75-91st) for IBD (p =
0.004, 0.006 respectively) and CD (p = 0.008, p = 0.002
respectively). Carrier status was also associated with higher
social class (DEPCAT score 1) for IBD (0.0005) but not CD
or UC (p = 0.06 for both). Carrier status was protective for
a lower social class (DEPCAT score 3) for IBD and UC (p =
0.02, 0.04 respectively). Mutation carriage was uncommon in
CD patients with joint extraintestinal manifestations (EIMs)
(5.9% vs. 24.9%), and skin EIMs (erythema nodosum) (9.5%
vs. 24.8%) but did not reach statistical significance (p = 0.07,
p = 0.11 respectively).
Summary: The DLG-5 113A mutation is associated with
susceptibilty to IBD in this population. It is associated with
higher height and weight centile at diagnosis of IBD and CD,
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
642 ESPGHAN 38TH ANNUAL MEETING
is commoner in the highest social class and is uncommon in
the presence of EIMs.
Conclusion: The DLG-5 113A mutation confers susceptibil-
ity to early onset IBD. In addition, the data reveal complex
gene-environmental interactions, and genotype phenotype
relationships.
PG2-06 ANTIGEN TARGETING TO MHC CLASS II-ENRICHED
ENDOSOMES OF COLONIC EPITHELIAL CELLS IN
INFLAMMATORY BOWEL DISEASE J Buning1,
M Schmitz2, G Hundorfean3, S Strobel4, KP Zimmer2,
A Gebert1, D Ludwig3. 1Institut fur Anatomie, Universitat zu
Lubeck, Lubeck, Germany. 2Klinik und Poliklinik fur Kinder-
heilkunde, UKM, Munster, Germany. 3Medizinische Klinik I,
UK S-H Campus Lubeck, Lubeck, Germany. 4Peninsula
Medical School, Universities of Plymouth and Exeter, Ply-
mouth, United-Kingdom.
Aim: In inflammatory bowel disease (IBD), colonic epithe-
lial cells (CECs) are suggested to be involved in stimulation
of proinflammatory CD4 + Th cells. There are few in vivo
data and MHC class II presentation pathways within these
cells still remain poorly understood. Our study was conducted
to examine antigen trafficking within CECs of IBD patients
under in vivo conditions. Methods: In patients with Crohns
disease (CD) (n = 6) and healthy controls (n = 6) undergoing
colonoscopy, ovalbumin (OVA)-solution (10 ml, 150 mg/ml
in saline) was sprayed directly onto inflamed or healthy
mucosa. Biopsies were taken before and 5, 10 and 20 minutes
after OVA exposure. HLA-DR, lysosome associated mem-
brane protein-2 (LAMP-2) and OVA were localised within
enterocytes using fluorescence and cryo-electron microscopy.
Results: Five minutes after application, OVA staining within
CECs was restricted to LAMP-2-negative small endosomes.
In healthy controls as well as in CD patients, sorting of OVA
to LAMP-2-positive endosomes was found 10 and 20 minutes
after exposure. HLA-DR expression in CECs was found in
CD patients but absent from CECs of healthy controls. In
CECs of CD patients, most of HLA-DR proteins were present
at basolateral membranes and in LAMP-2-labelled endo-
somes. These partially electron-dense endosomes contained
HLA-DR on their limiting membrane and within their inner
portion. A strong colocalisation of OVA and HLA-DR was
identified in these subcellular compartments at the 10 and
20 minutes time point.
Summary: Our in vivo data show antigen traffic into late
endosomal/lysosomal compartments in CECs of healthy
controls and CD patients. Targeting of antigens to HLADR-
enriched endosomes of CECs, with the bulk of intracellular
HLA-DR localised within late endosomes, is restricted to the
inflamed mucosa of CD patients. Conclusion: We suggest that
the endocytic pathway of CECs in the context of IBD
intersects class II loading compartments (MIIC). This has
been previously described for classical antigen presenting
cells and thought to be responsible for class II-associated
antigen processing and presentation. Our study strongly
suggests that CECs may be implicated in the pathogenesis of
IBD by stimulating proinflammatory CD4 + Th cells.
PG2-07 SOLUBLE IL-2 RECEPTORS AND LYMPHOCYTE
TRANSFORMATION IN ULCERATIVE COLITIS, AND
COLON PREDOMINANT CROHNS DISEASE JR Poley1,
A Wenger2, D Brassil1. 1Brody School of Medicine, East
Carolina University, Greenville, USA. 2Childrens Hospital,
Department of Informatics, Norfolk, USA. 3Eastern Virginia
School of Medicine, Department of Immunology, Norfolk,
USA.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
Aim: To determine, if soluble IL-2 receptors (SIL2R) and
lymphocyte transformation (LT) tests can differentiate
ulcerative colitis (UC) from colon- predominant Crohns
Disease (CD) and irritable bowel syndrome (IBS). SIL2R are
a reflection of lymphocyte activation, whereas LT indicates
the ability of lymphocytes to respond to stimulation with
a mitogen.
Patients: Between 1989 and 1998 a total of 104 children
were evaluated: 58 IBS (non-celiac; mean age 13.7y, SD 4.9;
32m); 29 CD (mean age 13.0y, SD 3.7; 15m); 17 UC (mean
age 13.7y, SD 3.8y; 10m). No patient was on corticosteroids
or had undergone surgery at the time of testing. Diagnosis
was established by determination of routine laboratory tests,
radiologic, and endoscopic exams.
Methods: SIL2R, LT tests and lymphocyte counts were
reviewed retrospectively. SIL2R were computed by commer-
cially available enzyme immunoassay (U/ml). LT was
analyzed by incorporating 3H thymidine into T-lymphocytes
(cpm), using Concanavalin A (Con-A) as the mitogen. Mean
differences were determined using ANOVA. Sensitivity and
Specificity of SIL2R and LT were computed.
Results: Descriptive statistics are expressed as the mean and
95% CI for each variable: SIL2R: IBS 578 (521635); CD
1532 (11771888); UC: 878 (5721184). LT: IBS 26794
(2038833201); CD 7139 (424410035); UC: 19666 (14318
25014). Abs. Lymphocyte counts: IBS 2713 (23363091);
CD: 1892 (15392245); UC: 2325 (20172634). Specify for
SIL2R with . 1100 U/ml was 92%, and sensitivity of LT
(with Con-A) with a value ,19,000 cpm was 95%.
Conclusion: Although the sample size is realtively small, the
test for SIL2R clearly distinguished CD and UC from IBS,
but not UC from CD. LT using Con-A further differentiated
CD from UC. While not detailed, the tests discussed, correctly
identified colonic disease as CD in 6 patients (all f) that were
diagnosed years earlier as UC. Finally, either test was not
helpful in evaluating CD restricted to the terminal ileum, but
did exclude colonic involvement beyond the cecum.
PG2-08 ANTI-SACCHAROMYCES CEREVISIAE ANTIBODIES
ARE ASSOCIATED WITH DISEASE ACTIVITY AND
LOW BODY MASS INDEX, BUT NOT WITH
NOD2/CARD15 MUTATIONS IN PEDIATRIC CROHNS
DISEASE D Urlep-Zuzej1, J Dolinsek1, B Zagradisnik1,
D Micetic-Turk1. 1Maribor Teaching Hospital, Maribor, Slovenia.
Introduction: Crohns disease (CD) is a polygenic multifac-
torial heterogeneous disease. Anti-Saccharomyces cerevisiae
antibodies (ASCA) are present in 5080% of CD patients.
The reason for the generation of ASCA remains unclear.
Recently, some studies have shown that ASCA occur in 25%
of healthy family members of patients with CD. Thus, the
generation of these antibodies may be genetically deter-
mined.
Mutations in the NOD2/CARD15 gene are found to be
associated with susceptibility to Crohns disease.
So far, there are no reports on the relationship between ASCA
and mutations of NOD2/CARD15 gene in the pediatric CD
population.
Aim: The aim of our study was to investigate the relationship
between ASCA, disease phenotype (location of the in-
flammation, disease activity and body mass index at the time
of diagnosis) and NOD2/CARD15 genotype in pediatric
Crohns disease patients.
Methods: 38 patients with CD (618 years, M/F = 2,3/1)
were tested for ASCA (IgA and/or IgG) by enzyme-linked
immunosorbent assay. Disease location, body mass index and
disease activity at the time of diagnosis were determined. All
patients had genotyping performed using sequence specific
 ESPGHAN 38TH ANNUAL MEETING
PCR directed against the wild tipe and the R702W, G908R
and 3020insC variants of NOD2/CARD15 gene.
Results: 26 of 38 (68,4%) CD patients were ASCA positive
(IgG and/or IgA). 12 of 38 (31,6%) CD patients had at least
one of the three major mutations of NOD2/CARD15 gene.
ASCA positive patients had significantly lower BMI (p =
0,012) and higher pediatric Crohns disease activity index -
PCDAI (p = 0,022) at the time of diagnosis compared to
ASCA negative patients. We found a negative association
between the presence of ASCA and ileal location of the
disease (p = 0,001). There was no association between
positive ASCA and the R702W, G908R and 3020insC
mutations of NOD2/CARD15 gene (p = 0,926).
Summary: In our study ASCA positive patients had
significantly lower body mass index and higher disease activity
at the time of diagnosis, however, we didnt find the positive
correlation between ileal location of the disease and positive
ASCA. There was also no association between positive ASCA
and the three major mutations of NOD2/CARD15 gene.
Conclusion: Although some studies have shown that
generation of ASCA may be genetically determined, our
study did not confirm the association between the presence of
ASCA and the major mutations of NOD2/CARD15 gene in
the pediatric CD population. This finding suggests some other
genetic factors may be involved in the generation of ASCA.
PG2-09 CONTRIBUTION OF THE MUCOSAL IMMUNE SYS-
TEM TO METHOTREXATE INDUCED INTESTINAL
DAMAGE BAE De Koning1, DJ Lindenbergh-Kortleve1,
JM Van. Dieren1, T Matsumoto3, R Pieters2, AWC
Einerhand1, JN Samsom1, E Nieuwenhuis1. 1Erasmus
MC-Sophia childrens hospital, laboratory of pediatric
gastro-enterology, Rotterdam, The Netherlands. 2Erasmus
MC-Sophia childrens hospital, laboratory of pediatric
oncology, Rotterdam, The Netherlands. 3Toho University
School of Medicine, department of microbiology, Tokyo,
Japan.
Background: Methotrexate (MTX) induced intestinal dam-
age is associated with increased exposure of the mucosal
immune system to vast amounts of microbial stimuli.
Because it is unclear how the mucosal immune system
responds to this increased antigen load, and whether it
contributes to mucositis, we have analyzed innate and
adaptive immune responses in MTX induced mucositis.
Methods and Results: To assess whether microbial stimuli
contribute to the severity of MTX induced intestinal damage,
LPS responsive and non-responsive mice were treated on day
1 and day 0 with 100- and 50 mg/kg MTX respectively. LPS
responsive mice exhibited a more prolonged weight loss than
non-responsive mice. Moreover, pre-treatment of WT mice
with 5 mg LPS prior to MTX, resulted in a more profound
weight loss and enhanced morphological damage, establish-
ing a contributive role for luminal LPS to the severity of MTX
induced mucositis. To elucidate the nature of the innate
immune response upon MTX treatment, cytokine release by
macrophages was measured after co-culture with MTX and
LPS. Treatment with increasing concentrations of LPS, in the
presence of MTX resulted in higher basal levels of TNF-a and
actively induced IL-10 production. In vivo, this IL-10 release
may have restricted the severity of mucositis as MTX treated
IL-10-KO mice lost significantly more weight, and showed
more severe morphological damage than WT controls. Not
only the innate but also the adaptive intestinal immune
system was affected by MTX as lamina propria lympho-
cytes, isolated from MTX-treated WT mice and re-
stimulated in vitro produced significantly more TNF-
a and IL-10 compared to cells from non-treated mice.
643
Conclusion: During MTX induced mucositis the enhanced
release of microbial stimuli affects both the innate and the
adaptive immune system leading to concomitant TNF-a and
IL-10 release. Despite ongoing inflammation, IL-10 restricts
excessive damage. These data may provide new targets in
mucositis therapy.
PG2-10 THE INCIDENCE OF ULCERATIVE COLITIS HAS
DOUBLED SINCE 1999 D Devadason1, H Hussien1,
C Spray1, B Sandhu1. 1Department of Paediatric Gastroen-
terology, Bristol Royal Hospital for Children, Bristol,
United-Kingdom.
Introduction: The incidence of inflammatory bowel disease
(IBD) in children aged less than 16 years in the southwest of
England rose almost 5 fold between 198898. This observa-
tion led to a Bristol based study conducted jointly with the
British Paediatric Surveillance Unit (BPSU) (1) that docu-
mented that the incidence of IBD in the UK during 1998
1999 was 5.1 per 100,000, 3.1 for Crohns Disease (CD), 1.4
for Ulcerative Colitis (UC) and 0.6 for Indeterminate Colitis
(IC). The purpose of the present study was to investigate
incidences of IBD, before and after the BPSU study to decide
whether it would be appropriate to repeat the national study.
Aims: To compare the incidences of IBD in the greater
Bristol area, during the periods 19951999 and 20002004.
Methods: Data is collected prospectively on all children
referred to this centre with newly diagnosed IBD. Only
patients from Bristol are analysed as referral of regional
patients may not be 100%. The childhood population figures
are based on national census figures. Postcodes were used for
geographical purposes.
Results: The childhood population of Bristol (,16 years) is
175,000 and has not changed.
Incidence figures 9951999 20002004
per 100,000 children (N = 43) (N = 53)
Total IBD 4.9 6.1
CD 3.6 (31) 3.1 (24)
UC 1.1 (9) 2.7 (27)
IC 0.3 (3) 0.2 (2)
Conclusion: This data suggests that the incidence of IBD in
children may still be rising. The rise appears to be in
ulcerative colitis whilst the incidence of Crohns has
plateaued. This needs further validation and hence it would
be appropriate to repeat the national study.
References:
1. Sawczenko A, Sandhu BK et al. Prospective survey of
childhood inflammatory bowel diseases in the British
Isles. The Lancet 2001;357:109394.
PG2-11 HYPERHOMOCYSTEINEMIA IN 56 CHILDREN WITH
INFLAMMATORY BOWEL DISEASE: PREVALENCE,
GENETIC DEFECTS AND VITAMINS B12, B9, B6
STATUS S Ganousse1, A Morali1, I Gastin2, JL Gueant2,
M Vidailhet1. 1Gastro-Pediatrics Children Hospital CHU
Brabois, Nancy, France. 2Biochemistry and Molecular
Biology CHU Brabois, Nancy, France.
Aim: IBD are associated with a risk of thrombotic
complications. Hyperhomocysteinemia is known to be an
independent risk factor for thromboembolic disease.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
644 ESPGHAN 38TH ANNUAL MEETING
Elevations in plasma total homocystein (Hcy) are either
explained by genetic defects in enzymes involved in Hcy
metabolism [5,10-methylenetetrahydrofolate reductase
(MTHFR) or methionine synthase (MS)] or by nutritional
deficiencies in vitamin co-factors such as folate, vitamins B12
and B6. The aim of this work was to evaluate the hyper-
homocysteinemia prevalence in children with IBD and to inves-
tigate the contribution of genetics defects and vitamin status.
Methods: IBD group: 56 children [40 Crohns disease (CD),
12 ulcerative colitis], 14.6 6 4.7 yr, in remission or relapse
were recruited. Plasma Hcy (n = 515 mmol/L), vitamins B6,
B9 and B12 levels were measured. MTHFR and MS
mutations were analysed by PCR, followed by the relevant
restriction enzyme digestions. Controls: 52 children, 12.7 6
4.3 yr, for homocysteinemia and vitamins assays; 227 healthy
adults for genetic study.
Results: Homocysteinemia was higher (9.7 6 3.2 mmol/L,
p = 0.012) in IBD patients and mainly during CD relapses
(p = 0.037) than in controls (8.2 6 2.8 mmol/L). A positive
correlation (p = 0.001) between age and homocysteinemia
could also be noticed. Nutritional factors were significantly
lower (without real deficiency) in IBD population than in
controls: plasma folate (IBD p = 0.06, CD relapse p = 0.007),
vitamin B12 (IBD p = 0.006, CD relapse p = 0.028), B6 (IBD
p = 0.013, CD relapse p = 0.025). Prevalence of the
homozygosity for the C677T or A1298C variants (MTHFR)
and the A2756G variant (MS) did not differ between IBD and
control groups.
Summary: IBD children have a higher prevalence of
moderate hyperhomocysteinemia in relation with folate,
vitamins B12 and B6 relative deficiency, particularly for CD
relapse.
Conclusion: A follow up of these parameters is recommen-
ded if previous thrombotic accidents are known, to assure an
effective supplementation.
PG2-13 ILEAL INVOLVEMENT IS AGE-DEPENDENT IN PAEDI-
PG2-12 OSTEOPOROSIS AND INFLAMMATORY BOWEL DIS-
EASE (IBD) IN CHILDREN: VARIABLES AFFECTING
BONE MINERAL DENSITY (BMD) M Paganelli1,
C AlbaneseF Civitelli1, M Cirulli1, V Labalestra1,
V Pannone1, R Passariello2, S Cucchiara1. 1Pediatric Gastro-
enterology Unit, University of Rome La Sapienza, Rome, Italy.
2
Department of Radiology, University of Rome La Sapienza,
Rome, Italy.
Osteopenia and osteoporosis commonly affect a large pro-
portion of patients (pts) with IBD; however, variables affecting
BMD in these pts are not yet fully identified.
Aims: To assess BMD in consecutively recruited pts with
Crohns disease (CD) and ulcerative colitis (UC) and to
investigate if variables such as nutritional status, inflamma-
tion and corticosteroids (CS) affect BMD in them.
Methods: All pts underwent bone age x-ray, anthropometric
and pubertal staging (height, weight and body mass index
expressed as Z-scores); lumbar spine (L1L4) BMD was
measured by dual- energy x-ray absorptiometry (DEXA).
Z-scores were obtained by comparison with age- and sex-
matched normal values. Due to frequent growth retardation in
children with IBD, z-scores were reassessed on bone age.
Disease activity (PCDAI for CD and Powell-Tuck for UC)
was calculated at DEXA evaluation and a mean value of 3
evaluations in the year prior to DEXA obtained. Endoscopic
disease activity index (CDEIS for CD and Rachmilewitz for
UC) was obtained in all pts. Serum and urine markers of bone
formation such as bone-specific alkaline phosphatase
(BALP), osteocalcin (BGP) and of bone resorption such as
C-terminal telopeptide of type I collagen (CTX) were
measured. Serum parathyroid hormone (PTH) and 25-(OH)-
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
vitamin D were dosed. Cumulative and daily dose of CS (mg
of prednisone) were calculated for total duration of the disease
and over the last 3 years. Therapy with anti-TNF-alpha
monoclonal antibody (infliximab) was considered in CD pts.
Results: (mean 6 SD): 61 subjects (18 CD, 16 UC, 27
healthy controls) were studied (mean age 13,3 yrs, range 3.0
19.3; no differences between groups). BMD (z-score for bone
age) was 21.1 6 0.8 in pts and 20.3 6 1.0 for controls (p ,
0.01). There was no difference in BMD between CD (21.16
6 0.7) and UC (21.0 6 1). Prevalence of low (z-score = ,
21) and very low (z-score = , 22) BMD was 50% and
11.1% for CD and 43.7% and 6.2% for UC, respectively. There
was a significant correlation between low BMD and low body
mass index (r = 0.46, p = 0.01) and low body weight (r = 0.47,
p , 0.01). No correlation was found between low BMD and
disease duration, age at diagnosis, dietary calcium intake,
family history for osteoporosis and disease activity indexes. No
relationship existed between BMD and low platelets count,
hypoalbuminemia, BALP, BGP, CTX. PTH and 25-(OH)-
vitamin D resulted within normal limits. Cumulative and daily
dose of CS and duration of therapy did not affect BMD in IBD
pts; CD pts who had received infliximab had higher BMD than
children who had not (p: 0.02).
Conclusions: In children with IBD, BMD is markedly
decreased as compared to controls, without significant
differences between CD and UC. Nutritional status is the
most important variable predicting a low BMD. We did not
find a significant correlation between CS and low BMD,
although CS are traditionally thought to contribute to
osteopenia. The higher BMD in CD pts on infliximab
suggests that inflammatory status has a role in reducing
BMD in these pts. Dosing plasma levels of PTH, 25-(OH)-
vitamin D and bone formation and bone resorption markers is
not useful to identify pts at higher risk for osteoporosis.
ATRIC CROHNS DISEASE U Meinzer1, M Idestrom2,
C Alberti4, M Peauchmaur3, JF Mougenot1, JP Cezard1,
Y Finkel2, JP Hugot1. 1Department of Paediatric Gastroenter-
ology, H^ opital Robert Debre, Assistance Publique, Paris,
France. 2Department of Paediatric Gastroenterology and
Nutrition, Astrid Lindgren Childrens Hospital, Stockholm,
Sweden. 3Department of Pathology, EA3102 Universite Paris 7,
Hopital Robert Debre, Paris, France. 4Department of Epide-
miology and Statistics, INSERM CIC-EC, H^ opital Robert
Debre, Paris, France.
Aims: Lymph follicles (LFs) have been suggested to play
a role at the early stage of Crohns disease (CD) lesions. In the
small bowel, LF are grouped forming Peyers patches (PPs)
which develop early in fetal life, grow in size and number
until puberty and then undergo involution. In contrast,
colonic LFs are isolated and undergo little change during
life. As a result, if LFs play a role in the occurrence of CD
lesions, the distribution of ileal and colonic lesions is
expected to be altered in small children.
Methods: Medical records of two independent French (n =
136) and Swedish (n = 55) cohorts of consecutive paediatric
CD were reviewed. Disease sites and age of onset were
recorded and the age-dependent probability to develop ileal
lesions was computed. The CARD15/NOD2 genotype was also
analysed when available (n = 99).
Results: The curves of disease occurrence were significantly
different in case of CD with or without ileal lesions (p ,
0.0001). At the age of 8 years, the probability (95% CI) of
small bowel involvement in CD patients is was 0.19 (0.07
0.39). It increases until 16 years to 0.61 (0.540.68). It was
slightly higher in patients carrying one or more CARD15/-
NOD2 mutations [0.75 (0.550.89)] than in wild-type
 ESPGHAN 38TH ANNUAL MEETING
patients [0.46 (0.340.58)]. CARD15 mutations also influ-
enced the age of onset of ileal disease (p , 0.02).
Summary and Conclusions: In children, ileal CD lesions
are delayed when compared to colonic lesions. This
observation is in agreement with the previously proposed
hypothesis of a pathophysiological role of PP in ileal CD. The
rarity of small bowel flesions incites to be cautious when
classifying chronic colitis in small children.
PG2-14 REDUCED NERVE CONDUCTION AFTER TREAT-
MENT WITH THALIDOMIDE IN CHILDREN AND
ADOLESCENTS WITH REFRACTORY CROHNS DIS-
EASE: IS IT JUSTIFIED TO CONTINUE? F Kiparissi1,
D Gindner1, M Elawad1, S Hill1, N Shah1, PJ Milla1, M Pitt1,
KJ Lindley1. 1Institute of Child Health and Great Ormond
Street Hospital for Sick Children, London, United-Kingdom.
Aim: To evaluate the safety of Thalidomide treatment in
patients with steroid dependent / resistant Crohns disease by
assessing nerve conduction velocity and clinical neurological
symptoms.
Methods: We retrospectively reviewed clinical and neuro-
physiological data of 21 patients (age range 7 years 4 months
to 23 years 6 months, median age 14 years 10 months) over
a 5-year period. All patients had received 50 mg Thalidomide
once daily, median follow up on treatment was 15 months
(range 6 36 months, n = 8). Nerve conduction studies were
performed on 15 patients pre-treatment and 10 during
treatment. 5 patients were only recently started on Thalido-
mide and no follow up studies have been performed yet. 15
were followed up in 6-monthly intervals for repeat nerve
conducting studies, 6 patients were either discharged into
adult care, lost to follow up or Thalidomide was discontinued.
Each study included up to 6 repetitive measurements with the
average result being determined, on up to three peripheral
PG2-16 INFLIXIMAB AS A FIRST-LINE THERAPY IN NEWLY
nerves. All 21 patients received standardized neurological
examinations and PCDAI scorings (pre-treatment .220, on
Thalidomide ,150, n = 8).
Results: All 21 patients had either steroid refractory Crohns
disease or severe side effects from previous treatment with
steroids and other immunosuppressants. Of the 15 patients 5
(33.3%) developed significantly reduced nerve conduction
velocities (nerve amplitude in mV reduced by over 50%) and
10 (66.6%) mild to moderate (no reduction to 50%). In the
most severe case reduction of nerve amplitude was from
52 mV to 0.7 mV. Results were unavailable in 6 patients. None
of the 15 patients became symptomatic and none stopped
Thalidomide.
Summary: Thalidomide treatment was used in 21 patients
with severe refractory Crohns disease despite reduced nerve
conduction velocities in 15 patients. All patients remained
clinically asymptomatic with no neurological symptoms and
a greatly improved quality of life.
Conclusion: The continuous use of Thalidomide in symptom-
free patients with reduced nerve conduction appears to be
safe and efficacious and may be justified on clinical grounds.
PG2-15 ANTI-INFLIXIMAB IMMUNIZATION AND CLINICAL
OUTCOME IN PEDIATRIC REFRACTORY CROHNS
DISEASE. A Mosca1, S Candon2, F Ruemmele2, O Goulet2,
L Chatenoud2, JP P. Cezard1. 1Hopital Robert Debre, Paris,
France. 2Hopital Necker-Enfants Malades, Paris, France.
Aim: Infliximab, a monoclonal antibody blocking TNF-a,
has proven effective as an inductio and maintenance therapy
for refractory Crohns disease (CD) in adult and pediatric
patients. However, infliximab therapy can induce the
645
appearance of neutralizing anti-infliximab antibodies (ATI),
potentially leading to loss of clinical response or immuno-
allergic reactions.
Methods: 28 patients with refractory CD received either 1
injection of 5mg/kg of infliximab for steroid resistance (n = 9)
or 3 injections at day 0, 15 and 45 for steroid dependence and
anoperineal lesions (n = 19). Upon relapse, 23 patients were
re-treated on an on-demand basis (n = 9) or sequentially every
2 months (n = 14). Blood TNFa, infliximab and ATI were
measured before each infliximab injection or every two
months.
Results: Remission after induction therapy was obtained in
96% of patients but a high rate of relapse was observed at one
year of follow up (96%). Maintenance therapy was efficient
in 64% of patients. Anti-infliximab sensitization occured in
35% patients. The presence of ATI was associated with a loss
of clinical efficacy: 33% of ATI positive vs 85% of ATI
negative patients still benefited from infliximab therapy at the
end of the study period (p = 0,026). A single infusion vs three
at induction increased the risk of ATI formation (77 vs 15 %,
p = 0,003). Severe infusion reactions (possibly IgE-
mediated) were observed in two immunized patients with
high titers of antibodies. We observed a strong correlation
between concentrations of TNF-a and infliximab: following
an infusion, the presence of circulating infliximab was
associated with a paradoxical increase of TNF-a. Some
patients with ATI displayed a faster clearance of infliximab
and the absence of such paradoxical increase of TNF-a.
Conclusion: Anti-infliximab sensitization occured in 35% of
patients leading to a significative loss of clinical efficacy and
severe infusion reactions. A single infusion at induction
increased the risk of sensitization. Monitoring serum TNF-a
and circulating infliximab could help to detect early anti-
infliximab immunization.
DIAGNOSED CROHNS DISEASE (CD) PROMOTES
LONG-TERM SUSTAINED REMISSION AND ALTERS
THE COURSE OF THE DISEASE. C Bascietto1, L De
Angelis2, O Borrelli1, B Bizzarri2, F Fornaroli2, F Viola1,
M Cirulli1, S Cucchiara1. 1Department of Pediatrics,
Gastroenterology Service, University of Rome, Rome, Italy.
2
Institute of Pediatrics, Gastroenterology Unit, University of
Parma, Parma, Italy.
Infliximab (IFX), a monoclonal antibody against TNF-alpha,
is currently indicated for children with CD unresponsive to
traditional treatment regimens consisting of corticosteroids
(CS), immunosuppressors, antibiotics, nutritional therapy.
IFX has never been used as first choice in children with
a recent diagnosis of CD.
Aim: We compared, retrospectively, the clinical course in
patients (pts) receiving IFX as initial therapy (group A) and in
those treated with a traditional therapeutic approach (group B).
Methods: At baseline all pts underwent ileocolonoscopy and
histology, as well as clinical assessment through PCDAI
(mean 6 SD). The latter was also measured after induction of
remission and after maintenance therapy. The number of
relapses (defined as PCDAI .15) during maintenance
therapy were also evaluated. In all, infectious and immuno-
logical disorders and malabsorption had been excluded by
appropriate diagnostic algorithm.
Results: 10 pts (group A: age range: 6.2-18.0 years) had
a baseline i.v. IFX schedule (5 mg/kg/dose) (0,2,6 weeks), 6
of them also underwent IFX retreatment for 12 months every
8 wks; all had azathioprine (AZA) or methotrexate (MTX)
during the IFX infusions and thereafter. Of the19 pts (age
range: 5-17 years) of group B, initial therapy for inducing
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
646 ESPGHAN 38TH ANNUAL MEETING
remission consisted of a nutritional course alone in 5, of CS in
14; then all received a maintenance therapy with AZA (or
MTX). At diagnosis all group A and B pts had an ileo-colonic
mucosal disease, 7 of group A and 3 of group B also had
perianal disease (fistulae and/or ulcers). After induction
therapy, clinical remission (PCDAI ,15) was achieved in
all pts of both groups (A: 8.1 6 2.8; B: 8.5 6 2.9; p , 0.01).
During the following 12 months, disease relapses were seen
in 1 of group A and in 17 of group B (Fishers exact test: p ,
0.01; number of relapses (mean 6 SD) were significantly
lower in A pts (0.1 6 0.31) than in B pts (1.89 6 1.1, p ,
0.01); 4 pts of group B also underwent surgery for intestinal
resection. The PCDAI at the end of the 12-month mainte-
nance treatment was markedly higher in group B (21.8 6
5.14) than in group A (10.1 6 2.33, p , 0.01).
Conclusions: Although traditional therapeutic regimens in
CD are able to induce remission, IFX as first line therapy in
newly diagnosed pediatric CD promotes long term remission
and seems to change the course of the disease. Our data
support the immunological view that the use of a potent
immunomodulator in early CD (typically occurring in child-
hood) may have a great potential therapeutic benefit.
(Inflammatory Bowel Disease, 2002;8:112).
PG2-17 INFLIXIMAB THERAPY RESTORES NORMAL
GROWTH IN CHILDREN WITH CHRONICALLY
ACTIVE SEVERE CROHN DISEASE REFRACTORY TO
IMMUNOMODULATORY THERAPY TD Walters1,
AR Gilman1, AM Griffiths1. 1Hospital for Sick Children,
University of Toronto, Toronto, Canada.
Background/Aims: Restoration of normal growth is a marker
of therapeutic success in paediatric Crohn disease (CD).
Although the efficacy of infliximab in adult patients with
otherwise refractory CD is well established, no studies have
examined the effects on linear growth. We reviewed the
outcome of infliximab therapy at a single pediatric institution,
specifically to assess if clinical response was associated with
improved linear growth.
Patients/Methods: From September 2000 to January 2004,
32 children and adolescents (63% males) (mean age 13.4 yrs,
range 4.7 to 17.3 yrs) with chronically active inflammatory CD
despite immunomodulatory and corticosteroid therapy com-
menced infliximab therapy. Mean duration of diagnosed CD
was 3.3 +/- 0.47 yrs. Standard deviation scores (SDS) of growth
parameters pre and post infliximab therapy were compared.
Results: 4 patients (12.5%) demonstrated no response to the
initial infusion. All others, who experienced partial (n = 6) or
complete (n = 22) response based on physician global
assessment, have continued to receive infliximab infusions
for 18 +/- 8.7 months. During follow-up all 28 responding
patients experienced a significant improvement in weight
(change in SDS 0.49, p = 0.002) and BMI for age (change in
SDS 0.42, p = 0.034). As shown in the Table, significant
changes in linear growth were limited by pubertal stage.
Pre-inflix Post Change
(SDS) inflix(SDS) in SDS
Height Velocity
Tanner I-III (n = 18) 4.2cm/yr (-0.43) 7.0cm/yr (3.2) +3.66 (p = 0.013)
Tanner IV&V (n = 7) 3.0cm/yr (1.33) 2.1cm/yr (-0.75) -0.59 (p = 0.74)
Height for Age
Tanner I-III (n = 21) -1.26 -0.91 +0.36 (p = 0.002)
Tanner IV&V (n = 7) -0.64 -0.71 -0.06 (p = 0.41)
Conclusions: Infliximab therapy restores a normal linear
growth velocity and increases height centile for patients
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
treated prior to or in early puberty. These data support the use
of infliximab in young patients with otherwise refractory
disease, and suggest that ultimate heights in this subset of
children with severe CD may be less compromised than with
previous therapies.
PG2-18 LONG TERM FOLLOW UP IN PEDIATRIC PATIENTS
AFFECTED BY CROHNS DISEASE TREATED WITH
AUTOLOGOUS ERYTROCYTES LOADED WITH
DEXAMETHASONE 21-PHOSPHATE D Knafelz1,
B Papadatou1, F Bracci1, L Rossi2, MI Ambrosini1, PL
Di Carlo3, M Magnani2, M Castro1. 1Gastroenterology Unit,
Ospedale Bambino Gesu`, Rome, Italy. 2Institute of Bi-
ological Chemistry, University of Urbino, Urbino, Italy.
3
Immunotrasfusional Service, Ospedale Bambino Gesu`,
Rome, Italy.
Aim: We previously described a new effective periodic
treatment with autologous erythrocytes loaded with dexa-
methasone 21-phosphate (dex 21-P) in paediatric patients
with Crohns disease (CD)1 and with cystic fibrosis2. Aim of
our study was to further demonstrate the efficacy of this form
of treatment and its long-term safety in a greater number of
patients and with a longer follow up.
Methods: We studied 11 patients (pts), (7F, 4M; mean age
16.3 yrs+-3.9) affected by CD. Inclusion criterium for
treatment with autologous erythrocytes loaded with dex 21-P
was corticodependence despite the use of azathioprine with
severe steroid induced side effects. Two pts that presented
serious adverse event to the use of Infliximab (anaphylactic
reaction and candida sepsis) were included as well.
Results: At the beginning of treatment 3 pts were in active
phase of the disease (mean PCDAI 58); 6 pts in a moderate
phase (mean PCDAI 15) and 2 were in complete remission
(mean PCDAI 5). A total of 277 infusions (one every three
week) were performed with a mean 25 infusions per patient
(range 12-39). Mean follow up was 72 weeks (range 36-117).
All pts could stop and did not need to start again conventional
steroid treatment either oral or e.v. Mean PCDAI decreased
from 25.2 to 11.5. In particular pts with active or moderately
active disease went into remission and the other maintained
remission. None of our pts showed any of the typical steroid
side effect (moon face, hypertension, stria rubre ect) and in
half of them in which we performed a DEXA-MOC at the
beginning of treatment and 6 months afterwards there was no
change in their Z-score.
Summary: We showed that periodic treatment with autolo-
gous erythrocytes loaded with dex 21-P in paediatric pts with
CD is effective and safe. The low dose of dexamethasone that
is continuously delivered in the blood stream by the
erythrocytes was able to induce remission and allow steroid
withdrawal without side effects.
Conclusions: We suggest the use of this treatment in selected
patients with steroid dependent CD.
1. JPGN 2003,36(4)
2. Blood cells, Mol and Disease 33(2004) 5763.
PG2-19 THE STRUCTURE, RELIABILITY AND VALIDITY OF
THE IMPACT QUESTIONNAIRE TO MEASURE QUAL-
ITY OF LIFE IN CHILDREN WITH INFLAMMATORY
BOWEL DISEASE. CA Davies1, J Abbott1, AK Akobeng2,
MR Sood2, AG Thomas2. 1University of Central Lancashire,
Preston, United-Kingdom. 2Booth Hall Childrens Hospital,
Manchester, United-Kingdom.
Introduction: Inflammatory Bowel Disease (IBD) can have
an enormous effect on the quality of life (QOL) of affected
 ESPGHAN 38TH ANNUAL MEETING
individuals. A disease specific measure of QOL in childhood
IBD (IMPACT) was developed in Canada then modified in
the Netherlands and the UK.
Aim: To determine the structure, reliability and validity of
the UK version of IMPACT.
Methods: 100 children (8-17yrs) with IBD completed the
IMPACT questionnaire and Child Health Questionnaire
(CHQ) while attending the outpatient clinic. Principal
Component Analysis (PCA) with varimax rotation was
performed to generate the factor structure of the instrument.
Cronbach alpha coefficients were computed to ascertain
internal reliability. ANOVA was used to calculate the
discriminant ability of IMPACT and Pearson correlation
coefficient was used to assess the concurrent validity of
IMPACT compared to the CHQ.
Results: The most clinically meaningful and statistically
robust solution of the PCA highlighted 5 factors: concerns
about IBD (alpha 0.88), IBD symptoms (alpha 0.83), body
image (alpha 0.79), embarrassment (alpha 0.79) and energy
(alpha 0.74). IMPACT showed good discriminant validity
between mild/moderate & severe disease activity. There were
significant correlations between several domains of IMPACT
and corresponding CHQ domains.
Summary: The factor structure of IMPACT has been altered
to suit British children with IBD. It has been shown to be
a reliable and valid instrument to measure their QOL.
Conclusions: IMPACT can now be piloted in clinical trials
and clinical practice. Further research is required to de-
termine the most effective way(s) to improve QOL in children
with IBD.
PG2-20 COPING STRATEGIES AND FAMILY FUNCTIONING
OF CHILDREN WITH INFLAMMATORY BOWEL DIS-
EASE AND THEIR FAMILIES C A. Davies1, J Abbott1,
AG Thomas2. 1University of Central Lancashire, Preston,
United-Kingdom. 2Booth Hall Childrens Hospital, Man-
chester, United-Kingdom.
Introduction: Inflammatory Bowel Disease (IBD) can have
an enormous effect on the quality of life (QOL) of affected
individuals and their families. Little is known about the effect
of IBD on family functioning, the coping strategies employed
and the effect on QOL.
Aims: To evaluate the ways in which children with IBD and
their parents cope with their disease and to assess their family
functioning.
Methods: 90 children (51 boys) with IBD (58 Crohns
disease, 12 ulcerative colitis, 20 indeterminate colitis) aged
817 yrs completed the Kidcope questionnaire. 87 parents
completed the Ways of Coping scale and 84 completed the
McMaster Family Assessment Device.
Results: Children with IBD used cognitive restructuring as
the principle way of coping (boys(b) 86%, girls(g) 85%).
Other popular coping strategies included wishful thinking
(b 84%, g 82%), distraction (b 73%, g 81%) and seeking
social support (b 71%, g 77%). When evaluated by age
(young (y) 812 yrs, old (o) 13+ yrs) cognitive restructuring
(positive reinterpretation of events) was used by 88% (o) and
83% (y), wishful thinking was used by 78% (o) and 90% (y)
and distraction techniques were used by 72% (o) and 83% (y).
Adolescents were more likely to accept their disease with
88% adopting resignation compared to only 23% of the
young group (z = 26.7, p = 0.001). Parents engaged in
problem solving strategies (78%), seeking social support
(77%) and self control (49%) as principle ways of dealing
with their childs IBD. Mean scores for domains of family
functioning were generally healthy and similar to previous
studies of normal families.
647
Summary: Children with IBD and their parents use many
different coping strategies. These are similar in boys and girls
and at different ages except that older children were more
likely to be resigned to their illness than younger children.
Family functioning appears to be healthy and similar to that
in families who do not have a chronically ill child.
Conclusion: More research is needed to determine whether
the QOL of children with IBD can be improved by teaching
positive coping strategies.
PG3-01 EFFICIENCY OF EXTRACORPOREAL SHOCK WAVE
LITHOTRIPSY OF PANCREATIC STONES IN TREAT-
ING CHILDREN WITH CHRONIC PANCREATITIS.
G Oracz1, B Oralewska1, M Teisseyre1, J Ryzko1,
A Borowka2, J Socha1. 1The Childrens Memorial Health
Institute, Warsaw, Poland. 2Central Railway Hospital,
Warsaw, Poland.
Objectives: Extracorporeal shock wave lithotripsy (ESWL)
of pancreatic stones is a treatment option for patients with
chronic pancreatitis (CP), when pancreatic stones cannot be
removed by endoscopic procedures during retrograde chol-
angiopancreatography (ERCP). The aim of the study was to
evaluate if ESWL of pancreatic stones is effective in treating
children with chronic pancreatitis.
Methods: 8 children (6 girls and 2 boys; mean age 10.4
years, range: 5.5 to 17 years) with severe CP, hospitalized
since 1998 to 2002, were treated by extracorporeal shock
wave lithotripsy for endoscopically irretrievable obstructive
stones. The medical records of these patients were reviewed
for data on the presentation, diagnostic findings, ESWL and
endoscopic treatment. ESWL was performed with an
electromagnetic lithotriptor. The etiology of chronic calcify-
ing pancreatitis was gene mutations (PRSS1, CFTR) in all but
one patient.
Results: Eight patients had 10 endoscopic-ESWL sessions.
ESWL was administered twice in two children. Pancreatic
stones fragmentation was achieved in all patients. The
procedures were well tolerated by all children. There were
no complications or failures related to ESWL. Early pain
relief occurred in all cases. At a mean follow-up of 49 months
(range: 2678 months) pain improved in 5 children (62.5%).
Acute episodes of CP after endoscopic- ESWL treatment
were observed in 3 patients (37.5%). Recurrence of
endoscopically irretrievable pancreatic stones was revealed
in two cases.
Conclusions:
1. Extracorporeal shock wave lithotripsy is a safe and
effective therapy in treating children with chronic pancre-
atitis and irreversible pancreatic stones.
2. Extracorporeal shock wave lithotripsy should be consid-
ered complementary and not alternative therapy to endo-
scopic drainage.
PG3-02 ESCIN ATTENUATING THE GASTRIC ULCEROGENIC
EFFECT OF DICLOFENAC SODIUM F Fu1, W Jiang1.
1
School of Pharmacy, Yantai University, Yantai, China.
Aim: Diclofenac is one of the most potent non-steroidal anti-
inflammatory drugs (NSAIDs) with adverse action of
gastrointestinal tract. Orally administered diclofenac is
therefore poorly tolerated and causes stomach ulcerations.
Escin is a natural mixture of triterpene saponins from horse
chestnuts. It has been reported that escins IaIIb (10
50 mg/kg) potently inhibited ethanol-induced gastric mucosal
lesions, and the gastroprotections were reduced partially by
pretreated with indomethacin.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
648 ESPGHAN 38TH ANNUAL MEETING
Methods: In this paper the gastro-protection of escin on
gastric mucosal lesions induced by diclofenac sodium (DS),
one of NSAIDs, was investigated. Forty rats were divided into
5 groups. First group animals were given intragastrically DS
solely at dose of 45 mg/kg. Other group animals were given
escin by i.v. at dose of 2 mg/kg 120 min, 60 min or 0 min prior
to administration of DS, or even 60 after DS given as above
respectively. The animals were killed 5 h after DS
administration. The stomachs were removed and the lesion
scores were recorded.
Results: The results showed that escin given simultaneously
with DS or 60 min prior to DS could reduced the gastric
mucosal lesion effectively but it would be no any effect on
mucosal damage induced by DS if escin was given 120 min
prior to DS administered or 60 min later than DS given.
Conclusion: It suggests that the gastroprotections of escin
are associated with the time of it given to DS administered
which means that enough blood concentration of escin is
necessary for its antagonizing side action of DS.
PG3-03 VIGOROUS REHYDRATION, WITH GLUCOSE, RICE
OR AMYLASE-RESISTANT STARCH-CONTAINING
ORAL REHYDRATION SOLUTION IN SEVERELY MAL-
NOURISHED CHILDREN WITH CHOLERA NH Alam1,
S Islam1, S Sattar1, JF Desjeux2. 1ICDDR,B: Centre for Health
and Population Research, Dhaka, Bangladesh. 2CNAM:
Conservatoire National des Arts et Metiers, Paris, France.
Fast rehydration is often considered risky in severely
dehydrated and malnourished children.
The aim: Of the study was to test in such children the efficacy
of a vigorous rehydration, with glucose, rice (both absorbed in
the small intestine) or amylase-resistant starch (metabolised
in the colon) - containing oral rehydration solution.
Methods: In a prospective randomised study, 174 children,
mean 6 SE age = 27.6 6 0.9 months, were included as
severely malnourished (Weight/Height = 68.5 6 5.5 with or
without pedal oedema) and dehydrated because of cholera.
After IV fluid therapy (602 6 46 ml) for 84% of them, they
were randomly assigned one of the three high potassium
(40 mEq/l) oral rehydration solutions, containing glucose, rice
or amylase-resistant starch. On the main parameters, there was
no significant difference between groups at inclusion.
Results: Although risk factors of death were frequently
present (55% not breast fed; 84% severe dehydration; 9%
severe hypoglycaemia), all the children recovered from
dehydration. In the first 2 days, stool volume and ORS intake
was significantly different in the 3 groups, but number of
unscheduled IV therapy (15%), duration of diarrhoea (66.3 6
2.2 hrs), weight gain (0.8 6 0.1 kg) and time to recover 80%
of the W/H (7.1 6 0.2 days) were not different.
Glucose- AR Starch + Rice-
Stool ORS Glucose- ORS ANOVA
volume (ml) (n = 58) ORS(59) (n = 57) P-value
1st 24 hrs 2357 6 1496 2177 + 1133 1639 + 1023 0.006
2nd 24 hrs 1676 6 1939 1437 + 1210 956 + 1087 0.031
3rd 24 hrs 836 6 1003 813 + 934 564 + 1093 0.321
Total in 72 hrs 4915 6 4294 4519 + 2932 3187 + 2942 0.025
Conclusion: In severely malnourished children presenting
with dehydration secondary to cholera, vigorous rehydration
is effective, with full and rapid recovery. In addition,
reduction of stool output by rice or amylase-resistant starch
in ORS does not alter the speed of recovery for rehydration or
renutrition.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
PG3-04 NOVEL SLC39A4 MUTATION IN KINDRED WITH
ACRODERMATITIS ENTEROPATHICA Y Bujanover1,
N Gal1, J Abu-Moch1, A Vardi1, Y Anixter1. 1Edmond &
Lili Safra Childrens Hospital, Ramat Gan, Israel.
Introduction: Acrodermatitis enteropathica (AE) is a rare
autosomal recessive disorder affecting early infancy. The triad
of dermatitis, alopecia and diarrhea is its diagnostic hallmark.
Recently the human gene encoding an intestinal zinc trans-
porter of the ZIP family, SLC39A4, was identified as the
mutated gene in AE.
Aim: The aim of the present study was to characterize the
molecular basis of the deficiency in the intestinal zinc
transporter in a large kindred of a family in which two index
cases with AE were diagnosed.
Subjects and Methods: Initially a 4 yr old girl and her 13 yr
old male cousin were studied. The first presented with
congestive heart failure, lymph node enlargement and
dermatitis. The second with dermatitis. DNA was extracted
from peripheral blood samples from both children and 30
members of their arab-muslim family. Genomic DNA of the
subjects was PCR amplified for each exon of the SLC39A4
gene and analyzed by direct nucleotide sequencing.
Results: We identified a novel 1223 C deletion, this frame
shift at amino acid A408 created after 76 amino acid, a stop-
codon (A483X). This deletion in exon 7 destroyed BsuR1
restriction site in the mutant amplicon and created a new Aci
1 restriction site. This mutation was found in the two children
with AE which were initially studied and in 17 carriers of
their family.
Conclusion: The results of the present study brings to 22 the
number of reported SLC39A4 mutations in AE families. The
data can be useful for the prenatal diagnosis and carrier
detection.
PG3-05 ASSOCIATION BETWEEN PYROSIS AND A POLY-
MORPHISM IN THE IL1RN GENE IN HELICOBACTER
PYLORY-POSITIVE CHILDREN I Aguiar1, M Santana2,
L Pena2, P Saavedra3, JC Ramos2, F Sanchez1.
1
U. Immunology. Hospital Dr. Negrn de Gran Canaria,
Las Palmas de Gran Canaria, Spain. 2U. Gastroenterology &
Nutrition. Hospital Universitario Materno Infantil de
Canarias, Las Palmas de Gran Canaria, Spain. 3Dpt.
Mathematics. Universidad de Las Palmas de Gran Canaria,
Las Palmas de Gran Canaria, Spain.
Aim: To study possible associations between host genetic
factors (polymorphisms at IL1B-511, IL1B+3953, TNFA-
308 and IL1RN), susceptibility to Helicobacter pylori
infection, epidemiological and socioeconomic data, and
clinical parameters (symptoms such as abdominal pain,
vomiting, pyrosis, nausea, abdominal burning or abdominal
distention).
Methods: Case-control study comprising 102 H. pylori-
positive patients aged 37178 months (mean = 105,97;
58 male, 44 females) and 96 H. pylori-negative patients aged
37178 months (mean = 104,87; 54 males, 42 females),
respectively. H. pylori diagnosis was undergone in triplicate
by serology, breath test and a stool antigen test. The different
polymorphisms were typed by PCF-RFLP or SSP as
appropriate. Socioeconomic and epidemiological data were
collected following an interview with the subjects and their
parents. This study was approved by our hospitals Ethics
Committee and informed consent was obtained from all
subjects in this study. Statistical study was performed by the
SPSS statistical software package.
Results: Socioeconomic factors were important in H. pylori
infection as low income and the high household density of
 ESPGHAN 38TH ANNUAL MEETING
children were identified as risk factors. No association was
found between any of the polymorphisms studied and
susceptibility to infection. The only association found
between the studied polymorphisms and symptomatology
appeared for IL1RN, where 75% of infected patients with
pyrosis had 1 or 2 copies of allele 2 while 73% of infected
children without pirosis were homozygotes for allele 1 (p =
0.000). Patients with the genotype 12 at the IL1RN gene
showed a tendency to suffer from abdominal pain: 37% from
the total of infected patients suffering from abdominal pain
had this genotype, genotype only found in 15,7% from
patients free of abdominal pain. However, this significance
was weaker (p = 0.007).
Summary: H. pylori is probably the most common human
infectious agent worlwide. Twin studies, ethnic differences
and inbred mouse strains have indicated a genetic influence
on the susceptibility to H. pylori. For the subset of patients
and variables here studied, it was found an association
between the variable number tandem repeats polymorphism
at the IL1RN gene and abdominal pain and specially pyrosis
in infected patients.
Conclusion: IL1RN gene polymorphisms appear to have
a role in the symptomatology of H. pylori infection.
PG3-06 NEW PATHOGENICITY MARKERS FOR THE PEPTIC
ULCER DISEASE FOUND IN HELICOBACTER PYLORI
STRAINS ISOLATED FROM PORTUGUESE CHILDREN
M Oleastro1, A Carvalho1, B Nunes2, L Monteiro1,
A Menard3, F Megraud3. 1Instituto Nacional Saude Dr.
Ricardo Jorge, Lisboa, Portugal. 2Observatorio Nacional
Saude, Lisboa, Portugal. 3Laboratoire de Bacteriologie,
Universite Victor Segalen, Bordeaux, France.
Aim: To search for new markers and potential virulence
candidates of H. pylori.
Methods: Comparison of 2 H. pylori strains isolated from
a peptic ulcer and a gastritis from 2 young children, by PCR-
based subtractive hybridization, showed the presence of 2
genes ulcer-associated and J99-specific. We investigated by
PCR and sequencing the presence of these 2 genes, jhp562
and jhp870, on H. pylori strains isolated from 72 Portuguese
children, mean age 10.8 years, 19 with peptic ulcer and 53
with gastritis only. We also evaluated if these genes where
related to previously described virulence-associated genes.
Results: Both jhp562 and jhp870 were significantly associ-
ated with peptic ulcer (p , 0.01, odds ratio (OR) of 9.48 and
6.87, respectively). The presence of the jhp562-positive
genotype was strongly associated with vacAs1, cagA and
babA2-positive status and oipA on status, while jhp870 was
associated with vacAs1, cagA and babA2-positive status,
hopQ I allele and oipA on status. The risk for peptic ulcer
increased when jhp562-positive genotype was combined with
cagA-positive genotype (OR = 8.84 vs OR = 6.72), vacAs1
(OR = 13.93 vs OR = 12.04) or both cagA-positive and
vacAs1 genotypes (OR = 10.54 vs OR = 7.73). The
association of jhp870 with cagA or vacA didnt improve the
discrimination between disease outcome.
Conclusions: These results suggest that jhp562 and jhp870
genes are candidates as virulence markers of H. pylori.
PG3-07 IDIOPATHIC THROMBOCYTOPENIC PURPURA AND
HELICOBACTER PYLORI INFECTION MG Marzano1,
G Loffredo2, R Migliorati1, E Miele1, S Avilia1, A Staiano1,
V Poggi2. 1Department of Pediatrics, University of Naples,
Naples, Italy. 2Department of Onco-Hematology, Pausilipon
Hospital, Naples, Italy.
649
Introduction: It has recently been reported that a relationship
between Idiopathic Thrombocytopenic Purpura (ITP) and
Helicobacter Pylori (HP) infection exists in adult series. A
link between the eradication of the infection itself and the
remission of thrombocytopenia has also been observed. The
aim of our study was to assess the prevalence of HP infection
in children affected by chronic ITP and to verify the influence
of the treatment on platelets counts.
Patients and Methods: Our study lasted a minimum of 12
months and involved 31 children (Mean age: 136 months;
range: 53216 months; M/F: 10/20), all affected by chronic
ITP. Patients were followed for 12 months: 6 months to assess
chronic behaviour of ITP and 6 months for the follow-up.
Unsplenectomized patients affected by chronic ITP un-
derwent: Urea Breath Test (UBT), a search for HP antigen
in stool samples as well as a search for HP antibodies in
serum. In order for a diagnosis of HP infection to be made,
two of the 3 results in the above mentioned tests had to be
positive. Patients affected by HP infection were randomized
into two groups: one group was treated with amoxicilline,
claritromicine and omeprazole; the second group did not
receive any therapy: Six weeks after the end of treatment
patients underwent a UBT to assess their recovery from HP
infection. In both groups, treated and untreated, for the 6
months following the diagnosis of chronic ITP, platelet counts
were performed weekly (for Platelets ,20000/mmcu), every
23 weeks (for Platelets between 20000 and 50000/mmcu)
and monthly (for Platelets .50000/mmcu).
Results: In our study the prevalence of HP infection in
patients affected by chronic ITP was 22.5% (7/31 patients).
Three of 7 patients were treated (Mean age: 174 months), and
the mean platelet count at the onset of ITP was 72000/ mmcu
(range 33000128000/mmcu); 1/3 patients who received
treatment did not recover from HP infection. In the 4
untreated patients (Mean age: 143 months) the mean platelet
count was 94000/mmcu (range: 63000124000/mmcu). At
the follow-up the platelet count difference between treated
and untreated patients and between infected and uninfected
patients was not statistically significant.
Conclusions: Our preliminary data do not suggest any
relationship between ITP and HP infection.
PG3-08 NEW ONSET TYPE 1 DIABETES MELLITUS (T1DM) IN
CHILDRENPREVALENCE OF SILENT GASTROPATHY
ESTABLISHED BY MEANS OF ELECTROGASTROG-
RAPHY AND ITS CORRELATION WITH METABOLIC
CONTROL E Toporowska-Kowalska1, K Wasowska-Kroli-
kowska1, A Szadkowska2, J Bodalski2. 1Department of
Pediatric Gastroenterology and Allergology, Medical Uni-
versity of Lodz, Lodz, Poland. 2Department of Children
Diseases, Medical University of Lodz, Lodz, Poland.
Many studies have aimed at validating the use of electro-
gastrography (EGG) measurements as a noninvasive indicator
of gastropathy and high prevalence of gastric myoelectrical
dysrhythmias in diabetic subjects has been reported. Al-
though little is known as regards the GI status in the early
stage of diabetes, there is no available data concerning gastric
myoelectrical activity in children with newly diagnosed
T1DM.
The aim: Was to evaluate the gastric myoelectrical activity in
children with new onset (,1 year) T1DM in respect of
metabolic control.
Material and Methods: 42 children with T1DM duration of
,1 year, aged 518 yrs (mean 12,87 6 3,09 yrs) and 35
healthy controls were enrolled. In all subjects precutaneous
EGG (PC Polygraff Synectics Medical Inc.) was performed.
Fasting (30 min) and postprandial (60 min) periods were
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
650 ESPGHAN 38TH ANNUAL MEETING
analyzed in terms of: the percentage of normogastria (2,43.6
cpm), bradygastria (,2.4 cpm), tachygastria (3,710 cpm),
dominant frequency instability coefficient DFIC and power
ratio PR. In subjects with T1DM hemoglobin A1c (HbA1c)
(HPLC method) and blood glucose levels were measured
(GLUKOTREND BOEHRINGER Mannheim). The evalua-
tion of ANS was based on deep breath test and heart rate
variability monitoring (HRV). The study protocol was
approved by the Medical University Ethics Committee.
Results: In all subjects, but one (2,38%), the screening for
neuropathy was negative. In 28,57% of T1DM and 91.42%
controls (p , 0.001) normal EGG criteria were fulfilled. The
fasting and postprandial percentage of gastric dysrhythmias
were significantly higher in T1DM subjects than in controls
(p , 0,001). The significant difference between T1DM and
controls was observed in he percentage of fasting bradygas-
tria (53,13 6 32,74% vs. 20 6 17,81%; p , 0.01) and normal
pre-prandial rhythm (36,97 6 30,65% vs. 71,8 6 13,74%;
p , 0,01). In the postprandial period in T1DM normogastria
was present in 50% subjects vs. 28,57% preprandially (p ,
0,001), mean normogastria percentage increased (71,79 6
22,89% vs. 636,97 6 30,65) and that of bradygastria
decreased (22,04620,44% vs. 53,13 6 32,74%); the EGG
signal proved to be more stabile (DFIC 57,25 6 35,74 vs.
31,93 6 22,11; p , 0,05). PR was comparable in T!DM and
in controls (2,01 6 2,33 vs 2,54 6 1,18; NS). The percentage
of fasting bradygastria and normogastria were correlated with
glycemia (r = 20,55 and r = 0,51; p , 0,05), and postprandial
DF with postprandial glycemia (r = 0,41; p , 0,05). There
was no correlation between HbA1c and EGG.
Conclusions:
1. Our results proved that the derangement of the gastric
myoelectrical activity is present in a high proportion of
children with early stage of type 1 diabetes without
neuropathy
2. Glucose level influences gastric myoelectrical activity,
whereas long-term metabolic control (HbA1c) does not
correlate with EGG exponents in new onset T1DM
children.
PG3-09 ANALYSIS OF INTESTINAL BACTERIAL DEVELOP-
MENT IN PRETERM INFANTS BY USING MOLECU-
LAR AND CULTURAL METHODS A Suau1,
F Campeotto2, F Magne1, AJWaligora-Dupriet3, C Aubert-
Jacquin4, C Dupont2, P Pochart1, MJButel3. 1Genie Biol-
ogique, CNAM, EA 3199, Paris, France. 2Neonatologie,
H^opital St Vincent de Paul, Paris, France. 3Microbiologie,
Universite Rene Descartes, Paris, France. 4Bledina SA,
Villefranche-sur-Sa^
one, France.
Aim: Limited information is available regarding the micro-
biota in preterm infants. Moreover, they relied mainly on the
application of culture-based methods. The purpose of this
study was to compare the analysis of the intestinal bacterial
establishment in preterm infants using both molecular and
culture methods.
Patients and methods: This monocentric prospective study
enrolled hospitalised preterm infants. Faecal samples were
collected at least once a week during the first month of life.
After extracting total DNA, bacterial 16S rDNAs were
amplified using PCR and analysed using temporal tempera-
ture gradient gel electrophoresis (TTGE). Aerobic and
anaerobic flora were analysed by quantitative cultures on
various media.
Results: 20 infants were included (gestational age 33.7 6 1.1
weeks). Seven were vaginally born, all were formula-fed, and
14 of whom received human milk supplementation. TTGE
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
profiles indicated that the faecal bacterial communities of
preterm infants are simple (1 to 15 bands per subject) with
a high inter-individual variation (no band common to all
samples). Sharp changes can be observed during the first
week. With culture method, 3 to 8 species by infant were
isolated. Both methods underscore the delayed colonisation
by a normal flora. Only 2 infants were colonised by
bifidobacteria and Bacteroides. However two third of the
infants were colonised by clostridia. The molecular method
allowed detecting the dominant species without determina-
tion of the population size whereas the culture method
allowed quantifying the main bacterial groups, even present
in low levels. In contrast with adult, uncultivable part of the
preterm intestinal microbiota appears very small.
Conclusion: Microbiota in preterm infants was very simple
and the inter-individual variability high. The bacterial
establishment is delayed as compared to full term infants.
The two methods, i.e. molecular tools and culture, each have
their advantages, and their linked use appears important to get
the most complete picture of an ecosystem and to improve
both methods.
PG3-10 GUT GRAFT VERSUS HOST DISEASE: THE ROLE OF
INFECTION AND IMPLICATIONS FOR TREATMENT
A Hassan1, M Elawad2, D Rampling3, V Smith3, P Veys1,
P Milla2. 1Department of Immunology and Bone Marrow
Transplant, Great Ormond Street Hospital, London, United-
Kingdom. 2Department of Paediatric Gastroenterology,
Great Ormond Street Hospital, London, United-Kingdom.
3
Department of Histopathology, Great Ormond Street
Hospital, London, United-Kingdom.
Aim: The aim of the study was to define factors in the
subgroup of patients that develop g-GVHD and to identify
possible environmental triggers including pre-existing gut
disease, infections, conditioning, type of HSCT and immu-
nosuppressive therapy.
Methods and Results: 130 children underwent HSCT
between January 2000 to December 2003. 24/45 (52%)
children with pre transplant GI disorders developed g-GVHD
compared to 28/85(32%) children with no pre-existing GI
disease (p , 0.02) who did develop g-GVHD. 10/24 has sex
miss-match HSCT and underwent X-Y FISH analysis of
intestinal biopsies. Three patients age 12, 4 and 3 developed
severe g-GVHD. Two were transplanted from a matched
unrelated donor, one from a matched family donor. 2/3
developed a gram positive central line infection at 3 and 4
days preceding the onset of GVHD. 1/3 had a pre-existing
adenoviral gut infection that has flared up after HSCT prior to
the development of g-GVHD.Patient 1&2 were treated with
methylprednisolone and cyclosporine. Patient 3s post trans-
plant immunosuppressive therapy was reduced due to
adenoviraemia. Two of the patients died and the third one
has ongoing gut inflammatoey disorder. Gut biopsies showed
typical features of GVHD with obvious apoptosis of enter-
ocytes. X-Y FISH analysis showed that donor cells (25%
range 2040%) and recipient immunocytes populated the
lamina propria of the gut, however only recipient enterocytes
were present in the crypt/villous axis.
Conclusion: The data showed that pre-existing gut in-
flammatory disease and infection are potent risk factors for
the development of g-GVHD. Reconstitution of the mucosal
immunocyte population with donor immuno-competent cells
as shown by our FISH analysis results in the ability of the
Toll like receptors to recognise extra cellular microbial
components which may activate the NF-kB pathway leading
to the activation of apoptotic pathway and production of
g-GVHD. Blockade of NF-kB is a potential therapeutic
option.
 ESPGHAN 38TH ANNUAL MEETING
PG3-11 MANAGEMENT OF ACUTE GASTROENTERITIS IN
FRANCE BY PAEDIATRICIANS AND GENERAL PRAC-
TITIONERS:WHATS RECOMMENDED AND WHATS
PRACTICED. JP Olives1, AM Gayral2, R Roche3.
1
Department of Paediatrics. H^
opital des Enfants., Toulouse,
France. 2CRP Consulting Conseil, Balma, France.
3
Laboratoire Pierre Fabre Medicament, Castres, France.
Aim: The ESPGHAN Working Group on acute diarrhoea and
the French Speaking Group (GFHGNP) published recom-
mendations with specific emphasis on oral rehydration, early
refeeding and avoidance of ineffective drugs. We conducted
a study to determine how closely physicians in France follow
the treatment guidelines.
Methods: Observational prospective study: 1 November
200329 February 2004. Children were included if they
presented at least 3 watery stools per day. Physicians were
fully free for their prescriptions
Results: 1602 children (,4 months: n = 88) (5 months to
3 years: n = 605) (315 years: n = 909) were treated by 332
general practitioners (GP) and 206 paediatricians (P). For
children ,18 months of age, dehydration was graded: ,5%
in 78.7%, 5 to 10% in 20.6% and .10% in 0.6%. Oral
Rehydration Solutions were prescribed in solely 70% of the
cases. 62% of responding physicians extend the rehydration
period to 12 to 24 hours. Refeeding was initiated within 4 h in
54.7%, within 48 h in 21.8%, within 812 h in 12.6%, within
1224 h in 8% and after 24 h in 2.7%. The current milk was
stopped in 66.4% of children. In these cases, formula was
changed for a lactose-free formula (63.8%), a low containing
lactose formula (14.9%), a protein hydrolysate (13.3%) and
various formulas including probiotics (8%). Regarding all
children (1month-15 years; n = 1602), one drug at least was
prescribed in 96% of cases with a mean of 2.4 drugs per child:
domperidone (86.4%), racecadotril (35.1%), lactobacillus
acidophilus (27.1%), smectite (25.2%),nifuroxazide (6.8%)
and antibiotics (0.7%)
Summary: Globally, P and GP in France all act alike in the
management of acute diarrhoea in children with two
exceptions: significantly more GP stop milk feeding until
1224 h and change formula when significantly more P
advance to a full strength concentration with the same
formula within 8 h.
Conclusions: One more time, this study demonstrates that we
are still far from the ESPGHAN guidelines for optimal
management of acute gastroenteritis, particularly regarding
ORS and drugs prescription.
PG3-12 CAMPYLOBACTER JEJUNI CAPSULAR POLYSAC-
CHARIDE AND INTESTINAL INNATE IMMUNITY
M Zilbauer1, N Dorrell2, P Boughan1, B Wren2, N Klein1,
M Bajaj-Elliott1. 1Institute of Child Health, London, United-
Kingdom. 2London School of Hygiene and Tropical Medi-
cine, London, United-Kingdom.
Aim: To investigate the potential role and contribution of the
C.jejuni capsule in modulation of intestinal innate immunity.
Methods: We have previously shown dynamic cross-talk
between wild-type C.jejuni (WT) and intestinal epithelial
defensin expression and function. In the present study an
isogenic capsule-deficient mutant of C.jejuni (kpsM) was co-
cultured with human colonic intestinal cell lines (Caco-2/HT-
29) and IL-8 and human beta-defensin (hBD) gene expression
was quantified. Transcriptional regulation of innate defence
genes was further delineated by specific promoter-luciferase
reporter assays. Bactericidal potency of recombinant hBD
peptides against kpsM was evaluated by broth-dilution
assay.
651
Results: A marked time-dependent induction of IL-8 and
hBD3 was observed with maximal expression at 8 hours post-
infection. In contrast hBD2 gene expression was modest.
Detailed transcriptional analyses showed similar degree of
NF-kB activation between wild-type and the mutant strain.
Importantly the mutant was found to be highly susceptible to
the antimicrobial action of hBD-3 amongst the peptides tested.
Summary: No significant differences were observed be-
tween the ability of the WT and the isogenic kpsM mutant in
eliciting host innate response. More importantly, defensin
peptides were equipotent in their killing capacity towards
both strains.
Conclusion: The capsule polysaccharide of C.jejuni plays
a minimal role in modulating intestinal epithelial innate defence.
PG3-13 RANDOMISED-CONTROLLED TRIAL OF RAPID VS
24-HOUR NASOGASTRIC REHYDRATION IN CHIL-
DREN WITH ACUTE GASTROENTERITIS AND MOD-
ERATE DEHYDRATION CVE Powell1, RG Heine1,
SJ Priestley2. 1Royal Childrens Hospital, Melbourne,
Australia. 2Western Hospital Sunshine, Melbourne, Australia.
Background: Oral or nasogastric rehydration is considered
the treatment of choice in children with acute gastroenteritis
and moderate dehydration.
Aims: To compare the efficacy, cost and parent satisfaction of
two nasogastric rehydration regimens in children with acute
gastroenteritis.
Methods: The study was conducted at two metropolitan
paediatric teaching hospitals. Children (aged 6m to 6 yrs)
with acute gastroenteritis and moderate dehydration present-
ing to the Emergency Department (ED) were enrolled in
a randomised-controlled trial of two nasogastric rehydration
regimens. For standard rehydration (SR), the estimated fluid
deficit was replaced in hospital over 24 hours, using oral
rehydration solution (ORS; Na=60mmol/L). Rapid rehydra-
tion (RR) consisted of ORS 100 mls/kg over 4 hours in the
ED, followed by discharge from hospital and a nurse visit at
home after 24 hours. Physiological data, including the body
weight, were collected before and after the initial rehydration
phase, at 24 hours and at 1 week. Failure of rehydration was
defined as further weight loss .2%, compared to the
admission weight. Data on parental satisfaction and cost to
families were collected.
Results: Of 199 patients (mean age 25m, range 7 to 69m),
106 (53%) were randomised to RR, and 93 (47%) to SR.
There were no significant group differences in baseline
characteristics for age, gender, tympanic temperature, weight
at presentation, and final weight one week after treatment. At
24 hours, treatment failure rates were similar for RR 12.2%
(95%CI; 6.218.2%) and SR 8.6% ( 95%CI; 2.614.6%).
Parent satisfaction was similar for both regimens. There was
no difference in cost to families at 24 hrs, but after one week
parents reported significantly greater travel expenses for SR
than RR. Parents in the RR group required significantly more
time off work.
Conclusions: Both rehydration regimens were safe and
efficacious. Parents reported similar satisfaction with both
regimens. Cost to the families were higher for those admitted
to hospital for SR, and parents treating their child at home
after RR needed more time off work. RR should be the
preferred treatment of children with acute gastroenteritis and
moderate dehydration.
PG3-14 SYNDROMATIC DIARRHEA IN CHILDREN. REPORT
OF 8 CASES C Martinez-Vinson1, O Goulet3, D Berrebi2,
L Ferkadji2, JP Hugot1, M Bellaiche1, F Ruemmele3,
JP Cezard1. 1Pediatric Gastroenterology H^
opital Robert
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
652 ESPGHAN 38TH ANNUAL MEETING
Debre, Paris, France. 2Pathology Department EA 3102
H^opital Robert Debre, Paris, France. 3Pediatric Gastroen-
terology H^opital Necker Enfants Malades, Paris, France.
Introduction: Syndromatic diarrhea (SD) is a rare etiology
of intractable diarrhea of infancy. The 1st serie of 8 patients
was published in 1994. SD associated: neonatale intractable
diarrhea, liver disease with cirrhosis in 2, low birth weight,
phenotypic abnormalities and a functionnal immundeficiency
in all. The pronostic was severe with death in 5 of them. Since
this 1st publication six new reports describe SD among 8
children.
Aim: To report a serie of 8 new SD collected in 2 centers
between 1976/01.
Methods and Results: All the children presented with
intractable diarrhea in the 1st months of life. All were born
small for their gestational age, consanguinity was found in 3.
Facial dysmorphism was present in all with proeminant fore
head, flat nose and abdnormal hairs. Mental retardation was
observed in 7. Liver disease was present in 3 with biliary
cirrhosis in 1. All of them had an functionnal immunodefi-
ciency with absence or low response to vaccines. B and T
lymphocytes phenotypes were normal in 5. Hypogammaglo-
bulinemia was noted in 6 with normalisation in 2. Infectious
complications occurs in all children and were severe in 5
(bronchopneumonia, pneumocystis, EBV, chicken-pox) de-
spite regular IV immunoglobulins and prophylactic antibio-
therapy in 3. Small intestinal biopsies showed severe (n = 2)
or partial villous (n = 5) atrophy without (n = 5) or minor
infiltration of the lamina propria. HLA DR expression was
normal. One child died from infection, 7 are still dependant of
partial or total parenteral nutrition (PN) (mean 6 years). One
has been weaned of PN at 5 years of age. Tolerance to enteral
nutrition appeared to be correlated to persistance of villous
atrophy.
Conclusion: Syndromatic diarrhea is a rare cause of
intractable diarrhea of infancy due to villous atrophy. The
high rate of consanguinity suggest a genetic etiology. Liver
failure and infectious secondary to the immuno deficiency are
the main causes of death. PN dependance seems to be
secondary to the persistance of villous atrophy. The pronostic
appears to be better than previously published with PN
wearning in few cases.
PG3-15 INTESTINAL INFLAMMATION AND RESIDUAL SUB-
CLINICAL DYSFUNCTION IN CHILDREN WITH HIV
INFECTION ON LONG TERM HAART E Bruzzese1,
A Russo. Raucci1, C Postiglione1, MT Russo1, LR Assanto1,
G Polito1, A Guarino1. 1Department of Pediatrics, Univer-
sity, Naples, Italy.
Aim: HIV-infected children show a wide pattern of intestinal
dysfunction, with nutrient malabsorption, and increased
permeability. We previously reported that a 6 month course
of highly active antiretroviral therapy (HAART) was effective
in restoring intestinal function in HIV-infected children. Aim
of this study was to determine whether long term antire-
troviral therapy is effective in maintaining a normal intestinal
function.
Methods: Thirty-five children with HIV infection were
enrolled. All were in stable condition without intestinal or
extraintestinal acute disease and all were on HAART. The
majority of children enrolled (28/35) were treated with
protease inhibitors (PI). Mean duration of treatment was 43
months (range 1073 months). The following parameters
were collected: sex, age, body weight, class of HIV infection,
viral load and number of CD4+ lymphocytes, duration of
HAART. The following markers of intestinal function were
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
determined: steatocrit, serum xylose levels, and fecal a1
antitripsin (a1AT). Intestinal inflammation was evaluated by
fecal calprotectin concentration (CLP) and rectal nitric oxide
production (NO).
Results: There was no major clinical evidence of intestinal
disease in any of the children. However abnormal values of
steatocrit, serum xylose and a1AT were found in 11/35
(31%), 5/35 (14%) and 2/35 (5%) children, respectively.
Increased CLP and NO concentrations were observed in
12/35 (34%) and 30/33 (90%) children respectively. A weak,
but significant correlation was found between viral RNA and
CLP (r = 0.52, p , 0.01). Mean CLP concentration was
significantly higher in children treated with PI than in those
who received non PI drugs (109 6 78 vs 46 6 29 mg/gr, p ,
0.05). There was no relationship between intestinal function
tests and the other parameters.
Conclusions: Despite the absence of clinical signs, intestinal
inflammation, as judged by non invasive markers, is a frequent
finding in HIV infected children and is associated with HIV
load and/or PI therapy. In addition, a consistent number of
children show residual abnormalities of digestive-absorptive
function.
PG3-16 TREATMENT OF ACUTE INFECTIOUS DIARRHEA IN
CHILDREN WITH A MIXTURE OF THREE NEW
LACTOBACILLUS RHAMNOSUS STRAINS (573L/1,
573L/2, 573L/3). A RANDOMIZED, DOUBLE-BLIND,
PLACEBO CONTROLLED TRIAL H Szymanski1,
J Pejcz1, M Jawien2, A Kucharska2, M Strus2, P Heczko2.
1
Department of Pediatrics, St.Hedwig of Silesia Hospital,
Trzebnica, Poland. 2Chair of Microbiology, Jagiellonian
University Medical College, Krakow, Poland.
Background: Multiple studies documented that Lactobacil-
lus GG is effective in treating infectious diarrhea (ID) in
children. However, the positive effect of these probiotics in
ID cannot be extrapolated to other L.rhamnosus strains.
A trial was conducted to evaluate the efficacy of L.rhamnosus
three strain mixture (573L/1,573L/, 573L/3) with patients
with ID of different causes.
Methods: Children from 2 months to 5 years with acute
diarrhea were enrolled into double-blind, placebo-controlled
trial. They were randomly allocated either to the group
receiving L.rhamnosus (at least 1,2 3 1010 CFU) twice daily
for 5 days, or to the group receiving placebo. The primary
outcome measure was the diarrhea duration. The secondary
outcome measures were: weight gain after rehydration,
diarrhea lasting longer than 7 days, presence of adverse
events, and gut colonization by the applied strains. The
molecular methods (PCR, PFGE) were used to demonstrate
gut colonization by the applied strains.
Results: 93 patients were enrolled in the study. There were no
differences in admission between the groups in: age, sex,
previous types of feeding, previous diarrhea duration,
antibiotic usage, weight, fever prevalence, overall status and
dehydration degree. Diarrhea duration in probiotic treated
group was 83,6 h 6 55,6 versus 96h 6 71,5 (p = 0,36) in the
control one. In children with rotavirus infection getting
probiotics (n = 22/87) it was 77,5h 6 35,4 versus 115h 6 67
(p = 0,03) in the control group (n = 17/87). Presence of the
used strains was detected in stools of 37/46 (80,4%) children
after 5 days and 19/46 (41,3%) after 14 days, after the
treatment initiation.
Summary: Diarrhea duration was insignificantly shorter in
a whole probiotic treated group but significantly shorter in
patients with rotavirus diarrhea. No differences in secondary
clinical outcomes were found among the study groups. We
proved, using molecular methods, the ability of applied
probiotic strains to gut colonization.
 ESPGHAN 38TH ANNUAL MEETING
Conclusion: Treatment of children with ID, with L.rhamno-
sus three strain mixture is safe and results in shorter duration
of rotavirus diarrhea.
PG3-17 PROBIOTIC INTERVENTION IN THE NEONATAL PE-
RIOD: LONG-TERM EFFECTS ON GUT MICROBIOTA
M Rinne1, M Kalliomaki2, S Salminen1, E Isolauri2.
1
University of Turku, Turku, Finland. 2Turku University
Central Hospital, Department of Pediatrics, Turku, Finland.
Probiotics may promote the gut defence mechanisms but the
long-term impact on the composition of the gut microbiota
remain unknown.
Aim: To assess the impact of 6 month postnatal probiotic
administration on gut microbiota, specifically the Bifidobac-
terium, Lactobacillus/Enterococcus, Bacteroides and Clos-
tridium counts up to the age of 24 months, representing the
most important species of early gut microbiota.
Methods: 132 infants from atopic families participating in
a placebo controlled double-blind study. Lactobacillus GG
was administered for 24 weeks before expected delivery to
mothers and for 6 months after the delivery to infants.The
growth of the infants and any signs and symptoms of disease
were evaluated at ages 3,6, 12 and 24 months. Fluorescent in
situ hybridization was used to evaluate the bacterial counts in
faecal samples.
Results: The numbers of Clostridia in placebo group
increased from 6 to 24 months of age, P = 0.01. A significant
difference between the numbers of Lactobacillus/Enterococ-
cus and Clostridia were detected in probiotic and placebo
groups at 24 months of age, P = 0.01 ans P = 0.03,
respectively (Table).
TABLE Gut microbiota at 24 months of age
Lactobacillus/Enterococcus Clostridia
Placebo 1.4 3 108 (1.0 3 1082.1 3 108) 1.8 3 108 (1.1
Probiotic 8.4 3 107 (6.9 3 1071.0 3 108) 9.8 3 107 (7.6
The growth of the infants did not differ between placebo and
probiotic groups and no signs of chronic disease were
detected. Furthermore, according to a special symptom chart
on vomiting, faeces content and crying, no differences were
detected between probiotic and placebo groups.
Summary: Probiotic administration in the neonatal period
did not affect the diversity, quality or quantity of the gut
microbiota as characterized using fluorescent in situ hybrid-
ization method.
Conclusion: The probiotic preparation was safe and did not
affect the compositional development of gut microbiota.
PG3-18 PROBIOTICS DECREASE THE INCIDENCE AND SE-
VERITY OF NECROTIZING ENTEROCOLITIS IN
PRETERM NEONATES M Wilschanski1, A Ben Nun2,
R Bromiker2, M Kaplan2, M Caplan3, C Hammerman2.
1
Hadassah University Hospital, Jerusalem, Israel. 2Shaare
Zedek Medical Center, Jerusalem, Israel. 3Northwestern
University, Chicago, USA.
Background: Necrotizing enterocolitis (NEC) is the most
common gastrointestinal catastrophe affecting premature
infants. The preterm newborn gut, in contrast to that of the
healthy, full term breast feeding infant, is predominantly
colonized by potentially pathogenic micro-organisms, which
predispose the preterm infant to develop NEC. We therefore
postulated that normalizing the intestinal flora of premature
653
infants via the administration of probiotics would offer
a natural protection against NEC in this population.
Objective: We sought to study the effect of prophylactic
probiotics on the incidence and severity of NEC in low birth
weight neonates.
Methods: Preterm neonates ,1500 gm birth weight were
randomized to receive either placebo or daily supplementa-
tion of enteral feeds with a probiotic mixture of 109 cfu/day
consisting of 350 million Bifidobacteria infantis, 350 million
Streptococcus thermophilus, and 350 million Bifidobacteria
bifidus. Clinical parameters relating to NEC were monitored
and recorded. NEC was graded according to Bells staging
criteria.
Results: 143 infants completed the study; 72 received
placebo and 71 received probiotics. There were no differences
in mean birth weight (1.170 6 0.272 vs. 1.152 6 0.264 kg
respectively); mean gestational age (29.3 vs 30.3 wk); or in
time to reach full feeds (181 vs 159 days respectively;
p = 0.121). The incidence of NEC was significantly reduced
in the probiotics group 16.6% (12/72) vs 4% (3/71); p =
0.031; Relative Risk 0.25; [ 95% CI: 0.075 to 0.86], and the
NEC which did develop was less severe in the probiotic
treated group by Bell staging criteria [1.33 6 0.47 vs. 2.33 6
0.47 respectively; p=0.016]. Two of the 15 babies with NEC
died, both from the control group.
Conclusions: Supplementation with probiotics was effective
in reducing both the incidence and severity of NEC in our
population of low birth weight infants.
PG3-19 CYTOMEGALOVIRUS COLITIS IN PATIENTS WITH
INFLAMMATORY BOWEL DISEASE IN CHILDREN
B Ghidini1, M Bellaiche1, D Berrebi2, J Viala1, JP Hugot1,
JF Mougenot1, M Peuchmaur2, JP Cezard1. 1Pediatric
Gastroenterology H^opital Robert Debre, Paris, France.
2
Pathology Department H^ opital Robert Debre, Paris,
France.
Objective: To determine the prevalence, diagnosis and
3 1082.8 3 108)
outcome of cytomegalovirus (CMV) infection complicating
3 1071.3 3 108)
the course of inflammatory bowel disease (IBD) in children.
Methods: The records and clinical courses were reviewed for
all IBD patients followed at our pediatric IBD center. All
patients resistant to oral and IV steroid treatment were noted
as well as the children who developped concomitantly CMV
infection.
Results: 135 children affected by IBD were followed in our
IBD center between 20002004. During this period 21 of
them (15%) received steroid IV at 11.5 mg/kg/d because of
resistance (.15d) to oral steroids for a severe flare up. Five
out of 21 (24%) had a documented CMV colitis (3 ulceratives
colitis (UC), 2 crohns colits (CC), one undetermined colitis
(UC). 5 of them were receiving Azathioprine and one CMV
was inaugural of her UC and associated to CMV gastritis.
CMV coinfection was documented by CMV inclusion, and
immunostaining of colon biopsies in all associated to CMV
antigen in blood and urine as well as CMV antibodies. No
other location of CMV was found in these children.
Gancyclovir IV was then administred at a dose of 10 mg/kg/d
for 1521 days without change of the immunosuppresor
treatment. A rapid remission (within 5d) was obtained in all.
2 patients treated 15d relapse one month later and were
treated again 3 weeks and did not relapse after.
Conclusion: CMV co infection can occur in pediatric IBD as
in adults with a prevalence of 2% of IBD and 24% of steroid
resistant patients. Its diagnosis should be done systematically
in all steroid resistant IBD patients. IV Gancyclovir treatment
improved dramatically the evolution of the apparent re-
fractory IBD flare up avoiding an increase of the immuno-
suppressive treatment (cyclosporine, infliximab) or surgery.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
654 ESPGHAN 38TH ANNUAL MEETING
PG3-20 GENETIC POLYMORPHISMS OF TLR4 AND CARD15
ARE NOT ASSOCIATED WITH NECROTISING EN-
TEROCOLITIS IN VERY LOW BIRTH WEIGHT IN-
FANTS B Szebeni1, G Veres2, A Dezsofi2, K Rusai1,
Vannay1, A Treszl2, B Vasarhelyi1, A Arato2. 1Research
A availability of PUFAs after digestion. The PUFA profile of the
Group for Paediatrics and Nephrology, Hungarian Academy
of Sciences, Budapest, Hungary. 2First Department of
Pediatrics, Semmelweis University, Budapest, Hungary.
Aim: Inadequate reaction of innate immune system to
bacterial antigens from the intestinal flora plays a central
role in the development of necrotising enterocolitis (NEC).
Toll-like receptor 4 (TLR4) and caspase-recruitment domain
15 (CARD15) bind bacterial lipopolysaccharide and pepti-
doglycan and their activation leads to the production of
inflammatory cytokines. Our aim was to analyse whether
functional single nucleotide polymorphisms (SNPs) of TLR4
and CARD15 genes are associated with the risk of NEC in
very low birth weight (VLBW) infants (birth weight 1500
grams).
Methods: In this retrospective analysis we enrolled dried
blood samples of 77 VLBW singleton infants without NEC
and 41 singleton infants with NEC. TLR4 A+896G, G+1196T
and CARD15 G+2722C SNPs were tested with polymerase
chain reaction followed by restriction fragment length
polymorphism (RFLP) analysis. The genotype distribution
was compared with that obtained in 146 healthy term
newborns.
Results: The prevalence of TLR4 A+896G, C+1196T and
CARD15 G+2722C SNPs was similar in VLBW infants with
and without NEC (0.10 vs 0.08, 0.10 vs 0.08, 0.05 vs 0.03,
respectively, p = NS). Carrier state of the tested SNPs was not
associated with the risk of other perinatal complications
(respiratory distress syndrome, patent ductus arteriosus,
severe hypotension, sepsis, intracranial haemorrhage, bron-
chopulmonary dysplasia). Logistic regression analysis re-
vealed independent association between sepsis and NEC
(OR: 3.96, CI: 1.1014.2, p = 0.036).
Summary: Carrier state of tested TLR4 and CARD15
genetic variants are not associated with perinatal morbidity
and NEC-risk in Hungarian VLBW infants.
Conclusion: Genetic polymorphisms of lipopolysaccharide-
signaling receptors do not influence the development of NEC
in very low birth weight infants.
PG4-01 SPECIFIC POLY-UNSATURATED FATTY ACIDS SUP-
PORT INTESTINAL BARRIER INTEGRITY AND RE-
DUCE IL-4 MEDIATED BARRIER DISRUPTION
LEM Willemsen1, MA Koetsier1, C Beermann2, EAF Van
Tol1. 1Numico Research B.V., Wageningen, The Netherlands.
2
Numico Research B.V., Friedrichsdorf, Germany.
Background: Besides nutrient and water uptake the in-
testinal mucosa functions as a barrier against potentially
harmful luminal compounds. Tight junctional complexes
prevent para-cellular permeability for macromolecules and
play an important role in barrier maintenance. Both allergic
and inflammatory mediators (e.g. IL-4, IFN-g) are known to
compromise epithelial resistance by affecting tight junctions.
Poly-unsaturated fatty acids (PUFAs) are commonly used in
infant formulas but their effect on intestinal barrier integrity
is poorly understood.
Aim: We investigated the effects of PUFAs (n6: GLA,
DGLA, AA; n3: EPA, DHA) as well as a comprehensive fat
blend on epithelial barrier integrity, i.e. transepithelial
resistance (TER) and permeability.
Methods: Monolayers of T84 intestinal epithelial cells (two
weeks post confluency) were pre-incubated for 48 h with
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
individual PUFAs as well as a lipase treated fat blend
(containing 15w% PUFAs) followed by additional 48 h
incubation. The fat blend was co-incubated with epithelial
cells in combination with lipase to mimick the in vivo
lipase treated fat blend was determined by GC-MS.
Furthermore the effect of PUFAs and fat blend was tested in
IL-4 exposed T84 cells. Barrier integrity of T84 cells was
evaluated by measuring TER and 4kDa-FITC-dextran fluxes.
Results: AA, DHA, EPA and to a lesser extend GLA and
DGLA dose and time dependently enhanced TER. Interest-
ingly, AA, DHA and EPA could also strongly reduce the IL-4
induced permeability in T84 cells. Moreover, the lipase
treated fat blend dose dependently reduced the IL-4 mediated
barrier disruption up to 50% (p , 0.001) whereas control
lipase incubation alone had no effect. Lipase treatment of the
blend indeed resulted in release of the relevant PUFAs as
revealed by GC-MS.
Conclusion: Specific n6 and n3 PUFAs can support barrier
integrity by improving barrier resistance and reducing IL-4
mediated permeability. AA, DHA and EPA were particularly
effective. Specific fat blends that generate these PUFAs in
vivo upon digestion may improve intestinal barrier integrity
thereby supporting natural resistance.
PG4-02 CAPSULE ENDOSCOPY: THE PREFERRED METHOD
OF LUMINAL SURVEILLANCE IN CHILDREN WITH
PEUTZ-JEGHERS SYNDROME? J Epstein1, G Schofield2,
C Fraser2, G Brown2, JMEFell1, W Hyer3. 1Department of
Paediatric Gastroenterology, Chelsea & Westminster Hospi-
tal, London, United-Kingdom. 2Wolfson Unit for Endoscopy,
St Marks Hospital, Harrow, United-Kingdom. 3Department
of Paediatric Gastroenterology, St Marks Hospital, Harrow,
United-Kingdom.
Aims: The natural history of Peutz-Jeghers syndrome (PJS) is
still poorly understood. The immediate childhood risk is
midgut intussusception, 68% of patients requiring laparot-
omy before their 18th birthday. The St Marks screening
protocol in PJS currently includes 23 yearly barium follow
through (BFT). This is the first clinical trial to prospectively
compare wireless video capsule endoscopy (VCE) and BFT
as screening tools for midgut polyposis in asymptomatic
children with PJS.
Methods: VCE was performed in 5 asymptomatic children
with PJS (median age 9 years, range 711), a median of 11
months (range 123) after BFT. Findings at VCE were
reported by investigators blinded to BFT results.
Results: In all patients, VCE was well tolerated and the study
was complete to terminal ileum or caecum. 2/5 patients
swallowed the capsule; in 3/5 it was inserted at concurrent
surveillance gastroscopy.
In 4/5, small polyps were identified on VCE (median number
per patient 2.25, range 25; median polyp size 2.75 mm,
range 1.53.5), whilst BFT detected no polyps in any of these
4 cases.
In 1/5, 23 midgut polyps of 2 cm were identified at BFT but
not on VCE. In this patient laparotomy is delayed pending
repeat VCE in 6 months.
Summary: We have identified a high incidence of small
bowel polyps in asymptomatic children with PJS. In 4/5, VCE
was at least as sensitive as BFT, and small polyps below the
detection threshold for BFT were visualised on VCE in all 4
cases.
Conclusion: VCE is at least as sensitive as BFT in the
identification of midgut polyps in children with PJS, and
provides more detail, without the radiation exposure. The
discovery of small polyps in 4/5 cases informs our un-
derstanding of the natural history of the disease in childhood
 ESPGHAN 38TH ANNUAL MEETING
and is itself a justification for screening. Greater subject
numbers are needed to quantify sensitivity and specificity and
thus define VCE as a gold standard screening test. The St
Marks protocol for midgut surveillance in PJS is likely to be
modified to include routine use of VCE with less reliance on
BFT.
PG4-03 THE USE OF CAPSULE ENDOSCOPY IN PEDIATRIC
PATIENTS Y Sutas1, ML Lahdeaho2, T Ruuska1. 1Univer-
sity Hospital of Tampere, Department of Pediatrics, Tampere,
Finland. 2University of Tampere, Medical School and
University Hospital of Tampere, Department of Pediatrics,
Tampere, Finland.
Aim: to assess the use of capsule endoscopy (CE) in
a heterogenic group of patients with gastrointestinal problems.
Patients: CE was used to screen the involvement of small
intestine in 32 cases (18 girls, 14 boys with median (range)
age 13.3 (1.217.2) years; weight 44 (10.984.0) kg. Main
indications for CE were i. (n = 11) suspicion of Crohns
disease (CD) undetermined by conventional methods, ii. (n =
17) evaluation of response to the medication in 12 CD
patients and 5 with ulcerative colitis (UC), iii. (n = 4) the
extent of small intestinal involvement in two patients with
lymphangiectasia and in one with Peutz-Jeghers polyposis
and in one infant with suspicion of persistent upper
gastrointestinal bleeding.
Results: The M2A capsule, 26 mm 3 11 mm (FCCID#=08P-
GIVEN IMAGING, Given Imaging Ltd, US) was swallowed
by 29/32 patients while in one (age 1.2y) has been planted by
gastroscopy. All excreted the capsule with no complication.
The median (range) gastric transit time was 14 (1195)
minutes while the median (range) intestinal transit time was
199 (135335) minutes. CE visualized lesions suggestive of
active inflammation in 3/11 cases with suspicion of CD as
well as in 10/12 CD patients and in 1/5 UC patients. The
findings were distributed in stomach (5/29;17%), duodenum
(11/29;38%), jejunum (14/29;48%) and ileum (15/29;52%).
CE revealed the extent of lymphangiectasia in two patients. In
3/11 cases with suspicion of CD was observed prominent
lymphoid hyperplasia with no diagnostic implications. A
single polyp with no sign of inflammation was seen in one
patient with CD and in one with UC. Villous atrophy was not
pertinent in any.
Summary: With respect to these findings, new therapeutic
strategies were adjusted in 15/29 cases.
Conclusion: In addition to its supplementary role in
diagnostics, CE may serve to optimize the medical manage-
ment of inflammatory bowel disease without the burden of
repeated invasive procedures in pediatric patients.
PG4-04 DIAGNOSTIC SENSIBILITY OF ILEOSCOPY WITH-
VIDEOCAPSULE IN PAEDITRIC-JUVENILE AGE.
B Bizzarri1, F Fornaroli2, C Del Rossi3, R Dalla Valle4,
F Vincenzi5, V Maffini6, G De Angelis7. 1Paediatric
Gastroenterology and Endoscopy Unit, Parma, Italy. 2Pae-
diatric Gastroenterology and Endoscopy Unit, Parma, Italy.
3
Paediatric Surgery, Parma, Italy. 4General Surgery and
Transplantations, Parma, Iceland. 5Paediatric Gastroenter-
ology and Endoscopy Unit, Parma, Italy. 6Paediatric
Gastroenterology and Endoscopy Unit, Parma, Italy. 7Pae-
diatric Gastroenterology and Endoscopy Unit, Parma, Italy.
Aim: The aim of this study is to demonstrate the high
diagnostic sensibility of ileoscopy with videocapsule even in
paeditric-juvenile age.
Methods: The wireless capsule endoscopy used was the
M2A capsules (Given Workstation imaging). Children over
655
6 years usually swallowed spountanously the capsule after
a training with hard candies. In infants not able to swallow the
capsule, it can be brought in the stomach or in the proximal
duodenum endoscopically with a rot net for foreign body in
general anaesthesia. Sixtytwo patients (38 male, 24 female;
between 2 and 21 years) afferent to Gastroenterology and
Endoscopic Unit of University of Parma with suspected small
bowel disease uderwent ileoscopy with videocapsule between
October 2001 and December 2004. Negative upper end lower
endoscopy was requested. The ileoscopy with videocapsule
was performed in 23 patients (37%) with suspected or
confirmed IBD, in 15 patients (24%) with acute or chronic
anaemization, in 16 patients (25%) with confirmed polyposis,
in 6 patients (10%) with suspected polyposis, in 1 patient
(2%) with malabsorption and in 1 patient (2%) with
generalized cutaneous angiomatosis and anaemization.
Results: Six exams were negative (10%), and 2 exams (3%)
technically failed (gastric retention of the capsule). In the
other 54 exams (87%) the videocapsule showed pathological
findings: 20 ileal Crohns lesions, 2 ileal lymphoid nodular
hyperplasia, 3 NSAIDs ileal bleeding lesions, 24 ileal/colic
polyps,1 diffuse ileal-colic angiomatosis, 1 ileal angiodys-
plasia, 2 ileal bleeding ulcerations, 1 ileal malignant neo-
formation. In two patients the videocapsule was retained in
the ileum: in an ileal occlusion due to an adenocarcinoma and
in an acquired blind loop with multiple bleeding ulcerations.
During surgeries the videocapsule was easily removed.
Summary: The videocapsule, if used in selected paediatric
patients, demontrated a very high sensibility in small bowel
pathologies, even higher than in adults.
Conclusions: Ileoscopy with videocapsule has to be per-
formed when there is the indication to study the small bowel
even in paediatric age. It is safe and its diagnostic sensibility
is very high if the patients are selected in an accurated way.
PG4-05 MITOMYCIN C: AN ALTERNATIVE CONSERVATIVE
TREATMENT FOR REFRACTORY OESOPHAGEAL
STRICTURE IN CHILDREN? S Uhlen1, P Fayoux2,
F Vachin2, D Guimber1, D Turck1, F Gottrand1,
L Michaud1. 1Division of Gastroenterology, Hepatology
and Nutrition, Department of Pediatrics, University Hospital
and Faculty of Medecine, Lille, France. 2Department of
Otolaryngology-Head and Neck Surgery, University Hospital
and Faculty of Medecine, Lille, France.
Aim: Mitomycin C is an antiproliferative agent that has been
successfully used as an adjuvant treatment in several
ophtalmological procedures, and for laryngeal and tracheal
stenosis. It has been recently suggested in 2 case reports that
local application of mitomycin C after dilatation may be of
help to prevent the recurrence of caustic oesophageal
strictures in children. The aim of this study was to the assess
efficacy and safety of local application of mitomycin C in
refractory oesophageal stricture in children.
Methods: We performed a prospective preliminary study of
local application of Mitomycin C in five children (age: 2, 3, 4,
6 and 11 years) with refractory oesophageal stricture (caustic
[n = 3], anastomotic after surgical repair of congenital
oesophageal atresia [n = 2]). Previous to mitomycin C
application, they required during a period varying from 3 to
24 months (mean: 15 months) 4, 4, 6, 10 and 10 sequences of
oesophageal dilatations, respectively. Mitomycin C was
applied locally on the dilatation wound using a rigid
endoscope immediately after an endoscopic dilatation.
Results: No complication was observed following the pro-
cedure. One child required a second application of mitomycin
C two weeks later because of recurrence of dysphagia. No
further dilatation was necessary for any of these children with
a follow-up varying from 9 to 23 months (mean: 17 months).
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
656 ESPGHAN 38TH ANNUAL MEETING
All children remained asymptomatic on follow-up. Radio-
logical control revealed no recurrence of oesophageal
stricture. No dysplasia was found on oesophageal biopsies
performed during the follow-up.
Summary and Conclusion: Local application of mitomycin
C could be an alternative to iterative dilatations, to surgery or
to stent placement in refractory oesophageal stricture in
children. However, long-term outcome assessment on a large
prospective series is required before any definitive conclu-
sion can be drawn on the usefulness of mitomycin C in these
patients.
PG4-06 THE EFFECT OF STEROID THERAPY ON SEVERE
CORROSIVE OESOPHAGEAL BURNS IN CHILDREN:
A MULTICENTRIC PROSPECTIVE STUDY Y Dogan1,
M Gulcan2, N Urganci3, Z Onal1, T Erkan1, F Cullu
Cokugras1, T Kutlu1. 1Department of Pediatrics, Division of
Pediatric Gastroenterology Hepatology and Nutrition, Cer-
rahpasa Medical Faculty, Istanbul University, Istanbul,
Turkey. 2Department of Pediatric Gastroenterology, SSK
Bakirkoy Maternity and Children Hospital, Istanbul, Turkey.
3
Department of Pediatric Gastroenterology, Sisli Etfal
Education and Research Hospital, Istanbul, Turkey.
Background and Aims: Ingestion of caustic agents consti-
tute a major public health problem in developing countries.
The most important complications are oesophageal and gastric
burns, which may result in severe strictures. The use of
steroids to prevent these complications is controversial. The
purpose of this study is to investigate whether the corticoste-
roid treatment is effective in preventing stricture development.
Methods: Between 2002 and 2004, oesophagogastric endos-
copies were performed to all of the patients who were
admitted to our hospital for suspected corrosive ingestion
within the first 2448 hours. Caustic lesions were graded
according to Zargars classification. Three hundred and sixty-
two cases with grade IIb and grade III oesophageal lesions
were prospectively evaluated in two randomised groups.
In the group I, patients received steroid (2 mg/kg/day, max:
60 mg/day/ period for 3 weeks), ranitidine and ceftriaxone.
In the group II, patients received ranitidine and ceftriaxone.
The patients in the both groups were followed up for six weeks.
Results: Oesophagogastric endoscopies were performed in
936 children. Oesophagus lesions were seen in 727 patients
and gastric lesions in 149 patients. In 365 of these patients,
only slight oesophageal lesions (grade I and IIa) were present;
whereas in 362 patients, the lesions were more severe (grade
IIb and III). Two hundred and fourteen (59.1%) of 362
patients were male. In the group I and group II, there were
223 and 139 patients, respectively. Oven cleaners (36.6%)
and lime solvent (23%) were the most frequently encountered
corrosive agents in both groups. During the follow up,
oesophageal stricture developed in 27 and 27 cases, gastric
outlet obstruction (GOO) in 4 and 2 cases in group I and II,
respectively. Oesophageal perforation developed in one case
in group I. There was no statistically significant difference in
oesophageal stricture and GOO developing between two
groups (p = 0.505). During the follow-up, there was no
mortality in this study.
Conclusions: Oven cleaner is the most caustic agent that
leads to oesophageal stricture. Systemic steroid treatment has
no beneficial effect on oesophageal stricture formation
following caustic oesophageal burns.
PG4-07 MOVEMENT DISORDER AND ENCEPHALOPATHY
IN CYCLICAL VOMITING SYNDROME IS A CONSE-
QUENCE OF DISORDERED CNS AMINE METABO-
LISM AND NITRIC OXIDE DEFICIENCY S Heales1,
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
P Milla2, P Andrews3, R Surtees2, K Lindley2. 1Institue of
Neurology, University College London, London, United-
Kingdom. 2Institute of Child Health and Great Ormond
Street Hopsital for Children, University College London,
London, United-Kingdom. 3St Georges Hospital Medical
School, London, United-Kingdom.
Aim: Cyclical vomiting syndrome (CVS) is characterised by
periodic episodes of intense emesis during which affected
individuals not uncommonly exhibit a stereotyped movement
disorder, encephalopathy and severe hypertension. We un-
dertook to investigate the biochemical basis of movement
disorder and encephalopathy in CVS.
Methods: 2 children (aged 14 and 15 years) with intractable
CVS and a stereotyped frequently recurring cycle refractory
to conventional treatments were investigated according to
standard in house clinical service guidelines. Both children
developed cogwheel rigidity and bradykinesia during emetic
phases. Cerebrospinal fluid was taken from both children
during convalescent phases and cycles of emesis for
measurement of CNS neurotransmitters.
Results: CSF concentrations of HVA, 5HIAA and dopamine
were depressed in both individuals during cycles of emesis
and normalised during convalescent phases. CSF concen-
trations of phenylalanine were also increased. These bio-
chemical findings can be explained by an acquired reduction
in tetrahydrobiopterin (BH4) reductase activity. These bio-
chemical abnormalities would explain the parkinsonian like
movement disorder in these individuals. BH4 deficiency may
result in functional nitric oxide deficiency which was
manifest in both these individuals as drug resistant hyperten-
sion. Prescription of a nitric oxide donor led to rapid control
of the hypertension and early resolution of the emetic cycle in
both individuals suggesting the NO deficiency was instru-
mental in perpetuating the hypertensive encephalopathy of
CVS.
Summary: Reduced BH4 activity during emetic cycles in
CVS results in NO deficient hypertension and a Parkinsonian
like movement disorder.
Conclusions: Disturbed CNS amine metabolism explains the
movement disorder and hypertensive encephalopathy of
severe CVS.
PG4-08 A POPULATION BASED ASSESSMENT OF THE ROLE
OF PSYCHOSOCIAL FACTORS IN THE DEVELOP-
MENT OF RECURRENT ABDOMINAL PAIN IN CHILD-
HOOD. D Forbes1, G Kendall2, F Stanley2. 1School of
Paediatrics & Child Health, University of Western Australia,
Perth, Australia. 2Institute for Child Health Research, Perth,
Australia.
Childhood recurrent abdominal pain (RAP) is believed to
result from the interaction of multiple bio-psychosocial
factors. In a population based study we have previously
reported associations between RAP and gender, other pain
sensitivity syndromes, defecation pattern and parental con-
cerns regarding health (Forbes D, Gastroenterology 2002 (4),
S1:122;723). We hypothesise that early psycho-social char-
acteristics will predispose to and predict later RAP.
Aim: To describe the relationship between psychosocial
factors throughout the first 8 years of life, with the reporting
of RAP at age 8 in a population based cohort.
Methods: Children of mothers prospectively enrolled by
week 18 of pregnancy were followed for 8 years with regular
data collection regarding child health and gastrointestinal
function. Psychological testing was undertaken over this
period using the Infant Temperament Scale, Child Behaviour
Checklist (CBCL), Self-Efficacy Scale (SES), and Family
 ESPGHAN 38TH ANNUAL MEETING
Assessment Device (FAD). Differences in psychosocial
scores were compared for children with and without RAP
(defined as 3 or more episodes of abdominal pain within the
last year) at age 8 years,
Results: 2979 women were enrolled and 2860 offspring
assessed at birth, and 2162 at age 8. 142 (6.9%) children had
RAP. Families of children with RAP were similar in structure
and income to other families. Children with RAP had a higher
mood score at age 2, but no other identifiable temperament
differences, and no differences in self-efficacy or family
function. Mean CBCL scores at year 2, 5 and 8 were within
the normal range, although lower for children with RAP, than
for other children.
Conclusion: Children with RAP at age 8 have only minor
psychosocial differences in comparison with children without
RAP. Physiological status and parental attitudes to health
appear to be more important than psychological status in the
development of RAP.
PG4-09 PREVALENCE AND RISK FACTORS ASSOCIATED
WITH ORGANIC DISEASES (OD) IN CHILDHOOD
ABDOMINAL PAIN SUBTYPES BASED ON ROME II
CRITERIA AM Magista`1, A De Canio1, T Chiarelli1,
N Billanzone1, G Leone1, E Lionetti1, L Cavallo1,
R Francavilla1. 1CIRGEEE (Centro Interdipartimentale di
Ricerca di Gastroenterologia ed Epatologia Pediatrica
dellEta` Evolutiva), University of Bari, Bari, Italy.
Background and Aim: The suspect of an OD in the
Paediatric Rome II Criteria is based on the presence of
alerting symptoms known as red flags; however, no data on
their efficacy in detecting the OD have been published. The
aims of our study were: a) to determine the frequency of OD
in different abdominal pain subtypes clinically based on
Rome II Criteria; b) to identify factors associated with
organic and functional disorders.
Patients and Methods: In a 30 months period 263
consecutive patients [(84%); M 105 (40%); mean age 10,2
yrs (range 4, 2 y17,9 y)] were referred to our Department for
RAP. All patients were firstly classified in symptoms subtypes
based on Rome II criteria and then were submitted to an
extensive laboratory and instrumental evaluations as appro-
priate. Generic and specific for each functional gastrointes-
tinal subtype risk factors were evaluated to assess their
sensitivity, specificity, positive and negative predictive values
for suspecting an organic cause.
Results: 213 (78,7%) were classifiable in: functional
dyspepsia (FD) (31.6%), irritable bowel syndrome (IBS)
(26.2%), functional abdominal pain (FAP) (20.9%), abdom-
inal migraine (1.9%) and aerophagia (0.4%). An organic
abnormality was found in 58 (27.2%), in particular 45.7% in
FD, 16.3% in FAP and 15.5% in IBS (table).
Both generic and specific risk factors were highly specific but
not sensible for the presence of an organic cause. The
presence of at least a specific risk factor was significantly
associated with OD in IBS subtypes (64% vs. 28%; p , 0.03)
but not in FD and in FAP. IBS in parents was significantly
associated with IBS in children (21% vs. 8%; p , 0.01). No
children among those who were unclassifiable by Rome II
criteria had OD (nil vs. 27.2%; p , 0.002).
Summary and Conclusion: In our experience an OD is
found in approximately 20% of RAP with a higher prevalence
in FD subtype, followed by FAP and IBS. The presence of
a risk factor is highly suspicious for an OD however their
absence do not exclude it. IBS in a parent is a risk factor for
IBS in their child.
657
Lactose Celiac Crohn
GERD Gastritis intolerance disease Giardiasis disease
IBS (N: 69) 0 0 4 (6%) 4 (6%) 3 (4%) 0
FD (N: 83) 22 (26%) 12 (14%) 4 (5%) 2 (2%) 0 0
FAP (N: 55) 4 (7%) 3 (5%) 0 0 1 (2%) 1 (2%)
PG4-10 A SYSTEMATIC REVIEW OF THE DIAGNOSIS AND
TREATMENT OF CHILDHOOD IDIOPATHIC CONSTI-
PATION JS Gordon1, HR Jenkins2, DC Wilson3. 1Napier
University, Edinburgh, United-Kingdom. 2University Hospi-
tal of Wales, Cardiff, United-Kingdom. 3Child Life and Health,
University of Edinburgh, Edinburgh, United-Kingdom.
Introduction: Childhood idiopathic constipation may appear
to be a simple condition, but it is frequently unrecognised or
undermanaged, resulting in ineffective management and
conflicting advice to families. An objective critical appraisal
of the literature is required to assess the appropriate evidence
base and thus to support high quality practice.
Aim: We aimed to systematically review the literature on
diagnosis, treatment, and outcomes for children with a di-
agnosis of childhood idiopathic constipation, and therefore
produce evidence-based recommendations.
Methods: Search criteria included Reviews, Randomised
Controlled Trials (RCTs), cohort studies, case-control
studies, and cross sectional surveys in childhood constipation
(English language). Databases searched included AMED,
Medline, CINAHL, ClinPSYCH, The Cochrane Database,
and reference lists (from 1975 to 2004). All papers were
critically appraised by 3 people, who judged their evidence
level (EL), and so aimed to provide graded recommendations,
based on the SIGN criteria (BMJ 2001;323:3346).
Results: Nine RCTs, 5 systematic reviews, 1 narrative review
and 40 other studies were appraised. The evidence base for
diagnosis, treatment and outcome is of poor quality - only one
review and one RCT were of evidence level 1+, whereas the
remaining 5 reviews and 8 RCTs were of low methodological
quality (EL 1-); all other studies are EL 3 and 4. There is no
agreement on definition of terms for diagnosis. Inclusion
criteria, interventions used, and methods of assessment of
outcomes are inconsistent between the studies. Methodolog-
ical problems abound, such as lack of details on random-
isation, lack of intention to treat analysis, and small sample
sizes. No evidence-based recommendations could therefore
be made.
Summary and Conclusions: Currently, there is insufficient
evidence to support recommendations for practice in
management of childhood idiopathic constipation. There is
a need for international guideline development, based on both
currently available evidence and consensus, in order to
standardise current practice and to permit clinically useful,
high quality research.
PG4-11 COWS MILK-INDUCED ALLERGIC COLITIS
IN SCHOOL AGED CHILDREN E Jarocka-Cyrta1,
M Ucinowicz2, M Sobaniec-otowska3, M Kaczmarski4. 1III
Department of Pediatrics Medical University of Bia3ystok,
Bia3ystok, Poland. 2III Department of Pediatrics Medical
University of Bia3ystok, Bia3ystok, Poland. 3Department of
Madical Pathomorphology Medical University of Bia3ystok,
Bia3ystok, Poland. 4III Department of Pediatrics Medical
University of Bia3ystok, Bia3ystok, Poland.
Cows milk protein intolerance is a well recognised cause of
colitis in the first year of life. By the age of 2 most affected
children have become tolerant of the milk allergens.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
658 ESPGHAN 38TH ANNUAL MEETING

Aim: To evaluate the prevalence of allergic colitis in children A) elimination of the cows milk from the diet resulted in an
over 6 years of age. increase of CD8+ cells percentages in peripheral blood and
Methods: The study covered children aged 618, referred to a reduction of CD4+/CD8+ ratio (1.65 6 0.46 V vs 1.5 6
our department from January 2002 to December 2003 due to 0.48, p = 0.0164). Percentages of B cells showed trends to
rectal bleeding. Patients were scheduled for rectosigmoido- decrease and percentage of NK cells to increase. This was not
scopy or colonoscopy with mucosal biopsy, laboratory blood the case in group B patients.
and stool examination. Children with bacterial infection, anal Conclusion: Our study indicated that despite a lack of
fissure, polyps, other surgical condition, inflammatory bowel clinical sign of food intolerance in children who have
disease were excluded. If histological examination showed outgrown cows milk allergy, the disturbances in lymphocytes
the number of eosinophils greater than 60 per 10 high-power subset in peripheral blood have persisted and can be restored
fields, the immunological assessment was performed (total by elimination diet.
IgE, IgM, IgA, IgE specific for cows milk, skin prick tests, This rises the questions if the children really achieved full
atopy patch tests) and a cowmilk-free diet was introduced for tolerance.
2 months, followed by control sigmoidoscopy and open food
challenge. The criteria for a diagnosis of allergic colitis PG4-13 FECAL ALPHA-1 ANTITRYPSIN EXCRETION IN CHIL-
were 1/characteristic endoscopic appearance, 2/more than 60 DREN WITH GASTROINTESTINAL OR CARDIAC
eosinophiles per 10 high power fields in colon biopsy DISEASE AND THEIR CORRELATION WITH SERUM
specimens, 3/resolution of symptoms on a milk-free diet, ALBUMIN CONCENTRATION C Sierra1, J Blasco1,
4/positive result of a food challenge. MIVicioso2, G Colome1, A Barco1, L Del Ro1. 1Pediatric
Results: Out of 29 children (mean age 12 years, 19 girls) with Gastroenterology and Nutrition Unit, H. Materno-Infantil,
rectal bleeding 4 patients revealed the number of eosinophils HRU Carlos Haya, Malaga, Spain. 2Biochemical and
in lamina propria greater than 60 per 10 high power fields. Laboratory Unit, H. Materno-Infantil, HRU Carlos Haya,
Only 2 patients fulfilled the criteria for allergic colitis- Malaga, Spain.
resolution of clinical symptoms and colonoscopic abnormal-
ities after the elimination of a cows milk from the diet,
positive result of a food challenge. In patients with allergic Introduction: Protein-losing enteropathy includes a large
colitis the results of skin prick tests and patch test with cows group of diseases characterized by enteric loss of plasma
milk, RASTs were negative. In both cases the symptoms of proteins in abnormal amounts, being a potential mechanism
colitis persisted from infancy. for a significant decrease of plasma albumin. This may
Conclusion: Cows milk allergy may be a causing factor of contribute to further impairment of the nutritional status in
colitis in school-aged children. The most effective means of mildly malnourished patients.
establishing a definitive diagnosis in such patients is a food Objective: To establish the differences of fecal alpha1
challenge. antitrypsin (F-AT) and plasma clearance of alpha1 antitrypsin
(C-AT) between healthy children and patients with gastroin-
testinal or cardiac disorders and their correlation with serum
PG4-12 IMMUNE RESPONSE TO ELIMINATION DIET IN albumin concentration.
SCHOOL AGED CHILDREN WHO HAVE OUTGROWN Material and Methods: We studied 80 children distributed
COWS MILK ALLERGY E Jarocka-Cyrta1, A Stasiak- in 20 healthy ones (3.5 6 3.3 years), 12 cardiac patients
Barmuta2, M Kaczmarski3. 1III Department of Pediatrics (3.4 6 4.9 years) and 48 patients with gastrointestinal
Medical University of Bia3ystok, Bia3ystok, Poland. 2De- problems (5.9 6 3.6 years). F-AT concentration were
partment of Allergology Medical University of Bia3ystok, measured by immunonephelometry and expressed as mg/g
Bia3ystok, Poland. 3III Department of Pediatrics Medical of dry weight of stool. C-AT was calculated from the product
University of Bia3ystok, Bia3ystok, Poland. of dry fecal weight (g/day) and F-AT concentration (mg/100 g
dry fecal weight), divided by the serum
1 antitrypsin
The outcome of cows milk allergy has been a subject of concentration (mg/dL).
controversy. Results: There is a highly significant correlation between
Aim: We aimed to investigate whether in children with cows F-AT and serum albumin concentration (r = 20.689, p ,
milk allergy of infancy period immunity changes persisted 0.01) as well as between C-AT and serum albumin
despite the lack of clinical sign of food intolerance. concentration (r = 20.716, p , 0.001). The regression line
Methods: We studied 20 children with cows milk allergy crosses a serum albumin concentration of 3.0 g/dL when
(CMA) diagnosed and treated before 2 years of age (group A, F-AT is 4.1 mg/g and C-AT 134 mL/day.
age range 616 years, mean10.5, 11 girls) and 24 children Conclusions: Excessive enteric protein loss is detected in
without any evidences of food allergy (group B, age range a high proportion of children affected by cardiac or
617 years, mean 11.6) referred to our department due to gastrointestinal disorders. It should be included in the
recurrent abdominal pain. All the children had recently been diagnostic work-up in patients with hypoproteinemia.
on a normal diet. The patients were scheduled for gastro-
duodenoscopy, skin prick tests, laboratory examination (IgM, Serum
IgA, total IgE, IgE specific for cows milk). Peripheral blood F-AT C-AT albumin
lymphocyte subpopulations (CD19+, CD3+, CD4+, CD8+, mg/g (mL/day) (mg/dL)
CD3 HLADR+, CD16+CD56+) were determined using Group n mean 6 SD mean 6 SD mean 6 SD
a cytometric method. In all the children cows milk-free diet
Control 20 0.6 6 0.4 10.9 6 7.8 4.0 6 0.3
was introduced for 6 weeks, followed by a double blind Cardiac patients 12 3.6 6 5.6 74.3 6 133.0* 3.3 6 1.0*
placebo controlled food challenge ( DBPCFC). Further blood Crohns disease 13 2.5 6 2.3** 128.3 6 167.1* 3.1 6 0.6**
samples for immunological analysis were taken after 6 weeks Acute diarrhoea 12 1.7 6 1.5* 11.6 6 12.4 3.9 6 0.2
of elimination diet. Diarrhoea associated 7 5.4 6 4.5* 40.4 6 37.5* 2.7 6 0.3**
Results: In all the patients from both groups the results of with chemoterapy
skin prick tests, DBPCFC were negative. There were not Schonlein-Henoch Purpura 4 4.1 6 2.9 27.5 6 23.8 2.9 6 0.8
differences in IgA, IgM and IgE concentrations on normal Functional abdominal pain 12 0.6 6 0.5 14.2 6 6.6 3.7 6 0.1
and on milk free diet. In children with CMA in infancy (group
*np , 0.05 ** p , 0.005.

J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005

 ESPGHAN 38TH ANNUAL MEETING
PG4-14 OMEPRAZOLE AND ASTHMA OUTCOME IN CHIL-
DREN WITH ASTHMA AND GASTROESOPHAGEAL
REFLUX DISEASE: A RANDOMISED CONTROL TRIAL
K Strdal1, GB Johannesdottir2, O Closs3, M Handeland4,
HK Holm2, B Bentsen2, KL Carlsen3. 1stfold Central
Hospital, Fredrikstad, Norway. 2Ullevaal University Hospi-
tal, Oslo, Norway. 3Ahus University Hospital, Oslo, Norway.
4
Vestfold Central Hospital, Tnsberg, Norway.
Aim: Gastroesophageal reflux disease (GERD) is prevalent
in asthma patients, and esophageal acid perfusion may cause
bronchial constriction and asthma symptoms under experi-
mental conditions. To assess whether acid reflux may induce
asthma symptoms in a clinical setting, we performed
a randomised control trial with omeprazole in children with
concomitant asthma and GERD.
Methods: Children between 7 and 16 years old with asthma
and symptoms suggestive of GERD were offered an
esophageal pH-monitoring, and those with pathological
reflux (reflux index .5.0) were randomized to 12 weeks of
treatment with omeprazole 20 mg daily or placebo. Primary
outcome measures were asthma symptoms (daytime wheeze,
symptoms at night, in the morning and during exercise) and
the Pediatric Asthma Quality of Life Questionnaire
(PAQLQ). Changes in lung function from baseline (FEV1%
and FEF2575) and the use of short-acting bronchodilator
were secondary endpoints. pH-monitoring was repeated at the
end of the study to confirm the efficacy of acid suppression
with omeprazole.
Results: 38 children (mean age 10.8 years; 7.216.8,
29 males) were allocated to omeprazole or placebo treatment,
19 in each group. The groups were similar in age and gender
distribution, and mean reflux index was 8.8 (O) vs 9.7 (P).
Two patients withdrew because of suspected side effects, one
in each group. The improvement in total symptom score were
identical in the two groups (21.38), and no significant
differences were found for the subscores. The total PAQLQ
score improved by 0.62 (O) and 0.50 (P), p = 0.51. The
FEV1% decreased by 1.4 (O) and 2.0 (P), p = 0.76, whereas
FEF25-75 changed by -0.07(O) vs 0.04 l/sec (P), p = 0.12.
The changes in need for short-acting bronchodilators were
similar (21.9 doses/2 weeks). Adequate acid suppression was
achieved in the omeprazole group (mean RI 3.2).
Conclusion: Omeprazole did not change asthma symptoms
and lung function in children with asthma as compared to
placebo.
PG4-15 PHARMACOKINETICS OF ESOMEPRAZOLE IN ADO-
LESCENT PATIENTS AGED 12 TO 17 YEARS INCLU-
SIVE WITH SYMPTOMS OF GASTROESOPHAGEAL
REFLUX DISEASE T Andersson1, J Zhao1, J Li1,
JE Hamer-Maansson1, M Illueca1, P Lundborg2. 1AstaZe-
neca R&D, Wilmington, DE, USA. 2AstraZeneca R&D,
Molndal, Sweden.
Aim: To determine the pharmacokinetics (PK) of esomepra-
zole after single and repeated oral doses in adolescent patients
(age, 1217 years inclusive) with gastroesophageal reflux
disease (GERD) symptoms.
Methods: In this single-center, randomized, open-label study
(D961400094), 28 adolescents (13 girls) received esomepra-
zole 20-or 40-mg capsules once daily for 8 days. Blood
samples were taken at 0, 0.5, 1, 1.5, 2, 3, 4 and 6 hours post
dose on days 1 and 8. Normal-phase liquid chromatography
and ultraviolet detection (limit of quantification, 0.025 mmol/L)
were used to determine plasma esomeprazole concentrations.
PK parameters were calculated by a noncompartmental
approach using WINNONLIN software.
659
Results: The PK parameters of these adolescents and
published values (Physicians Desk Reference Electronic
Library [pdrel.thomsonhc.com]) of adults with suspected or
established GERD are shown below.
Summary: Esomeprazole seems to have both dose-and time-
dependent PK changes in these patients. A doubled dose
results in more than a doubled overall exposure (AUC) and
Cmax for both single and repeated doses. The T was also
approximately 50% longer for the higher or repeated dose
compared with the lower or single dose. The PK parameters
in adolescents are generally comparable with those observed
in adults. The few observed adverse events were of mild
intensity.
Conclusion: The PK profile of esomeprazole in adolescent
patients with symptoms of GERD is similar to that in adults.
Esomeprazole 20 mg Esomeprazole 40 mg
Single dose Repeated dose Single dose Repeated dose
1217 y 1217 y Adult 1217 y 1217 y Adult
AUC (mmol
h/L)a 1.58 3.65 4.2 5.57 13.86 12.6
Cmax (mmol/L)a 0.67 1.45 2.1 2.78 5.13 4.7
Tmax (h)b 1.86 1.96 1.6 2.07 2.04 1.6
T (h)a 0.55 0.82 1.2 0.86 1.22 1.5
a
Geometric mean, bArithmetic mean.
PG4-16 ANALYSIS OF THE MOLECULAR MECHANISMS
RESULTING IN THE IMMUNOALLERGIC PREDISPO-
SITION IN IPEX F Ruemmele1, B Begue1, O Goulet1.
1
INSERM EMI 0212, Necker-Enfants Malades Hospital,
Paediatric Gastroenterology, Paris, France.
Immune dysregulation, polyendocrinopathy autoimmune
enteropathy X-linked syndrome is the first known human
disorder of regulatory T cells. Recently disease-causing
mutations were identified in the FOXP3 gene located on the
X-chromosome. FOXP3 acts as a transcription repressor
allowing a negative control of T cell activation via reporters
containing multimeric FKH binding sites. Besides the various
autoimmune symptoms, this disorder is also characterized by
massive immuno-allergic reactions. The aim of the present
study was to analyze the regulatory mechanisms in the
intestinal mucosa implicated in these immuno-allergic
reactions.
Material and Methods: Duodenal biopsies were analyzed in
2 patients with IPEX before and under immunosupressive
treatment. The expression of the regulatory factors which
prime a nave T cells towards a Th1 (Tbet) or Th2 response
(GATA3 and c-maf), as well as Th1 (interferon g, IL2, TNFa)
and Th2 cytokines (IL-4,5,10) was analysed using semi-
quantitative RT-PCR analyses.
Results: At the onset of disease a clear Th2 predominance
was observed seen in high IL4 transcripts which dropped
under tacrolimus/steroid/azathioprine therapy. In contrast
Th1 cytokines IL-2 and to a lower extend TNFa transcripts
were upregulated under therapy, whereas IFNg remained
unaltered. In both patients, the ratio IL-4/IL-2 was inversed
under therapy, indicating a shift from Th2 (in the native state)
to Th1 predominance (under immunosuppressive treat-
ment). In the native state the pro-Th2-regulatory transcription
factors GATA3 and c-Maf were clearly expressed. GATA3
levels further increased under tacrolimus, whereas c-Maf
expression remained unaltered. Pro-Th1 regulatory T-bet,
was almost absent at the onset of disease, but increased under
treatment.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
660 ESPGHAN 38TH ANNUAL MEETING
Summary and Conclusions: The immune dysregulation in
IPEX results in a Th2 predominance in the intestinal mucosa.
This Th2 primed response is only partially suppressed by the
remission inducing immuonsuppression. Persistent and high
GATA-3 levels indicate the potential to generate a Th2
response in the gut explaining the tendency to immuno-
allergic symptoms.
PG4-17 CLINICAL EFFECT OF A PREBIOTIC-SUPPLEMENTED
FORMULA IN HEALTHY INFANTS DURING THE
FIRST SIX MONTHS OF LIFE D Fuentes1, LA Fuentes1,
C Lopez2, L Suarez3, H Escobar3. 1Facultad de Ciencias de
la Salud, Universidad Autonoma del Carmen, Ciudad del
Carmen, Mexico. 2Departamento de Infectologa Pediatrica,
Hospital Infantil de Mexico Federico Gomez, Ciudad de
Mexico, Mexico. 3Departamento de Pediatra, Seccion de
Gastroenterologa, Hospital Universitario Ramon y Cajal,
Madrid, Spain.
Currently there is limited data evaluating supplementation of
prebiotics in infant formula. It is already known that
administration of oligosaccharides can modify infant gut
flora but there is scarse published evidence of their alleged
clinical benefits.
Aim: To evaluate clinical outcome measures as well as
nutritional efficacy and bifidogenic characteristics of
an infant formula containing prebiotic oligosaccharides in
healthy full-term infants during the first six months of
life.
Methods: 60 healthy vaginally delivered full-term newborns
(3742 weeks gestational age, birth weight between 1090
percentiles) whose mothers could not or choo se not to
give breast milk for any reason were recruited in one
regional medical center and randomized to receive either
an infant formula (PF) with a mixture of prebiotic
oligosaccharides (0.8 g/dL 90% galacto-oligosacchar-
ides (GOS) and 10% fructo-oligosaccharides (FOS)),
partially hydrolyzed whey protein, starch and high beta -
palmitic acid (OmneoO ` , Nutricia, Cuijk, Holland) or
a standard formula (SF) (Nutrilon PremiumO ` , Nutricia,
Zoetermeer, Holland). In addition, 30 other newborns
receiving exclusive breast milk were also included as
reference group. All infants were assessed seven different
times by the same pediatricians (study inclusion and
monthly until age six months). Introduction of solid food
started until six months of age in all cases. Clinical
endpoints included: growth and anthropometric mea-
surements, number of infections requiring antibiotics
(upper/lower respiratory tract or gastrointestinal), addi-
tional visits to pediatrician, hospital outpatient visits,
hospital admissions, signs or symptoms of atopic dis-
ease, presence of constipation, colic, bloating, excessive
crying or vomiting. Mothers were asked to complete a
diary and questionnaire which was assessed every month.
Stool examinations included fecal pH, volume, consis-
tency and analy sis for bifidobacteria with standard plate
technique.
Results: 90 newborns were enrolled in the study. Only 68
completed the 6-month trial: 23 receiving SF, 25 receiving PF
and 20 receiving breast milk. No significant difference
regarding growth was found between the three groups.
However, weight gain, height and head circumference was
greater in infants receiving PF during the first 3 months (p =
NS). Mean number of stools was greater in the PF group
compared to SF group. From second evaluation until the
end of the study, PF stools had a higher proportion of
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
bifidobacteria (expressed as a percentage of the total number
of bacteria in feces) and a lower pH compared with SF, but
similar characteristics to the exclusive breastfed reference
group (p = 0.03). These significant differences increased with
time. Both formulas were well tolerated. None of the infants
receiving PF experienced dehydration at any point due to the
presence of softer stools nor any other known complication.
There were no significant differences between groups in the
number of upper/lower respiratory tract or gastrointestinal
(GI) infections requiring antibiotics, nor in additional visits to
pediatrician, hospital outpatient visits or hospital admissions.
However, presence of excessive crying, spitting-up, bloating,
constipation and atopy was higher in infants receiving SF
(p = 0.02).
Conclusions: FOS/GOS-supplemented infant formula is
safe, well tolerated and promotes adequate growth in healthy
infants. Furthermore, it can modify the gut microflora
resulting in an increased proportion of bifidobacteria in the
stools, lower fecal pH, and soften stool consistency, similarly
to breastfed infants. Infants receiving prebiotics have lower
prevalence of GI and atopic signs and symptoms. Clinical
differe nces might be related to changes in fecal flora but
further research with a longer follow up and larger numbers is
required to confirm these preliminary results.
PG4-18 BENEFICAL EFFECTS OF MINIMAL ENTERAL NU-
TRITION ON FEEDING INTOLERANT VERY LOW
BIRTH WEIGHT PRETERM INFANTS G Terrin1,
A Passariello1, G De Santo1, R Berni Canani1,
P De Luca1, MT Russo1, R Maiorino1, C Cascioli1. 1Dept.
of Pediatrics, University, Naples, Italy.
The best nutritional approach in very low birth weight
(VLBW) preterm infants presenting gastric residuals is still
debated. We tested the hypothesis that minimal enteral
feeding (MEF) could be effective to determine a shorter
duration of central venous access without additional risks for
necrotizing enterocolitis (NEC) in these subjects.
Methods: The clinical charts of VLBW preterm infants
presenting gastric residual .40% of daily fed volume
consecutively admitted to our Neonatal Intensive Care Unit
from September 2001 to July 2003 were reviewed. According
to nutritional management adopted the subjects were divided
in two groups: Group 1) 49 subjects in witch enteral feeding
was suspended for at least 24 hours; Group 2) 48 subjects in
witch MEF (612 cc/Kg/d) was started without increase in
feeds until gastric residual was .30% of total daily enteral
nutrition input.
Results: Gestational age (Group 1: 29 wks, IQR 3; Group 2:
29 wks, IQR 3), birth weight (Group 1: 1100 g, IQR 415;
Group 2: 1095 g, IQR 405), sex (Group 1: 26; Group 2: 21
male) and median time to start enteral fed (Group 1: 9 h, IQR
4; Group 2: 8 h, IQR 2) were similar in the two groups.
Neonates receiving MEF achieved full enteral nutrition and
regained their birth weight earlier, whenever the incidence of
NEC (defined by standardized criteria plus fecal calprotectin
determination) was comparable in both groups. In addition,
a significant reduction in late-onset sepsis episodes and
a shorter median length of hospital stay were observed in
Group 2 (Table). Finally, duration of mechanical ventilation
(MV) were comparable in both groups (Table).
Conclusions: Our data suggest that MEF could be a success-
ful nutritional strategy in infants presenting gastric residual
alone as clinical predictive sign of NEC. It reduces duration
of prolonged central vascular access and length of hospital-
ization, limiting the risk of late onset-sepsis and the costs of
hospital care.
 ESPGHAN 38TH ANNUAL MEETING
Group 1 Group 2 p Plymouth and Exeter, Plymouth, United-Kingdom.
Median time to reach full enteral
feeding (days, IQR) 11 (5) 8 (4.8) ,0.0001
Median time to regained birth
weight (days, IQR) 12 (4) 9 (1.5) ,0.0001
Neonates presenting NEC (n, %) 1 (2) 1 (2.1) 0.747
Infants with late-onset sepsis (n, %) 17 (34.7) 8 (16.6) 0.047
Death (n, %) 2 (4.1) 3 (6.3) 0.490
Median duration of Hospital
stay (days, IQR) 80 (32) 65 (21) 0.001
Median duration of MV (day, IQR) 4 (3) 4 (3) 0.260
PG4-19 A CELL CULTURE MODEL FOR SMALL INTESTINAL
STEM CELL FUNCTION AND DIFFERENTIATION
E Myrsky1, K Lindfors1, H Kainulainen2, M Maki3.
1
Paediatric Research Center, University of Tampere, Tam-
pere, Finland. 2Institute of Medical Technolgy, University of
Tampere, Tampere, Finland. 3Tampere University Hospital,
Tampere, Finland.
Aim: The stem cells located in small intestinal crypts are
responsible for the continuous renewal of the epithelium and
thus have a crucial role in maintaining tissue homeostasis.
Since at present there is no cell culture model for small bowel
epithelial stem cell function and differentiation, our aim is to
establish culture conditions that allow intestinal stem cell
propagation and proliferation as well as differentiation into
all four epithelial cell types present in the small bowel.
Methods: Single crypt epithelial cells and small organoids
were isolated from mouse small intestine and cultured under
specific conditions on collagen with fibroblasts seeded within
the gel as feeder layer. Cultures were observed daily and
studied immunohistologically.
Results: The isolated small intestinal epithelial cells form
organized clusters that remained viable for at least 10 weeks.
The clusters were characterized by a large central lumen and
well-deposited basement membrane. A portion of the cells
were proliferative as evinced by Ki-67 positivity. In addition
the clusters also harboured polarized columnar cells that had
obviously differentiated as indicated by alkaline phosphatase
activity and mucin profile typical for small intestine. When
these parental clusters were broken into single cells that were
cultured under identical conditions they formed daughter
clusters with similar characteristics as the parental ones.
Summary: At this point we have cultured the clonogenic
cells derived from mouse small bowel for 10 weeks - the
longest period ever published. During this time the cells form
clusters containing both differentiated cells and undifferen-
tiated proliferating cells that can also be subcultured.
Conclusion: We have developed a long-term cell culture
system that models the in vivo small intestinal crypt villus
axis in terms of stem cell proliferation and differentiation. If
applicable to human, the development of this culture system
will eventually allow the use of small intestinal stem cells in
regenerative medicine as a source for transplantation in
diseased patients to correct dysregulated or prolonged
mucosal regeneration.
PG4-20 STATINS MODULATE IFN-GAMMA INDUCED HLA-DR
EXPRESSION ON INTESTINAL EPITHELIAL CELLS
J Buning1, M Schmitz2, K Nohroudi1, KP Zimmer2,
D Ludwig3, M Nitschke3, S Strobel4. 1Institut fur Anatomie,
Universitat zu Lubeck, Lubeck, Germany. 2Klinik und
Poliklinik fur Kinderheilkunde, UKM, Munster, Germany.
3
Medizinische Klinik I, UK S-H Campus Lubeck, Lubeck,
661
Germany. 4Peninsula Medical School, Universities of
Aim: Statins have been shown to attenuate IFN-gamma
stimulated HLA-DR expression on various cells types. There
are no reports investigating effects of statins on intestinal
epithelial cells (IECs). In this study we analyse the influence
of statins on induced HLA-DR expression on IECs and
evaluate toxic effects on these cells.
Methods: HT29 cells were grown unstimulated or stimu-
lated with hrIFN-gamma (50 U/ml) for 72 h. Cells were
simultaneously incubated with atorvastatin (040 mM) or
simvastatin (040 mM). Co-incubation with mevalonic acid
(100400 mM) served to identify statin specific effects. Cell
surface expression of HLA-DR, cell proliferation rates and
viability using propidium iodide staining of unfixed or
ethanol fixed cells were assessed by flow cytometry.
Results: HLA-DR expression, constitutively absent on HT29
cells, was markedly upregulated by IFN-gamma. Ator- (0.1
10.0 mM) as well as simvastatin (0.13.0 mM) reduced IFN-
gamma stimulated HLA-DR expression on HT29 cells. The
effect was most pronounced at 10.0 mM for atorvastatin and
3.0 mM for simvastatin. Unexpectedly higher concentrations
of both statins were less effective in lowering IFN-gamma
induced HLA-DR and even further increased the induced
HLA-DR expression. Co-incubation with mevalonic acid
completely abrogated all statins effects, thus indicating the
decrease as well as the exacerbation of HLA-DR to be
mediated by the inhibition of HMG-CoA reductase activity.
Except of simvastatin used at 40 mM, atorand simvastatin did
not affect the proliferation and viability of HT29 cells.
Summary: Low concentrations of Atorvastatin and Simvas-
tatin reduce IFN-gamma mediated HLA-DR expression on
HT29 cells. Unexpectedly, higher concentrations of these
statins not only seemed to be less effective in attenuating
HLA-DR stimulation but they even further increased the
effects of IFN-gamma. Our results indicate that the effects
observed are not related to toxic properties of statins on HT29
cells.
Conclusion: The immunmodulatory action of statins on IECs
in combination with its influence on other immunocompetent
cells may be used as antiinflammatory tool in therapeutic
strategies for inflammatory bowel disease in the future.
However increased HLA-DR expression on IECs due to high
concentrations of statins may be in part responsible for gut
associated side effects after statin medication.
PG5-01 TNF PROMOTER POLYMORPHISMS MODULATE
GROWTH RETARDATION AND DISEASE SEVERITY
IN PEDIATRIC CROHNS DISEASE A Levine1,
R Shamir2, E Wine1, B Weiss3, R Shaoul4, S Reif5,
E Leshinsky-Silver6. 1Pediatric Gastroenterology Unit, E.
wolfson Medical Center, Holon, Israel. 2Pediatric Gastroen-
terology Unit, Meyer Childrens Hospital, Haifa, Israel.
3
Pediatric Gastroenterology Unit, Safra Childrens Hospital,
Tel Aviv, Israel. 4Pediatric Gastroenterology Unit, Bnei Zion
Medical Center, Haifa, Israel. 5Pediatric Gastroenterology
Unit, Dana Childrens Hospital, Tel Aviv, Israel. 6Molecular
Biology Lab, Wolfson Medical Center, Holon, Israel.
Background and Aims: Delayed growth is common in
pediatric Crohns disease ( CD). Multiple factors have been
shown to affect growth in this situation, the most prominent
being presence and severity of inflammation and inadequate
nutritional intake. Inflammation, anorexia and weight loss
may are all manifestations of circulating TNF alpha, which is
elevated in CD. The ability to secrete TNF alpha may be
affected by polymorphisms in the TNF alpha promoter. The
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
662 ESPGHAN 38TH ANNUAL MEETING
aim of our study was to determine whether growth retardation
and disease severity in pediatric onset Crohns disease are
affected by TNF promoter genotype.
Methods: Genotyping for TNF alpha single nucleotide
polymorphisms (SNPs) was performed in 87 patients with
detailed growth records. Parameters including disease
location, disease severity and were recorded, and the effect
of these polymorphisms on z-scores for height and weight at
disease onset and during follow-up until age 16 for height and
18 for weight were analyzed.
Results: Z scores for height ,1 S.D. occurred in 44% of
patients, ,2 SD in 19%. Lower age of onset was linked to
more height retardation, while presence of colonic disease
and absence of ileal disease were more likely to predict
absence of growth retardation (p = 0.004). Mean height scores
were increased with 238 G/A (p = 0.003) while Height
retardation was reduced by two polymorphisms reported to
decrease circulating TNF(2238 G/A, 2863 C/A).The effect
on height was not associated with lower z scores for weight.
Disease severity was modestly associated with the pro-
inflammatory polymorphisms 2308 G/A and inversely
associated with 857 C/T.
Conclusion: Polymorphisms in the TNF alpha promoter may
modulate disease severity and growth retardation in pediatric
onset CD, though this effect appears to be modest. The effect
of genotype on growth retardation is not associated with TNF
mediated weight loss.
PG5-02 THE 2174 G/C INTERLEUKIN-6 POLYMORPHISM
DETERMINES GROWTH FAILURE IN PAEDIATRIC
ONSET CROHNS DISEASE A Sawczenko1, M Idestrom2,
AB Ballinger1, NM Croft1, IR Sanderson1. 1Adult and
Paediatric Gastroenterology, Barts and The London, London,
United-Kingdom. 2Astrid Lindgren Childrens Hospital,
Stockholm, Sweden.
Background: Growth impairment is a significant clinical
problem for children with Crohns disease. The illness is
characterised by raised levels of Interleukin-6 (IL-6). In
a rodent colitis model, we have previously shown that IL-6
mediates growth failure, with reversal by an anti-IL-6
antibody. The IL 6 2174 G.C polymorphism is reported to
affect IL-6 transcription. We therefore hypothesised that the
IL-6 GG genotype would induce growth failure in children
with Crohns disease, as this genotype is associated with
increased IL-6 transcription compared to GC and CC
genotypes.
Patients and Methods: The IL-6 2174 genotype was typed
in 153 Northern European Caucasian children with Crohns
disease and height and clinical data abstracted from case
records. Height at diagnosis was available in all cases and for
a subset of 66 that had finished growing. Plasma C reactive
protein (CRP) level, at diagnosis, was used as an indirect
measure of circulating IL-6 activity. Genotypes of the cases
were compared to 351 controls without Crohns disease.
Results: At diagnosis children were significantly growth
impaired (p = 0.012). However, children with the IL-6 GG
genotype were more growth retarded (Height standard
deviation score 0.51 vs. 0.1; p = 0.031) and had higher
CRP levels (36 vs. 18 mg/dl; p = 0.037) than the GC or CC
genotypes. Final adult height of the GG genotype cases was
also less than the GC or CC cases (p = 0.053). The frequency
of the IL-6 genotypes in the cases was the same as in controls
(p = 0.7).
Conclusions: These results suggest that the IL-6 2174
genotype determines growth failure, but it is not associated
with a risk of developing the disease. These findings, if
replicated in a distinct population, will target which children
require growth-sparing therapy.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
PG5-03 GENOTYPE AND PHENOTYPE ANALYSIS IN PEDIAT-
RIC-ONSET INFLAMMATORY BOWEL DISEASE AND
A COMPARISON TO ADULT-ONSET INFLAMMATORY
BOWEL DISEASE. L De. Ridder1, PCF Stokkers2,
MA Benninga1, JAJ Taminiau1, I Pronk2, JH Van
Deventer2, DW Hommes2. 1Emma Childrens Hospital/Aca-
demic Medical Center, Amsterdam, The Netherlands. 2Aca-
demic Medical Center, Amsterdam, The Netherlands.
Aim: Genetic susceptibility probably plays a more important
role in early-, than in late-onset IBD. If so, a higher frequency
of the NOD2/CARD15 gene mutation can be expected in
pediatric CD patients. We determined the frequency of the 3
major NOD 2/CARD15 and 2 TLR 4 mutations in a Dutch
pediatric IBD cohort. Thereby, genotype and phenotype of
pediatric-onset and adult-onset IBD were compared.
Methods: Genomic DNA from 62 pediatric IBD patients
(45 CD, 16 UC, 1 indeterminate) and 472 adolescent CD
patients was screened for the R702W, G908R and 1007fs loci
of NOD2/CARD15 gene. The Asp299Gly and Thr399Ile
polymorphisms in TLR4 were assessed for the pediatric
cohort and an adult cohort of 407 CD patients and 226 UC
patients. The screening was accomplished by a PCR and
restriction fragment-length polymorphism assay, with spe-
cific primers and restriction enzymes for each polymorphism.
Phenotypic data were assessed.
Results: In the pediatric CD cohort 56,8% had ileal disease
localization versus 63,3% of the adult CD patients. Colonic
involvement was seen in 68,2% of the pediatric CD patients
and in 37,9% of the adults. In the pediatric cohort carriage
of two mutated alleles was associated with ileal disease
(x2 = 6.3, p = 0.012).
Homozygosity for the 1007fs mutation was noted in 6,5% of
the pediatric cohort whereas 0,4% of the adult patients
were homozygous (x2 = 16,1 p , 0.0004). Three patients of
the pediatric cohort (6,5%) were compound heterozygote
compared to 20 adult CD patients (4,2%). In total 7 (15,5%)
pediatric CD patients and 36 adult CD patients (7,6%)
were either compound heterozygous or homozygous for
NOD2/CARD15 mutations. Three out of 16 pediatric UC
patients were heterozygous for the two TLR4 polymorphisms
versus 25 of the 226 adult UC patients tested. These
differences were not statistically significant.
Conclusion: We observed a higher frequency of the CD
related genotypes in the pediatric cohort, although this find-
ing was statistically significant only for the 1007fs mutation.
PG5-04 COLONIC SALVAGE IN FULMINATING STEROID
RESISENT ULCERATIVE COLITIS USING ANTI-CD25
THERAPY I Ricciardelli1, S Kirkham1, M Furman1,
M Elawad1, N Shah1, P Milla1, M Londei1, K Lindley1.
1
Institute of Child Health and Great Ormond Street Hospital
for Children, London, United-Kingdom.
15% of individuals with ulcerative colitis (UC) present at
some stage with a severe exacerbation of whom 6% fail to
respond to intensive therapy with intravenous steroids and
cyclosporine. Surgery in these individuals has a mortality of
approximately 3%. CD25 is an activation marker for T cells
(expressed on TH1, TH2 and TReg cells but not TH3 and TR1
cells) which drive the maladaptive immune response in UC.
Aim: To assess the efficacy of anti-CD25 therapy in acute
steroid and cyclosporine resistant ulcerative colitis.
Methods: 3 children (aged 1416 years) with fulminating
UC unresponsive to combination therapy with 10 days of IV
methylprednisolone (1.7 mg/kg) and 7 days IV cyclosporine
(24 mg/kg/day) (Truelove and Witts criteria) were studied at
 ESPGHAN 38TH ANNUAL MEETING
a single centre. Pre-treatment colonoscopic biopsies were
taken for histology (scored blindly), CD25 immunostaining
and cytokine PCR and peripheral blood lymphocyte subsets
were determined. Humanised chimeric anti-CD25 therapy
(Basiliximab) was administered as 2 doses of 2g given
14 days apart and repeat colonoscopic biopsies obtained
between days 2128.
Results: 3/3 individuals responded promptly within 48 hours
to anti-CD25 with cessation of gastrointestinal bleeding
diarrhoea and abdominal pain and improvement in inflam-
matory markers and albumin. Peripheral blood CD25 +ve
lymphocytes were undetectable within 48 hours. Colonic
histology was normalised at follow up biopsy and CD25
staining (strongly positive pre Basiliximab) was negative
following Basiliximab. Complete ensdoscopic and histologicl
mucosal healing was achieved in all within 2128 days.
Summary: 3/3 children with drug resistant fulminating UC
responded favourably to anti-CD25 therapy with improved
clinical scores, improved histological scores and favourable
alterations in mucosal cytokine profiles.
Conclusion: Anti-CD25 therapy is efficacious in steroid and
cyclosporin resistant fulminating UC, prevents the need for
emergency colectomy and results in rapid mucosal healing.
Early use of anti-CD25 in fulminating UC appears justifiable.
PG5-05 EARLY BACTERIAL INFECTIONS AND CAESAREAN
SECTION AS RISK FACTORS FOR CROHNS DISEASE
H Hildebrand1, P Malmborg1, SM Montgomery2. 1Paediatr.
Gastro Units, Karolinska University Hospital, Stockholm,
Sweden. 2Clinical Epidemiology Unit, Department of
Medicine, Karolinska Hospital, Karolinska Institutet, Stock-
holm, Sweden.
Aim: To evaluate if infections in early life and Caesarean
section, both known to effect bacterial colonisation of the
colon, may increase the risk of Crohns disease (CD) later in
life.
Methods: 1098 patients with CD and 6550 matched controls
born between 1973 and 1997 were investigated for infections
during their first five years of life. The diagnosis of
pneumonia, otitis media, cystitis and sepsis were analyzed.
In a subset of the material (pediatric onset only) association
between Caesarean section and CD was analyzed. The
patients were identified through Swedish population registers
and prospectively recorded data from the Medical Birth and
Inpatient Registers were analyzed using conditional logistic
regression. Linkage between registers was possible using the
unique individual identity number that is issued to all
Swedish residents from birth.
Results: Pneumonia by age five years was statistically
significantly associated separately with both pediatric and
adult CD, odds ratio: 2.74 (1.047.21) and 4.94 (1.8313.34)
respectively, with 95% confidence interval. No association
with pneumonia after age 5 years was found. Other infections
were not associated with CD. Birth by Caesarean section and
CD was statistically significantly associated with odds ratio
of 3.00 (1.048.69).
Discussion: Early exposure to micro-organisms may in-
fluence subsequent immune functioning, including the host
immune response to gut flora. The first colonisation occurs in
the birth canal and Caesarean section may influence the
pattern of colonisation. Severe bacterial infections by their
self or through antibiotic therapy have effects on intestinal
bacteria.
Conclusions: Caesarean section and pneumonia in early life
were associated with subsequent CD disease. Disruption of
bowel colonisation is one hypothesis to explain these
findings.
663
PG5-06 GUT ASSOCIATED BACTERIAL MICROFLORA IN
PATIENTS WITH INFLAMMATORY BOWEL DISEASE
(IBD) O Borrelli1, MP Conte2, S Schippa2, I Zamboni2,
A Callari2, F Chiarini2, L Seganti2, S Cucchiara1. 1Pediatric
Gastroenterology Unit, University of Rome La Sapienza,
Rome, Italy. 2Department of Public Health, University of
Rome La Sapienza, Rome, Italy.
Patients (pts) with IBD have higher amounts of bacteria
attached to their intestinal epithelial surfaces than do healthy
people. Although increasing evidence suggests that intestinal
flora is involved in the initiation and amplification stages of
IBD pathogenesis, and the presence of intestinal bacteria is
essential in animal models of colitis, to date no specific
bacterial pathogens have been identified the precise role of
intestinal bacteria in IBD remains elusive.
Aim: Investigation of the predominant mucosa-associated
bacteria in ileum, colon and rectum biopsies obtained during
colonoscopy of IBD pts and controls.
Methods: 36 pts (age range: 216 years) (9 with Crohns
disease (CD), 5 with ulcerative colitis (UC), 5 with
indeterminate colitis (IC), 10 with ileal lymphonodular
hyperplasia (LNH) and 7 controls) were studied. Colono-
scopic biopsies were taken from terminal ileum, colon and
rectum and processed according to Swidsinski et al.
(Gastroenterology 2002;122:44). Briefly, mucosal flora was
investigated by conventional culture technique (culture for
aerobic and anaerobic-facultative bacteria was obtained from
all pts) and PCR procedures based on 16S rRNA gene
sequences specific for anaerobic bacteria predominating in
the human intestinal tract.
Results and Summary: Concentration of aerobic and
anaerobic facultative bacteria (mean 6 SD (3 103 cfu/ml)
of bacteria detected after lysis) was markedly higher in IBD
(CD: 269.9 6 279, p , 0.03; UC: 125.2 6 137, p , 0.04; IC:
408.3 6 249; p , 0.01) than in controls (5.9 6 15). A high
heterogeneity of species was found in ileal mucosa of CD pts,
but not among other IBD groups. The main gram-negative
aerobes found were Enterobacteriaceae (primarily E.Coli);
the main gram-positive aerobes were Streptococcus spp and
Staphylococcus spp. Among anaerobic bacteria, there was an
overall decrease of species in IBD biopsies; Bacteroides
vulgatus was always undetectable in CD: this is in agreement
with a suggested protective role of this species against E. coli-
induced colitis (Gastroenterology. 2003;125:162).
Conclusions: This is the first report on mucosa-associated
bacteria in pediatric pts with IBD. Our data, according to
previous reports (Gut 2004;53:1), support the concept of
dysbiosis, that is breakdown balance between putative
species of protective versus harmful intestinal bacteria.
Knowledge of differences in bacterial flora among IBD pts
and healthy subjects could be useful for therapeutic
manipulation of the gut flora through probiotic or prebiotic
strategies.
PG5-07 INCREASED RATIO IFN GAMMA/TGF BETA IN THE
INTESTINE OF UNTREATED COELIAC DISEASE
PATIENTS A Leon1, JA Garrote1, A Blanco-Quiros1,
C Calvo2, L Fernandez-Salazar2, A Del Villar3, A Barrera3,
E Arranz1. 1Dept Pediatrics & Immunology-University of
Valladolid, and Institute of Genetics and Molecular Biology,
Valladolid, Spain. 2Hospital Clinico Universitario, Vallado-
lid, Spain. 3Hospital Universitario Rio-Hortega, Valladolid,
Spain.
Cytokines have a central role in the regulation of the
intestinal immune response to gluten. Previous studies have
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
664 ESPGHAN 38TH ANNUAL MEETING
used ex vivo gluten stimulation of either biopsies or isolated
T cell subpopulations, and age differences have not been
considered.
To study differences in cytokine expression in non-stimulated
small intestinal biopsies from children and adult CD patients,
and to investigate whether other cytokines may be responsible
for TH1 differentiation and IFNg production in absence of
IL12.
Specimens from 40 CD patients (16 adults, 24 children), and
25 controls (17 with normal intestine, 8 with altered histol-
ogy). Quantitative PCR was used to determine mRNA expres-
sion of IFNgamma, IL12p35/p40, IL4, IL23, TNFalpha,
TGFbeta1, and IL-10.
Untreated CD patients show higher IFNgamma levels (p ,
0.05) and IFNgamma/TGFbeta ratio (p , 0.001) than
controls, and these have higher TGFbeta1 expression than
untreated CD patients (p , 0.05). IFNgamma levels show
a positive correlation with the degree of mucosal lesion
(p , 0.02). The cytokine pattern was similar in adults and
children with CD.
The cytokine pattern in untreated CD patients is characterized
by an increased IFNgamma/TGFbeta1 ratio, similar in adult
and children with CD. This ratio may reflect an altered
immunoregulation within the coeliac intestine.
PG5-08 THE LINK BETWEEN COELIAC AND OTHER AUTO-
IMMUNE DISEASES: THE PUZZLING QUESTION
G Guariso1, S Conte1, F Brotto1, F Presotto2, S Chiarelli3,
L Zancan1, L Freo1, C Betterle2. 1Department of Pediatrics,
University of Padua, Padua, Italy. 2Department of Medical
and Surgical Sciences, University of Padua, Padua, Italy.
3
Department of Pathology, University of Padua, Padua, Italy.
Aim: Study of the prevalence of autoimmune (AI) disorders
among children at diagnosis of coeliac disease (CD) and after
gluten free diet (GFD).
Methods: We enrolled 236 children (152 F, 84 M; mean age
66 6 48 months) with CD diagnosed by serological markers
and typical hystological lesions. 149 (96 F, 53 M; mean age
61 6 44 months) were followed for a mean time of 43 6 30
months (range 12118). All were in complete remission on
a GFD. 220 age-matched children who underwent gastros-
copy for dyspepsia or abdominal pain served as controls. All
had normal intestinal histology and no serological markers of
CD. All patients and controls were tested for the presence of
organ and non-organ specific autoantibodies (Abs) and were
investigated for the presence of AI diseases. Statistical
analysis was performed by Chi-square test. A p value ,0.05
was considered to be significant.
Results: Of the 236 children, 54 (22.9%, 41 F, 13 M) had
clinical or latent (i.e., only serological) AI disorders at CD
diagnosis vs 1.4% of controls (p , 0.0001). 24 (10.2%) had
clinical AI diseases: 10 had type 1 diabetes mellitus (T1DM),
6 Hashimotos thyroiditis (HT), 2 HT+T1DM, 2 juvenile
chronic arthritis, 2 alopecia, 1 vitiligo, 1 AI thrombocytope-
nia, while 30 (12.7%) had Abs without clinical manifes-
tations: 12 had antinuclear Abs (ANA), 10 thyroid Abs, 4
parietal cell Abs (PCA), 2 PCA+ANA, 1 PCA+ islet cell Abs
(ICA), 1 ICA+ANA. Among the 149 children followed, 37
(24.8%) had AI disorders at diagnosis of CD. 16 patients
(10.7%) had clinical AI diseases, while 19 (12.7%) had latent
AI disorders. At the end of the follow-up, 48 (32.2%) had
clinical or latent AI disorders (p = 0.2): 14 (9.4%) had clinical
(p = 0.8) and 34 (22.8%) had latent autoimmunity (p = 0.07).
Summary: The prevalence of AI disorders is significantly
increased among children at diagnosis of CD. Over a period
of GFD, clinical AI diseases did not increase, while latent
autoimmunity showed a trend to increase.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
Conclusion: The prevalence of AI disorders is much higher
in children at diagnosis of CD. GFD seems to hold back the
development of new clinical AI diseases despite an increase
in serological autoimmunity.
PG5-09 IS THE INTESTINAL BARRIER FUNCTION INFLU-
ENCED BY GLUTEN INTRODUCTION AT WEANING?
PRELIMINARY RESULTS OF A PROSPECTIVE, RAN-
DOMIZED STUDY. R Buglia1, L Zampini1, G Natalini1,
A Sapone2, D Kryszak2, GV Coppa1, A Fasano2, C Catassi1.
1
University Department of Pediatrics, Ancona, Italy. 2Mu-
cosal Biology Research Center, University of Maryland,
Baltimore, USA.
Background: Recent data suggest that gluten peptides can
increase the para cellular permeability of the small intestine
through a zonulin mediated process. This increased intestinal
permeability could trigger the immunological cascade of
events in the lamina propria eventually leading to the celiac
enteropathy.
Aim of the Study: To investigate the relationship between
cereal introduction at weaning and intestinal permeability in
healthy children at family risk of food allergy.
Study Design, Patients and Methods: This is a prospective,
randomized study on healthy infants. After receiving only
milk (breast or artificial) during the first four months of life,
all infants underwent a small intestinal permeability (SIP) test
and a serum zonulin assay (T0). They were then randomised
to: Group 1 (n = 5): continued milk feeding; Group 2 (n = 5):
weaning with gluten containing cereals; Group 3 (n = 5):
weaning with gluten free cereals. After three weeks of this
dietary regimen (T1) infants underwent the same evaluation
than at T0. SIP was evaluated by measurement of 1-hour
serum lactulose/mannitol (l/m) after oral load, and sugars
were measured by an HPLC method. Serum zonulin was
measured by an ELISA method.
Results: At T0 the mean l/m was 0.065 6 0.048 in Group 1;
0.085 6 0.025 in Group 2; 0.092 6 0.051 in Group 3. At T1
they were 0.096 6 0.042 in Group 1; 0.097 6 0.059 in Group
2; 0.082 6 0.02 in Group 3. At T0 the mean serum zonulin
(ng/mL) was 0.26 6 0.18 in Group 1; 0.62 6 0.34 in Group 2;
0.35 6 0.08 in Group 3. At T1 zonulin levels were 0.3 6 0.15
in Group 1; 0.52 6 0.25 in Group 2; 0.42 6 0.4 in Group 3.
Conclusions: So far we did not find significant differences in
the intestinal barrier function between 5-months old infants
receiving exclusive milk feeding or weaned with either
gluten-containing or gluten-free cereals. A wider sample of
patients will be required to rule out minor changes between
different dietary groups.
PG5-10 SALIVARY ANTI-TRANSGLUTAMINASE AUTOANTI-
BODIES IN COELIAC DISEASE AT THE DIAGNOSIS
AND DURING FOLLOW-UP R Nenna1, C Tiberti2,
M Ferri1, E Thanasi1, S Mura1, A Verrienti2, B Fiore2,
M Bonamico1. 1Department of Paediatrics, La Sapienza
University, Rome, Italy. 2Department of Clinical Sciences,
La Sapienza University, Rome, Italy.
Aim: We have already demonstrated that saliva, a biological
fluid easily obtainable without any discomfort, can be used to
screen coeliac disease (CD) children (J Pediatr, 2004;144:
6326). The aim of this study was to evaluate salivary tTGAb
presence on a large series of CD patients at diagnosis and
during follow-up on a gluten-free diet.
Methods: 76 coeliacs on a free diet at the first biopsy (Group
1a: 27m; 1.926.8 yrs), 28 of them on a gluten-free diet of at
least 6 mos (Group 1b: 13m, 225.7 yrs), 74 gastroentero-
logical controls (Group 2: 35m; 1.318 yrs) and 40 healthy
 ESPGHAN 38TH ANNUAL MEETING
controls (Group 3: 11m; 21.941.5 yrs) were enrolled in the
study. The ROC analysis was used to detect the limit of
positivity of tTGAb saliva method. IgA-tTGAb presence on
serum and saliva of each subject were detected with a fluid-
phase radioimmunoprecipitation method. IgAEMA were
tested by indirect immunofluorescence method.
Results: Percentages of Ab positivity in CD children are
shown in the table:
Salivary tTGAb index+ Serum tTGAb index+
% (mean 6 SD) % (mean 6 SD) EMA
Group 1a 96% (0.41 6 0.41) 98.7% (0.78 6 0.33) 92%
Group 1b 71.4% (0.10 6 0.12) 64.2% (0.19 6 0.27) 34.4%
Mean salivary or serum tTGAb indexes were significantly
lower in Group 1b respect to Group 1a coeliac patients (p ,
0.0001). All Group 3 subjects were found EMA and tTGAbs
negative, both in saliva and serum assays.
Conclusion: This study confirms that it is possible to detect
salivary tTGAbs in CD subjects with high sensitivity, and in
particular also after beginning of the gluten-free diet. The
sensitivity of the salivary detection in coeliac patients on
a gluten-free diet appears to be even higher respect to that
found using serum. Salivary tTGAb presence could therefore
reveal a non strict adherence to the diet, particularly frequent
in adolescents, with possible nutritional and immunological
implications. The salivary tTGAb index reduction could be
a useful tool to monitor the diet of coeliac subjects.
PG5-11 SUSCEPTIBILITY TO DEAMIDATION BY TISSUE
TRANSGLUTAMINASE AS A TOOL TO IDENTIFY
IMMUNOGENIC GLIADIN PEPTIDES IN THE WHOLE
GLIADIN EXTRACTS G Mamone1, A Camarca1,
F Addeo2, L Longobardo2, P Ferranti2, S Auricchio3,
R Troncone3, C Gianfrani1. 1Institute of Food Science and
Technology -CNR, Avellino, Italy. 2Dept Food Science,
University of Naples, Federico II, Naples, Italy. 3Dept Pediatrics
and European Laboratory for the Investigation of Food-Induced
Diseases, University of Naples, Federico II, Naples, Italy.
Aim: Celiac disease (CD) is an enteropathy of the small
intestine induced by dietary gliadin. Tissue transglutaminase
(tTG) plays a direct role in the pathogenesis of CD by
deamidating specific glutamine (Q) residues of gliadin
peptides and increasing their immunogenicity. Based on
tagging with a monodansylcadaverine fluorescent probe and
nanospray tandem mass spectrometry, we previously identified
6 peptides containing tTG-susceptible Q (Q) residues within
a pepsin-trypsin digest of wheat gliadins (see Table) [1].
These six gliadin/glutenin peptides and the immunodominant
33-mer (a-gliadin 5789) peptide were synthesized and tested
for recognition by CD patients.
Methods: Gliadin-specific T cells lines (TCL) were gener-
ated from intestinal mucosa of 4 adult CD patients. Both
proliferation and cytokine (g-IFN, IL-4, IL-2, IL-10) pro-
duction were analyzed in response to either native or tTG-
treated peptides.
Results: The 18-mer, 25-mer and 14-mer-1 peptides were
recognized by TCLs from all 4 CD patients. 25-mer peptide
entirely contained the 18-mer and had a high sequence
homology with the 33-mer. 18-mer, 25-mer, and 33-mer
induced proliferation, g-IFN and IL-10 productions in either
native and deamidated forms, these latter being more potent
in stimulating proliferation: (SI 6 SD) 17.1 6 5.4 vs 8.1 6
665
9.7; 17.0 6 4.2 vs 7.9 6 9.1; 20.2 6 3.2 vs 9.9 6 11.4;
and g-IFN production: (net value 6 SD) 3449 6 1788.3 vs
402.5 6 72.8; 3536 6 1530 vs 607 6 346.5; 3511 6 1735 vs
765.5 6 317.5pg/ml, respectively. By contrast, IL-2 and very
low IL-4 were produced only in response to the tTG-treated
peptides. 14-mer-1 peptide was active only after the tTG-
treatment and exerted a lower stimulatory capacity compared
to 18-mer and 25-mer. No responses were observed against
the 14-mer-2, 19-mer, and 21-mer despite the tTG-treatment.
Summary: A high percentage (3 out 6, 50%) of gliadin
peptides naturally occurring in the extracts of whole gliadins
and identified on the basis of their susceptibility to be tTG
substrate, were recognized by CD patients. The tTG-treated
peptides were largely more immunogenic than in native form.
Conclusion: This study offers an important tool to detect
within wheat cultivars and other edible cereals, the immuno-
logically active gliadin peptides relevant for CD pathogenesis.
1. Mamone G. et al. FEBS letter 2004;62:177.
Parent protein &
Peptide Amino acid sequence peptide extension
18-mer QPQPY PQPQL PYPQ PQPF a/b-gliadin 7289
25-mer PQLPQFL QPQPY PQPQL a/b-gliadin 6689
PYPQ PQPF
14-mer-1 PQQQTL QPQQ PAQL g-gliadin 105117
14-mer-2 PQQPFP SQQQQ PLI g-gliadin 173185
19-mer LGQQQ PFPPQ QPY PQPQPF a/b-gliadin 3149
21-mer SHILG PERP SQQQ PLPPQQTL LMW-glutenin 1939
PG5-12 AUTOANTIBODIES AGAINST SOLUBLE AND IMMO-
BILIZED HUMAN RECOMBINANT TISSUE TRANS-
GLUTAMINASE IN CHILDREN WITH CELIAC DISEASE
D Agardh1, I Dahlbom2, T Daniels3, SA Ivarsson1,
Lernmark4, T Hansson2. 1Department of Pediatrics,
A
Malmo University Hospital, Lund University, Malmo,
Sweden. 2Pharmacia Diagnostics, Uppsala, Sweden. 3RH
Williams Laboratory, Department of Medicine, University of
Washington, Seattle, USA. 4Department of Endocrinology,
Wallenberg Laboratory, Lund University, Malmo, Sweden.
Aim: The conformation of tissue transglutaminase (tTG)
might influence the performance of immunoassays that are
used to detect autoantibodies in celiac disease (CD). We
investigated how recombinant human tTG kept in a liquid
phase or fixed to a solid support affected the binding of IgA
and IgG autoantibodies from children with untreated and
treated CD.
Methods: Included were 50 controls, 73 untreated and 80
treated CD children. IgA-tTG and IgG-tTG were measured
with solid phase ELISA and liquid phase radioligand binding
assays (RBA). Detection of IgG-tTG with RBA was made
both with anti-IgG sepharose and protein A-sepharose (PAS).
Results: The ELISA and RBA detected IgA-tTG in the same
65/73 untreated CD children and 2/50 controls. One
additional control child was detected with RBA alone. The
agreement between the two IgA-tTG assays was 99%.
The ELISA, RBA and PAS detected IgG-tTG in the same
40/73 untreated CD children and 2/50 controls. The PAS
assay detected elevated antibodies in 20/73 additional
untreated CD children and in one control child. The
concordance was 95% between IgG-ELISA and IgG-RBA,
83% between IgG-ELISA and PAS, and 75% between IgG-
RBA and PAS. However, when the PAS results were
compared to those obtained with IgA-RBA a 99% agreement
was found.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
666 ESPGHAN 38TH ANNUAL MEETING
Of the treated CD children, RBA detected 21/80 subjects with
elevated IgA-tTG whereas only 3/80 children were detected
with ELISA. A total of 6/21 treated CD children with
increased IgA-tTG were also found to have elevated IgG-tTG
that were detected with IgG-ELISA, IgG-RBA or PAS.
Summary: The RBA and ELISA bound IgA-tTG and IgG-
tTG from untreated CD children equally well. However, the
PAS assay seemed not to assess IgG antibodies only.
Discrepancies in the binding of IgA anti-tTG from a subgroup
of treated CD children indicated that alterations of tTG might
occur upon fixation of the antigen.
PG5-13 SENSITIVITY AND SPECIFICITY OF SIMPLE AND
RAPID COMMERCIAL ASSAYS FOR THE DIAGNOSIS
OF COELIAC DISEASE. G Nemec1, S Martelossi1,
V Baldas1, S Gamen2, E Bravi3, A Tommasini1, T Not1,
A Ventura1. 1Pediatric Department, University of Trieste,
IRCCS, Trieste, Italy. 2Operon s.a., Zaragoza, Spain. 3Euro-
spital S.p.A., Trieste, Italy.
Background: Given the high prevalence of the coeliac disease
(CD) and its protean nature a simple rapid diagnostic test
would represent a great step forward in the diagnosis of CD.
Aims: We evaluated two rapid immunochromatographic
assay (Stick-CD1 and Simple-CD1-WB, Operon s.a., Zara-
goza, Spain) for measuring the IgA-IgG-IgM anti-human-
transglutaminase antibodies (anti-h-tTG Ab) in the diagnosis
of CD, suitable for use by the general paediatrician in the
ambulatory setting. The sensitivity and specificity of this test
were compared with those of an enzyme linked immunosor-
bent assay (ELISA).
Methods: Serum sample were analyzed from: 43 healthy
controls and 31 diseased controls (20 Crohns disease, 5
ulcerative colitis, 6 failure to thrive) both tested negative by
ELISA assay, 75 biopsy confirmed CD patients tested
positive by ELISA test which were diagnosed between June
and November 2004. The sera were diluted 1/5, incubated for
5 minutes with Stick-CD strip and the colorimetric reaction
was promptly examined. Furthermore we analyzed 130 saliva
specimens from 16 biopsy proven celiac children and 114
children tested negative for anti-h-tTG Ab in which the CD
was excluded. The saliva was diluted 1/2, incubated for 5
minutes with Stick-CD1 strip and the colorimetric reaction
was promptly examined. We consecutively examined a whole
blood drop from 35 biopsy proven CD patients (tested
positive by ELISA) and 117 healthy controls (tested negative
by ELISA) using the Simple-CD1-WB test. In this case the
colorimetric reaction was examined after 15 minutes. The
immunological test were performed by four operators who
were unaware of the clinical and laboratory findings of the
subjects tested.
Results: The Stick-CD1 strip identified 73/75 CD serum
sample (sensitivity 97.4%) and 6/74 serum controls (speci-
ficity 92%) and it identified 13/16 CD patients (sensitivity
81.2%) and 0/114 controls (specificity 100%). The Simple-
CD1-WB test identified 32/35 CD whole blood samples
(sensitivity 91.4%) and all 117 controls were tested negative
(specificity 100%). The assays identified a CD child with total
IgA deficiency, by means the specific IgG.
Conclusions: The commercial assay described here are quick
and requires minimal handling. The test based on the serum
samples seems to produce an acceptable sensitivity for an
initial screening of CD at the medical office. The test based
on saliva also by using this assay is not a useful specimen for
detecting anti-h-tTG Ab. The test based on whole drop has
very high specificity but low sensitivity so further observation
on the reliability of this simple tool for the diagnosis of CD
are needed.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
PG5-14 CELIAC DISEASE AND PLACENTAL EXPRESSION OF
IGF-IR AND TTG IN RELATION TO INTRAUTERINE
GROWTH RETARDATION F Hadziselimovic1. 1Kinder-
tagesklinik Liestal, Liestal, Switzerland.
Introduction: Intrauterine growth retardation (IUGR) and
lower insulin like growth factor-I (IGF-I) levels predict
reduced compensatory insulin secretion and increased risk for
Type 2 diabetes in adulthood. Gluten ingestion in children
with celiac disease induce a significant decline in IGF-I
which was related to the degree of intestinal mucosa
inflammation. IUGR was found in newborns from mothers
with CD and those newborns from healthy mothers who later
in life developed CD(HCD); thus, gluten intake during the
pregnancy has a significant impact on the fetal growth.
Diminished expression of IGF-IR was found in villous
trophoblast of IUGR indicating that IGF-IR expression may
play an important role in the pathogenesis of IUGR. Aim of
the study was to analyse the expression of IGF-IR and tTG in
correlation to fetal growth and gluten ingestion.
Patients and Methods: 16 placentas from mother with CD, 6
placentas from HCD and 6 control placentas were analysed.
Biopsies from the centre and periphery of the placenta were
fixed in formaline and embedded in paraplast for histopatop-
logical examinations. Monoclonal antibodies for tTG and
IGF-IR were utilized for immunohistochemical staining and
the sections were analysed in blinded fashion.
Results: Six out of sixteen newborns from mothers with CD
had IUGR (median: 2676 g birth weight) 5 out of six mothers
did not to comply to gluten free diet during pregnancy.
Placentas from IUGR newborns had more frequently multiple
types of lesions; however, chronic villitis and increased
perivillous fibrin (immunological protective function) were
found frequently. There was a significant increase in
expression of tTG in the villous trophoblst of IUGR placentas
compared to the controls. In contrast, a diminished immu-
nostaining for IGF-IR was observed in IUGR villous
trophoblast.
Conclusion: Gluten intake resulted in chronic placental
inflammation and increased expression of tTG as a compen-
satory repair mechanism as well as a reduction of IGF-IRs
and IUGR indicating an increased risk for developing
a metabolic syndrome in adulthood and development of
growth hormone abnormalities in childhood.
PG5-15 BETTER RECOGNITION OF CHILDHOOD COELIAC
DISEASE IN THE NETHERLANDS AND ITS CHANG-
ING CLINICAL PICTURE: A NATIONAL PROSPECTIVE
STUDY 19932000 RFR Steens1, C Csizmadia1, E George1,
M Ninaber1, R Hirasing2, ML Mearin1, ML Mearin3.
1
Paediatric Gastroenterology, University Medical Centre,
Leiden, The Netherlands. 2TNO-Prevention and Health,
Leiden, The Netherlands. 3Paediatric Gastroenterology, Free
University, Amsterdam, The Netherlands.
Retrospective studies showed an increasing incidence of
diagnosed childhood coeliac disease in the Netherlands from
19751990. In this period the most frequent symptoms were
chronic diarrhoea, abdominal distension and failure to thrive.
Aim: To investigate prospectively whether the incidence of
diagnosed disease is still increasing and whether the clinical
presentation has changed.
Methods: All newly diagnosed cases of childhood coeliac
disease throughout the Netherlands were registered pro-
spectively from 1993 to 2000 by means of the Dutch Pediatric
Surveillance Unit (NSCK). The diagnoses of coeliac disease
19932001 were ascertained by means of the data of the
Dutch National Database of Pathology (PALGA). The
 ESPGHAN 38TH ANNUAL MEETING
clinical picture of coeliac disease was compared to the one in
19751990.
Results: We found an overall incidence rate of 0.85/1000 live
births with a significant linear increase from 19932000, as
well as from 19751990. In 19932000 there was a significant
increase in the diagnoses with partial villous atrophy of the
small bowel mucosa. The symptoms at presentation have
significantly changed in comparison to 19751990: abdom-
inal distension, chronic diarrhoea and failure to thrive (,P10
height and weight) have significantly decreased, but more
children presented with weigth loss (,P10 for height),
abdominal pain and lassitude. There is a significant lineair
trend in the median age at diagnosis in the period 19932000.
Conclusion: The incidence of diagnosed childhood coeliac
disease in the Netherlands has significantly increased during
the last years and the clinical picture has changed as well.
Both phenomenons are most likely being caused by the
increasing awareness of the Dutch pediatricians and their
ability to recognise subtler expressions of the disease.
PG5-16 GENETIC RISK OF FIRST DEGREE RELATIVES OF
COELIAC PATIENTS MG Limongelli1, O Esposito2,
C Marano3, L Sacchetti4, N Tinto5, D Gennarelli6,
R Troncone7, L Greco8. 1Department of Pediatrics and
European Laboratory for Food Induced Diseases, University
Federico II, Naples, Italy. 2Department of Pediatrics and
European Laboratory for Food Induced Diseases, University
Federico II, Naples, Italy. 3Department of Pediatrics and
European Laboratory for Food Induced Diseases, University
Federico II, Naples, Italy. 4Department of Biochemistry and
Biotechnology, University Federico II, Naples, Italy. 5De-
partment of Biochemistry and Biotechnology, University
Federico II, Naples, Italy. 6Department of Biochemistry and
Biotechnology, University Federico II, Naples, Italy. 7De-
partment of Pediatrics and European Laboratory for Food
Induced Diseases, University Federico II, Naples, Italy.
8 significant between the two groups. Hospital stay in the
Department of Pediatrics and European Laboratory for
Food Induced Diseases, University Federico II, Naples, Italy.
Aim: 45 to 60% of first degree relatives share the genetic risk
of Class II HLA DQ2 or DQ8 with their affected relatives.
Jeanin and coll. (Tissue Antigen 2004; 63: 5627) recently
showed that the genetic risk associated to HLA DQ2
(DQA1*05 DQB1*02) is not the same for all patients.
They proposed to stratify the patients in 5 risk classes,
according to the linkage of DQ with DR: G1 with DR3/3-
DR3/7 risk = 1; G2 with DR5/7 risk = 0.68; G3 with DR3/X
risk = 0.23; G4 with DR7/7-DR7/4-DR4/4 risk = 0.10; G5
with other DR risk = 0.02. The aim of the present study is to
evaluate the risk, derived from cases, to the first degree
relatives, to identify those with highest risk to develop the
disease since birth.
Methods: 205 families with at least one confirmed coeliac
were screened for Antiendomisium and anti-Transglutami-
nase antibodies, and Class II HLA status molecular staging
was performed.
Results: We screened 910 individuals: 205 celiac probands,
43 new cases identified by the serological screening and
confirmed by small intestinal biopsy. The prevalence of
coeliac disease in the first degree relatives is 43/705 = 6.1%,
obtained by 8/205 fathers (4%), 17/205 mothers (8.3%),
18/294 sibs (6.1%). The distribution of unaffected first degree
sibs into DR based G risk classes is: very high risk G1:
19/276 (6.9%); high risk G2: 38/276 (13.7%); moderate risk
G3: 43/276 (15.7%); low risk G4: 24/276 (8.8%); no risk G5:
152/276 (54.9%). We have identified 20.6% of sibs with a risk
above 40% to develop disease, 24.5% with a risk between 10
and 20%, 55% with practically no risk.
667
Conclusion: In first degree relatives of coeliac cases, by an
ordinary full molecular typing of HLA Class II, it is possible
to identify a cohort (about an half) with practically no risk to
develop the disease and a cohort of about 20% with a very
high risk (comparable to a mendelian recessive disease) to get
the disease. This study increases the chances to locate at birth,
in families with a know case, infants likely to develop
disease: strategies to anticipate the diagnosis and avoid the
clinical damage now are possible.
PG5-17 PRIMARY LAPAROSCOPIC-ASSISTED PULL-THROUGH
COMPARED WITH OPEN THREE-STAGE PROCEDURE
FOR HIRSCHSPRUNGS DISEASE A Vander Auwera1,
D Mekahli1, V Janssens1, F Penninckx1, M Miserez1,
I Hoffman1. 1University Hospitals Gasthuisberg, Leuven,
Belgium.
Background: Primary laparoscopic assisted pull-through
appears to become the new gold standard in the treatment of
Hirschsprungs disease (HD). We compared retrospectively
the early and medium-term results of the earlier open three-
stage technique with current primary laparoscopic assisted
pull-through (PT) in a group of matched patients.
Methods: Eight patients (7 boys, 1 girl) with HD operated on
by primary laparoscopic assisted Swenson pull-through have
been compared with eight patients treated by the open three-
stage Swenson procedure. Sex ratio, length of the aganglio-
notic segment, associated anomalies, familial occurrence and
preoperative enterocolitis are similar for the two groups. The
mean age and weight of the laparoscopic group at surgery are
respectively 4 months (range: 9 days to 2.5 years) and 6 kg
(range: 3.4 kg to 14.7 kg) versus 11 months (range: 4 to 13
months) and 9 kg (range: 6.2 kg to 11.3 kg) in the other group.
We have analysed operating time, hospital stay, early and
medium-term complications.
Results: The difference in operating time is not statistically
laparoscopic group (mean: 13 days; range: 1033) is
significantly shorter (P , 0,01) in comparison with the three
hospitalisations summed for the open group (mean: 28 days;
range: 1672). There was no need for conversion to
laparotomy. One patient needed a protective colostomy. Early
follow-up (0 to 3 months) of the laparoscopic group seems
more favourable than those of the standard procedure:
anastomotic stricture (2 vs. 3), enterocolitis (2 vs. 1), sub-
obstruction (1 vs. 2), anastomotic leak (0 vs. 1), wound
infection (0 vs. 2), stoma prolaps (0 vs. 4), reoperation (2 vs.
2), and others such as urinary infections, pneumonia, sepsis
caused by an indwelling catheter (0 vs. 5). In the medium
follow-up (3 months to 3 years), defecation difficulties seem
less frequent in the laparoscopic group: constipation (1 vs. 2),
soiling (3 vs. 4) and incontinence (0 vs. 2). Late follow-up
(more than 3 years) is too short in the laparoscopic group to
conclude functional results.
Conclusions: The combination of the benefits of the one
stage procedure (avoiding colostomy, two general anaes-
thetics and two hospitalisations) and those of the minimally
invasive surgery makes the primary laparoscopic pull-through
in the management of HD an attractive approach. However
longterm follow-up is necessary to assess the functional
results and the quality of life.
PG5-18 LONG TERM OUTCOME AND QUALITY OF LIFE IN
CHILDREN BORN WITH GASTROSCHISIS C Roman1,
L Viard2, MC Simeoni3, A Loundou3, A Delarue4, J Sarles1,
JM Guys4. 1Service de Pediatrie Multidisciplinaire, Mar-
seille, France. 2Service de reanimation pediatrique, Mar-
seille, France. 3Laboratoire de Sante Publique, Marseille,
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
668 ESPGHAN 38TH ANNUAL MEETING
France. 4Departement de chirurgie pediatrique, Marseille,
France.
Aim: The purpose of this study was to determine the long
term outcome and quality of life (QOL) of children born with
gastroschisis.
Methods: We conducted a retrospective, monocentric study
among the 38 children treated for gastroschisis in our center
between 1985 and 2000. Two died early and 5 were lost for
follow-up. The 31 remaining children were evaluated through
outpatient visits (n = 17) or telephone interviews (n = 14).
Three parameters were analysed : 1) general health and bowel
function, 2) growth, 3) psycho-motor development and
school integration. Furthermore, a validated and age adapted
QOL questionnaire was sent to each patient.
Results: There were 19 males and 12 females (mean age
9.5 years, range 417 years). All children but 3 (a girl with
short bowel syndrome requiring parenteral support during
9 years, and 2 children with mental retardation), had normal
general health and required no medical care. 25 children
(80%) never complained of recurrent abdominal pain. 26
(84%) had a normal intestinal transit. Growth was reduced
during the first 3 years of life, but catching up was reached
between 3 and 6 years. 7 children (22.6%) presented a delay
to acquire language, walk, sitting without support, or to
acquire anal continence. 11 children (35.5%) experienced
school retardation due to a delayed start or first classes repeat.
26 QOL questionnaire were returned by the patients (84%).
The QOL score was not statistically different in children with
gastroschisis compared to controls (67.9 6 15.4 vs 67.9 6 13,
p . 0.05). Similar results were obtained with the teenagers
(67.7 6 11.3 vs 60 6 13.3, p . 0.05). Low QOL was
correlated to prematurity (p = 0.014), a Schuster procedure
(p = 0.008), and a stay in intensive care unit for more than 10
days (p = 0.007) during the neonatal period.
Conclusion: This study demonstrates that gastroschisis can
be completely corrected in the neonatal period without any
sequelae. Favorable outcome is quickly achieved, except in
case of short bowel syndrome. Quality of life of survivors is
not compromised. Accordingly, prenatal counselling can be
optimistic.
PG5-19 FUNCTIONAL NON-RETENTIVE FAECAL SOILING IN
CHILDREN: 12 YEARS OF LONGITUDINAL FOLLOW-
UP. M Benninga1, W Voskuijl1, J Reitsma2, R Van Ginkel1,
J Taminiau1, H Buller3. 1Academic Medical Center, department
of pediatric gastroenterology, Amsterdam, The Netherlands.
2
Academic Medical Center, department of biostatistics, Am-
sterdam, The Netherlands. 3Sophia Childrens Hospital,
Rotterdam, The Netherlands.
Background: Functional non-retentive faecal soiling
(FNRFS), encopresis in the absence of signs of faecal
retention, is a frustrating phenomenon in children and
difficult to treat. It is assumed that FNRFS will resolve
spontaneously beyond puberty, however no data on long-term
outcome are available.
Aim: To investigate the very long-term outcome of FNRFS
patients after intensive medical treatment.
Methods: Between 1990 and 1999, 119 patients (96 boys)
with FNRFS were enrolled in 2 prospective, randomised
trials investigating the effect of biofeedback training and/or
laxative treatment. Thereafter, follow-up (FU) was performed
at 6 months, 1 year and thereafter annually until the end of
data collection in September 2004. A standardised question-
naire was used, either during clinical visit or by telephone, to
evaluate symptoms. Success was defined as having less than 1
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
encopresis episode in 2 weeks while not using medication for
more than 1 month.
Results: Median age at entry (25th75th percentiles) was 9.2
(7.911.6) years and the median duration of symptoms before
intake (25th75th percentiles) was 4.4 (3.06.7) years. A 90%
follow-up was achieved at all stages of the study. After 2 years of
intensive behavioural and medical therapy, 33 out of 112 (29.5%)
patients were successfully treated. The cumulative success
percentage after 7 years of FU was 80%. At the biological ages
of 12 and 18 years, 49.4% (40/81) and 15.5% (9/58) of the
patients still had encopresis, respectively. Age at intake younger
than 6 years in combination with secondary encopresis was
associated with a lower chance of achieving success (HR: 0.51
(95% CI: 0.270.98), P = 0.04). Relapse occurred in 37% of
patients (cumulative percentage after 7 years), and occurred most
likely in the first two years after an initial success.
Conclusions: Only 29% of the patients with FNRFS are
successfully treated after two years of intensive treatment.
Thereafter, a steady increase in success is observed. Neverthe-
less, at the age of 18 years, 15% still have encopresis.
PG5-20 IS THE RISK OF UPPER GASTROINTESTINAL BLEED-
ING INCREASED WITH NONSTEROIDAL ANTI-
INFLAMMATORY DRUG USED AS ANALGESIC IN
CHILDREN? A CASE CROSSOVER STUDY. L Bensouda1,
L Michaud2, AP Jonville-Bera1, B Giraudeau1, O Mouterde3,
E Autret-Leca1. 1Department of Pharmacology, University
Hospital and Faculty of Medicine, Tours, France. 2Depart-
ment of Paediatrics, University Hospital and Faculty of
Medicine, Lille, France. 3Department of Paediatrics, Univer-
sity Hospital and Faculty of Medicine, Rouen, France.
Aim: Many case-control studies have evaluated the risk of
upper gastrointestinal bleeding (UGIB) with nonsteroidal
anti-inflammatory drug (NSAID) in adults while no study has
evaluated this risk in children. Our aim was to assess the
hypothesis that the risk of UGIB after NSAID intake is
increased in children.
Methods: We conducted a case-crossover study in which
each child enrolled served as his/her own control. All the
children seen for UGIB and aged between 2 months and 16
years old were enrolled. Information on exposure to NSAID
during the month preceding the onset of UGIB was collected
by a phone interview to the parents. The relative risk (RR) of
NSAID related UGIB was estimated by comparing exposure
to NSAID during a risk period preceding the onset of UGIB
(Day-7 to Day 0), with exposure during a control period from
Day-28 to Day-21.
Results: A total of 178 children with UGIB were enrolled
between January 2002 and January 2004. Upper gastrointes-
tinal bleeding was associated with a recent intake of NSAID
65 cases (38%): ibuprofen (61%), acetylsalicylic acid (30%),
miscellaneous (9%). The RR of UGIB due to NSAID intake
was 16.5 [6.045.3]. NSAID were always used as analgesic
or antipyretic, with a dosage in accordance with the
recommendations of the company.
Summary and Conclusion: Upper gastrointestinal bleeding
due to NSAID intake represents a significant part of
paediatric UGIB. Despite a short study period which
overestimated the risk of UGIB, this study showed that the
risk of UGIB after NSAID intake, even at a normal dosage, is
increased in children.
On behalf the French-Speaking Group of Paediatric Hepatol-
ogy, Gastroenterology and Nutrition.
PH1-01 LIVER TRANSPLANTATION IN ARGININAEMIA
E Santos Silva1, E Martins1, ML Cardoso2, C Barbot1,
L Vilarinho2, M Medina3. 1Hospital de Crianc
xas Maria Pia,
 ESPGHAN 38TH ANNUAL MEETING
Porto, Portugal. 2Instituto de Genetica Medica Jacinto de
Magalhaes, Porto, Portugal. 3Hospital Geral de Santo
Antonio, Porto, Portugal.
Deficiency of arginase, which converts arginine to urea and
ornithine, is a rare autosomal recessive disease caused by
mutations in the ARG1 gene on chromosome 6q23. Onset is
usually in childhood and clinical manifestations include
progressive spastic paraparesis and mental slowing. In
contrast to other urea cycle disorders, liver involvement is
less frequent and usually not as severe as, for instance, that
observed in OCT deficiency or in citrullinemia. Consistently,
patients with argininemia are scarcely considered as candi-
date for orthotopic liver transplantation (OLT), mainly due to
the presence of neurological progressive disease with major
handicap at diagnosis and to minor liver involvement.
However, the rationale for OLT still stands in this condition,
because it would replace 50% of the deficient enzyme with an
active hepatic arginase. We present two patients with
argininemia submitted to OLT for different reasons. Case 1-
diagnosis at 2 months old during a neonatal cholestasis work-
up study. Other diseases have been excluded by appropriate
testing. An appropriate diet and oral sodium benzoate were
started immediately with good compliance. The disease
progressed to liver cirrhosis with portal hypertension and
marked hypercholesterolemia, xanthomatosis and pruritus in
the absence of neurological lesions. She underwent OLT at
7 y old with complete normalization of plasmatic arginine
levels 24 hours after, under free diet. Five years after OLT she
maintains normal neurological examination. Case 2
diagnosis at 20 months old due to persistent elevation of
transaminases after infectious mononucleosis. Despite an
appropriate treatment she progressed to spastic paraparesis
with marked limp. Liver involvement was asymptomatic. The
patient was submitted to OLT at 10 years old with complete
normalization of plasmatic arginine levels 24 hours after
OLT. Almost 3 years after OLT there is neither progression of
neurological lesions nor of MRI features. Conclusion We
conclude that OLT may be an alternative treatment for
selected patients, and must be always considered when
neurological lesions progress despite an adequate diet
compliance, and before major handicapping lesions contra-
indicate it.
PH1-02 DISEASE PROFILE, SELECTION CRITERIA, AND OUT-
COME OF PATIENTS REFERRED TO INTESTINAL
TRANSPLANTATION (ITX): A CENTER EXPERIENCE.
J Pirenne1, V Janssens1, R Lombaerts1, G Veereman1,
D Monbaliu1, I Hoffman1. 1University Hospitals Gasthuis-
berg, Leuven, Belgium.
Intestinal transplantation (Itx) has evolved from an experi-
mental endeavour into a life-saving treatment for pediatric &
adult patients with short-bowel and TPN- induced severe
complications. In Europe -and in stark contrast with the US-
the referral pattern to Itx is less standardized and fewer cases
are performed/reported. To better characterize and bring new
insight into the potential Itx activity in our region, we
evaluated the disease profile, the selection process, and the
outcome of short-bowel patients referred to Itx at our center.
A multidisciplinary adult & pediatric Itx program was
launched in 1998. Since then, a candidacy for Itx was
formally discussed in 31 patients (9 pediatrics/22 adults).
Criteria for Itx listing included: a) irreversible intestinal
failure; b) life-threatening TPN-complications; & c) suffi-
ciently preserved condition to tolerate Itx (surgery-immuno-
suppression). Of these 31 patients, 11 were in too good
669
condition (potential for reversibility of bowel failure and/or
no life-threatening complication) and their survival with
conservative therapy is 100% at last follow-up (1 received
a liver Tx at another center). On the contrary, 4 were too
sick to tolerate Itx, and of them, 3 (75%) rapidly succumbed
(sepsis). 6 candidates from abroad were excluded (4 due to
terminal/hopeless condition; 2 to extra-intestinal carcinoma-
tosis). Of the 31 initial patients, 10 (32%) were deemed
suitable for Itx: 2 females (56yo and 57yo, bowel resection-
TPN liver-failure) are well 2 & 4 yrs after liver/Itx; a 2 yo boy
with volvulus is recovering from a recent liver/Itx; 1 neonate
with volvulus in whom family had refused Itx succumbed
shortly thereafter to sepsis; a 53yo male with crohn/amyloi-
dosis succumbed to pneumonia while waiting for kidney-Itx;
a 50yo female with acute/diffuse splanchnic ischemia rapidly
succumbed to sepsis/MOF while being prepared for multi-
visceral transplant; a 11mth yo boy with enterocolitis/liver
failure succumbed to GI bleeding while being prepared for
liver/Itx; 3 patients are currently waiting {32yo male with
crohn for Itx, 25yo female with pseudo-obstruction for Itx,
and 51yo female with bowel resection- renal failure for
kidney/Itx}. In conclusion, 2 thirds of short-bowel patients
referred to Itx do not fulfill the criteria (either too-sick or
too-good). In 1 third, Itx is indicated and is life-saving -
when done in due time- whereas the survival without Itx
(natural history) is very poor. These data validate the
selection criteria used and justify wider application of Itx in
this selected group. Itx is limited by an -unexpected- shortage
of available grafts and it is therefore essential that intestinal
donors are identified and referred to procurement organiza-
tions. More short-bowel patients could be saved by an Itx if
they were referred before being too sick to tolerate surgery,
immunosuppression, and an unavoidable waiting period.
PH1-03 SEROLOGICAL MARKERS OF EBV INFECTION AND
REACTIVATION IN PAEDIATRIC LIVER TRANSPLAN-
TATION M Del Rizzo1, C Mengoli2, U Cillo3, G Guariso1,
L Zancan1, L DAntiga1. 1Paediatric Department, Padova,
Italy. 2Virology Department, Padova, Italy. 3Department of
surgical and gastroenterological sciences, Padova, Italy.
Aim: EBV infection and reactivation are the main causes of
post-transplant lymphoproliferative disease (PTLD). We
aimed to study serological markers of EBV infection in
pediatric liver transplant (OLT) recipients.
Methods: Retrospective review of the files of 33 children
transplanted from 1986 to 2004. 20 patients were EBV-IgG
seronegative (group A) and 13 seropositive (group B) before
OLT. We performed serology (19861997) then also Real-
Time Polimerase Chain Reaction (RT-PCR). Primary
infection was the appearance of VCA-IgM or VCA-IgG or
RT-PCR in patients previously not infected; reactivation was
the appearance of VCA-IgM or EA-IgG or RT-PCR in
patients previously infected; positive VCA-IgM or EA-IgG or
RT-PCR lasting longer than 6 months were defined as
persistent infection.
Results: 17/20 patients of group A had a primary infection.
At least one viral reactivation occurred in 12/17 of group A
and 9/13 of group B (p = N.S.); in group B first reactivation
developed mostly in the first 3 months after OLT, while in
group A occurred also several years after OLT. 13/17 patients
in group A and 1/13 patients in group B had a primary
infection or a reactivation persisting for more than 6 months
(p = 0.0004, Fishers exact test). Viral load greater than 500
copies occurred in 11/17 patients in group A and 0/13 patients
in group B (p = 0.001). During primary infection and
reactivation there was a correlation between seroconversion
to VCA-IgM or EA and a RT-PCR .500 copies. 3 patients
developed PTLD; all patients were seronegative before OLT
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
670 ESPGHAN 38TH ANNUAL MEETING
and presented a persistent infection before or after developing
PTLD.
Conclusions: EBV seronegative patients before OLT are less
able to control EBV infection as compared to seropositive
subjects. Although serology on its own may lead to under-
diagnose EBV infection in this setting, it allowed us to
demonstrate that infection and/or reactivation in seronegative
patients is characterized by a significantly longer persistence
as compared to seropositive subjects. Studies in larger series
of patients could verify the usefulness of EBV serology and
confirm a correlation between persistence of infection and
risk of developing PTLD in paediatric OLT recipients.
PH1-04 COMPARISON OF CHILD-TURCOTTE-PUGH AND
PELD SCORES FOR PREDICTING SHORT TERM OUT-
COME OF LIVING- RELATED PEDIATRIC LIVER
TRANSPLANTATION F Ozcay1, B Saygili1, H Karakayali2,
A Dalgic2, M Haberal2. 1Baskent University Department of
Pediatric Gastroenterology, Hepatology, and Nutrition, Ankara,
Turkey. 2Baskent University Department of General Surgery,
Ankara, Turkey.
Introduction: Several prognostic parameters for chronic
liver diseases in children have been advocated during the past
few decades. The Pediatric End- Stage Liver Disease (PELD)
model is a new model for scoring pediatric liver disease
severity. The PELD scoring system uses objective and
standardized laboratory values rather than clinical findings
to avoid interobserver or interinstitutional variability. We first
aimed to compare PELD scoring system with Child-Turcotte-
Pugh (CPT) scoring system to evaluate the severity of liver
disease before liver transplantation. Our second aim was to
test the validity of PELD and CPT scores to predict short-
term outcome of living- related liver transplantation (LRLT)
in children.
Patients and Methods: 23 children (8 female) underwent
LRLT between 20012004. Patients transplanted for fulmi-
nant liver failure were excluded. PELD score was calculated
using the United Network for Organ Sharing formula. CPT
and PELD scores were calculated the day of transplantation
and correlated with patient survival. Patients were allocated
into three groups according to PELD scores namely less than
15, 1525 and higher than 25.
3 m survival % 6 m survival %
CPT class A 100 100
CPT class B 84.6 83
CPT class C 100 100
PELD , 15 88,9 85,7
PELD 1525 80 80
PELD . 25 100 100
Results: Mean6SD age at transplantation was 81.7663.4
months (range 12204 months). Indications of LRLT were
Wilsons disease (n = 9), genetic cholestatic liver diseases
(n = 6), biliary atresia (n = 5), tyrosinemia and hepatocellular
carcinoma (n = 2), Caroli syndrome (n = 1). The numbers of
patients according to CPT classification were as following;
class A (n = 4), class B (n = 14), and class C (n = 5). Eleven
patients had a PELD score less than 15. PELD scores were
between 1525 in 6, and higher than 25 in 6 patients. Mean 6
SD values of PELD and CPT scores were 17 6 14 (range 8
to 58) and 8 6 2 (range 5 to 12) respectively. There was
a strong positive correlation between PELD and CPT scores
to assess severity of liver disease (r = 0.80, p = 0.000). Median
follow-up time was 7 months (2 to 40 months). During this
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
period 2 patients died because of sepsis. Overall 3 and 6
months survival was 90% and 88.9% respectively. Post
transplantation survival at 3 and 6 months did not signifi-
cantly different among the 3 PELD subgroups. CPT class A,
B, C patients were also similar in terms of 3 and 6 months of
survival.
Conclusion: PELD scoring system is comparable to CPT
scoring system to evaluate severity of end stage liver disease.
Both pretransplant PELD and CPT scores did not predict
short-term outcome after pediatric living related liver trans-
plantation.
PH1-05 LONG-TERM RESULTS OF BASILIXIMAB INDUCTION
IMMUNOSUPPRESSION IN PEDIATRIC LIVER GRAFT
RECIPIENTS R Ganschow1, E Grabhorn1, A Richter1,
A Schulz1, A Von Hugo1, X Rogiers2, M Burdelski1.
1
University of Hamburg, Dept. of Pediatrics, Hamburg,
Germany. 2University of Hamburg, Dept. of Hepatobiliary
Surgery, Hamburg, Germany.
It has been shown that induction therapy with the monoclonal
anti- Interleukin-2 receptor antibody basiliximab (Simu-
lect) is capable to reduce the incidence of acute graft
rejection in adult and pediatric liver transplantation (Ltx).
However, data on long-term results using basiliximab in
children are still pending.
Aim: Therefore, the objective of our study was to report on
the long-term results of basiliximab induction therapy in
pediatric liver transplant recipients.
Patients: 54 children received two single doses of basilix-
imab in addition to cyclosporine and prednisolone following
Ltx in our pilot study. We analyzed the incidence of acute and
chronic graft rejection, that of PTLD, and patient and graft
survival. The follow-up was 32 to 56 months. The historical
control group (matched controls) consisted of 54 patients
treated with a cyclosporine and prednisolone dual therapy.
Results: Patient survival was 53/54 in the treatment group
and 51/54 in the controls. One patient was re-transplanted in
the treatment group versus 3 patients in the control group.
The incidence of acute graft rejection was 16.6% compared to
53.7% in the control group (p , 0.001), that of chronic
rejection was comparable in both groups (1/54 vs. 1/54). The
incidence of steroid resistant rejection was 4/54 vs. 6/54, that
of PTLD was 1/54 vs. 0/54. There were no adverse effects
seen which could be related to the antibody treatment.
Conclusions: We conclude that basiliximab provides safe
and effective induction immunosuppression in pediatric liver
graft recipients. Short- and even long-term results are excellent.
PH1-06 MANAGEMENT AND OUTCOME OF EXTREME END
STAGE VENOUS ACCESS IN CHILDREN WITH IN-
TESTINAL FAILURE (IF) ASSESSED FOR SMALL
BOWEL TRANSPLANTATION (SBTX) AF Rodrigues1,
IDM Van Mourik1, K Sharif1, J Bennett1, S Protheroe1,
DJ Barron1, J DeGiovani1, S Beath1. 1Birmingham Child-
rens Hospital, Birmingham, United-Kingdom.
3- year survival after SBTx has improved to between 73% and
88%.
Impaired venous access is an indication for SBTx in children
with chronic IF, whereas thrombosis of the superior vena cava
(SVC) was a contra-indication to SBTx, historically.
Aim: To report our experience in management of children
with extreme end stage venous access.
Subjects: 6 children (all males), mean age of assessment
30 months (range 13 52). Diagnosis: Total intestinal
aganglionosis1, protracted diarrhoea1, short bowel syndrome4
 ESPGHAN 38TH ANNUAL MEETING
of which gastroschisis2 and malrotation with mid gut
volvulus2. All presented with thrombosis of the SVC and or
inferior vena cava and malnutrition because of interruption of
parenteral nutrition. All had venograms and/or MR angio-
grams to access venous access. All had a documented history
of 618 central venous line (CVL) insertions previously.
Methods: Venous access was achieved as follows: Trans-
hepatic venous catheters (THVC) (5), Right atrial (RA) CVL
via midline sternotomy4, Azygous venous system2, dilatation
of left subclavian vein following passage of a guide wire and
then placing a CVL to reach the RA1 and SVC dilatation,
stent insertion and placement of a CVL in situ1. Complica-
tions included pleural effusion, which resolved after chest
drain insertion, following RA insertion1, displacement of
THVC needing repositioning2, and balloon dilatation of SVC
stent after 16 and 22 months to restore patency.
Outcome: 4 children with permanent IF on assessment were
offered SBTx, 3 of which were transplanted and were
established on full enteral nutrition while the family of one
child declined the procedure (and he died 3 years later). In the
remaining 2 children in whom bowel adaptation was still
a possibility, attempts were made to provide adequate venous
access as feeds and manipulations of intestinal motility drugs
were undertaken. One child received SBTx 3 years after his
first assessment, as bowel adaptation failed and the other
child (the sixth subject) died in the immediate post operative
period following a THVC insertion, but the cause of death is
inconclusive. RA lines in transplanted children proved
adequate for the management of uncomplicated transplanta-
tion, although the usual infusion protocol had to be modified
to take account for the lack of access.
Conclusion: It is possible to re-establish central venous
access using unconventional techniques. However it is
difficult and time consuming to assemble a skilled team
consisting of one of more: surgeons, cardiologists, interven-
tional radiologist, transplant anaesthetists. If SBTx is in-
evitable it is imperative as well as easier and safer with
adequate venous access and we advocate liaison with a SBTx
centre at an early stage.
PH1-07 HIGH INCIDENCE OF DUCTOPENIC REJECTION IN
CHILDREN TRANSPLANTED FOR IDIOPATHIC ACUTE
LIVER FAILURE RML De Bruyne1, B Portmann1,
CI Kortsalioudaki1, RM Taylor1, N Heaton1, M Rela1,
G Mieli-Vergani1, A Dhawan1. 1Kings College Hospital,
London, United-Kingdom.
Introduction: Liver transplantation is an accepted mode of
treatment of acute liver failure (ALF). Patient survival
however is poorer compared to transplantation for chronic
liver disease. The aetiology of ALF may affect the outcome of
liver transplantation but the data are limited.
Methods: The hospital notes of 204 children (101 male,
median age 3.97 y, range 1 d17.4 y) who presented with
ALF between 1989 and September 2004 were reviewed.
Results: Out of 143 (70.1%) for whom aetiology could be
established 41 underwent liver transplant. 3 (7.3%) of them
required retransplantation, 2 due to ductopenic rejection (DR)
and 1 due to hepatic artery thrombosis (HAT). 61 (29.9%)
patients had idiopathic ALF, of whom 36 (59%) were
transplanted. 11 (30.6%) of these needed a second transplant.
In 6 (54.5%) of the 11 cases DR was the cause of graft loss.
The other causes of retransplantation were biliary problems3,
HAT1 and primary nonfunction1. 4 of the 6 patients with DR
subsequently had a 3rd transplant. All transplants were ABO
matched. Initial immunosuppression in the patients with DR
consisted of tacrolimus and steroids in 3 patients, the other 3
were started on cyclosporine A, azathioprine and steroids but
were subsequently converted to tacrolimus and steroids.
671
Mycophenolate mofetil was added to the treatment in 4 cases,
sirolimus in 1. Two patients received basiliximab, one OKT3
and another cyclophosphamide. One child was treated
peroperatively with ATG at the time of the 3rd transplant.
DR was diagnosed histologically and defined as duct loss in
more than 50% of portal tracts in more than one biopsy. The
median duration to develop signs of DR was 40 days (range
3093 days). 4 of 6 patients with DR died. Causes of death
were sepsis and multi-organ failure 3 and liver failure due to
DR where further transplantation was not considered because
of severe hypoxic brain damage1.
Conclusion: Children transplanted for idiopathic ALF have
poorer graft and patient survival, ductopenic rejection being
the dominant cause of graft loss.
PH1-08 CYCLOSPORINE MONITORING IN THE FIRST DAYS
AFTER LIVER TRANSPLANTATION: TROUGH OR
2-HOURS-POSTDOSE LEVEL DO NOT CORRELATE
WITH DRUG EXPOSURE E Frauca1, G Munoz-Bartolo1,
L Hierro1, A De La Vega1, C Daz1, C Camarena1,
C Sanchez-Peinado2, P Jara1. 1Pediatric Hepatology Service.
La Paz Univ. Hospital, Madrid, Spain. 2Biochemistry
Department. La Paz Univ. Hospital, Madrid, Spain.
Aims: To investigate cyclosporine absorption and optimal
monitoring in the immediate period after liver transplantation
(LT).
Methods: Twenty children were studied (age ,3 yrs n = 15,
living donor n = 7). All had a Roux-en-Y biliary anastomosis,
done at LT in 10 cases. Immunosuppression consisted of
steroids+azathioprine+cyclosporine (CsAME). First dose of
CsA-ME was 10 mg/kg, followed by 15 mg/kg/day divided
in 2 doses, then modified as needed. The target trough CsA
(C0) was 250350 ng/ml. Intravenous perfusion of CsA
2 mg/kg/day was added if C0 , 200 ng/ml. Samples were
obtained on 1st, 3rd and 5th day post-LT, at moments 0, 1.5 h,
2h, 4h, 6h and 8h post-dose to determine whole-blood
CsA concentration (by EMIT) and AUC (area under the
concentration-time curve).
Results: CsA concentration is given as mean values in ng/ml.
On the first day, patients with a recent Roux loop displayed
a pattern of CsA malabsorption (C0: 71 C2h: 171 C4h: 181),
and those without recent intestinal surgery showed a slow
CsA absorption (C0 = 168 C2h = 255 C4h = 516). On days 3rd
and 5th recent or previous intestinal surgery did not determine
differences. At day 3 mean values were C0 = 310 C2h = 753,
at day 5: C0 = 337 C2h = 888.CsA-ME dose was increased
from 16.8 mg/kg/day (day 1) to 29.5 mg/kg/day (day 3) and
36.5 mg/kg/day (day 5). Half of the patients needed iv
supplement. Mean AUC (hr x ng/ml) was 4989 (day 1), 6698
(day 3) and 7108 (day 5). C0 or C2h did not correlate with
AUC in any of those days.
All patients survived, 19 out of 20 with the first graft.
Rejection affected 7 patients (35%), none had refractory
rejection. C0, C2h or AUC at 5th day did not differ between
children with or without rejection.
Summary: Immediately after LT CsA-ME is poorly
absorbed, dose requirements are high, target C0 was achieved
by 3rd day, but C2 was lower than the theoretical optimum.
Drug exposure does not correlate with C0 or C2h.
Conclusions: The efficacy of CsA-ME in prevention of
rejection was high, but drug exposure cannot be estimated by
single C0 or C2h determinations in the first days after liver
transplantation.
PH1-09 PAEDIATRIC AUTOIMMUNE LIVER DISEASE: INDI-
CATIONS AND OUTCOME OF LIVER TRANSPLANTA-
TION PF Chai1, RM Brown2, JL McPartland2, K Foster3,
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
672 ESPGHAN 38TH ANNUAL MEETING
P Davies4, P McKiernan1, DA Kelly1. 1Liver Unit, Birming-
ham Childrens Hospital, Birmingham, United-Kingdom.
2
Histopathology Unit, Birmingham Childrens Hospital,
Birmingham, United-Kingdom. 3Radiology Department,
Birmingham Childrens Hospital, Birmingham, United-King-
dom. 4Statistical Advisory Department, Birmingham Child-
rens Hospital, Birmingham, United-Kingdom.
Autoimmune liver disease(AILD)includes sclerosing chol-
angitis(SC), autoimmune hepatitis type 1 & 2(AIHT1&AIH-
T2)and overlap syndrome(OS).
Aim: To review indications and outcome of liver transplant
(LT) in paediatric AILD.
Methods: Retrospective review of all paediatric AILD
presenting from 19812004.
Results: 102 patients had confirmed AILD, (62 F). 66 had
AIHT1, 18 AIHT2, 9 SC and 9 OS. Median age at disease
onset was 11.9 yr (range 1.218.3). 98/102 were treated with
prednisolone (PNL) and azathioprine (AZA). Table:
Treatment response and outcome AIHT1 AIHT2 SC OS
Fail to response to PNL&AZA 16/64 3/16 3/9 2/9
Fail 2nd line treatment(MMF/CyA/FK) 4/12 3/3 2/2 1/1
Liver transplantation 9 5 4 1
Median age at transplant was 14.3 yrs (range 1.7 to 21.8).
1 child with AIHT1 and 3 with AIHT2 had emergency LT.
Median duration of treatment to LT for the remaining 15
children was 29 mo (range 892). Indications for LT were
acute liver failure (22%), disease progression despite treat-
ment (39%), poor quality of life (22%), complications of
portal hypertension (22%) and hepatopulmonary syndrome
(5%). Main postoperative complications were biliary com-
plications4, GI bleed1 and steroid induced psychosis1. 11
patients (57%) had 19 episodes of acute rejection and 2 (11%)
had chronic rejection. 5 patients (26%) had disease re-
currence (1AIHT1, 3AIHT2 ,1ASC). Median time from LT to
disease recurrence was 27 mo (range 551). 4 had repeat LT
(2 chronic rejection, 2 disease recurrence) and 1 had a third
LT (hepatic artery thrombosis). There were 4 deaths due to
sepsis1, surgery for ulcerative colitis1and disease recurrence2.
Conclusion: Most paediatric AILD respond to medical
therapy, only a minority require LT. Counselling for LT
should indicate the morbidity and mortality related to graft
rejection and disease recurrence.
PH1-10 LIPID AND CARBOHYDRATE METABOLISM, AND
ANTIOXIDANT STATUS IN CHILDREN AFTER
LIVER TRANSPLANTATION. P Socha1, A Wierzbicka1,
I Jankowska1, M Teisseyre1, H Ismail1, E Skorupa1,
J Socha1, J Pawlowska1. 1The Childrens Memorial Health
Institute, Warsaw, Poland.
Organ transplantation is a risk factor of atherogenesis that
may be related to immune suppression therapy. Increased free
radical generation may even aggrevate atherogenesis.
The aim of the study was to assess lipid metabolism in
relation to risk factors of atherogenesis as well as carbohy-
drate metabolism and antioxidant status in children after liver
transplantation.
Material: We investigated 35 children 35 y after liver
transplantation aged 10.6 6 4.7. We did not observe major
liver injury in the patients studied (normal bilirubin and INR).
Results: 95% conf. interval of total cholesterol (150
191 mg/dl), LDL-C (83111 mg/dl), VLDL-C (1218 mg/dl),
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
HDL-C (4454 mg/dl), apo AI (1.271.49 g/l), apo B (0.6
0.89 g/l), alpha-tocopherol (7.88.83 mg/ml) serum concen-
trations were within the normal limits.
GSH was decreased in transplanted children when compared
to 28 aged matched healthy controls (527.6606.6 mmol/ml
vs. 667.5763.5), whereas GPx activity was similar in the
study and control group (28.332.8 U/gHb vs. 30.634.5).
Kruskall Wallis analysis in subgroups of children treated with
cyclosporine (Cs) only (n = 8), tacrolimus (Tac) only (n = 12)
and mycophenolate mofetil (MMF; n = 8) showed differences
only in total cholesterol (Cs: 131.6285.6; Tac: 144.0181.6l;
MMF: 132.1181.2) and LDL-C (Cs: 79.4126.9; Tac: 42.2
118.8; MMF: 74.2117.3).
Conclusion: Lipid metabolism is not significantly disturbed
in children after liver transplantation that does not point to the
high risk of atherogenesis. Cyclosporine seems to have the
strongest untoward effect on cholesterol metabolism. GSH
concentration is decreased after liver transplantation that may
be related to slightly impaired liver function, but GPx activity
and alpha-tocopherol concentration remain normal.
PH111 RENAL DYSFUNCTION IN PAEDIATRIC LIVER
TRANSPLANT RECIPIENTS ON CYCLOSPORINE:
A COMPARISON BETWEEN NEORAL AND SANDIM-
MUN FORMULATION. PF Chai1, IDM Van Mourik1,
P Davies2, C LLoyd1, DA Kelly1. 1Liver Unit, Birmingham
Childrens Hospital, Birmingham, Birmingham, United-
Kingdom. 2Statistical Advisory Department, Birmingham Child-
rens Hospital, Birmingham, Birmingham, United-Kingdom.
Cyclosporin(CyA)is an effective immunosuppressive drug.
Microemulsified Neoral(NEO) with improved bioavailability,
replaced Sandimmun(SI) as first line immunosuppresant for
paediatric liver transplant(LTx) recipients from 1995.
Patients were transferred from SI to NEO in 1996.
Aim: To compare the incidence of renal dysfunction (RD) in
long term LTx survivors on Neo, SI and those transferred
from SI to NEO.
Methods: Retrospective study of 177 (male 89) LTx
recipients between 19852001. RD defined as calculated
glomerulo-filtration rate(cGFR) # 60 ml/min/1.73m2 using
Schwartz formula. All patients had CyA for .1 yr and
baseline cGFR . 60 ml/min/1.73m2 at LTx or transfer.
Results: At 1, 3 and 5 years post LTx there were 89, 78, 52
patients on NEO and 88, 82, 61 on SI respectively. There were
61 patients transferred from SI to NEO. At 1,3 and 5 years
post transfer there were 61, 56 and 49 patients respectively.
Duration of exposure to SI prior to transfer was 50 mo (range
16216). Median age(mo) at LTx for patients on Neo and SI
was 26 (range 0.5226) and 31 (range 0.5215) respectively.
% of patients witn renal dysfunction at 1,3 and 5 years post LTx.
Or transfer
1 year 3 years 5 years
NEO (%) 19 23 40
SI (%) 49 55 75
p/Odds Ratio 0.001/0.30 0.001/0.29 0.002/0.25
SI to NEO transfer (%) 4.9 14 18
A significant higher percentage of patients on SI developed
RD as compared to those on NEO at 1, 3 and 5 yrs. Patients
transferred from SI to NEO continue to develop RD but at
a lower rate.
Conclusion: Long term clinical use of NEO had a signifi-
cantly lower risk of RD despite having better bioavailability
 ESPGHAN 38TH ANNUAL MEETING
as compared to SI. Patients transferred from SI to NEO are
still at risk of RD and require continual renal monitoring.
PH1-12 CHILDHOOD CIRRHOSIS, HEPATOPULMONARY SYN-
DROME AND LIVER TRANSPLANTATION G Tumgor1,
C Arikan1, HA Yuksekkaya1, E Levent2, C Dorak1,
D Goksen1, M Kilic3, S Aydogdu1. 1Ege University
Department of Pediatric Gastroenterology Hepatology and
Nutrition, izmir, Turkey. 2Ege University Pediatric Cardiol-
ogy, izmir, Turkey. 3Ege University Transplantation Research
center, izmir, Turkey.
Background: The hepatopulmonary syndrome (HPS) is
characterized as a triad: liver disease, intrapulmonary
vascular dilatatiton, and arterial hypoxemia. Hepatopulmo-
nary syndrome have developed as a consequence of hepatic
dysfunction.
Aim: To investigate pulmonary complications in children
with cirrhosis
Methods: 52 children (26 boys, 29 girls) with cirrhosis were
included to study. Age of children ranged from 6 months to
22 years with median age of 10 years. Cirrhosis and its
complications were determined by biochemical, serologic,
histopathologic, sonographic and endoscopic investigations.
Among 52 cases; 27 had noncholestatic and 25 had
cholestatic hapatic failure signs. According to Child Pugh
scores; 15 of patients were Child A, 15 of them were Child B
and 18 of them were Child C (4 cases fulminant hepatic
failure). All patients were evaluated by chest radiography,
arterial blood gas analysis, contrast echocardiography
(ECHO). To determinate pulmonary arteriovenous (AV)
shunts, %20 galactose solution and saline were used as
a contrast material during ECHO.
Results: In this study HPS established in 17 patients (%32)
by contrast ECHO. Among HPS; 5 were Child A (%33), 3
were Child B (%17) and 9 were Child C(%50). 9 patients
(%16.3) were hypoxemic (PaO2 , 70 mmHg). 4 of them had
severe hypoxemia.(PaO2 , %50). 9 of patients with HPS had
liver transplantation in which the ECHO findings regressed in
all of them postop on 6th month. Arterial blood gas analysis of
these patients were improved postop on 6th. None of them had
pulmonary hypertension. HPS was determined in %37
(10/27) non cholestatic patients and in 7 of 25 (%28)
cholestatic patients. There was no statistically difference
between cholestatic and noncholestatic patients with respect
to the risk of HPS developed.
Conclusions: HPS is a serious and important complication of
cirrhotic children that leads to tissue hypoxi and santral
ciyanosis. To determine HPS before transplantation is very
important to follow up postoperative intensive care and to
evaluate hypoxia and hypocapnia. HPS seems reversible after
liver transplantaion in many patients.
Clinical Investigations
Diagnose N findings n of HPS
ntra-ekstra hepatic 17 Failure to thrive 21 Abnormal X ray
bilier problems
Criptogenic cirrhosis 14 Esophageal Var. 23 Hypoxemia
Metabolic diseases 10 Digital clubbing 14 Hypocapnia
Autoimmune hepatitis 6 Cyanosis 3 Abnormal O2 sat.
Neonatal hepatitis 2 Platipne 1 Orthodeoxia
Viral infection 2 Shunts in ECHO
PH1-13 NFLUENCE OF PATIENT AND DONOR MACROPHAGE
MIGRATION INHIBITORY FACTOR (MIF) 173 G/C,
INTRACELLULER ADHESION MOLECULE 1 (ICAM-
1) G241R POLYMORPHISM ON LIVER ALLOGRAFT
673
REJECTION: EVIDENCE FROM A SING C Arikan1,
A Berdeli1, M Kilic2, R Yagci1, S Aydogdu1. 1Ege University
Pediatric Gastroenterology, Hepatology and Nutrition, Izmir,
Turkey. 2Ege University General Surgery, Izmir, Turkey.
Background: Cytokines are key immune mediators and it
has been suggested that cytokine gene polymorphisms
affecting expression influence rejection or tolerance. Macro-
phage migration inhibitory factor is a proinflammatory
cytokine that plays role in cell mediated immunity and
delayed type hypersensitivity. MIF expression is upregulated
along with another cytokines such as ICAM-1. The
significance of these molecules in the pathogenesis of liver
allograft rejection and rejection treatment outcome is not
clear.
Aim: To determine whether an association exists between
gene polymorphisms and liver allograft rejection and re-
jection therapy outcome.
Method: Patients who received a primary liver graft from
2000 onward and were seen at the transplant outpatient clinic
since then were included on this study. Frequency data for the
polymorphisms in the transplanted group (N = 52) and their
matched donors, where available (N = 33), were divided into
biopsy proven rejectors (n = 8) and non -rejectors (n = 44).
They also compared with a group of healthy controls (n =
140). After informed consent, blood sample was collected
and DNA was obtained. Using amplification-refractory
mutation system polymerase chain reaction, ICAM-G241R
and MIF173G/C gene polymorphisms were performed.
Rejection grading was assessed according to Banff criteria.
Associations were assessed by using Fishers exact test.
Results: Primary indication for liver transplantation was
biliary atresia (n= 12), metabolic liver disease (n = 24),
autoimmune hepatitis (n = 6) and the others (n = 10). The
median age of patients was 7.7 (ranged; 0.516 years). Primary
immunosuppression consisted of cyclosporine A in 12 patients
and FK-506 in 40 children. Clinically diagnosed rejection was
observed in 21 children, 8 of whom were biopsy-proven acute
rejection. Median rejection time was 11 days (ranged 6121).
Rejectors most often showed the GG or AG genotype,
whereas no rejectors possessed the AA genotype. Univariate
analysis showed that polymorphism of ICAM-G241R was
significantly related to liver graft rejection. No significant
difference was observed in the presence of any genotypes of
recipient and donor MIF be tween nonrejectors and rejectors
(p = 0.44 and p = 0.27, respectively). When patients were
analyzed according to biopsy proven rejectors and non
rejectors, we found that polymorphism of recipient and donor
ICAM-G241R was significantly correlated to allograft re-
jection (p = 0.05 and p = 0.05) respectively. The donors
whose recipients did not develop rejection had a significant
increase of ICAM-G241R GG genotype compared with
donors, whose recipients developed rejection episode. On
the other hand rejector recipients did not have ICAM-G241R
AA genotype compared to nonrejectors. Despite steroid
n responsive rejectors (n = 13) had more MIF GC genotype
than steroid nonresponsive rejectors (n = 8), it did not reach
(-) statistically significance( %87.5 vs. %12.5; p = 0.056).
9
Conclusion: There is a relationship between a genetic
7/17 characteristics and susceptibility to rejection after liver
4/17 transplantation. According to our preliminary result, recipient
9 ICAM-G241R AA and donor ICAM-G241R GG genotype
17 has preventive role for acute liver allograft rejection. MIF
genotype detection may be useful to estimate patients
response to steroid therapy in acute liver allograft rejection.
PH1-14 EFFECTIVE RESCUE OF PATIENTS WITH UNRESECT-
ABLE HEPATOBLASTOMA BY LIVER TRANSPLAN-
TATION. A SINGLE CENTRE EXPERIENCE IN ITALY
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
674 ESPGHAN 38TH ANNUAL MEETING
F Vallortigara1, U Cillo2, G Guariso1, L Zancan1,
G Perilongo1, L DAntiga1. 1Paediatric Department, Padova,
Italy. 2Department of Gastroenterological and Surgical
Sciences, Padova, Italy.
Aim: Some 30% of cases of hepatoblastoma (HB) are still
unresectable after chemotherapy. For this reason the progno-
sis of high risk HB before the era of liver transplantation
(OLT) was very poor. The aim of this study was to review
features and outcome of patients transplanted for HB
followed at our transplantation centre and to compare them
to children who underwent a simple resection of the mass.
Methods: We reviewed the notes of patients diagnosed with
HB at our centre in the last 20 years, dividing patients who
received a OLT (Group A) from those who underwent tumour
excision (Group B). We recorded: age at presentation, pre-
treatment extent of the tumour (Pretext), alpha-fetoprotein
(AFP) level, chemotherapy regimen, age at operation, liver
function tests, left ventricle shortening fraction, glomerular
filtration rate (GFR) by the Schwartz formula, graft and
patient survival.
Results: Group A: 9 patients, 6 males, median age at
presentation 6.3 years (range 0.235). Seven classified
Pretext 4, 2 Pretext 3. Median age at OLT was 9.2 years
(0.635). One patient had a primary resection followed by
OLT whereas 8 had a primary OLT. Six patients received
postoperative chemotherapy. After a median follow up time
of 4.7 years (0.517.5) overall patient and graft survival is
75%. One patient is alive with recurrence of the disease that
presented 10 years after OLT. Two patients, died at 0.5 and
5 years post-OLT from recurrence of the disease. All patients
have normal liver function tests on low doses of calcineurin
inhibitors +/2mycophenolate mofetil. At the last follow up
50% of the survivors had low GFR and normal cardiac
function. Group B comprised of 18 patients, 12 males,
median age at presentation 0.6 years (range 0.036.5) (Group
A vs Group B two tails T-Test p = 0.003). After a median time
of 4.8 years (0.116) overall patient survival is 73%. Five
patients died and 1 is alive with recurrence of the disease. At
the last follow up all the survivors had normal GFR and
normal cardiac function.
Conclusions: Liver transplantation for high risk, unresect-
able HB rescues a group of patients with very poor prognosis.
Age at diagnosis of HB is significantly higher in patients with
an unresectable tumour. Patients transplanted for HB are at
risk for long-term renal dysfunction. Their overall survival is
comparable to patients who had a standard risk HB and
underwent primary resection.
PH1-15 A SIMPLE NONINVASIVE INDEX (APRI) PREDICTS
ADVANCED LIVER FIBROSIS IN CHILDREN WITH
CHRONIC HEPATITIS B D Lebensztejn1, E Skiba1,
M Sobaniec-otowska2, M Kaczmarski1. 1IIIrd Department
of Pediatrics, Medical University, Bia3ystok, Poland.
2
Department of Clinical Pathomorphology, Medical Univer-
sity, Bia3ystok, Poland.
Aim: Liver biopsy is thought mandatory for management of
patients with chronic HBV infection, especially for staging
fibrosis, however there are no serum parameters that reliably
predict the stage of liver fibrosis in children. Therefore we
studied an aspartate aminotransferase (AST) to platelet ratio
index (APRI) in assessment of fibrosis degree in children
parallel to biopsy and prior to antiviral treatment for chronic
hepatitis B.
Methods: We determined APRI in 71 children (age range 4
17, mean 10 years) with biopsy-verified chronic hepatitis B.
APRI was calculated according to Wai et al. (Hepatology,
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
2003; 38: 518526). Fibrosis stage was assessed in a blinded
fashion according to Ishak et al. (J Hepatol, 1995; 22: 696
699) We defined advanced liver fibrosis as a score .2.
Receiver operating characteristics (ROC) analysis was used
to calculate the power of the index to detect advanced liver
fibrosis (AccuROC, Canada).
Results: APRI . 0.59 had a sensitivity of 76.5% and
a specificity of 70%; AUC(95% CI) = 0.748(0.630.87), p =
0.003, PPV = 70.3%, NPV = 76.5% in predicting advanced
fibrosis. Correct classification proportion (efficiency) of
staging fibrosis was 73.2%.
Conclusion: APRI is a suitable, simple noninvasive index in
predicting advanced liver fibrosis in children with chronic
hepatitis B. Application of this index may decrease the need
for liver biopsy in this group of children.
PH1-16 EARLY SERUM MARKERS OF HEPATIC FIBROGENE-
SIS IN CYSTIC FIBROSIS LIVER DISEASE RA Munoz1,
R Codoceo2, L Suarez3, L Maiz4, P Jara5, H Escobar6.
1
Hospital la Paz, Madrid, Spain. 2Hospital la Paz, Madrid,
Spain. 3Hospital Ramon Y Cajal, Madrid, Spain. 4Hospital
Ramon Y Cajal, Madrid, Spain. 5Hospital la Paz, Madrid,
Spain. 6Hospital Ramon Y Cajal, Madrid, Spain.
Background: As cystic fibrosis (CF) patients increasingly
survive longer, hepatobiliary manifestations of the disease
have become a diagnostic therapeutic challenge. Based on
clinical criteria, liver disease is found in 1.4% to 7% of
patients with CF. However, when biochemical indicators and
ultrasound imaging are included, the prevalence increases
significantly. The limitations of clinical examination, and the
controversy surrounding biochemical screening and liver
biopsy, make of interest the development of new serum
markers for the early diagnosis of this hepatopathy.
Aims: To evaluate the utility of serum markers (TGF beta,
TIMP 1, and fecal and serum bile acid levels), in the detection
of hepatic fibrosis in children with CFLD vs. CF patients
without liver disease and controls.
Patients and Methods: An experimental, transversal and
observational study of 73 patients with CF (246 years) has
been made. Each patient was assessed for biochemical
markers of cholestasis, citolisis, pancreatic and lung function,
nutritional status (BM), fat vitamins, serum TGF beta, TIMP
1 levels, and fecal and serum bile acid levels as early serum
markers of hepatic fibrogenesis in CF. All patients were also
assessed by magnetic cholangyoresonance (NMCR).
Results: TGF beta and TIMP 1 serum levels were higher in
cystic fibrosis patients (p = 0.078), with a positive non-
statistically significative correlation with those patients
showing NMCR liver alterations. Serum and fecal bile acid
levels were significative higher in CF patients. CFLD patients
had increased toxic bile acids serum levels. Serum total bile
acid levels showed a negative correlation with TGF beta (p =
0.066). Toxic serum levels of bile acid had also a positive
correlation with TIMP 1 (p = 0.018). NMCR detected 33% of
CF patients without liver disease studied by biochemical and
clinical parameters. They present a significative positive
correlation with TIMP 1 (p = 0.064). Vitamin A serum levels
had a negative non significative correlation with TIMP 1 as
a stellate cell activation (fibrogenesis hepatic cell).
Conclusions: TIMP 1 is the most discriminative serum
marker of hepatic fibrogenesis in cystic fibrosis liver disease.
PH1-17 NORMAL EXPRESSION OF BSEP IN GRAFTS FROM
CHILDREN WITH BSEP DISEASE:DOES IT JUSTIFY
LOW-GGT CHOLESTATIC DYSFUNCTION OF GRAFTS
RESPONSIVE TO HIGHER IMMUNOSUPPRESSION?
L Hierro1, L Alvarez2, C Daz1, C Camarena1, A De La
 ESPGHAN 38TH ANNUAL MEETING
Vega1, E Frauca1, G Munoz-Bartolo1, P Jara1. 1Pediatric
Hepatology Service. La Paz Univ. Hospital, Madrid, Spain.
2
Research Unit.La Paz Univ. Hospital, Madrid, Spain.
Aims: To evaluate canalicular expression of BSEP in grafts
showing low- GGT cholestasis.
Methods: Standard immunohistochemistry was used to
detect BSEP, MRP2 and MDR3 in biopsies obtained during
5 episodes of low-GGT cholestasis.
Results: Nine children underwent cadaveric liver trans-
plantation (LT) for BSEP deficiency. All patients survived,
but 3 of them developed episodes of cholestasis mimicking
the pre-LT disease. Case 1 presented jaundice and pruritus at
12th year (45th day: TBil:30.2 mg/dl DBil: 21.6 mg/dl
GGT:35 U/L). Biopsy showed cholestasis (IC) and giant-cell
transformation (GCT). On UDCA, steroid boluses (SB) and
conversion to tacrolimus (Tac), he evolved to near-normal
function at 4th month. Case 2 developed jaundice at 3rd year
(30th day: TBil:6.7 DBil:4.6 GGT:9). Biopsy showed
ductopenia, IC, prominent GCT, Mallory hialine. Conversion
to Tac +MMF+SB led to normal function in 5 months. At 5th
year, after switch to cyclosporine for EBV infection, jaundice
and pruritus appeared (TBil:2.7 DBil:1.5 GGT:8) and
reverted with Tac+SB. Jaundice and pruritus reappeared after
steroid withdrawal at 8th year (TBil:4.5 GGT:33). Biopsy
showed GCT, IC, periportal hepatitis. Addition of ster-
oids+MMF resolved dysfunction in 3 months. Case 3 started
pruritus at 2nd year (30th day: TBil:2.5 DBil:1.7 GGT:7).
Biopsy showed ductopenia. Conversion to Tac+SB and
UDCA normalized function. She was converted to cyclo-
sporine at 4th year for food allergy; she developed pruritus,
followed by jaundice, 1 year later (60th day: TBil:9.4 DBil:7
GGT:21). Biopsy showed IC and prominent GCT. Conversion
to tacrolimus reverted dysfunction completely. GGT in-
creased upon recovery in Cases 1 and 3. BSEP, MRP2 and
MDR3 were expressed normally in all samples.
Summary: 3 out of 9 children transplanted for BSEP disease
developed graft dysfunction resembling the disease. Clinical
features reverted with higher immunosuppression. Canalicu-
lar expression of BSEP in grafts was normal.
Conclusions: In view of normal BSEP expression, the
possibility of an immune recognition of BSEP triggering
these low-GGT processes can not be ruled out.
PH1-18 IS URSODEOXYCHOLIC ACID BENEFICIAL FOR
SUCCESSFULLY OPERATED CHILDREN WITH
BILIARY ATRESIA? S Willot1, S Uhlen1, G Briand2,
M Bonnevalle3, L Michaud1, F Gottrand1. 1Department of
pediatric gastroenterology, hepatology and nutrition, Lille
university childrens hospital, Lille, France. 2Structural
biochemistry laboratory, faculty of medicine, Lille, France.
3
Department of pediatric surgery, Lille university childrens
hospital, Lille, France.
Introduction: Although few data are available, ursodeox-
ycholic acid (UDCA) is used for several years in the
treatment of various cholestasic liver diseases in children,
including biliary atresia (BA). The aim of our study was to
assess efficacy of UDCA in children successfully operated for
BA.
Methods: We prospectively studied 15 children presenting
BA who had a successfull portoenterostomy (post-operative
conjugated bilirubinemia , 20 mg/l) and treated from time of
surgery onwards with UDCA (600 mg/m2/d), for at least 18
consecutive months. UDCA was then discontinued, and
clinical and biological assessment (ALAT, GGT, bilirubin,
total biliary acids, cholicchenodeoxycholic- and UDCA) was
675
performed at the time of discontinuation (T0), on follow-up
(every 612 months) (T1), and 612 months after reinstitu-
tion of UDCA (T2) if clinical or biological status worsened.
Results: One patient remained with unchanged normal
clinical and biological status 1 year after stopping UDCA.
The 14 remainders needed to start UDCA again 6 to 33
months after discontinuation. One patient presented recur-
rence of jaundice 3 months after UDCA was stopped, that
disappeared when treatment was restarted. No other clinical
modification (pruritus, occurrence of bacterial cholangitis)
was observed in the remaining children. Liver function tests
worsened significantly when UDCA was discontinued
(T1 vs T0, median [range], N: normal): ALAT: 3.1xN[0.8
7] vs 1.5xN[0.55.4] (p , 0.02), GGT: 7.9xN[1.830.2] vs
3.7xN[0.527.4] (p , 0.02) and serum cholic and cheno-
deoxycholic bile acids: 53 mmol/l[5279] vs 44 mmol/l[2-
130] (p = 0.02). When UDCA was reinstitued, ALAT and
GGT significantly decreased in all the patients studied (n = 7)
(p , 0.02). At T2, all these biochemical markers returned to
T0 levels (T2 vs T0, NS). Bilirubinemia did not change at any
time of the study.
Conclusion: Our results clearly demonstrate the beneficial
effect of UDCA on liver function tests in children success-
fully operated for BA. Effect on long term prognosis remains
to be assessed.
PH1-19 THE POSITIVE ASSOCIATION OF MIF GENE 2173 G/C
POLYMORPHISM WITH BILIARY ATRESIA IN TURK-
ISH PATIENTS C Arikan1, A Berdeli2, F Ozgenc1,
S Aydogdu1, R Yagci1. 1Ege University Pediatric Gastroen-
terology, Hepatology and Nutrition, IZMIR, Turkey. 2Ege
University Pediatric Molecular Research Center, IZMIR,
Turkey.
Macrophage migration inhibitory factor (MIF) is a pleiotro-
phic lymphocyte and macrophage cytokine; it is likely to play
an important role in innate immunity. Despite the importance
of MIF in innate and acquired immune responses, until
recently, few mechanisms had been reported that could
adequately explain MIFs functional contribution to inflam-
matory and autoimmune disease.
Aim: To analyse the association of the 2173 G/C poly-
morphism of the MIF gene genotype and allele frequencies
with biliary atresia.
Method: Between February 2003 and November 2004, 12
patients (mean age 0.6 6 0.4) diagnosed as biliary atresia.
After informed consent, blood was collected and DNA was
obtained. MIF 173 C/G polymorphism was detected using the
PE ABI Prism 310 Sequence Detection System (SDS) for
multicolor real-time or endpoint fluorogenic polymerase
chain reaction detection. They also compared with children
with chronic liver disease (n = 42) and a group of unrelated
healthy controls (n = 133). Associations were assessed by
using Fishers exact test.
Results: MIF 173 gene genotype and allele distribution
according to groups is summarized in table 1. Univariate
analysis showed that MIF-173G/C polymorphism was
significantly related to biliary atresia. When compared with
the control group the frequency of the MIF-173 C allele was
significantly higher in biliary atresia patients than chronic
liver disease and healthy controls.(p = 0.04). Further analysis
confirmed that MIF-173C allele was independent risk factor
for biliary atresia (p = 0.03, OR = 5.1; 95%CI 1.320.36).
Conclusion: Our results strongly suggest that 2173G/C
polymorphism of MIF gene might be associated with the
susceptibility of biliary atresia.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
676 ESPGHAN 38TH ANNUAL MEETING

TABLE 1. Distribution of MIF-173 gene genotype Alagille Syndrome (AS) is diagnosed in the presence of at
according to groups. least 3 major syndromic features. To study the clinical and
histological features at presentation, we performed a retro-
MIF-173G/C gene Biliary atresia Chronic liver Healthy spective analysis of the records of 121 children with AS.
genotype group disease group control group Cholestasis was seen in 118/121[97.5%], pale stools in
102/121[84.2%], characteristic facies in 94/121[77.6%],
GG 5 29 107
posterior embryotoxon in 73/121[60.3%], butterfly vertebrae
G/C 7 8 26
in 45/121[37.1%], heart disease (most often peripheral
CC 0 0 0
pulmonary stenosis), in 108/121[89.2%], renal disease in
Total 12 37 133
28/121[23.1%], birth weight 2.5kg or less in 55/108 [50.9%].
Cholesterol of .3.5 mmol/L was seen in 36/48 [75%]
children before 8 wks old and in 51/63[80.9%] , 16 wks old.
PH1-20 ICAM-1 GENE G241R POLYMORPHISM IS ASSOCI- Between 16wks and 1 year serum cholesterol of .5mmol/L
ATED WITH BILIARY ATRESIA A Berdeli2, C Arikan1, was seen in 7/10[70%] mean level 9.84mmol/L, and at
G Tumgor1, S Aydogdu1, R Yagci1. 1Ege University presention after1year of age in 12/13[92.3%], mean
Pediatric Molecular Research Center, IZMIR, Turkey. 2Ege 7.26mmol/L. Liver biopsy showed characteristic features of
University Pediatric Gastroenterology, Hepatology and paucity of interlobular bile ducts (PILBD) in 59/77 [76.6%]
Nutrition, IZMIR, Turkey. children below 16wks old, in 11/15 [73.3%] between 16 wks
and 1 year old and in 8/12 [66.66%] above 1 year old. Biopsy
Biliary atresia is the most common cause of pathologic infant findings other than PILBD were seen in 18/77 [23.3%]
jaundice and one of the most common reasons for liver children below 16wks of age of whom 12/77[15.5%] showed
transplantation. Through a number of hypothesis have been features of non-specific hepatitis and 6/77 [7.79%] had biliary
developed to account for this disease, its pathogenesis is features. Biliary features on biopsy were also seen in 4/14
poorly understood. There is a substantial evidence that this is [14.2%] between 16 wks and 1 year old, and in 3/12[25%]
an immune mediated disease which occurs in a genetically above 1 year. HIDA scans showed no excretion of isotope
susceptible host. The intercellular adhesion molecule-1 into the bowel after 24 hours in 21/35 [60%] and
(ICAM-1) is of paramount importance for the initiation and small/contracted/no gall bladder on ultrasound (U/S) was
propagation of various inflammatory conditions. The signif- seen in 30/105 [28.5%]. 12/121[9.91%] had a laparotomy
icance of this molecule in the pathogenesis of biliary atresia is and operative cholangiography, 2 proceeding to Kasai prior
not clear. to referral to our unit and 7/121[5.78%] had PTC/ERCP
Aim: To determine whether the Glu241Arg polymorphism in because of diagnostic difficulty. Clinical features of AS are
the ICAM-1 gene, which impairs inflammatory responses, is not as consistently informative as in the literature. Heart
associated with biliary atresia. disease/murmur and facial features are most likely to be noted
Method: Twelve patients (mean age 0.6 6 0.4) with biliary at presentation. Hypercholesterolaemia is an early feature.
atresia, 42 children with chronic liver disease and a group of Histology is misleading in 25%, HIDA scan and U/S suggest
unrelated healthy controls (n = 123) included in this study. a false diagnosis in 60% and 28% respectively, illustrating the
After informed consent, blood was collected and genomic difficulty of diagnosing AS and supporting the concept that
DNA was obtained. Genotyping was performed by amplifi- infants with liver disease warrant early specialist referral.
cation-refractory mutation system polymerase chain reaction
(ARMSPCR). Associations were assessed by using Fishers
exact test.
Results: Univariate analysis showed that polymorphism of
ICAMG241R polymorphism was significantly related to PH1-22 HYALURONIC ACID AS A NON-INVASIVE PREDIC-
biliary atresia. When compared with the control group the TOR OF FIBROSIS IN PAEDIATRIC LIVER DISEASE
frequency of the allele R241 and the heterozygous genotype JL Hartley1, RM Brown1, A Tybulewicz3, P Hayes3,
R/G241, were significantly increased in patient with biliary D Wilson2, P Gillett2, P McKiernan1. 1Birmingham Child-
atresia (p = 0.008). Also they had significantly more ICAM rens Hospital, Birmingham, United-Kingdom. 2Royal Hos-
R/G 241 genotype than patients with chronic liver disease pital for Sick Children, Edinburgh, United-Kingdom. 3The
(p = 0.03). University of Edinburgh, Edinburgh, United-Kingdom.
Conclusion: ICAM-1 gene Glu241Arg polymorphism is
associated with biliary atresia which might therefore Objectives: Hyaluronic acid (HA) is excreted by the liver via
represent a functional candidate genes. sinusoidal cell adhesion molecules. Movement of HA across
sinusoidal cells is impeded in fibrosis leading to a rise in
serum HA. As a marker of fibrosis, HA may obviate the need
chronic liver biliary atresia healthy control for liver biopsy, a porcedure that has a significant morbidity
disease group group group Total risk and may be inaccurate due to sampling error. In
paediatric practise HA has not previously been evaluated as
RR 23 4 89 116 part of a histologically controlled study in unselected
G/R 10 7 34 51 patients.
RR 3 1 0 4 Methods: 93 unselected consecutive children (median age
Total 37 12 123 172 7.5 years, range 0.8 months - 19 years) undergoing a liver
biopsy between April 2003 and March 2004 were pro-
spectively recruited.
Liver biopsy and fasting HA levels were taken simultaneously.
PH1-21 ALAGILLE SYNDROME - THE DIFFICULTY OF INI- The Ishak score was used to stage fibrosis and scores of three
TIAL DIAGNOSIS P Subramaniam1, A Kniseley1, G Mieli- or greater were regarded as significant fibrosis. HA levels
Vergani1, A Baker1. 1Paediatric Liver Service, Variety Club were measured using an enzyme linked binding protein assay
Childrens Hospital, Kings College Hospital, London, (@ 2002 Corgenix, Inc)(adult reference range 0 75 ng/ml).
United-Kingdom. Liver function tests and anthropometric data were recorded.

J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005

 ESPGHAN 38TH ANNUAL MEETING
Results: 23/93 (25%) biopsies had significant fibrosis with 5
staged as cirrhotic (stage 6). The HA levels in this group was
significantly higher than the children with mild fibrosis (less
than 3), (median level 72ng/ml vs. 30ng/ml, Mann Whitney U
test p,0.005).
Using a stepwise logistic regression analysis of laboratory
and anthropometric measurements HA was the only variable
predictive of significant fibrosis (p = 0.007). HA level
.50ng/ml had a sensitivity of 65% and specificity of 68%
for significant fibrosis.
Summary: Despite 75% of this study cohort having mild
fibrosis, HA was a significant non-invasive predictor of
histological fibrosis. In this unselected study group the
clinical use of HA is limited by sensitivity and specificity.
Conclusion: In clinical practise HA can not yet replace
histiological examination to evaluate hepatic fibrosis. Further
evaluation is needed to ascertain the utility of serial
hyaluronic acid levels in targeted patient groups.
PH1-23 PREHEPATIC PORTAL HYPERTENSION: 43 YEARS OF
FOLLOW-UP IN SINGLE INSTITUTION AND 20 YEARS
AFTER INTRODUCTION OF SCLEROTHERAPY
J Vukovic1, R Grizelj1, M Dujsin1, T Brkic1, R Stern-
Padovan1, A Antabak1, T Luetic1, S Batinica1. 1University
Hospital Rebro, Zagreb, Croatia.
Introduction: Surgery was treatment of choice for children
with complications of prehepatic portal hypertension until
sclerotherapy and band ligation has occurred. Introduction
of octerotide was another landmark in treatment. We report a
43-year (first 26 retrospective and next 17 prospective) anal-
ysis of 64 patients.
Methods: The charts of all patients treated before 1987 were
reviewed retrospectively and from that year on a prospective
evaluation based on database and registry has been started.
Results: Between 1960 and Dec 2003 64 children were
followed up between 2 and 43 years with a median of 17.4
years. We have divided our patients in two groups. First
consisted of all patients (No 20) treated before the in-
troduction of sclerotherapy (1984. in our hospital), and in the
second group were remaining 44 patients. First symptom in
historical group was hematemesis in 17 of 20 patients (85%).
Thirteen palliative surgical interventions, and 4 shunt
(spleno-renal) procedures were done in 14 patients (mortality
rate 35%), a 6 patients were treated medically (mortality rate
33%). In group which was treated initially with sclerotherapy
hematemesis was leading symptom in 54% of patients, and
overall mortality rate is 9%. In this group, 20 patients along
the course of their disease, due to failure of conservative
therapy or other reasons were assigned for 25 operations.
Mortality rate in combination therapy group is 12.5%. Rest of
the patients (24) were treated only with sclerotherapy or
octreotide. Mortality rate in this subgroup is only 5%. In the
last 2 years, there was any new case of prehepatic portal
hypertension and we are the only hospital in our country
equipped for dealing with such a problem. There is a steady
decline in incidence that has been observed for years
probably due to disappearance of so far unknown etiological
factor and improving neonatal practices.
Conclusion: There is a decline in incidence of prehepatic
portal hypertension. Sclerotherapy and octreotide have
improved outcomes considerably, but there is still subset of
patients who have to be operated. Our data support thesis that
exist subgroup of patients who should be candidates for
primary surgical procedure.
PH1-24 6-THIOGUANINE RELATED HEPATOTOXICITY IN
CHILDREN WITH ACUTE LYMPHOBLASTIC LEU-
677
KAEMIA - A DUAL CENTRE EXPERIENCE
M Ravikumara1, F Hill2, DC Wilson4, PM Gillet4,
R Brown3, A Thomas5, P Darbyshire2, P McKiernan1.
1
Department of Hepatology, Birmingham Childrens Hospi-
tal, Birmingham, United- Kingdom. 2Department of Haema-
tology, Birmingham Childrens Hospital., Birmingham,
United-Kingdom. 3Department of Pathology, Birmingham
Childrens Hospital., Birmingham, United-Kingdom. 4De-
partment of Paediatric Gastroenterology, Royal Hospital for
Sick Children, Edinburgh, United-Kingdom. 5Department of
Haematology, Royal Hospital for Sick Children, Edinburgh,
United-Kingdom.
Introduction: In children with Acute Lymphoblastic Leu-
kaemia (ALL), who had received 6-Thioguanine (6-TG) as
part of their treatment, there are reports that a significant
minority developed hepatic complications. Some children
developed portal hypertension attributed to Nodular Re-
generative Hyperplasia (NRH), which progressed despite
discontinuation of 6-TG treatment.
Aim: To describe a dual centre experience of chronic
hepatotoxicity following 6 TG treatment in children with
ALL.
Methods: Retrospective review of children who had 6-TG
treatment for ALL between October 1997 and March 2004
and had evidence of liver disease.
Results: A total of 9 children (7 boys and 2 girls) were
identified. Age at presentation ranged from 5 yr to 15 yrs
(median 11 yrs). All but one had clinically detectable
splenomegaly and one child had signs of chronic liver
disease. Two children had experienced upper gastrointestinal
bleed. All had persistent thrombocytopenia but only three had
abnormal liver function tests at presentation. Ultrasonogra-
phy (USG) showed coarse echo texture of liver and
splenomegaly in all. At endoscopy, 7 had oesophageal
varices. Eight children had liver biopsy and all showed some
degree of sinusoidal dilatation with varying degrees of
fibrosis and inflammation. Only one showed classical NRH
but four children had subtle features compatible with NRH.
One showed other Non Cirrhotic Portal Hypertension
(NCPHT) type changes. In two children neither NRH nor
NCPHT changes were seen.
At follow-up ranging from 7 to 37 months (median 26
months), all had persistent splenomegaly and 7 had persistent
thrombocytopenia.
Conclusions: Some children treated with 6-TG develop
chronic hepatotoxicity with severe portal hypertension. Not
all children had histological features of NRH. Clinically this
represents a significant morbidity for which long term follow
up and monitoring is indicated.
PH2-01 PREVALENCE OF HEPATITIS C INFECTION IN TEEN-
AGERSWHO HAD RECEIVED BLOOD TRANSFU-
SIONS AS NEONATES PRIOR TO IMPLEMENTATION
OF BLOOD DONOR SCREENING IN ISRAEL M Kori1,
O Flidel-Rimon1, E Granot1. 1Kaplan Medical Center,
Rehovot, Israel.
Aim: To determine presence and rate of occurrence of HCV
infection in teenagers who had received blood products after
birth, prior to HCV blood donor screening and study the
natural history of HCV infection acquired in the neonatal
period.
Methods: Using blood bank records we identified 509
neonates who had received blood products during 1988-
1990. Letters recommending HCV antibody testing were sent
to 415 surviving teenagers. 355 recipients were contacted and
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
678 ESPGHAN 38TH ANNUAL MEETING
invited to undergo testing. Testing was performed on 269
(74.7%). Nine (2.5%) refused, 8 (2.2%) were unavailable and
69 (19.1%) agreed to testing at a later date.
Results: 269 teenagers underwent HCV antibody testing; 154
born at term and 115 preterm. Birth weights ranged from
6705000gr. (mean 2281gr). Number of blood product units
transfused ranged from 136 (mean 3.8). Nineteen children
received more than 10 units. Three children (1.1%) tested
positive for HCV antibodies and were HCV-RNA positive by
PCR. No cases of spontaneous clearance of the virus were
detected. Birth weight of two of the three infected teenagers
was below 750 gr and both had received above 20 blood
product units. These two children had elevated liver enzymes
and underwent liver biopsy. Biopsies showed mild inflam-
matory activity and low fibrosis scores.
Conclusions: Children who had received blood products as
neonates, before the implementation of blood donor screen-
ing for HCV, had a relatively low risk of acquiring HCV
infection. In children who received transfusions of more than
10 blood units the risk of infection increased up to 10 fold.
PH2-02 FOETAL IMMUNOPROPHYLAXIS WITH HEPATITIS- B
VACCINE COULD BE A COST EFFECTIVE APPROACH
TOWARDS ERADICATION IN THE HIGH-RISK POPU-
LATION A Patwardhan1, P Jamjute2, G Peter3. 1Darlington
Memorial Hospital, Darlington, United-Kingdom. 2Royal
shrewsbury hospital, Shrewsbury, United-Kingdom. 3Dar-
lington Memorial Hospital, Darlington, United-Kingdom.
Objective: Antenatal vaccination in pregnant women with
recombinant DNAHepatitis-B Virus Vaccine that has the
Hepatitis-B Virus DNA- but not the core antigen (which is
responsible for infectivity), should be able to produce active
immunization in neonates, prevent vertical transmission
without producing HBV - infection. This assumption is
because carrier mothers can pass the HBV antigen to their
babies with resulting vertical transmission.
Hypothesis: Vaccination of pregnant women before 24
completed weeks of gestation with recombinant DNA HB
Vaccine which contains 22nm particle as an HBsAg as
opposed to 42 nm Virion (Dane particle) as an HBsAg
(hepatitis B surface antigen). which has got clearly the best
chance to pass through placenta due to its size to developing
immune system of baby and induce active and long lasting
immunity. Placenta becomes impermeable to antigen after
24/40 wks to antigen but continues to be permeable to
HBsAb.
Method: This is a Randomised Controlled Trial. 200 women
were randomly recruited from antenatal out patient. The cases
were recruited in to five groups
A. 44/200 Healthy women, vaccinated with HBVaccine
before conception.
B. 3/200 HBV carrier women who were vaccinated in third
trimester.
C. 3/200. HBV carrier women who were vaccinated before
24 /40 wks.
D. 37/200 healthy women who were vaccinated before
24/40 wks.
E. 38/200 healthy women who were vaccinated in third
trimester.
Group A acted as control group. After vaccination with
Recombinant Hepatitis B Vaccine Antibody to surface
antigen was measured in babies for one year. HBsAb
(Hepatitis B surface antibody ) was measured at birth, at
6/12 months, at 12/12 months and after a low dose of 0.1 ml
intradermal (1/10 th of standard dose ) booster at 12/12
months. In all the five groups.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
Results: All Hepatitis B surface antibody levels are in IU/L.
A. Babies born to this group of women, 32/44 had HBsAb
levels at birth between 10 IU/l-100 IU/l while 12/44
had levels below 10 IU/l at birth. 10/44 babies had HBsAb
levels between10 to 100 IU/l while 34/44 had levels
below 10 IU/l, at 6/12 months, All babies had HBsAb
levels below 10 IU/l at one year. and none showed
response to booster.
B. Babies born to In this group of women. Showed no
HBsAb at birth and no response to booster.
C. Babies born to in this group of women 3/3 babies had
HBsAb levels between1001000 IU/l at birth. The levels
remained in same range at 6/12 months and 12/12 months.
3/3 gave good response to booster.
D. Babies born to this group of women, 32/37 showed
HBsAb levels between 100 -1000 IU/l while 5/37 showed
HBsAb levels of 10100 IU/l at birth. At 6/12 months and
12/12 months, level stayed the same and all the babies
gave very good response to booster.
Babies born to in this group of women 20/38 had HBsAb
levels between 10100 IU/l while 18/38 had levels below
10 IU/l at birth.2/38 babies had HBsAb levels above 10 IU/l
while 36/38 had no detectable HBsAb levels at 6/12 months.
None had detectable HBsAb levels at 12/12 months and none
responded to the booster.
Conclusion: Group A and E were clearly inferior to group D
and F in their development of immunity.
In-group F, 96 % of women had HBsAb .100 IU/L at 12
months, compared to 86 percentage in-group D. These
proportions were compared using the Fisher 2-Tail Exact
Test giving p = 0.11 i.e. some evidence of a genuine
difference but not significant at the 5 % level.
In the group C, 100 % of women had HBsAb .100 IU/L at
1 2 months, compared to none in the group B. These
proportions were compared using the Fisher 2-tail Exact
Test, giving p = 0.10 i.e. that data alone provide some
evidence of a genuine difference, although not significant at
the 5 % level. This combined with the marked difference in
response to the booster, supports the hypothesis of superior
immunity with vaccination at 2024 Wks.Antenatal imuno-
prophylaxis when given in second trimester gives a lasting
active immunity to the newborn baby.
PH2-03 INTERFERON-ALPHA AND EXTENDED LAMIVUDIN
COMBINATION THERAPY IN CHILDREN WITH
CHRONIC HEPATITIS B INFECTION: EFFICACY AND
RATE OF EMERGENCE OF YMDD MUTANT VARIANT
Z Kuloglu1, A Kansu1, E Erden2, N Girgin1. 1Ankara
University School Of Medicine, Department of Pediatrics,
Division of Gastroenterology, Hepatology and Nutrition,
Ankara, Turkey. 2Ankara University School Of Medicine,
Department of Pathology, Ankara, Turkey.
Aim: To evalute the efficacy and rate of breakthrough of IFN-
alpha and extended LAM combination therapy in chronic
hepatitis B infection.
Methods: 38 children (9.8 6 4.1 years) received simulta-
neously interferon- alpha (510 megaunits /m2for six months)
and LAM (4 mg/kg/day, max 100 mg) for median 31 (12 -60)
months. LAM was administered until antiHBe seroconver-
sion and was continued for another 6 months in responders. In
nonresponders it was stopped after at least 2 years. Histologic
evaluation was performed at baseline and end of therapy.
Follow up time after end of therapy was median 12 (330)
months.
Results: Pretherapy median ALT and HBV DNA were
60 U/L and 2000 pg/ml. End of therapy ALT normalization
(71.1%), HBeAg loss with anti HBeAg seroconversion
 ESPGHAN 38TH ANNUAL MEETING
(34.2%), HBsAg clearance (5.2%), HBV DNA clearance
(36.8%), histologic (47.6%), complete (34.2% ) and sustained
(31.7%) response were achieved. Relapse rate was 7.6%.
Predictive factor in responders was high baseline ALT (p ,
0.01). Breakthrough and YMDD mutant rate were 65.8% and
55.2%.Baseline ALT was lower in patients with breakthrough
(p , 0.01) but no significant difference in sex, age, baseline
HBV DNA level and HAI score were found (p . 0.05). In
children with breakthrough baseline and end of therapy ALT
were not significantly different, but HBV DNA was lower
than baseline (p , 0.01). In follow-up, 22.2% of non-
responders showed ALT elevation and 8% ( one with YMDD
mutant) achived response.
Conclusion: Response rate of IFN and extended LAM
therapy was not higher than previously reported monotherapy
or combination therapies. Histologic response occured in
47.9% of patients. Relapse rate was low. High ALT level was
the predictor of succesful therapy. HBe Ag seroconversion
continued with extended therapy, but mutation rate also
increased. Breakthrough was associated with lowel ALT.
Hepatitis flare was not observed in nonresponders. Because of
development of YMDD mutants, the benefit versus risk of
LAM therapy must be weighed carefully.
PH2-04 LONG TERM LAMIVUDINE TREATMENT FOR
CHRONIC HEPATITIS B INFECTION IN CHILDREN
UNRESPONSIVE TO INTERFERON C Hartman1,
D Berkowitz1, O Eshach1, B Eno1, E Rozenthal1,
N Rimon2, T Kra-Oz2, R Shamir1. 1Division of Pediatric
Gastroenterology and Nutrition, Meyer Childrens Hospital,
Haifa, Israel. 2Laboratory of Virology, Rambam Medical
Center, Haifa, Israel.
Background: Three years ago we reported the results of an
open prospective study that investigated the efficacy and
safety of one year lamivudine treatment in children (n = 22)
with chronic hepatitis B (CHB) who failed interferon
treatment. We showed that one year lamivudine treatment
resulted in decreased HBV replication and improved ALT
values, but an exceptionally high rate (65%) of lamivudine-
resistant mutants.
Aims: The present study examined the efficacy and safety of
prolonged lamivudine therapy in this group of children (aged
8.519 years of age).
Methods: This cohort of 22 children (17 boys, 5 girls) was
prospectively followed for a median of 3.6 years (range 3 to
4 years). Children were treated with lamivudine 3 mg/kg/day
up to 100 mg/day, and were instructed to come back to follow
up every 4 months. At each visit children underwent physical
examination, compliance to treatment and side effects were
recorded and blood tests were performed, including blood
count, liver enzymes and bilirubin, HBeAg/antiHBe, HBV
DNA and HBV mutants.
Results: Of the 22 children that started the study, only five
were taking lamivudine after 4 years. The reasons for drop
out from the study were lack of adherence to treatment
(10/17), HBeAg/antiHBe seroconversion (5/17) and lack of
response to treatment (2/17). Children who maintained
lamivudine treatment at the end of 4 years had persistent
ALT normalization and low HBV DNA levels and YMDD
mutant was present only in one child. No clinical adverse
effects were recorded during lamivudine treatment or after
treatment termination. Transient ALT flare up (312 ULN)
was present in one patient. Two patients showed persistent
elevated CPK values (1753 IU/L to 478 IU/L) with normal
blood gases and serum lactate.
Conclusions: In this cohort, children with CHB who failed to
respond to previous IFN therapy and were treated with
lamivudine for up to 4 years, had high rates of drop out,
679
mainly because of lack of compliance to treatment and poor
HBeAg seroconversion rate, suggesting that prolonged
lamivudine treatment in children is hard to achieve and
probably not efficient.
PH2-05 CTLA-4 GENE POLYMORPHISM IN CHILDREN WITH
AUTOIMMUNE HEPATITIS M Wozniak1, H Witas2,
M Woynarowski1, J Socha1, J Michalkiewicz1. 1Childs
Health Memorial Institute, Warsaw, Poland. 2Dept. Cyto-
biochemistry, Lodz, Poland.
Background: A G transition in ekson 1 (+49) of CTLA-4
gene can influence T-cells activity and may trigger autoim-
mune reactions.
Aim of the Study: The aim of the study was to evaluate
CTLA-4 gene polymorphism in children with autoimmune
hepatitis (AIH).
Materials & Methods: CTLA-4 polymorphism was ana-
lyzed by restriction analysis. We tested 92 samples taken
from children (M-32, F-60, mean age 14 yrs) with
autoimmune hepatitis and the results were compared to the
CTLA-4 gene polymorphism in 223 healthy subjects.
Results: We found that allele G frequency was higher in
children with AIH than in controls (44% vs 33.4%, p , 0.02).
Homozygosity for CTLA-4 polymorphic allele G (GG) was
more represented in children with AIH than in controls (23%
vs 7.6%, p , 0.001). The distribution of genotypes is shown
in the table:
genotype AIH (N = 92) Controls (N = 223)
AA 32 (35%) 91 (40.8%)
AG 39 (42%) 115 (51.6%)
GG 21 (23%) 17 (7.6%)
Conclusion: We demonstrated that the distribution of poly-
morphic allele G of CTLA-4 in children with AIH is different
from its distribution in healthy subjects.
PH2-06 PREVALENCE OF LIVER STEATOSIS AND IMPACT ON
INTERFERON THERAPY IN CHILDREN WITH CHRONIC
HEPATITIS CA Giannattasio1, A Sepe1, C Della Corte1,
R Vecchione2, A Vegnente1, R Iorio1. 1Department of
Pediatrics, University Federico II, Naples, Italy. 2Depart-
ment of Pathology, University Federico II, Naples, Italy.
Hepatic steatosis is a common histopathologic feature in
adults with chronic hepatitis C (CHC), particularly in
presence of genotype 3. Aim of the present study was to
evaluate prevalence of histological signs of steatosis, its risk
factors and impact on response to IFN therapy in children
with CHC.
Methods: 64 consecutive children with CHC (29 males,
median age at liver biopsy 8.16 years, range 114) were
studied. 32 (50%) children were infected with genotype 1b, 4
(6.3%) with genotype 3, the remaining with other genotypes.
Liver biopsy was performed after a median duration of HCV
infection of 6.9 years (range 214) and was evaluated for
steatosis, and necroinflammation (grade) and fibrosis (stage)
according to Ishak. 47 children were treated with IFN after
liver biopsy. Clinical, auxological, laboratory, histological
features and response to IFN were compared between
children with and without evidence of liver steatosis.
Results: Histological evidence of mild-moderate steatosis
was present in 16 (25%) patients, while the remaining
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
680 ESPGHAN 38TH ANNUAL MEETING
children did not have steatosis. The analyses of liver biopsies
showed in children with steatosis and in those without
steatosis a median grade of 4.7 (range 28) and of 4.3 (range
27), and a median stage of 1.6 (range 04) and 1.9 (range
14), respectively. Obesity was found in 3 (18.7%) children
with steatosis and in 3 (6.3%) children without steatosis. No
patient showed other causes of liver steatosis. Sustained
complete response to IFN was obtained in 17 (35.5%)
patients without steatosis and in 3 (23%) children with
steatosis. No significant difference was found between the
two groups of patients for obesity, route of infection, HCV
genotype, liver function tests, histological grade and stage
and response to therapy.
Conclusion: 25% of children with CHC showed histological
evidence of liver steatosis. No risk factors for steatosis was
identified. Although no significant difference was found,
response rate to IFN was higher in patients without steatosis.
PH2-07 HIGH PREVALENCE OF AUTOIMMUNE HEPATITIS
AMONG CHILDREN WITH PRIMARY SCLEROSING
CHOLANGITIS M Wozniak1, M Woynarowski1, J Socha1.
1
Childs Health Memorial Institute, Warsaw, Poland.
Primary sclerosing cholangitis (PSC) is a rare disease in
children. We retrospectively analyzed the records of 12
patients (M-5/F-7) aged 216 (mean 12,3) years with PSC
diagnosed at our institution between 1996 and 2003. At the
beginning of the disease patients presented with high activity
of ALT (mean 225 U/l) and GGTP (175 U/l), increased
concentration of bilirubin (mean 2.6 mg/dl), IgG (2021 mg/dl)
and gammaglobulins (27.18 G/l). Autoantibodies (ANA,
SMA) were present in 10 children. 11 children had
inflammatory changes in liver biopsy. The initial diagnosis
was autoimmune hepatitis (AIH) (9 patients), ulcerative
colitis (UC) (1 patient), PSC (1 patient), PSC-AIH overlap
syndrome (1 patient). Patients with AIH and UC received
immunosuppressive treatment (prednisone with or without
azathioprine). ALT activity IgG and gammaglobulins con-
centration decreased but no improvement in cholestasis was
observed and thus patient were reevaluated for PSC. The
delay in PSC diagnosis was 062 (mean 20) months from
onset of disease symptoms. The diagnosis of PSC was based
on liver biopsy in 9 (75%), ERCP in 5 (42%) and MRI
cholangiography in 3 (25%) of children. The final diagnoses
in this group of children were: PSC-AIH overlapping
syndrome in 9 (75%), PSC-AIH-UC overlapping syndrome
in 1 and isolated PSC in 2 (17%) cases. All children received
UDCA treatment. The control evaluation done after 624
months (mean 16) showed decrease of mean bilirubin
concentration from 1.98 mg/dl on the day of PSC diagnosis
to 0.6 mg/dl and GGTP activity from 255 U/l to 159 U/l. No
further changes in ALT, IgG and gammaglobulins were noted.
Conclusion: PSC-AIH overlapping syndrome was very
common in our group of children. Children with AIH and
continuous or increasing cholestasis should be evaluated for
PSC. UDCA treatment may improve cholestasis.
PH2-08 LONG TERM EFFICACY OF INTERFERON TREAT-
MENT IN CHILDREN WITH CHRONIC HEPATITIS
CAUSED BY A AND D HBV GENOTYPES L Szenborn1,
I Kacprzak-Bergman1, I Zaleska1, P Wintermeyer2,
P Gerner2, S Wirth2. 1Department of Pediatric Infectious
Diseases, Wroclaw University of Medicine, Wroclaw, Poland.
2
Children & #8217;s Hospital, Helios Klinikum Wuppertal,
Witten-Herdecke University, Wuppertal, Germany.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
Hepatitis B virus (HBV) genotypes may be related to severity
of liver disease and treatment response. There are limited data
about such relation in children. The aim of our study was
investigation of influence HBV genotypes A and D on
interferon alpha treatment results.
Methods: The serum samples were purchased from 77
patients (49 males and 28 females) aged 8 to 24 years (median
13 years) with chronic viral hepatitis B. Duration of the
disease was 3 to 18 years. All examined patients were treated
with interferon alpha for 20 or 24 weeks (3 x 3 million units
weekly). Interferon treatment was finished 3 to 10 years
(median 7 years) before examination. We analyzed HBV
markers by EIA method and HBV DNA level by PCR (Roche
reagents, positive results - above 1000 copies per milliliter).
The HBV-DNA sequences were assigned to the appropriate
genotype based on the restriction fragment length poly-
morphism created by Ava2 and Dpn2 action on an amplified
segment of the pres-S-region.
Results: In our patients genotype A in 59 (76.6%) group I,
and genotype D in 18 (23.4%) - group II were identified. Loss
of HBsAg occurred in 7/59 (11.9%) in group I whereas in
group II in 1/18 (5.4%). Anti HBsAb was found in 8/59
(13.6%) in group I and in group II in 1/18 (5.4%). In group I
49/59 (83%) patients cleared HBeAg while in group II all
patient were HBeAg negative. Positive anti HBeAb were
found in 49/59 (83%) in group I and in group II in 16/18
(88.9%). These all differences were not significant. All
patients were positive for HBcAb IgG and negative for
HBcAb IgM. Reduction in HBV DNA to undetectable levels
(below 1000 copies) was achieved in 18/59 patients (30.5%)
of group I and in 8/18 patients (44.4%) of group II. These
differences were not significant. Median levels of numbers
HBV copies in booth groups were similar (in group I 13650
copies/ml and in group II 10769/copies/ml) so differences
were not statistically significant.
Conclusion: We do not found any influences of HBV
genotypes A and D on the response to interferon therapy
defined as loss of HBeAg, HBsAg, HBV elimination and
intensity of virus replication in children with chronic
hepatitis B.
PH2-09 THE EFFICACY OF SERUM HYALURONIC ACID
LEVEL IN PREDICTION OF HEPATIC HISTOPATHOL-
OGY. S Cam1, D Ertem1, E Kotiloglu. Karaa2, E Tutar1,
E Pehlivanoglu1. 1Marmara University School of Medi-
cine, Division of Pediatric Gastroenterology, Hepatology
and Nutrition, Istanbul, Turkey. 2Marmara University School
of Medicine, Department of Pathology, Istanbul, Turkey.
Introduction: Fibrosis associated with chronic liver diseases
is an insidious process involving inflammatory responses and
alterations in the extracellular matrix (EM). Hyaluronic acid
(HA) is a basic component of EM of the liver and it was
thought that increased serum levels of HA might indicate
hepatic fibrosis as a simple non-invasive marker.
Aim: The objective of the current study was to investigate the
efficacy of serum HA level to predict the histopathological
changes, observed in liver biopsies in children with chronic
liver diseases, and to determine the relationships between
serum HA level and age, sex, etiology of liver disease, and
biochemical parameters.
Method: The study group consisted of 34 children with
clinical and laboratory findings of chronic liver disease with
a mean age of 65.31 6 64 months. All children underwent
liver biopsies for diagnostic purposes. The biopsies were
stained by hematoxylin-eosin, reticulin and trichrome stain-
ing and evaluated blindly by the same pathologist. The degree
of fibrosis on biopsy specimens was graded 0 (no fibrosis)
to IV according to the Knodell hepatic activity index. The
 ESPGHAN 38TH ANNUAL MEETING
degree of inflammation and reticulin structure was also
graded semi-quantitatively. Simultaneously blood samples
were obtained just before the liver biopsy for the measure-
ment of HA levels and biochemical tests. Forty-one healthy
children were included into the control group.
Results: Histopathological examination of 34 biopsy sam-
ples revealed stage I fibrosis in 6 patients, stage II fibrosis in
4 patients, stage III fibrosis in 8 patients, stage IV fibrosis in
1 patients, and no fibrosis was found in remaining 15 patients.
The average serum level of HA was 79.25 6 72.65 ng/ml in
the study group, while serum HA level was 5.5 6 6.4 ng/ml in
the control group. The difference was statistically significant
(p , 0.0001). The average levels of HA regarding the severity
of fibrosis were 56.9, 52.6, 146 ng/ml in grade 0, I-II, III-IV
fibrosis, respectively. There was a statistically significant
correlation between the degree of fibrosis and the serum HA
levels (p , 0.05). When the patients were categorized
according to the etiologies of chronic liver disease (hepatitis
B, metabolic diseases, biliary pathologies, neonatal hepatitis,
autoimmune hepatitis and others) the average serum HA level
was higher in patients with biliary pathologies when
compared to the other groups (p , 0.01).
Since there was a statistically significant difference between
serum HA a level of children with chronic liver disease and
controls, the reticulin stained liver biopsies were evaluated
for condensation and collapse of reticulin fibers, and various
degree of collapse in reticulin meshwork was found in all
biopsy samples.
There was no statistically significant correlation between the
levels of HA and degree of inflammatory activity in biopsy
samples and the characteristics of study group such as age,
sex and liver function tests.
Summary: Current study suggests that serum HA level may
indicate the presence of chronic liver pathology as a non-
invasive marker. Patients with grade III-IV fibrosis had the
highest serum HA levels. Although serum HA levels were
higher in patients with mild fibrosis when compared to
controls, the statistical significance of serum HA level as
a noninvasive marker became more prominent in children
with moderate to severe fibrosis.
Conclusion: Detection of a higher serum HA levels in
children with liver fibrosis with respect to healthy controls
may indicate that serum HA level may be used as a marker in
chronic pathologies of the liver.
PH2-10 AUTOIMMUNE LIVER DISEASE/SYSTEMIC LUPUS
ERYTHEMATOSUS OVERLAP SYNDROME IN CHIL-
DREN M Samyn1, D Bogdanos1, Y Ma1, B Portmann1,
D Vergani1, G Mieli-Vergani1. 1Institute of Liver Studies,
Kings College Hospital, London, United-Kingdom.
Background: An overlap syndrome between autoimmune
liver disease (AILD) and systemic lupus erythematosus
(SLE) has been occasionally described in adults, but never
in children.
Patients: Between 1973 and 2004, 8 (7 female) children,
who presented to our centre with AILD, were also diagnosed
with SLE. Median age at presentation was 8.75 (range 2.7
12.4) years. Median duration of symptoms was 1 (range 0.5
12) month, the most common being jaundice (7), fever (5),
lethargy (5), vomiting (4), joint (3) and abdominal pain (3).
Five children had antinuclear antibody (ANA) and smooth
muscle antibody (SMA), 1 ANA only and 1 SMA only, while
1 was ANA, SMA and liver kidney microsomal1 (LKM1)
antibody positive. Liver histology showed severe inflamma-
tion in all 4 children biopsied at presentation, and moderate
(2) and mild (2) inflammation in children biopsied after
starting treatment. Fibrotic and cirrhotic changes were seen in
6 and 1 respectively. Treatment with Prednisolone was started
681
at a median time of 1 day after presentation (range 14).
Azathioprine was added in 4 patients. Mycophenolate Mofetil
replaced Azathioprine in 2 and Cyclosporin was used in 1.
SLE was diagnosed between 1 week and 6.9 years (median
7 mo) after AILD. 8 children had skin, 7 joint, 4 renal, 4
haematological and 3 pleural/pericardial involvement. ANA
became positive in all, anti-double-stranded DNA and
extractable nuclear antigens in 4 and 2 respectively. C4 was
low in 5. Soluble liver antigen (SLA) antibodies were positive
in 5/7, ribosomal P antibodies in 4/5 tested. SLE was treated
with high dose pulse steroids (5), Cyclophosphamide (4),
Hydroxychloroquine (3), Methotrexate (2) and plasmaphere-
sis (1). Median follow up is 3.7 (range 0.2- 8.7) years. Six
children have normal liver function tests while 2 continue to
have mildly raised AST. Two patients required further pulse
steroids because of SLE relapse.
Conclusion: AILD/SLE overlap syndrome does exist in
children with AILD. SLE can be present at diagnosis of AILD
or develop during follow up and must be timely and
aggressively treated.
PH2-11 METABOLIC DISTURBANCES IN CHILDREN WITH
NON-ALCOHOLIC FATTY LIVER DISEASE A Wierzbicka1,
P Socha1, E Skorupa1, R Janas1, M Dadalski1, E Dzik1,
M Litwin1, J Socha1. 1The Childrens Memorial Health Institute,
Warsaw, Poland.
Obesity, lipid diturbances, insulin resistance and free radical
injury are possible risk factors leading to fatty liver disease in
adults. Still both patomechanisms and risk factors of fatty
liver in children are not precisly described. The aim of the
study was to estimate metabolic disturbances in children with
non-alcoholic fatty liver disease (NAFLD) in relation to
healthy and obese (BMI . 95perc.) children.
We studied 25 children with NAFLD (diagnosis based on
ultrasound and increased ALAT activity, infectious and
metabolic disorders excluded) aged 12.4 6 4.4 y (10 children
were obese), 30 children with obesity and normal liver
function aged 15.6 6 2.9 y as well as 29 healthy aged
matched controls.
Results: ANOVA analysis of the three groups showed
differences in TBARS (lipid peroxide) serum conc., fasted
insulin conc., HOMA IR (insulin resistance indicator), LCAT
activity, ApoA1, ApoE, HDL-C serum conc., GSH ercs conc.,
GPx ercs activity, there were no differences in fasted glucose,
total cholesterol, TG, LDL-C, ApoB, VLDL-C values. The
lowest LCAT activity was found in obese children (104.6 6
58.8 nmol/ml/h), intermediate in NAFLD (145.3 6 68.9) and
highest in controls (166.7 6 58.1). ApoE conc. were similar
in NAFLD and controls (6.8 6 5.1 mg/dl vs. 8.3 6 3.7) and
lower than in obese pts. (14.4 6 5.9; p,0.01). The following
parametres increased in NAFLD compared to controls:
TBARS (0.41 6 0.14 nmol/ml vs. 0.24 6 0.05), fasted insulin
(17.7 6 13.1 U/l vs. 9.9 6 2.8). Apo A1 (1.23 6 0.3 g/l vs.
1.46 6 0.2), HDL-C (43.4 6 10.3 mg/dl vs. 55.0 6 9.5), GSH
(556.0 6 124.8 mcmol/ml vs. 720.5 6 113.2), GPx (28.8 6
4.6 U/gHb vs.34.8 6 8.8). Similar differences were found
between children with obesity and controls.
Conclusions: Children with NAFLD and obesity present
with similar metabolic disturbances that may cause liver
injury: increased lipid peroxidation and decreased antioxi-
dant status as well as hiperinsulinemia. Decreased HDL and
apoA1, and increased LCAT seem to be secondary changes to
obesity or fatty liver disease.
PH2-12 PREVALENCE OF LIVER STEATOSIS IN A COHORT OF
OBESE CHILDREN AND ITS RELATIONSHIP TO
BIOCHEMICAL AND AUXOLOGICAL PARAMETERS
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
682 ESPGHAN 38TH ANNUAL MEETING
W Kleon1, G Radetti2, K Pittschieler3. 1Department of
Pediatrics, Bolzano, Italy. 2Department of Pediatrics,
Bolzano, Italy. 3Department of Pediatrics, Bolzano, Italy.
Aim: To investigate the prevalence of liver steatosis assessed
by MRI in a group of obese children and the correlation
between severity of hepatic fatty infiltration, biochemical
findings and anthropometric parameters.
Patients and Methods: We studied 44 obese children
between 6 and16 years of age, with a BMI on the 97 th
centile. All were assessed for body fat and its distribution
(skinfold thickness, waist-to-hip ratio). Fasting blood sam-
ples were obtained to evaluate liver function, serum glucose
and insulin and lipid profile. A phase contrast MRI was
performed to quantify the degree of the liver steatosis.
Results: Liver steatosis was diagnosed in 14 patients
(31.8%); 8 of them (57.1%) also had an elevated ALT. Most
of the children (91%) showed insulin resistance, assessed by
HOMA and QUICKI, and an abnormal lipide profile. A
strongly positive correlation was found between the degree of
hepatic steatosis and liver function tests, such as AST (p ,
0.0001) and gGT (p , 0.0001). We did not find any
significant correlation between the degree of obesity and the
amount of hepatic fat infiltration. In our study patients height
(SDS) appeared to be a predictive factor of liver involvement
(p , 0.006). SUMMARY and
Conclusions: The low prevalence of hepatic steatosis in the
study group seems to be in contrast to the high prevalence of
liver steatosis in previous studies. This study shows a positive
correlation between patients height and fatty liver and
confirms the relationship between the degree of abnormal
liver enzymes and fatty infiltration of the liver.
PH2-13 DOES SMALL INTESTINE-LIVER AXIS PLAY A ROLE
IN PEDIATRIC OBESITY RELATED LIVER DAMAGE ?
A PILOT STUDY. P Vajro1, P Colicchio1, S Lucariello1,
B Aceto1, MI Spagnuolo1, MR Licenziati2, A Barile1,
G Vallone3. 1Dept Pediatrics Univ Naples Federico II, Naples,
Italy. 2Dept Pediatrics Cardarelli Hospital, Naples, Italy.
3 ages 515 years, presenting hepatomegaly and/or elevated
Dept Radiology Univ Naples Federico II, Naples, Italy.
Background: Studies in an obese animal model and in some
obese adults have recently suggested that a derangement of
the small intestine-liver axis may play a role in the
development of obesity related liver abnormalities.
Aim & Methods: To examine small intestinal data [basal &
lactulose H2 breath test (BT), permeability to mannitol/cel-
lobiose (spectrophotometric method), serum levels of circu-
lating bound endotoxins (LAL chromogenous method) and of
Abs to bacterial wall products (peptidoglycan-polysaccharide
(PG-PS) polymers; ELISA test) which reach the liver through
the portal flow] in obese children (age 613 y) with and
without hypertransaminasemia and/or ultrasonographic (US)
bright liver not due to other causes of hepatopathy.
Results: Up to now we have enrolled 27 obese children with
[group 1, n = 16, mean age 10.8 y; BMI 30.6; ALT 52 6
25 U/L) and without (group 2, n = 11, mean age 10.5 y (p =
NS); BMI 27.5 (p = NS), ALT 21 6 9 U/L (P = 0.001)] liver
disease. 100% pts had familial obesity; 75% vs. 36% had
familial history of liver disease (p = 0.06). Obesity duration,
food habits, skinfold tickness and body circumferences,
bioimpedance analysis of % lean and fat mass, visceral and
subcutaneous US fat, serum lipids, IL6, TNFa and iron status
were comparable in the two groups. As expected, fasting
insulin to glucose ratios (FGIR) and n. insulin-resistant
(FGIR ,7) pts were different between groups [(5.5 6 2.9 vs.
8.2 6 4.1 (p = 0.05) and 12/16 vs. 4/11 (p = 0.06),
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
respectively]. As for intestinal data, there were no differences
in prevalence of small intestine bacterial overgrowth
evaluated by H2 BT and abnormalities of intestinal perme-
ability (1 case/group) & serum levels of bound endotoxins
(9.2 6 4.0 vs 12.0 6 6.5 pg/ml). Serum IgA (but not IgG and
IgM) Abs to PG-PS were significantly higher in group 1
(1.0 6 0.4 vs. 0.7 6 0.2 pg/ml; p , 0.05). We are now
evaluating changes of the aforementioned data during
a double blind treament with probiotics vs. placebo.
Summary & Conclusions: Apart from Abs to PG-PS, so far
we have not found relevant evidences suggesting that
intestinal abnormalities may greatly contribute towards liver
damage in pediatric obese children.(Acknowledgements:
Italian MIUR 2003 & ELFID).
PH2-14 MEASUREMENT OF LIVER FAT BY FAST RMI IN
OBESE CHILDREN AND ITS RELATIONSHIP TO
ANTHROPOMETRIC AND BIOCHEMICAL PARAME-
TERSL L Pacifico1, C Anania1, A Laghi2, M Celestre2,
P Paolantonio2, M Matrunola1, C Chiesa3, R Passariello2.
1
Dpt of Pediatrics, La Sapienza Univ., Rome, Italy. 2Dpt of
Radiology, La Sapienza Univ., Rome, Italy. 3National
Researh Council, Rome, Italy.
Background: Nonalcoholic fatty liver disease (NAFLD) is
increasingly recognized as a common cause of chronic liver
disease in the pediatric population. Children with NAFLD are
uniformly obese and asymptomatic. The disease is conven-
tionally diagnosed by ultrasonographic appearances together
with an elevated level of alanine aminotransferase (ALT). MR
imaging (Fast MRI) has recently been shown to accurately
quantitate hepatic fat fraction, even at low or near-normal
levels. There are, however, very few studies in children.
Objective: The aim of this study was to investigate, in
a population of obese children, the association between the
hepatic fatty changes as established by fast MRI and the
anthropometric and biochemical parameters.
Design: Cross-sectional study.
Methods: 41 obese (BMI . 95th percentile for age) children,
aminotransferases and/or ultrasonographic appearance of fatty
liver disease were candidates for MRI assessment of hepatic
steatosis. All subjects underwent dual energy x-ray absorpti-
ometry DEXA scan measurement, and fasting serum mea-
surement of glucose, insulin, c-peptide, leptin and lipid profile.
Results: A diagnosis of fatty liver was established by MRI in
twelve (29%) patients with an hepatic fat fraction (HFF)
. 8%; of these, six had HFF of 9 to 11%, while the remaining
had a HFF of 20% or higher. HFF was not correlated to age,
sex, BMI and fat mass as well as to leptin concentrations.
Linear regression analysis showed that HFF was positively
associated with raised ALT (p , 0.01) and triglycerides
(p , 0.05) as well as with elevation of insulin (p , 0.05 ) and
C-peptide (p , 0.05). Insulin resistance (as defined by
HOMA- homeostasis model assessment index) and severe
steatosis represented a unique combination.
Conclusions: Fast RMI being noninvasive and easily to
perform could be used for the monitoring of the progression
of hepatic steatosis.
PH2-15 NON ALCOHOLIC FATTY LIVER DISEASE RISK
PROFILE IN A PAEDIATRIC OBESE POPULATION
H Antunes1, A Martins1, D Lemos1, C Santos2. 1Gastrenter-
ology, Hepatology and Nutrition Unit, Pediatric Dept,
Sao Marcos Hospital, Braga, Portugal. 2Department of
Biostatistics and Medical Informatics, Faculty of Medicine,
Porto University, Porto, Portugal.
 ESPGHAN 38TH ANNUAL MEETING 683

Introduction: Non alcoholic fatty liver disease (NAFLD) Age at admission and serum levels of lipid and glucose were
covers a spectrum from static disease to more aggressive not different in two groups. Male sex was predominant in fatty
forms that can progress to cirrhosis within childhood. liver group. In obese children, mean BMI, and mean levels of
Aim: To determine the prevalence of NAFLD in an obese serum insulin, transaminases, and HOMA-IR scores were
paediatric population and try to found a NAFLD risk profile. significantly higher in the fatty liver group. In the study
Methods: We collected all the protocol data of the patients population, serum ALT levels were found weakly correlated
referred to us between 1/2/199920/12/2004. Obese was with BMI, insulin and HOMA-IR scores (p = 0.02 r = , 167; p =
defined as BMI . 95th percentile (P) for age and gender. 0.01 r = , 186; p = 0.008 r = 0.194 respectively).
Hyperinsulinism: insulin .33 mU/mL. Insulin resistance was Conclusion: Juvenile obesity associated with higher BMI
determined using homeostasis model assessment of insulin and insulin levels brings much higher risk for NAFLD. We
resistance (HOMA-IR). We used the x2, Mann-Whitney-U conclude that obese children should be screened for
and t-test. hepatosteatosis, which might be associated with hyper-
Results: Of the 327 obese children and adolescents, 48.2% insulinemia.
were male. The median age was of 10 years (range 2 to 17
years) and the mean (sd) of BMI 27.3 6 4.3 with 6.7 %
having BMI . 35. At 3 months follow-up, the BMI decreased Patients Patients
to 26.6 6 4.3. They had hyperglycaemia in 4% (n = 250) and with NAFLD without NAFLD
hyperinsulinism in 8 subjects (n = 199). HOMA-IR was Mean 6 SD Mean 6 SD p-value
elevated in 50.2% and hyperinsulism (Tanner and gender) in
N (male/female) 40 (11/29) 166 (94/72) 0.001
15.9%. Acantose nigricans was present in 18.0% (59).
Age (years) 12.6 6 2.7 11.9 6 3.1 .0.05
Hypercholesterolemia 15.8% (247) and hypertriglyceridemia
BMI (kg/m2) 30.8 6 7.8 26.8 6 3.6 ,0.001
22.7% (242), 28.2% (234) had HDL , 40 mg/dl, mean (sd) of
Cholesterol (mg/dl) 166.1 6 32.8 171.7 6 29.4 .0.05
LDL 103.5 mg/dl (29.6), 13.5% with LDL . 130 mg/dl. In 67
LDL (mg/dl) 99.3 6 27.1 99.6 6 25.2 .0.05
of 211 subjects (31.7 %) we found NAFLD, 61.8% were male
HDL (mg/dl) 48.4 6 11.8 50.4 6 12.3 .0.05
(p = 0.012). In 222 and 226 registrations of AST and ALT,
VLDL (mg/dl) 21.1 6 12.9 21.8 6 11.1 .0.05
7.6% e 20.7% had elevated values; 118 children made
Triglyceride (mg/dl) 107.7 6 63.3 105.8 6 52.6 .0.05
abdominal ultrasonography (US) and 28.8% presented signs
Glucose (mg/dl) 86.8 6 6.2 87.1 6 7.6 .0.05
of hepatic steatosis. In NAFLD children the HOMA-IR
Insulin (mU/ml) 20.1 6 12.0 14.7 6 9.4 0.004
values were higher, 2.04 vs 1.96. In children with HOMA-IR
HOMA-IR 4.4 6 2.7 3.2 6 2.2 0.006
elevated 27.5% had NAFLD. The mean (sd) of HDL of non
AST (U/L) 32.3 6 20.2 22.6 6 7.0 ,0.001
NAFLD was 47.3 (10.9) mg/dl and in NAFLD children was
ALT (U/L) 42.0 6 35.7 20.3 6 9.0 ,0.001
42.1 (12.8) mg/dl (p = 0.003); the median of triglyceride of
non NAFLD population was 73.5 mg/dl and in NAFLD was BMI, Body mass index; HOMA-IR, Homeostasis model assessment
90.0 mg/dl (p = 0.042). of insulin resistance.
Conclusions: More than one third of this obese paediatric
population presented with NAFLD. It is yet unclear if all
overweight children should undergo testing and US to screen
NAFLD. We concluded that males with high triglyceride and
low HDL had risk profile and must be screened for NAFLD. PH2-17 PEDIATRIC H1069Q CARRIERS IN WILSON DISEASE
PRESENT WITH HIGHER CERULOPLASMIN AND
PH2-16 PREVALENCE OF NAFLD AND ALTERATIONS OF SERUM COPPER CONCENTRATIONS AND LOWER
LIPID AND CARBOHYDRATE METABOLISMS IN 24H URINARY COPPER EXCRETION M Dadalski1,
CHILDHOOD OBESITY S Kinik. Tulgar1, F Ozcay2, P Socha1, H Schmidt2, G Oracz1, B Muszynska1,
S Kanra1. 1Baskent University Department of Pediatric E Pietraszek3, J Ryzko1, J Socha1. 1Gastroenterology, Hepat-
Endocrinology, Ankara, Turkey. 2Baskent University De- ology and Immunology, The Childrens Memorial Health
partment of Pediatric Gastroenterology, Hepatology, and Institute, Warsaw, Poland. 2Gastroenterology, Charity Campus
Nutrition, Ankara, Turkey. Mitte, Berlin, Germany. 3Department of Laboratory and
Diagnostics, The Childrens Memorial Health Institute, War-
Introduction: Obesity is the most significant risk factor for saw, Poland.
the development of non-alcoholic fatty liver disease
(NAFLD) both in children and adults. The incidence of Background: Wilson disease results from heterogenous
NAFLD rising as childhood obesity becomes increasingly mutations within the ATP7B gene, which encodes the copper
prevalent. Previous reports have revealed that fatty liver in transporting enzyme ATPase 7B. H1069Q mutations account
obesity is closely related to hyperinsulinemia and dyslipide- for up to 60% of identified mutations. So far there is a lack on
mia. In this study we aimed to evaluate the frequency of data on the correlation of biochemical parameters of copper
NAFLD in the obese children and adolescents, and to metabolism and the corresponding genotypes.
compare obese children and adolescents with and without Aim: The aim of this study was to assess and compare the
fatty liver for carbohydrate and lipid metabolism parameters. results of ceruloplasmin [mg/dl], serum copper [mg/dl] and
Methods: 206 obese children and adolescents (101 male; age 24h urinary copper excretion [mg/24h] obtained before
12.1 6 3.0, mean 6 SD, range 5.818.3 years) were included therapy of WD had been started, and mutational analysis.
in our study. In all patients, levels of fasting serum lipids, Patients and Methods: 31 patients (12F, 19M, aged 918)
glucose, insulin and transaminases were studied and abdom- with mutations identified on both alleles were involved in this
inal ultrasonographic examinations were performed. An study. Eight patients were homozygous for H1069Q, 14
appearance of hyperechogenic (bright) liver in ultrasonogra- patients were heterozygous for H1069Q, and 9 patients
phy implicated steatosis. carried mutations different from H1069Q. The results of
Results: In all study groups, 40 obese children had ultrasono- biochemical parameters in copper metabolism were com-
graphically fatty liver appearance (19%, 40/206). The clinical pared among the group of H1069Q homozygous patients vs.
and laboratory characteristics and comparisons between remaining ones, and among the group of patients carrying at
patients with and without fatty liver were given in Table 1. least one H1069Q mutation vs. remaining ones, and among

J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005

684 ESPGHAN 38TH ANNUAL MEETING
all three groups vs. each other. The t-test, U-test, ANOVA and
Kruskal-Wallis tests were used in statystical analysis.
Results: Patients homozygous for H1069Q had significantly
higher serum copper concentrations (56.25 6 13.47 vs.
32.44 6 15.75; mean 6 SD). Patients carrying at least one
H1069Q mutation had higher ceruloplasmin (13.69 6 8.35
vs. 4.53 6 3.11) and copper (45.23 6 13.7 vs. 18.78 6 8.98)
concentrations and lower 24h urinary copper excretion
(169.22 6 344.2 vs. 389.84 6 490.23) than patients with no
H1069Q mutation. Besides patients carrying no H1069Q
mutation had lower ceruloplasmin (4.52 6 3.12) and serum
copper (18.78 6 8.98) concentrations when compared
with patients homozygous for H1069Q (14.98 6 8.35 and
52.25 6 13.47) as well as patients with one H1069Q mutation
(12.96 6 8.23 and 41.21 6 12.56).
Conclusions: Our data incline that the presence of at least
one H1069Q mutation results in higher ceruloplasmin and
serum copper concentrations and lower 24h urinary copper
excretion in patients with WD. This may implicate that
H1069Q WD carriers may present with less severe symp-
toms.
PH2-18 ZINC AS SOLE FIRST LINE THERAPY IN CHILDREN
WITH WILSONS DISEASE: TEN YEARS FOLLOW
UP STUDY V Nobili 1 , R Devito3 , D Comparcola 1 ,
M Sartorelli 1 , F Callea3 , V Diciommo2 , M Marcellini 1 .
1
Bambino Gesu Hospital - Liver Unit, Rome, Italy.
2
Bambino Gesu Hospital, Rome, Italy. 3 Bambino Gesu
Hospital - Pathology Unit, Rome, Italy.
Objectives: Wilsons disease (WD) is an inherited disorder
of copper metabolism characterised by a failure of biliary
excretion of copper. It is generally accepted that pre-
symptomatic patients diagnosed in childhood should be
treated prophylactically with zinc.
Methods: Here we report our results in 22 children with
continuous oral zinc therapy over ten years. Zinc sulfate was
used at a dose of 25 mg b.i.d. until the age of 6 years, 25 mg
t.i.d. from 7 to 16 years or to a body weight of 125 lbs, 50 mg
t.i.d.thereafter.
Results: Five years after starting zinc treatment an highly
significant lowering of alanine aminotransferase (ALT),
aspartate aminotransferase (AST) and urinary copper excre-
tion was observed, whereas white blood cells did not vary
significantly. 6 out of 22 patients continued to have ALT
above normal and only one patient showed ALT more than
1.5 times the upper normal limit. Further lowering of ALT,
AST and urinary copper excretion was observed at the end of
the 10-year follow-up. All histological scores were signifi-
cantly decreased after treatment.
Conclusion: The excellent clinical results in all our patients,
coupled with the improvement in liver histology in the vast
majority, indicate convincingly that zinc treatment can
control the disease effectively and safely, preventing its
progression over 10 years. Therefore, our findings indicate
that pediatric presymptomatic patients should be treated with
zinc as the treatment of choice.
PH2-19 SAFETY AND EFFICACY OF N-ACETYLCYSTEINE
IN CHILDREN WITH ACUTE LIVER FAILURE
NOT CAUSED BY ACETAMINOPHEN OVERDOSE
CI Kortsalioudaki1, S Bansal1, RM Taylor1, P Cheeseman1,
G Mieli-Vergani1, A Dhawan1. 1Institute of Liver Studies,
Kings College Hospital, London, United-Kingdom.
Introduction: ALF carries a high mortality without trans-
plantation. N- acetylcysteine (NAC) has been suggested to be
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
of benefit for nonparacetamol ALF and was introduced as
treatment of ALF since 1995 at our hospital.
Aim: Evaluate the safety and efficacy of NAC in children
with ALF not caused by acetaminophen overdose.
Method: A review of the medical records of 171 children
presenting with non-acetaminophen induced ALF between
1989 and September 2004 was made. ALF was defined as
either INR . 2 and abnormal liver function or INR . 1.5
with encephalopathy and abnormal liver function. Children
were divided into: Group 1 (19891994): standard care (n =
59, 34 (58%) male, median age 1.31 [range 0.00315.8] yrs)
and Group 2 (19952004): NAC administration (n = 111, 57
(51%) males, 3.51 [0.00517.4] yrs). Diagnoses were
metabolic in 11 (20%) in Group 1 vs. 16 (14%) in Group 2;
autoimmune liver disease in 6 (10%) vs. 7 (6%); neonatal
haemochromatosis in 6 (10%) vs. 7 (6%); infectious in 7
(12%) vs. 14 (13%); drug induced in 4 (7%) vs. 6 (7%);
miscellaneous causes in 6 (10%) vs. 18 (16%) and idiopathic
in 19 (32%) vs. 42 (38%). NAC was administered as a
continuous infusion (150 mg/kg/24h) until normal INR or
liver transplantation (LT).
Results: Median duration of NAC administration was 5
[range 177] days. Complications were noted in 8 (10.8%);
rash in 3, bradycardia in 3, dizziness in 1 and 1 had broncho-
spasm leading to discontinuation of NAC. The length of
hospital stay was 24 [1264] days in Group 1 vs. 18 [1201]
days in Group 2, p = ns. Survival with own liver occurred in
13 (22%) in Group 1 vs. 48 (43%) in Group 2, p = 0.005.
Thirty- six patients died without LT (23 were not LT
candidates). LT was performed in 32 (53%) in Group 1 vs.
42 (38%), p = 0.05. Seven (23%) in Group 1 were re-
transplanted vs. 6 (14%) in Group 2, p = ns. Death after
transplantation occurred in 15 (47%) in Group 1 vs. 8 (19%)
in Group 2, p = 0.01.
Conclusion: NAC administration in non-acetaminophen
ALF is safe and is associated with higher native liver
recovery without LT, and also better post- LT survival.
PH2-20 OUTCOME OF IDIOPATHIC ACUTE LIVER FAILURE
IN CHILDREN: 15 YEARS, SINGLE CENTRE
EXPERIENCE CI Kortsalioudaki1, T Dassios1, S Bansal1,
RM Taylor1, M Rela1, N Heaton1, G Mieli-Vergani1,
A Dhawan1. 1Institute of Liver Studies, Kings College
Hospital, London, United-Kingdom.
Acute liver failure (ALF) in children is uncommon but carries
high mortality without liver transplantation. The outcome
also depends on the aetiology, however in many children with
ALF the aetiology remains unknown.
Aim: To review clinical and biochemical characteristics in
children with idiopathic ALF and identify factors that could
determine outcome.
Method: Medical records of 204 children presenting with
ALF between 1989-Sept 2004 were reviewed. Of these 61
(32 male, age 4.6yrs [12 days17.5 yrs]) had idiopathic ALF.
All the known causes of ALF were excluded. ALF was
defined as either INR . 2 and abnormal liver function or
INR . 1.5 with encephalopathy and abnormal liver function
tests. Known causes of ALF were: drug induced in 44 (31%),
metabolic in 27 (19%), infectious in 21 (15%), neonatal
haemochromatosis in 14 (10%), autoimmune in 13 (9%) and
miscellaneous in 24 (17%).
Results: Presenting features were jaundice in 53 (87%); hepa-
tomegaly in 46 (75%); encephalopathy in 37 (61%). Grade of
encephalopathy was .II in 25 (41%), with invasive ICP
monitoring in 17 (28%). Forty-three (71%) required PICU
admission. Laboratory indices on admission: median AST,
1355 [range 6412860] iu/l; SBR, 368 [121018] umol/l;
 ESPGHAN 38TH ANNUAL MEETING
GGT, 43 [19175] iu/l; albumin, 32 [1349] g/l; INR 3.07
[1.2815]. Median length of hospital stay was 11 [1166]
days. Eighteen (30%) survived with their own liver. Seven
(12%) died without transplantation 4 [175] days from
admission. Thirty-six (59%) received a liver transplant.
Eleven (31%) children required re-transplantation due to:
chronic rejection in 5, biliary complications in 3, primary
non-function in 1, reoccurence of disease in 1, HAT in 1. Five
of them required a 3rd transplant. Thirteen (36%) died after
transplantion due to multi-organ failure and sepsis in 8, pri-
mary non-function in 4, chronic rejection in 1. Factors related
to survival without transplantation were no hepatomegaly
(p = 0.05) or encephalopathy (p = 0.03).
Conclusion: Idiopathic ALF accounts for a third of ALF
children and carries high mortality despite transplantation.
PH2-21 HIGH PREVALENCE OF ANTIBODIES TO SOLUBLE
LIVER ANTIGEN (SLA) IN CHILDREN WITH ACUTE
LIVER FAILURE Y Ma1, N Hadzic1, P Cheeseman1,
G Mieli-Vergani1, D Vergani1. 1Kings College Hospital,
London, United-Kingdom.
Aim: The pathogenesis of liver damage in acute liver failure
(ALF) is complex and incompletely understood. ALF has
a poor prognosis, often requiring emergency liver trans-
plantation. Antibodies to soluble liver antigen (SLA) are
characteristic of autoimmune liver disease (AILD), where
they represent markers of a particularly severe course.
Methods: We investigated presence of anti-SLA in 64 children
who presented with ALF to our tertiary referral centre. Sera
stored at admission were retrospectively analysed under code
for the presence of anti-SLA using a radioligand assay. The cut
off value was 1300 cpm, representing the mean +3SD of
a normal population. Thirteen children with acute presentation
of Wilson disease (WD) and AILD were also investigated.
Results:
Conclusions: SLA antibodies are present in a sizeable pro-
portion of patients with non A-E ALF, as well as in patients
with acute decompensation of WD and AILD. They could be
detected in non A-E ALF at a higher percentage than in other
disorders leading to ALF in children. This may suggest that
presumed viral aetiology of non A-E ALF could trigger
autoimmune mechanisms leading to liver injury. Whether this
observation has a genuine pathogenic role, or represents only
an epiphenomenon of liver damage requires further study.
Condition Number antisla pos. (%)
Non A-E hepatitis 5/40 (13%)
Paracetamol overdose 0/12
Fulminant HBV infection 1/3 (33%)
Systemic herpes infection 0/3
Mitochondrial cytopathies 0/4
Neonatal liver failure 0/3
Wilson disease 2/5 (40%)
AILD 4/8 (50%)
PH2-22 LONG TERM FOLLOW-UP OF CHRONIC HBV IN-
FECTION: ANY TREATMENT IS BETTER THAN NO
TREATMENT? A Figueiredo1, S Jacinto1, F Santos1,
I Afonso1, J Cabral1, I Po1, G Cordeiro-Ferreira1. 1Gastro-
enterology Unit - H. D. Estefania, Lisbon, Portugal.
Aim: To assess all the children with chronic Hepatitis B virus
(HBV) infection (defined as persistence of AgHbs for .6
months) followed in a tertiary care centre in order to
685
determine the pattern of transmission, natural history of the
disease and response to treatment.
Methods: A retrospective chart review of the children
mentioned above was conducted. The mean follow-up was 5
years. Epidemiological, past medical history, biochemical
and immunological data, liver biopsy changes and effects of
treatment with interferon alpha and/or lamivudine were
studied. Data were analysed statistically with Fishers exact
test (p , 0.05).
Results: The number of children studied was 187 (median
age at diagnosis 6,2 years); 65.2% were male. Transmission
occurred primarily in household settings (42.7%). Ten
children had extra-hepatic manifestations (4 Nephrotic S., 2
arthralgias, 2 glomerulonephritis, 1 macular rash and 1
Gianotti-Crosti S.).There was 1 co- infection with virus delta.
27.2% had liver biopsy showing evidence of fibrosis in 45%,
piecemeal necrosis in 29% and cirrhosis in 5.8%. 152
(81.2%) of the children remained without any treatment and
35 (18.7%) had been treated with interferon alpha (18/51.4%)
and/or lamivudine (12/34.2%); 5(14.2%) did both, sequen-
tially. In the former group 25 (13.3%) had a spontaneous
seroconversion and in the treated group only 2. Remission
rate was achieved in 78 (51.3%) in the first group, as opposed
to 20 (57.1%) in the second. Interferon alpha achieved 1
(5.5%) seroconversion and 14 (77.7%) remission rate.
Lamivudine was responsible for 1 (8.3%) seroconversion
and 4 (33.3%) remission rate. Both groups has a 50%
normalization of AST/ALT. Comparing the 2 drugs showed
a significant difference in the remission rate with interferon
(p 0.024).
Conclusions: In our series of patients the most frequent
pattern of transmission occurred in the household setting. In
a long term basis neither interferon alpha nor lamivudine
proved better than no treatment in achieving loss of AgHbs.
Treatment with interferon alpha achieved higher remission
rates with statistical significance but if we consider the end-
point of treatment, normal AST/ALT, no difference was
observed.
PH2-23 DOES VITAMIN K IMPROVE BONE MINERAL
DENSITY IN CHILDREN WITH OSTEOPENIA AND
CHRONIC LIVER DISEASE? C Veiga1, L Pereira2,
I Albuquerque1, I Gonc xalves1. 1Paediatric Hospital, Coim-
bra, Portugal. 2University Hospital, Coimbra, Portugal.
Background: Osteopenia is a common and multifactorial
problem in children with chronic liver disease (CLD). The
role of vitamin K as a factor preventing osteopenia has not
been demonstrated. In osteopenic patients optimal carboxyl-
ation of the bone proteins requires a higher vitamin K intake.
Aim: Addressing the effects of two different doses of vitamin
K in paediatric patients with CLD and osteopenia.
Material & Methods: A prospective study was carried out in
20 children with stable CLD and Osteopenia (Z score , 21).
Patients were randomized to receive either oral 10 mg (n =
12) or 20 mg (n = 8) of Vitamin K (phylloquinone in
a micellar state), weekly during 6 months. In addition calcium
and vitamin D were administered to all children according to
recommendations. Before treatment and 6 months later, bone
mineral density (BMD), anthropometric data, PIVKA, para-
thyroid hormone (PTH), osteocalcin (OC), and urinary deoxy-
pyridinoline/ creatinine (Dpy/Cr) were analysed. BMD was
measured by dual X-ray absortiometry (DXA) in the lumbar
spine. The serum osteocalcin and urinary Dpy levels were
determined by chemiluminescent immunometric assay.
Statistical analysis was done using the independent and
pared- samples t tests.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
686 ESPGHAN 38TH ANNUAL MEETING
Results: The median age of children was 1,1 years old (0.5
11.7 y) and 50% (10/20) were from female gender. The
aetiology of CLD was: biliary atresia-9, intrahepatic chole-
stasis-5, metabolic liver diseases-4 and autoimmune hepati-
tis-3. The mean improvement in BMD was 23.5% (216 to
62.4%). There were no significant differences in mean Z
scores of weight (20.37/ 20.05; p = 0.066) and height
(21.5/21; p = 0.118) in the two phases of the study. BMD
was significantly higher after the 6 months period when
expressed in Z score (23.4/ 22.7; p = 0.009) and
g/cm2(0.319/ 0.382; p = 0.001). No significant changes were
observed in the mean levels of OC (48/44 ng/ml; p = 0.622)
and Dpy/Cr (53/ 40; p = 0.444) during the study period. The
mean percentage of BMD gain was not significantly different
in children who received 10 mg of vitamin K (23.4%) when
compared with those who received 20 mg (24%) (p = 0.936).
Comments: Treatment with oral vitamin K may improve
bone mineralization in children with CLD. However, this
study could not demonstrate a beneficial effect of higher
doses.
PH2-24 HOURS WORKED AND PROFESSIONAL QUALITY OF
LIFE AMONG UK BSPGHAN MEMBERS - THE RE-
SULTS OF A QUESTIONNAIRE SURVEY AJ Baker1,
P Cheeseman2, M Morgan3. 1BSPGHAN Council, London,
United-Kingdom. 2Dept. of Child Health; Kings College
Hospital, London, United-Kingdom. 3Public Health Scien-
ces, Kings College, London, United-Kingdom.
In 1993 a questionnaire was sent to all 89 members of
BSPGHAN with clinical GI responsibilities to provide data
for job plans in keeping with the new consultant contract
(CC) and European Working Time Directive (EWTD). 41
(46%) replied including 2 trainees. We report working hours,
work related stress, best and worst aspects of job, and
predictors of Quality of Life score (QoL). Of 39 consultants,
9 had academic roles, 7 were maximum part time and 2 part-
time. Two did no on-call while median on-call frequency was
1 in 5 (range 1 in 1.8 to14). They spent median 50 hr/wk in
hospital (9- 64), with 25 (61%) exceeding 48 hr/wk (CC &
EWTD maximum) by median 8 hrs (216). They were
available, including on-call, median 65 hr/wk (22- 168) with
11 (27%) over 72 hr/wk. 36 (87%) experienced stress,
Median QoL was 7 (29) with 6,5. Those with QoL , 7, n =
14, are Gp1 compared with QoL . 7, n = 15, Gp2. Gp1 vs.
Gp2 were similar in 55 vs. 50 hrs at work, 70 vs. 55 hrs
available, 8 vs. 10 working .48 hr/week. All Gp1 reported
stress while 5 of Gp2 did not. Best aspects for Gp1 were:
team/colleagues, n = 9; clinical work, n = 9; patient
feedback, n = 7; and teaching, n = 4. For Gp2 they were:
team/colleagues, n = 7; research, n = 7; patient feedback, n =
6; and teaching, n = 5. Worst aspects for Gp1 were: resources,
n = 14; professional interactions, n = 7; own time/too busy,
n = 6; and admin/bureaucracy, n = 4. For Gp2 they were: own
time/too busy, n = 13; admin/bureaucracy, n = 6, professional
interactions, n = 6; and resources, n = 4. Regression analysis
showed predictors of QoL: Working hours recorded by diary
(p = 0.000); willingness of employer to comply with EWTD
(p = 0.000); support of employer (0.048); considering a
sabbatical (p = 0.000); time for clinical administration (p =
0.000), feeling it is enough (p = 0.000); protected time for
non-clinical roles (0.000); clinical lead role (p = 0.030).
Those with worse QoL were more likely to report stress,
valued clinical work more than research and saw the
problem as resources rather than use of their own time.
Employers support and time to perform roles are keys to
higher QoL. CC requires a radical review of priorities, roles
and resources.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
PN1-01 PREDOMINANT T-HELPER 1 CYTOKINE PROFILE IN
OBESE CHILDREN L Pacifico1, L Di. Renzo2, C Anania1,
E Schiavo1, R Parente2, F Ippoliti2, P Strappini1, C Chiesa3.
1
Dept of Pediatrics, La Sapienza Univ., Rome, Italy. 2Dept of
Exp. Medicine, La Sapienza Univ., Rome, Italy. 3National
Council Research, Rome, Italy.
Background: The recently discovered orexigenic hormone
of gastric origin, ghrelin and the adipocyte hormone leptin,
exert mutually antagonistic effects on food intake at the
hypothalamic level. Yet, leptin, a member of gp 130 family of
cytokines, induces a strong T-helper (Th) 1 response and is
regarded as a proinflammatory inducer. Ghrelin functions as
a counterregulatory signal in the immune system, controlling
leptin-induced expression of inflammatory mediators.
Objective: The aim of this study was to evaluate Th 1/Th 2
balance and its relation with circulating ghrelin and leptin in
obese children.
Methods: Thirthy-three children, ages 216 years (mean 6
SD, 10 6 3.4), with BMI . 97th percentile for age and ten
aged-matched healthy control subjects were included in the
study. The proportion of CD4 positive cells that expressed
intracellular cytokines as interleukin (IL)-2, interferon
gamma (IFN-g), and IL-4 were analysed by flow cytometry.
T-cell populations were defined as type 1 being IL-2+ and
IFN-g+, and type 2 being IL-4+.
Results: We found that in obese children the percent of IFN-g+
cells were significantly higher (mean 6 SEM, 19 6 2 %) in
comparison with the control group (5 6 1 %; p , 0001).
There were no significant differences in IL-2+ (19 6 2 % vs.
13 6 2 %) and IL-4+ (4 6 1 % vs. 0.5 6 0.2 %) expressing
T cells between patients and controls. Obese children had
significantly higher (21 6 8 ng/ml) serum leptin concen-
trations than healthy subjects (2 6 2 ng/ml; p , 0.01), while
ghrelin concentrations did not differed between the two
groups. No significant linear correlations were observed be-
tween Th1/Th2 response and the changes in serum levels of
leptin and ghrelin.
Conclusions: Our results suggest that there is Th1/Th2
imbalance in obese children with a shift to Th1-cytokine
profile dominated by the production of IFN-g, and that such
response may be in part related to the increase of leptin con-
centrations. The prevalent Th1 pattern of secreted cytokines
may be regarded as a mechanism contributing to inflamma-
tion in obese patients.
PN1-02 OBESITY AND NON-ALCOHOLIC FATTY LIVER DIS-
EASE L Pena-Quintana1, E Colino1, N Montesdeoca1,
D Gonzalez1, IA Aguiar1, P Saavedra2, JC Ramos1.
1
U. Gastroenterology and Nutrition Infantil. Hospital Uni-
versitario Materno-Infantil de Canarias, Las Palmas de GC,
Spain. 2Dpt Mathematics. University of LasPalmas de GC,
Las Palmas de GC, Spain.
Aim: To describe the prevalence, the clinical features and the
risk factors in obese children and adolescents with non-
alcoholic fatty liver disease (NAFLD).
Methods: Study comprising 104 obese children and adoles-
cents (BMI and skinfolds . P95) (52 boys, 52 girls) aged
414 years old (mean 10.8 +/2 2.50) (group I) and 99 healthy
controls (51 boys, 48 girls) aged 414 years old (mean
10.20 +/2 2.52) (group II) (Tanner 14). Additionally,
a sample of 20 obese children (15 boys, 5 girls) aged 514
years old (mean 11.5 +/2 2.50) under study for NAFLD
underwent a percutaneous liver biopsy (group III). A number
of measures and tests were performed: a detailed anamnesis
and a full clinical exploration; life-style, emotional state, and
 ESPGHAN 38TH ANNUAL MEETING
dietary questionnaires; and exercise and socioeconomic
levels. Hematimetry, biochemical parameters, leptin, lipids,
hepatocellular function, microalbuminury, insulin resistance
(HOMA) and insulin sensitivity (basal insulinemia and
glycaemia/insulin index) were determined, among other para-
meters. In group I and III, an abdominal ultrasonography and
an oral glucose tolerance test were also performed. The
presence of acanthosis nigricans was also studied. For pa-
tients with NAFLD, known causes of chronic hepatopathy
were excluded. This study was approved by our hospitals
Ethics Committee and informed consent was obtained from
all subjects in this study. Statistical study was performed by
the SPSS statistical software package.
Results: Prevalence of NAFLD by ultrasonography in obese
children was 18.8%, being more frequent in males (27.1% vs.
10.4%) (p = 0.036). The greater the evolution time of obesity,
the greater this proportion, reaching significance from 5 years
of evolution (25% vs. 10%) (p = 0.03). Only 27.7% of obese
children with NAFLD showed increased serum levels of
transaminases. Increases in GGT, AST and ALT levels were
clearly correlated with NAFLD (p = 0.056, p = 0.001, p =
0.000, respectively). When comparing obese children with
and without fatty liver, it was found a clear association with
hyperinsulinaemia (p = 0.000), triglyceride level (.150) (p =
0.018), and acanthosis nigricans (p = 0.006). No relationship
was found with total cholesterol, C-LDL, C-HDL, glycemia,
leptin levels or Tanner stage. In Group III, 55% of patients
showed steatohepatitis and 45% showed a simple steatosis.
All patients were clinically asymptomatic and none of them
had physical signs of chronic liver disease.
Summary: NAFLD is a frequent finding in obese children
and adolescents. We identified in this population a number of
risk factors, such as being male, evolution time of obesity
(significant from 5 years), hyperinsulinaemia and hyper-
triglyceridaemia, and so it can be considered as another
component of the metabolic syndrome. It correlates to
acanthosis nigricans by virtue of the hyperinsulinaemia. Less
than a third of patients present an increase in the figures of
aminotransferases and generally remain asymptomatic and
without signs of chronic liver disease, despite the fact that
more than 50% present steatohepatitis al at time of diagnosis.
Conclusions: NAFLD should be excluded in all obese chil-
dren by imaging methods as it is related to time of evolution,
hypertriglyceridemia and hyperinsulinemia. At diagnosis, a
high number of children present advanced hepatic injuries.
PN1-03 MANAGEMENT OF OBESITY IN DUCHENNE MUS-
CULAR DYSTROPHY: A LONGITUDINAL STUDY
C Morin1, JM Cuisset1, K Mention1, A Carpentier1,
L Jouannic1, F Gottrand1. 1clinique de pediatrie CHU J de
flandre, Lille, France.
Aim: Obesity occurs early in the course of Duchenne
muscular dystrophy (DMD), and is observed in more than
50% of patients after the age of 14 years. The aim of this
study was to evaluate efficacy and tolerance of a nutritional
program in a population of obese DMD patients.
Methods: From 1996 to 2004, all the children with DMD and
obesity (weight . 90% for age according to Edwards chart)
followed in our centre, were included in a nutritional program
which associated a 10% diet intake reduction including at
least 15% of protein, and a twice-a-year paediatrician and
dietician follow-up. We measured anthropometrical and
impedancemetry data to evaluate weight gain velocity, fat
mass and fat free mass. 24h dietetic recall was used to
evaluate food intake. A questionnaire gave information on
families characteristics and tolerance of the nutritional
program.
687
Results: 25 children, aged 9 years (range 5 to 16 years),
weighted 34 kg (range 19 to 62 kg), were included in the
study. Four were lost of follow-up. 62% of patients had at
least one relative (without DMD) presenting obesity. After
a follow-up of 4 years (range 0.5 to 12 years), weight gain
velocity (expressed as Z-score) decreased from 2.8 to 20.5
SD (p = 0.0004). Body composition analysis showed a mild
increase of fat mass (from 28 to 33 %) (p = 0.03) but no
significant variation of fat free mass (21 versus 26 kg). Food
intake decreased from 1727 to 1485 kcal/d (p = 0.03) with
a parallel decrease of lipids and carbohydrate intakes (p #
0.005) while proteins remained to 15% of caloric intake.
Sixteen children (64%) were consider as a success (weight
gain velocity , 0). No criteria were found significantly
associated to success excepted that families seemed to be
more implicated in the diet in this group (50 versus 33 %)
(ns). We were not able to observe any difference in evolution
of DMD among groups. This program was judged as well
tolerated by children and families.
Conclusion: Nutritional program in obese DMD patients is
well tolerated and can limit weight gain without apparent
deleterious effect on body composition.
PN1-04 PLASMA FATTY ACIDS AND INSULIN RESISTANCE IN
OBESE CHILDREN S Scaglioni1, E Verduci1, M Salvioni1,
C Raimondi1, M Bruzzese1, G Radaelli2, E Riva1,
C Agostoni1. 1Department of Pediatrics, San Paolo Hospital,
University of Milan, Milan, Italy. 2Unit of Medical Statistics,
San Paolo Hospital, University of Milan, Milan, Italy.
Aim: To examine whether an association of plasma fatty
acids may exist with the degree of obesity and insulin
resistance in obese children.
Design: Observational case-control study.
Methods: Sixty-seven obese children, aged 812 y, and 67
age- and sex-matched normal-weighing children were in-
cluded into the study. Obesity was defined in accordance with
the International Obesity Task Force. BMI z scores were
calculated and adjusted by using the LMS-method of Cole
and Italian reference data. Fasting blood samples were
analysed for insulin, glucose and fatty acids (FA) composi-
tion. Insulin resistance (IR) was estimated by the homeostatic
model assessment (HOMA).
Results: Compared with normal-weighing children, obese
children showed lower mean plasma total polyunsaturated FA
(38% vs. 40%), n-6 PUFA (35.5% vs. 37.2%), C18:2n-6
(26.1% vs. 27.5%), C22:6n-3 (1.5% vs. 1.8%), and higher
levels of total monounsaturated FA (26% vs. 25%), C18:3n-3
(0.33% vs. 0.25%), C20:5n-3 (0.46% vs. 0.38%), C20:3n-9
(17% vs. 14%), C20:5n-3/C20:4n-6 (0.07 vs. 0.05), and
(C18:3n-3 + C20:5n-3)/ C22:6n-3 ratios (0.58 vs. 0.41).
Fasting insulin levels (19 vs. 6 mU/ml) and HOMA (4 vs. 1)
were higher in obese children. In obese children BMI z score
was negatively associated with plasma levels of plasma
total polyunsaturated FA, n-6 PUFA, n-3 PUFA, C20:5n-3,
C22:6n-3, C20:5n-3 + C22:6n-3, C22:6n-3/C20:4n-6 ratio,
and positively with total saturated FA, n-6/n-3 PUFA ratio,
C20:3n-9 and (C18:3n-3 + C20:5n-3)/ C22:6n-3 ratio;
raised IR (i.e., HOMA . 75th percentile) was independently
associated with lower total monounsaturated FA (OR = 1.8,
95% CI 1.22.8) and C20:4n-6 (OR = 4.0, 95% CI 1.410).
Summary: Obese children showed lower total plasma PUFA
than normal-weighing children, although they exhibited
higher dietary intake of all the main FA families. Obese
children displayed also lower levels of C22:6n-3, in spite of
higher levels of the precursors, C18:3n-3 and C20:5n-3.
Obese children in the highest BMI z score quartile re-
markably differed from both normal- weighing children and
obese children in lower quartiles of BMI z score for many of
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
688 ESPGHAN 38TH ANNUAL MEETING
FA component. In obese children total monounsaturated FA
and C20:4n-6 were independently associated with insulin
resistance.
Conclusion: In obese children the plasma FA profile may be
associated with the degree of obesity and insulin resistance.
Biotransformation of C18:3n-3 to C22:6n-3, and the distri-
bution of C22:6n-3 should be investigated in obese children.
PN1-05 FATTY LIVER DISEASE AND INSULIN SENSIVITY
INDEXES IN OBESE CHILDREN AND ADOLESCENTS
H Ferreira Mansilha1, S Rodrigues1, I Valente1, R Gouveia1,
xo. Gomes1. 1Nutrition Unit - Pediatric Dept - Maria
J Lourenc
Pia Childrens Hospital, Porto, Portugal.
Non-alcoholic fatty liver disease (NAFLD) is now recognised
as an emerging clinical problem in children and adolescents.
The prevalence of NAFLD is probably increasing parallel to
prevalence of obesity. Paediatric NAFLD occurs most
frequently in conditions associated to insulin resistance.
Aims: (1) to describe the clinical and biochemical character-
istics of NAFLD in obese paediatric population; and (2) to
determine the association between NAFLD and insulin
sensitivity indexes and degree of obesity.
Methods: A total of 89 obese children/adolescents with
NAFLD (mean age: 10 years old; 4 to 17 years old range; 47
girls) were examined. Body mass index (BMI) and percent
body fat (%BF) were calculated. Serum levels of aspartate
aminotransferase (AST), alanine aminotransferase (ALT),
alkaline phosphatase, gamma-glutamyl transferase, fasting
insulin and glucose, lipid profile of all patients were recorded.
The diagnosis of NAFLD was based on ultrasound scan
criteria. Homeostasis model assessment of insulin resistance
(HOMA-IR), HOMA of percent b-cell function (HOMA-
B%), insulinogenic index and the quantitative insulin
sensitivity check index (QUICKI) were used to evaluate
insulin sensitivity in all subjects. A group of 81 age and sex
matched control obese subjects was included.
Results: Central pattern of fat distribution was present in
70.8% of the subjects. Acanthosis nigricans (AN) was
observed in 8 cases. Hypercholesterolemia, hypertriglycer-
idemia and raised ALT were found in 9 (10.1%), 41 (46.1%)
and 43 (48.3%) subjects, respectively. The criteria for IR
were met by 97.4% of subjects. The obese children with
NAFLD had significantly higher blood pressure, BMI Zscore,
%BF and IR when compared to the control group.
Logistic regression determined the combination BMI Zscore
and IR to be a strong predictor of NAFLD (p , 0.05). A
positive association between AN and IR (p = 0.019) was also
observed.
Conclusions: In this study, NAFLD was predicted by the
combination of quantitative insulin sensitivity indexes
(HOMA-IR and QUICKI) and BMI Zscore. Consistent to
other reports, acanthosis nigricans could be a reliable
cutaneous marker of insulin resistance in obese children.
Zscore. Consistent to other reports, acanthosis nigricans
could be a reliable cutaneous marker of insulin resistance in
obese children.
PN1-06 ADIPONECTIN, INSULIN AND C-PEPTIDE SERUM
LEVELS IN OBESE CHILDREN BEFORE AND AFTER
REDUCTION OF BODY WEIGHT J Bronsky1,
J Nedvidkova2, D Sramkova2, M Pechova3, R Prusa3,
J Nevoral1. 1Dpt. of pediatrics, University hospital Motol,
Prague, Czech Republic. 2Institute of Endocrinology,
Prague, Czech Republic. 3Dpt. of clinical biochemistry and
pathobiochemistry, University hospital Motol, Prague, Czech
Republic.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
Aim: Adiponectin is an adipocytokine secreted by adipose
tissue which increases insuline sensitivity. It is a biomarker of
metabolic syndrome in humans and its production is
stimulated by insulin and insulin-like growth factor 1.
Adiponectin serum levels are decreased in obese adults and
in patients with type II diabetes in comparison to healthy
controls. We measured adiponectin, insulin, and C-peptide
serum levels and various anthropometrical parameters in
obese children undergoing 5 weeks of weight-reduction
therapy in spa.
Methods: We measured serum adiponectin levels using
human ELISA kit in 69 obese children (40 girls, 29 boys;
mean age 13.1 6 0.38 years; mean weight 74.2 6 2.79 kg;
mean weight-loss 6.8 kg) and in 32 age and gender matched
healthy controls. We used the Matiegkas anthropometrical
method based on skinfold thickness to determine the amount
of body fat.
Results: Adiponectin levels before weight-reduction were
16.8 6 0.85 ng/ml and after weight-reduction 17.0 6 0.82
ng/ml (N.S., p = 0.6309). Levels in the control group were
22.4 6 1.80 ng/ml and differed significantly from levels in
obese children both before and after weight-reduction (p =
0.0017 and p = 0.0021 respectively). Adiponectin levels
did not differ significantly between girls (17.3 6 1.16 ng/ml)
and boys (15.7 6 1.07 ng/ml, p = 0.3385). We found
a negative correlation between adiponectin and insulin levels
(r = 20.3905; p = 0.0012), C-peptide levels (r = 20.3859;
p = 0.0014) age (r = 20.4143; p = 0.0012), body height (r =
20.3684; p = 0.0044), body weight (r = 20.3888; p =
0.0026), body mass index (r = 20.3664; p = 0.0047) and
amount of body fat (r = 20.272; p = 0.0388).
Conclusion: Adiponectin levels correlate negatively with
insulin, C-peptide, age, body height, body weight, BMI and
amount of body fat, however they are not affected by middle-
term changes of body weight in children.
Supported by research grants IGA MZ NE/7443-3 and GAUK
59/2004C.
PN1-07 IS AGE OF ONSET OF OBESITY A PREDICTOR OF
LATER CARDIOVASCULAR RISK? C Rego1, I Campos1,
D Silva1, M Fontoura1, A Aguiar1, A Guerra1. 1Department
of Paediatrics, Hospital S. Joao/Faculty of Medicine
University of Porto, Porto, Portugal.
Background: It has been observed that childhood obesity is
a relevant predictor of development of insulin resistance
syndrome (IRS). Age of onset of obesity could also play a
role in prevalence of risk factors of cardiovascular disease.
Aim: To evaluate risk factors of cardiovascular disease
according to age of onset and duration of obesity.
Methods: 293 subjects of both genders (148 M, 145 F) with
nutritional obesity according to Cole criteria were included in
the study. Height and weight were measured and BMI was
calculated. Body fat mass (FM) was evaluated by electrical
impedance (Tanita TBF 410x) and blood pressure (BP) was
measured by oscylometry according to recommendations of
American Heart Association. Blood samples were collected
for oral glucose tolerance test and fasting lipids assessment.
Insulin resistance was determined by homeostatic model
assessment (HOMAIR). Subjects were divided in 4 groups
according to age of onset of obesity: Group 1:
2 years;
Group 2: .2
6; Group 3: .6,10; Group 4:
10. Para-
metric and non-parametric test were used according to
variables distribution for correlations and comparative studies.
Results: Mean age was 10.3 6 3.2 years (1.7 up to 17.8).
Those with an early onset of obesity (groups 1 and 2) showed
a significantly higher BMI z-score (4.4 6 1.8 and 4.1 6 1.7)
compared to groups 3 and 4 (3.3 6 1.3 and 2.9 6 1.1) (p ,
0.001). No differences were observed between groups
 ESPGHAN 38TH ANNUAL MEETING
regarding BP (adjusting to age and gender), plasma lipids and
HOMAIR. BMI is significantly correlated to systolic (r =
0.489 - p , 0.001) and diastolic blood pressure (r = 0.290 -
p , 0.001), triglycerides (r = 0.152 - p , 0.05) and HOMAIR
(r = 0.344 - p , 0.001).
Conclusion: Childhood obesity is a powerful predictor for
development of cardiovascular risk factors that are not
dependent on age of onset of obesity. These risk factors seem
to be particularly dependent on the magnitude of Body Mass
Index and Fat Mass.
PN1-08 RESISTIN DECREASES AFTER WEIGHT LOSS IN
OBESE CHILDREN K Krohn1, C Boczan1, CP Bauer2,
B Koletzko1. 1Dr. von Hauenersches Kinderspital, Munich,
Germany. 2Fachklinik Gaissach, Bad Tolz, Germany.
Introduction: The peptide hormone resistin is secreted by
adipose tissue. Initial animal studies suggested that resistin
may mediate insulin resistance in obesity. However, human
studies have not been conclusive. The aim of our study was to
investigate the effect of weight loss on resistin and associated
changes in glucose/insulin metabolism in obese children.
Methods: We studied plasma resistin concentrations, glucose
and insulin in obese children (10 to 16 years) before and after
diet-induced weight loss over six weeks. Resistin was de-
termined after an overnight fast in the morning. Insulin and
glucose concentrations were determined before and after oral
administration of 75 g glucose at 0, 30, 60 and 120 min. Insulin
sensitivity was estimated using the whole body insulin
sensitivity index (WBISI). Fasting resistin, insulin and glucose
were measured in normal weight subjects (10 to 15 years).
Results: 23 obese and 11 normal weight subjects were
studied. In obese children mean plasma resistin concentra-
tions were higher than in normal weight subjects and
decreased significantly after weight loss (1.6 6 0.1 ng/ml
before vs. 1.35 6 0.07 ng/ml after weight loss, p , 0.001;
1.27 6 0.06 ng/ml in normal weight subjects, p , 0.05). The
auc (mean 6 SEM) in the insulin profile decreased by 41%
(17551 6 1334 mE 3 min/ml vs. 10429 6 825 mE 3 min/ml,
p , 0.001). Mean ( 6 SEM) auc in the glucose profile
decreased by 13 % (13318 6 390 mg x min/dl before vs.
11556 6 328 mg x min/dl after weight loss, p , 0.001).
WBISI increased from 2.3 6 0.3 to 3.7 6 0.4. There was no
significant correlation between BMI SDS and resistin (r =
0.18, n.s.). WBISI and resistin did not correlate significantly
(r = 20.16, n.s.). There was only a weak correlation between
resistin and glucose auc (r = 0.36, p = 0.085).
Summary: Diet-induced weight loss in obese children led to
a significant decrease in plasma resistin concentrations
associated with a decrease in AUC plasma insulin and
glucose concentrations and an increase in WBISI. However,
there was no significant correlation between resistin and
parameters of insulin sensitivity.
Conclusion: Resistin may be involved in the regulation of
body weight. However, our findings do not support the
concept of resistin mediating insulin resistance in obesity.
PN1-09 PULMONARY EMBOLI RISK ASSOCIATED WITH
LONGTERM PARENTERAL NUTRITION I Irastorza1,
P Pifarre1, I Gordon1, S Hill1. 1Great Ormond Street
Hospital for Children, London, United-Kingdom.
Aim: Long-term survival of children with severe intestinal
failure is dependent on treatment with parenteral nutrition
(PN) administered via a central venous catheter. One poten-
tially life-threatening complication of PN treatment is the
development of pulmonary emboli (PE). We aimed to review
the incidence of PE in children on PN.
689
Methods: Sample: 64 children aged 0 to 17 years who had
PN for a total of 5115 months [median 49, range 16 - 204] and
had VQ scans whilst on PN. Possible risk factors for PE i.e.
type of underlying intestinal disease, intestinal inflammation,
lipid content of PN, positive thrombophilia screen and
presence of anticardiolipin antibodies were recorded.
Results: Overall incidence of PE was 39% and similar in
children with an enteropathy (36%), pseudo-obstruction
(44%) and short gut (39%). 41% of patients with gut
inflammation developed PE vs 20% of those without (P =
0.21). 56% of patients with pathologic thrombophilia
screening developed PE vs 36% of those without (P = 0.28).
PE were more frequent in patients given lipid infusion
.2 days/week: 46% vs 20% (P = 0.40). 2 patients without
gut inflammation and with normal thrombophilia screen
developed PE.
Summary: 39% of children on long-term PN developed PE.
Indentified risk factors for PE were gut inflammation,
pathologic thrombophilia screening and lipid infusion with
PN but patients without inflammation and clotting abnormal-
ities also developed PE.
Conclusion: Lipid-containing PN with an indwelling central
venous catheter and no other risk factors can be associated
with PE. Contributing factors include intestinal inflammation
and abnormal thrombophilia screen. VQ scan and anti-
coagulation treatment would appear to be indicated in all
children on long-term PN.
PN1-10 PARENTERAL FEEDING OF PRETERM INFANTS WITH
TWO FAT EMULSIONS CONTAINING LONG AND
MEDIUM CHAIN TRIGLYCERIDES: EVALUATION OF
OXIDATIVE STRESS C D. Alfonso1, B Giuffre`1, A Orsi1,
V Puricelli1, P Roggero1, C Salis2, G Buonocore3, F Mosca1.
1
Neonatal Intensive Care Department University Medical
School, Milano, Italy. 2Pharmacy Service University Medical
School, Milano, Italy. 3Institute of Preventive Pediatrics and
Neonatology, University Medical School, Siena, Italy.
Background: Polyunsaturated fatty acid (PUFA), a major
component of fat emulsions, are susceptible to peroxidation.
Total hydroperoxides (TH), which are the intermediate
oxidative products of lipids, peptides, and amino acids,
represent a measure of overall oxidative stress.
Advanced oxidation protein products (AOPP) are reliable
markers of the degree of protein damage in oxidative stress.
Premature infants have limited ability to cope with oxidative
stress.
Aim: To evaluate the oxidative effects of short-term total
parenteral nutrition (TPN) with two lipid emulsions (soybean
and coconut oil-based ) in premature infants.
Methods: In a double blind randomised trial, 12 preterm
infants received either a standard soybean oil or a coconut oil
emulsion. Lipid intake represented 20-25% of the total non-
protein energy daily intake. Infants were included with
gestational age from 28 to 36 weeks and birth weight . 500 g.
All babies studied did not have blood group incompability,
infections, hypoxia and severe hyperbilirubinaemia. At
baseline and after 7 days we measured plasma levels of
(TH) and (AOPP). The procedure used has been reported by
G. Buonocore et al. (Pediatric Reasearch, 2000). The data
(expressed as means +/2 SD) were analyzed for statistically
significant differences by two-tailed t test for unpaired data
(SPSS/PC).
Results: 12 preterm infants (mean gestational age 32 weeks,
mean birth weight 1150 g), were randomized to receive
soybean oil-based emulsion (6 babies): LCT group and
coconut oil-based emulsion (6 babies): MCT group.
Conclusion: The significantly high TH plasma levels found
in preterm babies fed with the soybean oil emulsion (LCT
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
690 ESPGHAN 38TH ANNUAL MEETING
group) are an indirect evidence of an increase in generation of
free radicals (FR). The AOPP plasma levels did not signif-
icantly differ at T0 and T7, indicating low protein damage, as
AOPP have been described as markers of protein oxidative
stress.
Therefore, the generation of FR exposes the premature
infants, with a poorly developed antioxidant system, mainly
to a lipid damage. A partial replacement of PUFA by MCT in
fat emulsion could have a favourable influence on adverse
effects caused by FR, which are generated by lipid perox-
idation.
TABLE 1. Shows data related to TH ( Carr U/L) are showed
Groups Baseline (T0) p After 7days of TPN (T7) p
LCT 132 +/2 27.5 196 +/2 42.8
MCT 170.6 +/2 58.4 NS 170 +/2 87.4 .05
TABLE 2. Shows the data related to AOPP (Umol/dl)
Groups T0 p T7 p EMBASE, Cumulative Index to Nursing and Allied Health
LCT 11.9 +/1.91 14.5+/25.9
MCT 11.63+/3.7 NS 17+/25.5 NS
PN1-11 METABOLIC BONE DISEASE RELATED TO LONG
TERM PARENTERAL NUTRITION IN CHILDREN
S Miranda-Sanchez1, JC Ruiz2, C Talbotec1, O Corriol1,
P Taupin1, C Ricour1, O Goulet1, V Colomb1. 1Service de
gastroenterologie et nutrition pediatriques, h^
opital Necker-
Enfants Malades, 149 rue de Se`vres 75743, Paris, France.
2
Laboratoire dexplorations metaboliques 5 Av Daniel
Lesueur 75007, Paris, France.
Introduction: Metabolic bone disease (MBD) related to
long-term parenteral nutrition (LTPN), characterized by an
impaired bone mineralization, has been described in adults
and children. The etiology and natural history of MBD are not
well understood.
Aim: To assess MBD course and to correlate bone mineral
density (BMD) with growth in children and adolescents on
LTPN.
Methods: 26 patients (10 girls) on LTPN for short bowel
syndrome (SBS, n = 19) or functionnal disorder of the
gastrointestinal tract (FDGI n = 7) were included. BMD was
assessed by dual X-ray absorptiometry (DXA). Clinical data,
weigth, height, blood and urinary parameters, as well as PN
composition were retrospectively collected at the dates of
DXA (n = 78, i.e 3/patient). Statistics:linear regression
analysis and Wald tests were performed.
Results: Mean PN duration was 113 6 47 months. No
abnormal clinical or laboratory findings were detected.
The mean BMD Z score according to age and gender was
21.46 6 1.12. Z score was positively related to remaining
bowel length in SBS (p = 0.018), and inversely related to the
age of PN onset (p = 0.013), and to PN duration (p = 0.002).
BMD was positively related to growth velocity in girls (p =
0.027), and to height (p = 0.001), calcium PN intake (p =
0.006) and vitamin D PN intake (p = 0.01) in both genders.
Conclusion: This is the first study providing longitudinal
data of MBD in children on LTPN and the largest study made
on PN-related MBD in children. MBD can develop in chil-
dren while on LTPN without clinical or biological symptoms.
Thus, DXA is an essential tool for its detection and followup.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
As MBD is related to PN duration, followup is very important
during puberty, since MBD might impair peak bone mass,
and also in patients with SBS.
PN1-12 THE EFFECT OF BRIEF EXPOSURE TO WATER,
BREAST-MILK SUBSTITUTES, OR OTHER LIQUIDS
ON THE SUCCESS AND DURATION OF BREASTFEED-
ING: A SYSTEMATIC REVIEW OF RANDOMIZED
CONTROLLED TRIALS A Horvath1, B Koletzko2,
M Kalisz1, H Szajewska1. 1Dept of Paediatric Gastroenter-
ology & Nutrition, The Medical Univ of Warsaw, Warsaw,
Poland. 2Dr. von Hauner Childrens Hospital, Ludwig-
Maximilians-University of Munich, Munich, Germany.
Objective: To systematically evaluate the effect of supple-
mental fluids or feedings during the first days of life on the
overall breastfeeding duration and rate of exclusive breast-
feeding among healthy infants.
Design/search Strategy: Medical subject headings and free-
language terms were used to search the following electronic
databases for studies relevant to breastfeeding: MEDLINE,
(CINAHL), The Cochrane Library, and La Leche League;
additional sources were obtained from reference lists of
review articles. Only randomized controlled trials (RCT)
were considered for study inclusion.
Main Results: Of 56 potentially relevant clinical trials
identified, only one RCT (170 infants) met the inclusion
criteria for this systematic review. In this study, formula
feeding was significantly more frequent at 4 weeks in the ex-
perimental group in which breastfeeding had been supple-
mented with 5% glucose ad libitum during the first 3 days of
life (n = 83) than in the exclusively breastfed control group
(n = 87) (p , 0.05). At 16 weeks (5 months postpartum), the
percentage of mothers who continued breastfeeding, either
exclusively or partially, was significantly lower in the exper-
imental group than in the control group (p , 0.01).
Conclusions: On the basis of this systematic review, there
remains considerable uncertainty about the effect of brief
exposure to water, breast-milk substitutes, or other liquids on the
success and duration of breastfeeding. Future trials are needed.
PN1-13 OUTCOME OF BREASTFED INFANTS IN CYSTIC
FIBROSIS (CF): AN ITALIAN, SINGLE-CENTRE FOL-
LOW-UP SURVEY C Colombo1, V Bennato1, G Romano1,
D Costantini1, D Ghisleni2, M Giovannini2, E Riva2,
C Agostoni2. 1CF Centre, University of Milan, Milan, Italy.
2
Department of Pediatrics-San Paolo Hospital, University of
Milan, Milan, Italy.
Aim: We have investigated height achievement, rate of
infections in the first 3 yrs of age, incidence of Pseudomonas
(Psm) infections and lung function in a group of CF patients
according to the early type of feeding and CFTR genotype
(severe/mild).
Methods: Children referring to a single Center for CF have
been followed up over 20 years. Children underwent
established follow-up diagnostic protocols for CF including
genotyping (PCR method), periodic anthropometric measure-
ments with standardized techniques, evaluation of lung
function (spirometry: FVC, FEV1) and collection of dietary
and anamnestic data. Anthropometrics have been transformed
into z scores by means of the ANTHRO software program
(CDC, Atlanta, US). For this study the parameters of the most
recent clinical control have been considered. Chi square,
parametric and non-parametric tests were used as appropriate
(SPSS 11.5 for Windows).
 ESPGHAN 38TH ANNUAL MEETING
Results: One-hundred and five children have entered the
present analysis (M/F 52/53, mean age 8, SD 4, range 3-19,
yrs, severe/mild genotype 72/33). Among BF children
(68/105), the mean BF duration was 5, SD 4, range 1 to 30,
mos. BF was prevalent in patients with mild (26/33)
compared to severe (42/72) genotype (P = 0.04). Considering
all the 105 subjects, a trend of association was found between
BF duration and height z score attainment (P = 0.07), while
BF . 6 mos (n = 15) vs no BF (n = 36) was associated to
a reduced number of infections in the first 3 years of life (5,
SD 4, vs 9, SD 6, P = 0.04). The same trends were present in
patients with severe and mild genotype. Rate of Psm infection
and age at its first acquisition were connected to CF genotype
and not BF. As far as lung function, BF duration was
associated with better outcomes only in patients with mild
genotype, with clear trend of correlations (r = 0.38, P = 0.07,
for FEV1, and r = 0.46, P = 0.02, for FVC, respectively).
Summary: In 105 CF patients we have observed positive
associations between prolonged BF and lower rate of
infections, and between BF duration and lung function in
mild CF forms.
Conclusion: BF, particularly if protracted, may be associated
with better growth, health and functional outcomes in CF
children, particularly those with mild genotypes.
PN1-14 RELATION BETWEEN LEPTIN LEVELS IN BREAST-
FED INFANTS AND MATERNAL BMI F Savino1,
SA Liguori1, MF Fissore1, S Maccario1, EC Grassino1,
GE Nanni1, R Oggero1, L Silvestro1. 1Department of
Paediatrics, University of Turin, Regina Margherita Hospi-
tal, Turin, Italy.
Aim: Leptin concentration in breast milk has been found to
be correlated to maternal BMI and maternal plasma leptin
concentration, but little evidences are available about
maternal BMI and leptin levels in infants. The aim of the
study was to evaluate the relation between plasma leptin
concentration of breast-fed infants and maternal BMI.
Methods: We have conducted a cross sectional study on 75
AGA healthy infants aged 012 months, 51 exclusively
breast-fed for at least 4 months and 24 formula-fed, seen in
our Department, without any chronic and acute gastrointes-
tinal disease. Serum leptin concentration has been determined
at least 3 hours after feeding by RIA test (LEP-R44,
Mediagnost, Reutlingen, Germany). The same operator
Figure 1. Correlation between breast-fed infants leptin levels and
maternal BMI.
691
performed a single anthropometrical measurement of weight,
lenght, and cranial circumference for each infant. BMI was
calculated for each subject and their mothers using the
formula: weight(Kg)/height(m)2. Statystical analysis was
performed by Pearsons correlation and multiple regression
(p , 0.05). Leptin values have been normalized with natural
logarithm.
Results: In breast-fed infants we have observed a significant
positive correlation (r = 0.389; p = 0.005) (Fig. 1) between
infant leptin levels and maternal BMI; in a multiple re-
gression model adjusted for infants age and infants BMI,
maternal BMI was an independent determinant of infants
plasma leptin concentration ( = 0.065; p = 0.006; SE =
0.022). Furthermore in formula-fed infants we havent
observed a significant correlation between infant leptin levels
and maternal BMI.
Summary: In our cross-sectional study we have enrolled 75
healthy infants aged 012 months, 51 breast-fed and 24
formula-fed. In breast-fed infants we have observed a positive
correlation between infant leptin levels and maternal BMI.
Conclusions: Plasma leptin levels are higher in breast-fed
infants and are positively associated to maternal BMI at time
of delivery. Our findings confirm the same relation in breast-
fed infants also until the first year of life. Higher maternal
BMI values could probably influence higher infants leptin
levels through breast-feeding.
PN1-15 EFFECT OF AN INFANT FORMULA WITH PREBIOTICS
ON THE INTESTINAL MICROBIOTA AFTER AN ANTI-
BIOTIC TREATMENT O Brunser1, M Gotteland2,
S Cruchet2, D Garrido2, G Figueroa2, P Steenhout3. 1Cell
Structure Lab. INTA-U. of Chile, Santiago, Chile. 2Microbi-
ology Lab. INTA-U. of Chile, Santiago, Chile. 3Nestec SA.,
Vevey, Switzerland.
The intestinal microbiota of the newborn develops until about
two years of age. During this period it may be more
susceptible to the effects of a variety of factors, including
antibiotics. One way of regulating the homeostasis of the
microbiota is through the administration of prebiotics.
Aim: The aim of the study was to evaluate the effect of
a formula enriched with prebiotics on the modifications of the
faecal microbiota of children after an antibiotic treatment.
Subjects and Methods: In this prospective, monocentric,
randomized, controlled, double blind study, 140 children one
to two years of age were distributed into two groups after
a one-week treatment with amoxicillin (50 mg/kg/day) for
acute bronchitis. The children received daily .500 ml of
a control (C) or the same formula with 4.5 g/L of a mixture of
inulin and oligofructose (70:30) (Prebio1) for a total of three
weeks. Faecal samples were obtained on day -7 (before anti-
biotic administration), on day 0 (after the antibiotic treatment
had been completed and before formula administration), and
on days 7 and 21 of formula administration. The faecal counts
of Bifidobacterium, Lactobacillus-Enterococcus, the C.
lituseburiense cluster (which includes C. difficile), the C.
histolyticum cluster (which includes C. perfringens), E. coli
and Bacteroides-Prevotella were evaluated by FISH and flow
cytometry. Formula tolerance and the characteristics of the
faeces were assessed daily.
Results: Amoxicillin induced a slight but significant de-
crease of the total counts of the bacteria studied (p = 0.0000);
E. coli increased (p = 0.02) while the other species evaluated
in the study did not experience changes. The prebiotic
induced a significant increase of the bifidobacteria on day 7
(p = 0.014) without subsequent changes. The other bacterial
populations did not experience changes in any of the periods
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
692 ESPGHAN 38TH ANNUAL MEETING
evaluated. Prebiotic intake did not induce gastrointestinal
disturbances.
Conclusion: A formula with prebiotics helps maintain high
levels of colonic bifidobacteria after antibiotic treatment and
was not associated with digestive symptoms.
Supported by Nestec, Vevey, Switzerland.
PN1-16 IMPACT OF ATOPIC ECZEMA AND PERINATAL
ADMINISTRATION OF PROBIOTICS ON GROWTH:
FOLLOW-UP STUDY FROM BIRTH TO FOUR YEARS
K Laitinen1, M Kalliomaki1, E Isolauri2. 1Turku University
Central Hospital, Department of Paediatrics, Turku, Finland.
2
University of Turku, Department of Paediatrics, Turku,
Finland.
Food-derived antigens may initiate or exacerbate the
symptoms of allergic diseases, and consequently various
elimination diets comprise the management of choice.
Adverse effects on growth may ensue. The effects of a new
management regimen, administration of probiotics, again, on
the growth of children are unknown.
Aim: To evaluate the impact of atopic eczema (AE) and
perinatal supplementation of probiotics (Lactobacillus rham-
nosus strain GG; ATCC 53103) on growth of children.
Methods: A prospective follow-up study from birth to 48
months of children with a family history of allergic disease.
Probiotics were administered prenatally to mothers and
postnatally for 6 months to their infants. Weights and heights
were measured at study visits (6, 12, 24 and 48 months).
Results: The mean difference for height during the follow-up
period between children with (n = 60) and without (n = 38)
AE was 0.02 (95% CI: 20.30 to 0.35) SD scores (P = 0.890).
The mean weight for height was 23.5 % (26.5 to 20.5, P =
0.025) lower in children with AE compared to those without.
In the probiotic group the mean height was 0.1 SD scores
(20.4 to 0.2, P = 0.532) lower and the mean weight 2.9%
(26.0 to 0.1, P = 0.056) lower compared to the children with
perinatal administration of placebo.
Summary: The mean weights for heights were lowered in
AE. However, we showed that with careful monitoring and
management of AE in early childhood, the growth of patients
were maintained within population reference values. Perina-
tal administration of probiotics did not influence growth.
Conclusion: Perinatal administration of probiotics was safe
as evaluated by the effects on growth, whilst AE had
moderate adverse effect on growth. This needs to be
considered as the reasons for growth failure in AE are various
and failure to thrive may culminate in patients not being
regularly monitored.
PN1-17 INTESTINAL MICROBIOTA IN INFANTS SUPPLE-
MENTED WITH THE PROBIOTIC BACTERIUM LAC-
TOBACILLUS REUTERI T Abrahamsson1, T Jakobsson1,
G Sinkiewicz2, M Fredriksson3, B Bjorksten4. 1Dept
Paediatrics, Linkoping University, Linkoping, Sweden. 2Dept
Biomedical Lab Sciences, Malmo Univeristy, Malmo,
Sweden. 3Dept Occupational and Environmental Medicine,
Linkoping University, Linkoping, Sweden. 4Inst. Environ-
mental Medicine, Karolinska Institutet, Stockholm, Sweden.
Background: Probiotics have been reported to have effect on
atopic eczema, but there are no data on colonisation rates in
these reports.
Aim: To assess the colonisation rate in breast milk and stool
from mothers and their infants after supplementation with the
probiotic bacteria Lactobacillus reuteri (Lbr) from birth up to
1 year of age, and to evaluate the effects on colonisation with
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
total lactobacilli (tLb), bifidobacteria (Bf) and Clostridium
difficile (Cld).
Methods: 232 mothers with a family history of atopic disease
were randomised to a daily intake of either Lactobacillus
reuteri ATCC 55730 (1 3 108 colony forming units, CFU) or
placebo for the last 4 weeks of pregnancy. Their babies then
continued with the same study product from birth until 12
months of age. The numbers of Bf, Cld, Lbr and tLb were
assessed in faeces from the infants and in the breast milk from
the mothers at birth and 1 month after delivery, employing
conventional bacterial cultivation methods.
Results: At 5-6 days of age, the prevalence of Lbr in the stool
from infants in the actively treated group was higher than in
the placebo group, i.e. 80% vs 19% (p , 0.001). The
prevalence then declined to 63 % at 12 months of age in the
actively treated infants but remained at 23% in the placebo
group (p , 0.001). Lbr was isolated from 12% and 2% of the
colostrum samples (p , 0.05). The prevalence of total
lactobacilli was higher at every time point in the active group,
though only significantly so in colostrum, 88% vs 66% (p ,
0.05), and in stool at 56 days, (95% vs 74%, p , 0.01) and
12 months (91% vs 70%, p , 0.001). However, Lbr did not
affect Bf or Cld colonisation. 29% of the infants received
antibiotics during the first year, but this did not affect the
colonisation rate significantly. Breastfeeding at 12 months
correlated to higher Bf and tLb counts (p , 0.01). Adjusting
for possible confounders did not affect the results signif-
icantly.
Conclusions: Lbr colonizes neonates in a high degree but the
prevalence decreases despite continuous supplementation.
Lbr is able to colonize breast milk and increases tLb in stool
and breast milk but does not affect Bf or Cld.
PN1-18 CHEMOKINE PRODUCTION BY BUCCAL EPITHELIAL
CELLS AS A POTENTIAL PARAMETER FOR IMMU-
NOMODULATION BY PROBIOTICS J Hol1, L De Ruiter1,
HC Raatgeep1, JN Samson1, EHG Van Leer2, JC
De Jongste3, EES Nieuwenhuis1. 1Erasmus MC-Sophia
Childrens Hospital, Laboratory of Pediatrics, Rotterdam,
The Netherlands. 2Groene Hart Ziekenhuis, Department of
Pediatrics, Gouda, The Netherlands. 3Erasmus MC-Sophia
Childrens Hospital, Department of Pediatric Pulmonology,
Rotterdam, The Netherlands.
Background: Although supplementation of probiotics has
been shown to have beneficial effects in allergic disorders the
efficacy of this treatment is controversial. In this study we
have assessed whether the activation of buccal epithelium can
be used to assess the potential immunomodulatory capacity
by probiotics.
Epithelial cells play an important role within the mucosal
immune system and are equipped with various pattern
recognition receptors. We hypothesized that the buccal
epithelium, the first host cells that encounter microbial
components, may be able to respond to exposure with
probiotics.
To test this hypothesis we assessed whether buccal epithelium
from healthy individuals was able to respond to in vitro and/or
in vivo exposure to a range of commercially available dairy
products supplemented with probiotics.
Methods: In vitro incubation for 24 h of buccal epithelium
with probiotics did not result in any detectable induction of
CXCL-8 (IL-8); 3.2 pg/ml. However, 1 minute of in vivo
exposure of the epithelium within the buccal cavity and
subsequent isolation and culture of these cells resulted in
a significant production of CXCL-8 without the addition of
external microbial stimuli. Release of CXCL-8 was detect-
able within 4 hours (42.4 pg/ml, p = 0.003) of culture and
increased during the next 24 hours (68.4 pg/ml, p , 0.001).
 ESPGHAN 38TH ANNUAL MEETING
Results: All available probiotic products that were tested
elicited CXCL-8 release. However, when comparing the
response of epithelial cells derived from different subjects to
the same probiotic not all responded to the same degree,
suggesting host specific characteristics that may determine
the response. Moreover, the response elicited by different
probiotics within the same subject was variable, implicating
a role for microbial factors for the quality of response.
Conclusion: Measuring CXCL-8 release by buccal epitheli-
um upon in vivo stimulation may become an useful tool in
identifying the correct probiotic strain for the right individual.
PN1-19 EFFECTS OF OLIGOFRUCTOSE SUPPLEMENTATION
IN INFANTS 6 TO 24 MONTHS OF AGE ON GUT
MICROFLORA AND WELL-BEING: A DOUBLE-BLIND,
PLACEBO-CONTROLLED STUDY AJ Waligora-Dupriet1,
F Campeotto2, A Bonet1, P Soulaines2, I Nicolis3,
C Dupont2, MJ Butel1. 1Microbiologie, Universite Rene
Descartes, Paris, France. 2Neonatologie, H^
opital St Vincent
de Paul, Paris, France. 3Biomathematiques, Universite Rene
Descartes, Paris, France.
Aim: The purpose of this study was to assess the effects of
daily administration of oligofructose (OF), an inulin-type
oligosaccharide from the chicory root, on the intestinal
microflora (prebiotic effect), intestinal acceptability and
gastro-intestinal (GI-) illness symptoms in infants.
Patients and methods: 35 healthy infants (6 tot 24 months)
were randomly assigned to either the OF supplemented group
(2 g/d RaftiloseP95, Orafti) or a control group (maltodex-
trin). Exclusion criteria were antibiotic use within the previous
8-days or breast-feeding during the previous month. It was a
three period study: 8-d observation (D8), 21-d supplementation
(D28), and a 15-d run-out (D42). Antibiotic therapy during
study led to exclusion.
Results: Of the 35 infants enrolled, 10 infants in each group
completed the study. Levels of bifidobacteria rose from 9.1 to
9.5 log10 cfu/g of feces in the OF-group at the end of
supplementation (D28). Differences between OF- and control
groups at D28 were not significant. A trend towards a higher
increase in bifidobacteria was observed if initial levels of
colonization were lower. Parallel decreases in clostridia
levels (7.6 to 7.2 log10 cfu/g of feces) in the OF-group were
found at D28, with a significant decrease in the OF-group
versus controls during supplementation (p , 0.05). Flatu-
lence, diarrhea, vomiting and fever were significantly less
often observed in the OF than within the control group (p ,
0.001, except for fever: p , 0.05).
57% of the infants completed the study, pinpointing the
frequent use of antibiotics in day-care infants. Bifidobacteria
increased up to 9.5 log10 cfu/g of feces after 3 w of OF-
feeding, but significance was borderline due to their high
levels at the start of supplementation and the variability in age
and hence bacterial levels between infants. Additionally,
clostridia numbers decreased to a major extent. These
PN1-21 QUANTITATIVE REAL TIME PCR OF FECAL LACTO-
bacterial changes with OF-feeding were accompanied by
fewer febrile events and gastrointestinal illness symptoms in
the infants.
Conclusion: This study was able to demonstrate a clear
benefit of OF-feeding on functional well-being of infants with
fewer episodes of fever, flatulence, diarrhea and vomiting.
PN1-20 PREBIOTIC OLIGOSACCHARIDES IN INFANT NUTRI-
TION: ADDITION OF ONLY GALACTOOLIGOSAC-
CHARIDES DOES NOT INDUCE FAECAL ACIDIC PH
AND SCFA-SPECTRUM TYPICAL FOR BREAST FED
INFANTS AM Bakker-Zierikzee1, MS Alles2, J Knol2,
693
F Kok1, JJM Tolboom3, JG Bindels2. 1Wageningen Univer-
sity, Department of Human Nutrition, Wageningen, The
Netherlands. 2Numico Research B.V., Wageningen, The
Netherlands. 3Radboud Academic Hospital, Paediatric
Department, Nijmegen, The Netherlands.
Introduction: Recently, the ESPGHAN Committee on
Nutrition summarised available information on the effects
of adding prebiotic oligosaccharides to infant formulae. The
Committee commented that only the combination of gal-
actooligosaccharides (GOS) and fructooligosaccharides
(FOS) was tested in controlled infant feeding studies and
that future trials should define optimal quantity and quality of
oligosaccharides with prebiotic function. We present data on
a study with an infant formula supplemented with only GOS.
These data can be compared with those from a previous study
where a formula with a mixture (90% GOS and 10% FOS) is
investigated using an identical protocol, formula matrix and
analytical methods.
Methods: Before birth, infants from mothers intending to
formula feed were at random and double blindly allocated to
one of two formula groups. From birth until 16 weeks of age,
the GOS formula group (n = 17) received formula supple-
mented with 0.6 g/100 ml GOS. The standard group (n = 17)
received the infant formula without supplementation. Faecal
samples were taken from the diapers at postnatal days 5, 10,
and weeks 4, 8, 12 and 16. Percentage of bifidobacteria from
total number of bacteria (analysed by fluorescence in situ
hybridisation), total and relative amounts of short-chain fatty
acids (SCFA), lactate concentration and pH were measured in
faeces. Parallel to the two formula groups a breast-feeding
group was followed using the same protocol.
Results: Both formula groups revealed a highly similar
development of faecal bifidobacterial flora. Faecal pH (6.5
7.0), SCFA total concentration (7590 mmol/kg wet faeces)
and profile (acetate: 7080%) were also very similar
between both formula groups, but distinct from the breast
feeding group (pH: 5.55.7; SCFA: 5060 mmol/kg faeces;
acetate: .85%). Under highly similar conditions nearly
identical results were found earlier for a group fed standard
formula (n = 19) and a parallel breast-fed group. However, the
prebiotic group (n = 19) fed formula supplemented with
0.6 g/100ml GOS/FOS mixture clearly showed different faecal
characteristics (pH 5.56.0; SCFA: 6575 mmol/kg faeces;
acetate: .80%), close to the ones for breast fed infants.
Conclusions: In contrast to infant formula supplemented
with a GOS/FOS mixture, infant formula supplemented with
short chain GOS only, does not induce faecal acidic pH and
SCFA-spectrum typical for breast-fed babies. This result
supports the hypothesis that the long chain FOS components
of the prebiotic mixture are fermented rather slowly, similar
to polymeric human milk oligosaccharides, leading to
a typical acidic pH and acetate-dominating SCFA spectrum
in the distal part of the colon and the faeces.
BACILLUS SPECIES IN INFANTS RECEIVING A PRE-
BIOTIC INFANT FORMULA M Haarman1, J Wells2,
G Boehm3, J Knol1. 1Numico Research B.V., Wageningen,
The Netherlands. 2Royal Numico N.V., Schiphol Airport, The
Netherlands. 3Numico Research B.V., Friedrichsdorf,
Germany.
Aim: A healthy intestinal microbiota, like the microbiota of
breast-fed infants, is considered to be important for priming
of the infants mucosal and systemic immune system.
Generally Bifidobacterium and Lactobacillus predominate
the microbiota of breast-fed infants and several health effects
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
694 ESPGHAN 38TH ANNUAL MEETING
of these genera have been reported. However, the Lactoba-
cillus species distribution in breast-fed or formula-fed infants
has not yet been described in great detail. The aim of this
study is to quantitatively determine the species with newly
developed real time PCR methods.
Methods: For accurate enumeration of different lactobacilli,
duplex 5 nuclease assays, targeted on rRNA intergenic
spacer regions, were developed for Lactobacillus acid-
ophilus, Lactobacillus case, Lactobacillus delbrueckii,
Lactobacillus fermentum, Lactobacillus paracase, Lactoba-
cillus plantarum, Lactobacillus reuteri and Lactobacillus
rhamnosus. The designed and validated assays were used to
determine the relative amounts of different Lactobacillus
species in fecal samples of infants receiving a standard
formula (SF, n = 10) or a standard formula supplemented with
galacto- and fructooligosaccharides (OSF, n = 10). A breast-
fed group (BF, n = 10) was studied in parallel as a reference.
Results: During the 6-week intervention period a significant
increase was shown in total percentage of fecal lactobacilli in
the BF-group (0.82 6 0.29% versus 4.05 6 1.50%; p = 0.034)
and the OSF-group (0.79 6 0.32% versus 4.37 6 1.36%; p =
0.026). The Lactobacillus composition in the OSF-group was
directed towards that of breast-fed infants, with relative high
levels of L. acidophilus, L. paracase and L. case at the end
of the study. After intervention the microbiota of SF-fed
infants contains more L. delbrueckii and less L. paracase
compared to breast- or OSF-fed infants.
Conclusion: The prebiotic infant formula induces a fecal
microbiota that mimics the microbiota of breast-fed infants
even at the level of the different Lactobacillus species.
PN1-22 THE EFFECTS OF INFANT FORMULAS CONTAINING
EITHER PREBIOTICS OR PROBIOTICS ON THE FAE-
CAL LACTOBACILLUS POPULATION M Haarman1,
A Bakker-Zierikzee2, M Alles1, J Bindels1, J Knol1. 1Numico
Research B.V., Wageningen, The Netherlands. 2Wageningen
University, Wageningen, The Netherlands.
Aim: The intestinal microbiota of breast-fed infants is
generally dominated by the genera Bifidobacterium and
Lactobacillus. The intestinal microbiota composition is
regarded as an important factor for the infants health and
well being, and several Lactobacillus species have been
reported to inhibit pathogens or to stimulate the immune
system. In this study the effects of infant formulas containing
either prebiotics or probiotics on the percentage of faecal
lactobacilli were studied.
Methods: At birth, infants of whom the mother had decided
not to breast- feed, were at random and double blindly
allocated to one of the four formula groups. In total 36 infants
received standard infant formula; 19 received a prebiotic
formula containing a specific mixture of 0.6 g/100 ml
GOS/FOS (ratio 9:1), 17 received a prebiotic formula
containing 0.6 g/100 ml GOS and 19 received a probiotic
formula containing 6.0 3 109 viable cells/100ml Bifidobacterium
animalis Bb12 (ProF). A group of 101 breast-fed infants was
included as a reference group. Faecal samples taken on age d5
and w12 were analysed, using a newly developed duplex
5nuclease PCR assay to determine the percentage of lactobacilli.
Results: In the breast-fed group, the percentages of
lactobacilli increased significantly between d5 and w12 from
1.1 6 0.4% to 6.4 6 1.2% (mean 6 SEM; p = 0.001). The
GOS/FOS group also showed an increase of lactobacilli
resulting in a significantly higher percentage of lactobacilli
than in the standard formula group (6.1 6 2.6% vs. 0.9 6
0.4%; p = 0.007) at w12. No significant differences were
found between GOS (1.1 6 0.4%) or ProF (2.4 6 1.7%)
versus the standard formula group at w12.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
Conclusion: Addition of GOS/FOS to the standard infant
formula stimulates the percentage of faecal lactobacilli
similar to that found in breast-fed infants. In contrast no
stimulation of lactobacilli was shown with standard infant
formula or after the addition of 0.6g/100 ml GOS or viable
Bifidobacterium animalis Bb12 to the standard formula.
PN1-23 COMPARISON OF HUMAN MILK OLIGOSACCHAR-
IDES WITH OLIGOSACCHARIDES FOR USE IN IN-
FANT NUTRITION ON IN VITRO FERMENTATION
USING FAECES FROM BREAST-FED AND FORMULA-
FED INFANTS M Govers1, A Buco1, T Hendrich2, B Stahl2,
G Boehm2. 1Numico Research B.V., Wageningen, The Nether-
lands. 2Wageningen University, Wageningen, The Netherlands.
The beneficial properties of human milk oligosaccharides
(OS) are largely due to their fermentation by the infants
colonic microflora.
Aim: To study the fermentation pattern of human milk OS
(HMOS) in vitro, and to compare it to the fermentation of OS
(to be) used in infant nutrition.
Methods: An in vitro semi-dynamic fermentation system was
inoculated with fresh faeces from either breast-fed or
formula-fed infants (age ,1 yr). HMOS, a mixture of
galacto- & long-chain fructo-OS (GOS/FOS, 9:1), acidic OS
(AOS, derived from citrus pectin; equivalent to carrot soup
OS) and intact citrus pectin were added and samples were
taken at t = 3, 24 and 48 hr. Samples were analysed for short-
chain fatty acids (SCFA) by GC. Gas production was also
measured. In every experiment, glucose fermentations were
carried out as a reference.
Results: Total SCFA and gas production are expressed as
percentage relative to glucose (mean 6 SEM). Molar ratio
SCFA = acetate: propionate: butyrate.
Total SCFA (%) Ratio SCFA Gas (%)
3 hr 24 hr 48 hr 48 hr 48 hr
Faeces from breast-fed infants (n = 3)
HMOS 37 6 3 77 6 5 90 6 5 91:8:1 92 6 18
GOS/FOS 32 6 3 84 6 8 99 6 8 97:3:0 81 6 12
Faeces from formula-fed infants (n = 5)
GOS/FOS 26 6 3 81 6 7 94 6 8 82:14:4 83 6 10
AOS 13 6 2* 65 6 6 73 6 12 81:11:8 156 6 34*
*p , 0.05 vs. GOS/FOS (ANOVA).
In a separate experiment using faeces from a formula-fed
infant with similar results for GOS/FOS, AOS produced more
SCFA and gas than intact citrus pectin, but with an identical
SCFA ratio.
Summary & Conclusions:
1. GOS/FOS are fermented gradually and almost completely
by the colonic microflora of breast-fed and formula-fed
infants, in a similar way to HMOS.
2. AOS are also well fermented by the infants microflora:
(a) initially slower than GOS/FOS, but with a higher final
gas production,
(b) more completely than intact citrus pectin, and
(c) with an identical SCFA ratio.
PN1-24 PHYTOSTEROL LEVELS IN BLOOD CELL MEM-
BRANES OF CHILDREN RECEIVING PARENTERAL
NUTRITION A Campanozzi1, G Salvia3, P Pianese4,
F Campanozzi1, M Pettoello-Mantovani1, G & Corso2.
 ESPGHAN 38TH ANNUAL MEETING
1
Pediatrics, University of Foggia, Foggia, Italy. 2Clinical
Biochemistry, University of Foggia, Foggia, Italy. 3NICU
Fatebenefratelli Hospital, Napoli, Italy. 4Dept. of Bio-
chemistry & Medical Biotechnology, University Federico II,
Napoli, Italy.
Children with intestinal failure receive lipid emulsions which
are usually olive-oil or soy-oil based. In a significant number
of cases parenteral nutrition (PN) is complicated by liver
dysfunction whose pathogenesis remains poorly understood.
Data from the literature show a correlation between lipid
emulsion administration, higher serum phytosterols (Cam-
pesterol and Sitosterol) and cholestasis. However, no data
regarding phytosterol levels in blood cell membranes (BCM)
are currently available.
Aim of the Study: To evaluate phytosterol levels in plasma
and BCM of children receiving PN.
Patients and Methods: Plasma and BCM levels of
Cholesterol (Ch), Desmosterol (Ds), 7Dehydrocholesterol
(Dhc), Lathosterol (Lt), Campesterol (Cp) and Sitosterol (Sts)
have been evaluated in 3 newborn infants (mean weight:
2.8 6 1.1 kg) receiving PN and 3 controls, by using a gas
chromatography mass-spectrometry method. The blood
samples were taken 12 hours after the end of lipid infusion.
The duration of PN and the amount of intravenous doses of
lipids, glucose and nitrogen were assessed.
Results: in patients, the mean amount of glucose and amino-
acids administered were respectively 8.4 6 5.7 gr/kg/day and
1.9 6 1.1 gr/kg/day. The mean duration of lipid administration
was 4.3 6 2.5 days and the mean total amount of lipids infused
was 7.6 6 6 grams (1.1 6 1.1 gr/kg/day). Phytosterolemia (%
of total plasma sterols) in patients was 0.8 6 1.3 and in
controls was 0.2 6 0.05 (p = 0, 15). BCM level of Sts (% of
total BCM sterols) was 0.97 6 0.51 in patients and 0.08 6 0.01
in controls (p , 0.05). BCM levels (% of total BCM sterols) of
Ch, Ds, Dhc, Lt and Cp are reported in the table below:
Ch (%) Ds (%) Dhc (%) Lt (%) Cp (%)
Controls 99.36 6 0.13 0.03 6 0.01 0.03 6 0.01 0.38 6 0.1 0.11 6 0.06
Patients 97.22 6 1.9 0.04 6 0.04 0.04 6 0.04 0.26 6 0.1 0.55 6 0.31
Conclusion: Our data suggest that Sitosterol accumulation in
blood cell membranes is an early onset event in children
receiving lipid emulsions, preceding any significant incre-
ment of phytosterolemia. This could be a useful marker to
study endogenous metabolism of phytosterols and evaluate
their role in the pathogenesis of PN-associated liver disease.
PN2-01 MID-UPPER ARM CIRCUMFERENCE IS A RELIABLE
PREDICTOR OF BODY-MASS INDEX IN HEALTHY
DUTCH CHILDREN J Schweizer1, WJ Gerver2. 1Leiden
University Medical Center, Leiden, The Netherlands. 2Uni-
versity Hospital Maastricht, Maastricht, The Netherlands.
Background: To estimate the BMI as a measure of
nutritional status in paediatric patients, there is a need for
easy-to-use instruments that reflect body composition. Mid-
upper arm circumference (MUAC) is used for that purpose in
many underdeveloped countries.
Aim: To investigate the correlation between MUAC and BMI
in a large cohort of healthy Dutch children.
Methods: Participants were 2305 healthy Dutch children
(53% boys; median 10.8 years, range 0.0817.9) that
participated in a previous study. Data on height (cm), weight
(kg) and MUAC (cm) were available. Ponderal index (PI) was
calculated for children ,1 year as weight (gram)/height
695
(cm)3. BMI was calculated for all subjects as weight
(kg)/height (m)2. Pearson correlation and regression statistics
were calculated with S-Plus.
Results: In boys and girls ,1 year no correlation between
MUAC and PI was found. The Pearson correlation between
MUAC and BMI was 0.74 in boys and 0.79 in girls (p ,
0.0001). BMI was used for further analysis in all age
categories. The mathematical relationship between MUAC
and BMI was:
 Boys: BMI = 13.47762 + 0.09549717 * MUAC +
0.005446704*(MUAC-15)3 0.004294027*(MUAC-
18.1)3 0.007911969*(MUAC-20.6)3 + 0.006890421*
(MUAC-23.6)3 1.31129*10-4(MUAC-27.5)3
 Girls: BMI = 11.35804 + 0.2069558*MUAC +
0.004313624*(MUAC-15)3 1.701833*10-5 (MUAC-
18.1)3 0.01223151(MUAC-20.6)3 + 0.007855673*
(MUAC-23.6)3 + 7.923586*10-5*(MUAC-27.5)3
For practical use, the curves were also plotted in separate
graphs for each gender.
Summary: The correlation between MUAC and BMI was
studied in a large cohort of healthy Dutch children. We found
a strong non-linear correlation. Two easy-to-use graphs were
made for boys and girls.
Conclusion: MUAC may be used as a screening instrument
for malnutrition in paediatric patients in the Netherlands.
PN2-02 GHRELIN LEVELS AND WEIGHT GAIN IN THE FIRST
YEAR OF LIFE F Savino1, SA Liguori1, MF Fissore1,
S Maccario1, EC Grassino1, R Oggero1, L Silvestro1.
1
Department of Paediatrics, University of Turin, Regina
Margherita Hospital, Turin, Italy.
Aim: Ghrelin is a 28-amino acid peptide mainly secreted by
the stomach and involved in the regulation of food intake. It
has been described as a regulator of energy balance and in
infancy higher ghrelin levels have been detected in small for
gestational age (SGA) neonates. Information about the role of
ghrelin in the regulation of energy homeostasis and body
weight in healthy infants in the first year of life is scanty. The
aim of our study was to evaluate the correlation between
ghrelin levels and weight gain in the first year of life.
Methods: We studied 93 AGA healthy infants (52 males, 41
females) aged 112 months seen in our Department, without
chronic or acute gastrointestinal disease. Serum ghrelin
concentrations have been determined at least 3 hours after
feeding by RIA test (R-90, Mediagnost, Reutlingen, Ger-
many). Mean weight gain was calculated as the difference
between the weight at the day of blood sampling and birth
weight, divided by the age in days. The study was approved
by the hospitals Ethical Committee and parents gave their
written consent. Statistical analysis was performed by
Pearsons correlation (p , 0.05). Ghrelin values have been
normalized with natural logarithm (nl).
Results: Our study sample had a mean age of 197.52 6 116.19
days, with mean ghrelin level of 3026.01 6 1386.37 pg/ml and
mean ln of ghrelin of 7.92 6 0.44; the weight gain had a mean
value of 23.09 6 8 g/day. In the study group we have observed
a significant negative correlation between ghrelin levels and
weight gain (r = -0.302; p = 0.003) (Fig. 1).
Summary: In our cross-sectional study we have enrolled 93
healthy infants aged 112 months. We have observed
a negative correlation between ghrelin levels and weight gain.
Conclusions: Our findings show that infants who have gained
more gram for day have lower ghrelin levels than those who
have gained less. Considering that ghrelin levels are reported
to be increased in anorexia nervosa and after fasting, whereas
in obese patients and after feeding they are decreased, we
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
696 ESPGHAN 38TH ANNUAL MEETING
PN2-04 RELATIONSHIP BETWEEN SERUM LEPTIN CONCEN-
Figure 1. Correlation between ghrelin levels and weight gain.
could hypothise that also in healthy infants in the first year of
life ghrelin may change as part of an adaptive response to
a condition of positive energy balance.
PN2-03 EFFECT OF A LOW COST FEED ON RECOVERY AND
DEATH RATE IN MALNOURISHED CHILDREN L Di
Donato1, J Balungi2, K Amono2, R Iriso2, B Corrado2,
L Greco1. 1Department of pediatrics, University of Naples
Fededrico II, Naples, Italy. 2St Marys Hospital, Lacor, Gulu,
Uganda.
Aim: Evaluation of the efficiency of a low-cost nutritional
rehabilitation program carried out in a nutritional unit in
Northern Uganda.
Methods: The study design was a cohort study. Children
were admitted to the Nutritional Unit according to the WHO
malnutrition criteria. Children were fed with a thick cereal
based porridge with added proteins and fats made with cheap
local ingredients in addition to F100 milk. Evaluation of the
mean daily weight increments before and after the in-
tervention was carried out by randomly sampling the files of
100 cases dismissed in October, November and December of
the years 2001 (before the intervention), 2002 (soon after the
intervention), 2003 (more than one year after the interven-
tion). For each group of children the average length of stay in
the unit and the average weight gain reached at discharge was
computed. Differences between means were evaluated by the
Student t test. Additional outcome variables were obtained for
a three year period: (i) % Of cases cured /total admissions
for each month; (ii) % Of cases dead/ total admissions for
each month; (iii) % Of cases lost or escaped/total admissions
for each month.
Results: Mean weight increments and Confidence Intervals
(CI) went from 496 grams in 2001 (306587), before
NUTRICAM, to 798 grams in 2002 (5841011); in 2003
increments reached 1310 grams (11291491). Oedema- free
average daily weight gain went from 21 grams/day (1229)
before NUTRICAM to 35 g/day (2545) in 2002 up to
59 g/day (5165) in 2003.The % cases dismissed as cured
moved from 36/66 (54.5%) in Jan 2002 to 97/104 (93.3%) in
Aug 2004. Mortality and escape rates moved from 30/66
(45.5 %) in Jan 2002 to 7/104 (6.7%) in Aug 2004. Out of these
we had a 14/66 (21.2%) fatality rate in Jan 2002 versus 3/104
(2.9%) in Aug 2004.
Conclusions: The NUTRICAM feed supplement is locally
feasible at low cost (about 0.056 Euro/serving/child, in-
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
cluding labour and fuel), well accepted by local dwellers,
simple to prepare and very effective for the nutritional
rehabilitation. We estimated to have 216 less deaths/year after
the nutritional intervention compared to the previous period.
TRATIONS AND ANTHROPOMETRIC MEASURES IN
PRETERM NEONATES: FROM FETAL TO EARLY
POSTNATAL LIFE C DAlfonso1, A Orsi1, V Puricelli1,
P Roggero1, R Di Toro2, L Perrone2, F Mosca1. 1Dept. of
Neonatology, University Milan, Milano, Italy. 2Dept. of
Pediatrics, III Cl. Pediatrics, Naples, Napoli, Italy
Background: Leptin plays a key role in the regulation of
body fat mass and weight in adult life. The mechanisms by
which maternal and fetal/neonatal weight are regulated
during human pregnancy and in early postnatal life are
poorly understood. It was demonstrated that leptin can have
a potential role in regulating fetal growth and neonatal
development. Human milk leptin, secreted by breast adipose
tissue, has a positive effect on satiety and regulation of energy
intake.
Aim: To evaluate whether leptin influences growth and
nutritional status during gestation and early postnatal life of
preterm infants.
Patients and Methods: Fifty seven LBW newborns were
enrolled: 31 males (M), 26 females (F), gestational age (GA)
32.5 +/22.2 wks, birth weight (BW) 1448+/2324 g.
Infants with congenital malformations, severe perinatal
asphyxia, neurological problems, cardiac disease, chromo-
somal abnormalities, necrotizing enterocolitis, feto-maternal
group incompatibility and other haemolitic anemia, were
excluded. To assess the correlation between leptin and
nutritional status, body mass index (BMI), fat mass evaluated
by plicometry ( tricipital skinfold (TS) and subscapular
skinfold (SS) thickness ) and auxological measurements were
determined.
Two blood venous samples were collected: one from the
mother before delivery, the other from umbelical cord at
delivery. Thereafter, several blood samples for leptin
measurements were taken at 0, 4,10,30,90 days of life and
anthropometric data for defining nutritional status were
simultaneously recorded. Type of feeding were recorded for
each newborn.
Serum leptin levels were measured by radioimmunoassay
(Linco Research, St Charles, MO).
The data (expressed as means +/2 SD) were analyzed for
statistically significant differences by two-tailed t test for
unpaired data and by linear correlation (SPSS/PC).
Results: Leptin levels in venous cord blood was 1.33+/2
1.12 mg/ml (mean+/2SD) and range 0.4124.5 mg/ml.
Maternal leptin concentrations was 13.69 +/2 13.56 mg/ml
(mean+/2SD) and range 0.6861 mg/ml. There was no
significant correlation between umbilical leptin concentration
and maternal leptin concentration. There was a correlation
between umbilical leptin concentration and birth weight of
preterm newborns (p = 0.01).
Serum leptin levels increased from birth to the 90th day of
life: T0 2.89+/2 1.87 mg/ml; T90 4.55+/2 2.9 mg/ml (mean
+/2 DS) p = 0.001. Leptin correlated to weight (r = 0.40) and
to SS and TS thickness only at 90 days of life( SS r = 0.56; TS
r = 0.5). We observed no significative differences in serum
leptin levels between breastfed and mixed fed preterm
infants.
Conclusions: The association between umbilical serum
leptin and birth weight in this and other studies suggests
a pivotal role of fetal leptin in regulating fetal growth and
development. Our data, moreover, show a significative in-
crease of leptin serum levels from birth to the third month of
 ESPGHAN 38TH ANNUAL MEETING
life, and a good correlation with subscapular and tricipital
skinfold thickness and weight at 90 days of life. The low
leptin levels in the first period after birth could be important
for the stimulation of feeding behaviour and the acquisition of
energy homeostasis in the newborn. The preterm baby, which
notably presents difficulties in reaching his catch up growth,
probably needs an appropriate number of adypocites for the
synthesis of leptin.
PN2-05 DEPOT-SPECIFIC MRNA EXPRESSION OF 11 BETA-
HYDROXYSTEROID DEHYDROGENASE TYPE 1 AND
LEPTIN IN ADIPOSE TISSUE OF CHILDREN AND
ADULTS X Li1, S Lindquist1, R Chen3, T Olsson2,
O Hernell1. 1Department of Clinical Sciences, Pediatrics,
Umea University, Umea, Sweden.2Department of Public
Health and Clinical Medicine, Medicine, Umea University,
Umea, Sweden. 3Childrens Research Institute of Nanjing
Medical University, Nanjing, China.
Background: Visceral obesity confers greater risk for
associated diseases than overall obesity, and subcutaneous
(SC) and visceral adipose tissue depots are characterized by
metabolic differences. In adults, genes coding for some of the
proteins implicated in obesity and/or insulin resistance are
also differently expressed in SC and visceral, or omental
(OM) adipose tissue.
Objective: To compare expression of mRNA coding for the
cortisol regenerating enzyme 11-hydroxysteroid dehydro-
genate type 1 (11HSD1), and the adipocytokines leptin and
resistin in paired SC and OM adipose tissue biopsies from
children.
Design: Paired biopsies (SC and OM) were obtained from
54 children (age 0.1716 years, BMI 12.528.3 kg/m2) and
16 adults (2872 years, BMI 1946 kg/m2) undergoing open
abdominal surgery. mRNA levels of 11HSD1, leptin and
resistin were measured using quantitative real-time RT-PCR.
Results: 11HSD1 mRNA levels were higher in OM than
in SC (p , 0.05), whereas leptin mRNA was higher in SC
than in OM (p , 0.001). There was no difference between
SC and OM for resistin mRNA. These results were consistent
in children and adults. Furthermore, in children 11HSD1
mRNA in SC was positively associated with BMI (p , 0.05),
whereas 11HSD1 mRNA in OM was positively associated
with age (p , 0.05). Leptin mRNA was positively associated
with BMI (SC: p , 0.001, OM: p , 0.01). In adults, there
was a significant correlation between leptin and BMI (SC:
p , 0.01, OM: p , 0.05). When normal weight and over-
weight children were analyzed separately, 11HSD1 mRNA
levels were positively associated to those of leptin in OM
from the overweight group (p , 0.05).
Conclusion: Depot-specific differences in mRNA levels of
11HSD1 and leptin were present already in childhood. The
elevated level of 11HSD1 mRNA in OM may reflect a causal
role in visceral fat accumulation during growth. Increasing
11HSD1and leptin mRNA in SC with increasing BMI may
suggest that the risk of the metabolic consequences of obesity
occurs relative early in life.
PN2-06 DOCOSAHEXAENOIC ACID SUPPLEMENTATION
DURING PREGNANCY MODULATES THE GENE EX-
PRESSION OF SOME FATTY ACID TRANSPORT PRO-
TEINS IN HUMAN PLACENTA E Larque1, C Campoy2,
M Klingler1, M Cruz2, A Cano2, H Demmelmair1, B Bondy3,
B Koletzko1. 1Department of Nutrition and Metabolic
Diseases, LMU University, Munich, Germany. 2Department
of Pediatric, University of Granada, Granada, Spain.
3
Department of Neurobiochemistry, LMU University,
Munich, Germany.
697
Introduction: The placental transfer of fatty acids is
a complex process mediated by specific carrier proteins from
membranes and cytosol. Nevertheless, the underlying mech-
anisms for the preferential materno-fetal transfer of long-
chain polyunsaturated fatty acids (LC-PUFA) are still poorly
understood. The aim of this study is to evaluate the effects of
nutritional supplements with docosahexaenoic acid (DHA)
and folic acid on the placental mRNA expression of proteins
that may regulate fatty acid transfer.
Methods: We recruited 137 pregnant women from Granada
(Spain) who received from the week 20 until delivery
different nutritional supplements: placebo; MTHF supple-
mented with 400 ug folic acid; DHA supplemented with
500 mg DHA + 150 mg EPA; and DHA + MTHF (400ug folic
acid, 500 mg DHA, 150 mg EPA ). Placental tissue was
sampled at birth, fatty acid profile analyzed by gas-liquid
chromatography, and mRNA expression of placental fatty
acid transport proteins-1 (FATP-1) and-4 (FATP-4), heart-
fatty acid binding protein (H-FABP) by real time-PCR.
Results: No significant difference was found in the gene
expression of fatty acid transport proteins FATP-1, FATP-4 or
H-FABP among the 4 groups. There was a positive correlation
of DHA % in placental phospholipids with mRNA expres-
sion of membrane proteins FATP-1 (R = +0.364, P = 0.001)
and FATP-4 (R = +0.387, P , 0.001). Arachidonic acid/DHA
ratio (AA/DHA) was inversely correlated with FATP-1 and
FATP-4, respectively, in all placental lipid fractions except for
cholesterol esters (phospholipids: R = 20.287, P = 0.007; R =
20.335, P = 0.002; NEFA: R = 20.213, P = 0.005;
R = 20.235, P = 0.032; triglycerides: R= 20.310, P = 0.004;
R = 20.407, P , 0.001). In contrast, the cytosolic protein
H-FABP was not correlated with LC-PUFA.
Conclusion: In uncomplicated pregnancies the mRNA
expressions of membrane placental proteins FATP-1 and
FATP-4 are related to placental accumulation of DHA and
AA, respectively, whereas there is no correlation with
expression of the cytosolic protein H-FABP.
PN2-07 PEROXISOME PROLIFERATOR-ACTIVATED RECEP-
TORS (PPAR) AND RETINOID X RECEPTORS (RXR)
ARE RELATED TO THE GENE EXPRESSION OF SOME
FATTY ACID TRANSPORT PROTEINS IN HUMAN
PLACENTA E Larque1, H Dominic2, S Krauss-Etschmann2,
C Campoy3, S Pardillo3, H Demmelmair1, S De Jongue4,
B Koletzko1. 1Department of Nutrition and metabolic
diseases, LMU University, Munich, Germany. 2Department
of Pediatric Immunology, LMU University, Munich, Ger-
many. 3Department of Pediatrics, University of Granada,
Granada, Spain. 4Department of Neurobiochemistry, LMU
University, Munich, Germany.
Introduction: Peroxisome proliferators-activated receptor
(PPAR)
is a nuclear receptor that heterodimerizes with
retinoid-X-receptor (RXR) to regulate not only adipogenesis
but also trophoblast differentiation and maturation in
placental tissue. Since docosahexaenoic acid (DHA) might
modulate the RXR signaling pathway, we asked whether
DHA supplementation during pregnancy modifies mRNA
expression levels of placental fatty acid transporters via
activation of PPAR and RXR receptors.
Methods: Placental tissue samples were obtained from
healthy women with uncomplicated singleton pregnancies
aged between 18 and 40 years from Spain (n = 137). Subjects
had been assigned at random, double blind to one of four
supplementation groups: fish oil (500 mg DHA, 150 mg EPA,
daily), folate (400 mg, daily), both in combination or
a placebo during second half of pregnancy. We quantified
the mRNA levels in placental tissue of fatty acid transport
proteins-1 (FATP-1), FATP-4, heart-fatty acid binding protein
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
698 ESPGHAN 38TH ANNUAL MEETING
(HFABP) and the transcription factors PPAR-a, PPAR-g,
RXR-a and RXR-g by real time RT-PCR.
Results: No differences were found in mRNA expression of
FATP-1,-4, HFABP, PPARs and RXRs among the 4
supplemented groups, but significant positive correlations
among them were found (table).
PPAR-a PPAR-g RXR-a RXR-g
R P R P R P R P
FATP-1 0.313 0.001 0.154 n.s. 0.358 0.001 0.442 0.001
FATP-4 0.262 0.006 0.250 0.008 0.365 0.001 0.393 0.001
H-FABP 0.280 0.003 0.260 0.005 0.348 0.001 0.385 0.001
Conclusion: mRNA levels of FATP-1, FATP-4, and H-FABP
in human placental tissue might be modulated reciprocally by
PPARs and RXRs. This could contribute to the regulation of
materno-fetal fatty acid transport.
PN2-08 GENE EXPRESSION OF FATTY ACID TRANSPORT
PROTEIN-4 (FATP-4) IN HUMAN PLACENTA IS POSI-
TIVELY CORRELATED WITH DOCOSAHEXAENOIC
ACID CONTENT IN MATERNAL AND CORD BLOOD
PHOSPHOLIPIDS E Larque1, J Linde-Gutierrez2,
C Campoy3, V Dolz3, H Demmelmair1, S De Jongue4,
B Bondy4, B Koletzko1. 1Department of Nutrition and
Metabolic Diseases, LMU University, Munich, Germany.
2 10.4 mg of AA and 7.0 mg of DHA per serving. Children
Institute of Food Technology, University of Granada,
Granada, Spain. 3Department of Pediatrics, University of
Granada, Granada, Spain. 4Department of Neurobiochem-
istry, LMU University, Munich, Germany.
Introduction: Docosahexaenoic acid (DHA) is a major
constituent of fish oil and has beneficial effects on blood
cholesterol levels, insulin sensitivity and neuronal function.
Due to the limited rate of DHA synthesis from precursors, the
maternal supply of preformed DHA is of major importance
for foetal supply. The objective of the present study is to
evaluate the relationship of some fatty acid transport proteins
of the placenta in the content of long-chain polyunsaturated
fatty acids (LC-PUFA) in maternal and cord blood from
women receiving a DHA supplementation during the last
trimester of pregnancy.
Methods: 137 pregnant women from Granada, Spain were
participants of the NUHEAL study. Subjects were assigned at
random, double blind to one of four supplementation groups:
fish oil (500 mg DHA, 150 mg EPA, daily), folate (400 mg,
daily), both in combination or a placebo during second half of
pregnancy. We analyzed the fatty acid composition in cord
blood and maternal peripheral blood phospholipids by gas-
liquid chromatography. We also quantified the mRNA
expression in placental tissue of fatty acid transport
proteins-1 (FATP-1), -4 (FATP-4), heart-fatty acid binding
protein (H-FABP) by real time-PCR.
Results: Correlations among FATP-1, FATP-4, and H-FABP
mRNA expression in placenta and LC-PUFA % in maternal
and cord phospholipds.
Conclusion: The correlation of mRNA expression of
membrane placental proteins FATP-1 and specially of
FATP-4 with the maternal and cord DHA and arachidonic
acid (AA), respectively, suggests a role of these fatty acid
transport proteins in placental LC-PUFA transfer.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
Maternal
DHA Maternal AA Cord DHA Cord AA
R P R P R P R P
FATP-1 0.336 0.001 20.21 0.021 0.134 n.s. 20.44 0.001
FATP-4 0.235 0.010 20.28 0.002 0.201 0.032 20.20 0.031
H-FABP 0.102 n.s. 20.18 n.s. 0.044 n.s. 20.17 n.s.
PN2-09 CONSUMING AN AA AND DHA FORTIFIED BEVER-
AGE FOR 7 MONTHS IMPROVES VISUAL PERCEP-
TION AMONG CHILDREN 47 YEARS OLD V Lien1,
K Pramuk2, I MacDonald1, JR Mickelson1, YK Goh1,
MT Clandinin1. 1University of Alberta, Edmonton, Canada.
2
Wyeth Nutrition, Philadelphia, USA.
Arachidonic acid (AA) and docosahexaenoic acid (DHA) are
important for infant growth and development. There is
limited research investigating vision and brain development
beyond 2 years of age, despite the continuance of neurolog-
ical growth into the early years of childhood.
Aim: Healthy children between the ages of 47 years who
had previously been identified as consuming a diet with low
DHA were provided with either a milk based vitamin and
mineral fortified beverage or the same beverage supple-
mented with AA and DHA. The aim of the study was to
evaluate essential fatty acid status and tests of neuropsycho-
logical development between the two feeding groups.
Method: The study design consisted of a controlled, double
blind study where children who were attending the same
kindergarten were randomized into two feeding groups (four
formula codes). The LCPUFA supplemented milk contained
consumed 2 3 sachets of their assigned milk per day (at
home) for 7 months. Primary outcome measures included
complete ophthalmic exam(s), Test of Visual and Perception
skills (TVPS-R), and plasma phospholipid fatty acids. Four-
day food record (s), food frequency questionnaires, and
health diaries were also collected. Height and weight were
measured at baseline and study completion.
Results: Plasma phospholipid and red blood cell AA and
DHA levels were higher in the LCPUFA group than in the
control group at 7 months. The median visual assessment
scores for the group fed the LCPUFA fortified supplement
were significantly higher than those of the control group for 6
of the 9 visual assessments in the TVPS-R. Daily supple-
mentation of AA and DHA for a period of 7 months had
positive effects on development of visual perception.
Conclusion: Daily supplement with AA and DHA improved
development of visual perception in children who were
previously identified to have low dietary intakes of DHA.
PN2-10 INFANTS FED DOCOSAHEXAENOIC ACID AND
ARACHIDONIC ACID SUPPLEMENTED FORMULA
HAVE DECREASED INCIDENCE OF RESPIRATORY
ILLNESSES THE FIRST YEAR OF LIFE N Pastor1,
B Soler2, P Ferguson3, C Lifschitz4. 1Mead Johnson &
Company, Madrid, Spain. 2E-C-BIO S.L., Estudios Cientifi-
cos, Madrid, Spain. 3Mead Johnson & Company, Evansville,
USA. 4Baylor College of Medicine, Houston, USA.
Long chain polyunsaturated fatty acids may enhance immune
function (Uauy R., Proc. Nutr. Soc., 2000; 59: 3). To assess
the effects of docosahexaenoic acid (DHA) and arachidonic
acid (ARA) supplementation in infant formula on common
illnesses during the first year of life, 357 pediatricians
 ESPGHAN 38TH ANNUAL MEETING
throughout Spain enrolled 1388 term infants. Infants were
assigned to be fed either DHA (17 mg/100 kcal) and ARA
(34 mg/100 kcal) supplemented formula (DHA+, n = 1133)
(Enfalac Premium, Mead Johnson Company) or non-supple-
mented formula (Control, n = 255) (4.4:1 ratio) for 1 year.
Infants returned at 1, 3, 5, 7, 9, and 12 months of age for
follow up anthropometrics. At each visit, physician records
were reviewed for diagnoses of medical illnesses. Counts
(incidence of illnesses) were analyzed by chi-square. There
were no significant differences between the DHA+ and
Control formula groups for baseline measurements (age of
enrollment, weight, length, head circumference, and past
medical history) or follow-up measurements (weight, length,
and head circumference). The incidence of physician
recorded respiratory illness was lower in the DHA+ group,
particularly bronchitis/bronchiolitis:
PN2-12 EFFECTS OF THE MODE OF IRON ADMINISTRATION
Compared to DHA+, controls had a higher incidence of
pneumonia at 3 months (p = 0.03), upper respiratory
infections at 12 months (p = 0.011), and rhinitis at 1 month
(p = 0.02); and a lower incidence of cough at 7 months (p =
0.04). Infants fed DHA+ formula have a decreased incidence
of bronchitis/bronchiolitis, pneumonia and upper respiratory
infection in the first year of life compared to infants fed
Control formula.
PN2-11 SHOULD IRON REQUIREMENTS IN FIRST YEAR OF
LIFE BE DIFFERENTIATED BY GENDER? H Antunes1,
S Gonc xalves2, C Santos3, A Costa-Pereira3, R Tojo-Sierra4,
A Aguiar5. 1Gastrenterology, Hepatology and Nutrition Unit,
Paediatrics Dept, Sao Marcos Hospital, Braga, Portugal.
2
Psychology Dept, Minho University, Braga, Portugal.
3
Biostatistics and Medical Informatics Dept, Faculty of
Medicine, Porto University, Porto, Portugal. 4Paediatric
Dept, Faculty of Medicine, Hospital Clinics University,
Santiago de Compostela University, Santiago de Compostela,
Spain. 5Paediatric Dept, Medicine Faculty, Sao Joao Hospital,
Porto University, Porto, Portugal.
Introduction: In adolescence, the definition of iron de-
ficiency anaemia (IDA) differs in term of gender. The results
of previous studies on infants IDA revealed that most of
those were boys. According to some authors, such a gender
distinction should also be made in the first year of life.
Aim: To find answers to the question raised at the 2
WCPGHAN - Why is there a prevalence of boys rather than
girls suffering from IDA in the first year of life? Should we
also consider the definition of IDA in the first year of life in
terms of gender?
Methods: 226 parents allowed blood samples to be collected
from their 9-month old babies; 23 refused. In 2 of the babies
the sample was insufficient, 201 out of the 226 children were
studied. These childrens weight at birth and weight at nine
months was registered and blood count, iron, transferring, and
ferritin were carried out. The definition of IDA used was that
699
of the WHO, haemoglobin ,110 g/l, ferritin .12 ng/ml and
response to iron therapy. The x-square and t tests were used
for independent samples. Mann-Whitney U tests were used to
compare antropometric and hematologic parameters. All tests
used a level of significance of 5%. This study was approved
by the Ethical Committee of HSM.
Results: Of the 39 infants suffering from IDA (19.4%), 24
(61.5%) were male and of the 162 infants without IDA, 50%
were male (p = 0.195). The medium (minimum-maximum)
measurement of ferritin in male at nine months was of 9.8 ng/ml
(0.567.0 ng/ml) and in females 16.5 ng/ml (0.4574.5 ng/ml)
(p , 0.001). The average (standard deviation) weight increase
between birth and nine months was of 5 863.3 g (855.4 g) in
male and 5 556.9 (1 054.3 g) in female (p = 0.027).
Conclusion: Like in other studies, the results of our research
revealed that there was a prevalence of male infants with IDA,
although such results were not statistical significant. ID was
significantly more frequent in male infants, but this difference
may be explained by the fact that there was a greater weight
increase from birth to nine month in males than in females.
Therefore, we believe that it is not the definition of ID that
should be differentiated in terms of sex in the first year, but
the iron needs of this age group.
ON FERRITIN AND HEMOGLOBIN IN INFANTS
M Domellof1, T Lind1, B Lonnerdal2, LA Persson3,
KG Dewey2, O Hernell1. 1Dept of Clinical Sciences,
Pediatrics, Umea University, Umea, Sweden. 2Dept of
Nutrition, University of California, Davis, CA, USA. 3Dept
of International Maternal and Child Health, Uppsala
University, Uppsala, Sweden.
Iron-fortified foods (FF) and/or medicinal iron drops (MD)
are recommended for infants but little is known about
possible differences in effects on iron status and iron
metabolism.
Method: We performed a secondary analysis of pooled data
from one FF trial and one MD trial. We divided healthy, term,
non-anemic, Swedish infants into two groups depending on
the predominant source and amount of dietary iron at 69 mo
of age: 1) MD: Medicinal iron drops (1 mg/kg/day) 2) FF:
Predominant iron intake from FF, iron intake .1.3 mg/kg/day
and no iron supplements. 3) Control group: Predominant iron
intake from FF, iron intake ,1.3 mg/kg/day and no iron
supplements. The cutoff for iron intake was chosen to achieve
similar total iron intake in the MD and FF groups.
Results: Mean iron intake was similar in the MD (n = 37) and
FF (n = 29) groups (1.57 vs 1.54 mg/kg/day, p = 0.67) and signi-
ficantly lower in the control group (n = 260, 0.55 mg/kg/day,
p , 0.001). The recommended daily iron intake in this age
group is 12 mg/kg. There was no significant difference
between groups in sex distribution, birth weight, weight at 6
months, weight at 9 months, Hb at 6 months or ferritin at 6
monhts. Controlling for baseline Hb and ferritin at 6 mo,
infants in the FF group had significantly higher mean Hb at 9
months compared to the MD group (120 vs 115 g/L, p = 0.003)
and also compared to the control group (120 vs 115 g/L, p ,
0.001), while infants in the MD group had significantly higher
mean ferritin at 9 months compared to the FF group (41 vs
25 ug/L, p , 0.001) and also compared to the control group
(41 vs 25 ug/L, p , 0.001).
Conclusions: As far as we know, this is the first study directly
comparing the effects of MD and FF on iron status in infants.
Our results suggest that different modes of iron administra-
tion have different effects on iron metabolism and iron status
in healthy, term, non-anemic infants at 69 months of age:
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
700 ESPGHAN 38TH ANNUAL MEETING
Iron given as MD is primarily deposited into iron stores while
iron given as FF is primarily utilized for Hb synthesis. This is
a potentially important discovery which needs to be explored
in further studies.
PN2-13 ENERGY REQUIREMENTS AND DIETARY INTAKE IN
A PEDIATRIC POPULATION WITH JUVENILE CHRON-
IC ARTHRITIS A Morais. Lopez1, RA Lama. More1,
J Garca-Consuegra Molina2, R Merino Munoz2. 1Unidad de
Nutricion Infantil. Hospital Universitario Infantil La Paz.
Universidad Autonoma de Madrid, Madrid, Spain. 2Servicio
de Reumatologa Infantil. Hospital Universitario Infantil
La Paz, Madrid, Spain.
Introduction: Juvenile chronic arthritis (JCA) can lead, in
pediatric patients, to a poor nutritional status secondary to
several factors.
Aim: To analyse energy expenditure (EE) in a pediatric
population with JCA and check whether energy intake (EI) in
these children suits caloric and protein needs.
Subjects and Methods: 91 children admitted to the Nutrition
Unit along 12 months with diagnosis of JCA, with one-year
follow-up (43% pauciarticular, 26% polyarticular, 20%
systemic and 11% others). Activity markers, corticotherapy,
nutritional status and seven-day dietary intake were assessed.
EE was measured by indirect calorimetry and compared with
ideal values for age and gender according to Schofield
equations. Statistical analysis was made using SPSS 10.0.
Results: Mean activity time during the follow-up was 6.16 6
0.51 months. 68.1% of children received corticotherapy. In
18.7% of children growing rate was retarded, specially in the
systemic and corticoid-treated groups (p , 0.01). 11% of
children showed low Waterlow index. EE was decreased in
comparison with ideal values (86.77 6 1.34%), being
significantly higher in those subjects with long-time disease
and longer follow-up. Children with highest values of
interleukyna-6 and tumoral necrosis factor showed the lowest
values of EE and lean body mass. In most cases, total EI
suited WHO allowances, although protein intake was high
(15 6 2% of total calories). Concerning the group of children
who required an extra intake to reach an acceptable
nutritional status, up to 54% did not suit their needs.
Conclusions:
1. Children with systemic involvement and treated with
corticoid drugs showed lower growing rate.
2. Most of subjects showed an appropriate weight for their
height, but body composition was not always correct,
which might be related to EE values.
3. EI was acceptable, although a higher proportion of non-
protein calories would be more adequate.
4. Nutritional assessment may be specially useful in those
children who do not suit their energy requirements, in
order to prevent malnourishment situations.
PN2-14 IS IT REALLY NECESSARY TO RECOMMEND A
CALORIC INTAKE OF 120-150% OF RDA IN YOUNG
CYSTIC FIBROSIS CHILDREN WITH NORMAL RE-
SPIRATORY FUNCTION? N Wizla1, J Hays1, M Zenaidi1,
L Michaud1, A Deschildre1, C Thumerelle1, F Gottrand2,
D Turck1. 1CF Centre, Department of Paediatrics, Jeanne de
Flandre Hospital, Lille, France. 2Hepatogastroenterology
and Nutrition Unit, Department of Paediatrics, Jeanne de
Flandre Hospital, Lille, France.
Aim: To maintain an optimal nutritional status in cystic
fibrosis (CF) patients, daily caloric intake (DCI) is recom-
mended to be 120150% of RDA. The aim of the present
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
study was to evaluate DCI in CF children and adolescents,
and the association between DCI, nutritional status, pancre-
atic function and lung function.
Methods: Fifty-five CF patients (29 boys; mean age = 10.1 6
4.9 y; range: 118.5 y) followed in our paediatric CF centre
were included in the study. DCI was evaluated by a 3-day
prospective dietary questionnaire filled in by the caregiver at
home during the week preceding a scheduled consultation
(Bilnut software). During this visit, nutritional status was
assessed by measurement of weight, height. Lung function
was assessed using a spirometer Jaeger.
Results: Forced expiratory volume in 1 second was 102 6
24% predicted value [range: 57148]. Nutritional status was
as follows: Z score weight/age = 20.52 6 1.16, Z score
height/age = 20.63 6 1.45, Z score weight/height = 20.24 6
1.08, Z score BMI = 20.36 6 0.99. Mean DCI was 107.2 6
23.3 % RDA [range: 64172]. Repartition of nutrients was as
follows: carbohydrates = 51.4 6 5.4 %; proteins = 14.0 6
2.4 %; lipids = 34.3 6 5.6 %. DCI was significantly higher
in patients with pancreatic insufficiency (111 6 24.8 % RDA,
n = 40) than in pancreatic sufficient patients (97.1 6 15 %
RDA, n = 15, p , 0.05). Nutritional status was not
significantly different among 39 patients with DCI , 120%
RDA and 16 patients with DCI . 120% RDA. DCI was not
different according to age, sex, bronchial bacterial colonisa-
tion and lung function. Weight was significantly lower in 11
patients chronically infected by Pseudomonas aeruginosa
(20.91 6 1.18 SD vs 20.86 6 1.61 SD; p = 0.031) and in
36 patients chronically infected by Staphylococcus aureus
(21 6 1.7 SD vs 20.63 6 1.11 SD; p = 0.048). Other
anthropometric parameters were not different according to
age, sex, and lung function.
In this transversal study, no association was found between
caloric intake, nutritional status and lung disease. The advice
of a DCI from 120150%RDA might be useless in young CF
patients with a normal respiratory function. This should be
more better evaluated in a longitudinal study.
PN2-15 CEREBRAL PALSY SPECIFIC GROWTH CHARTS
(CPSGC): JUST MASKING THE UNDERNUTRITION
PROBLEM? G Marakis1, D Zafeiriou2, V Katri1,
E Vargiami2, A Savvidou1, T Lampoudi2. 1Nutrition-
Dietetics Dept, School of Food Technology, Nutrition, TEI.,
Thessaloniki, Greece. 21st Pediatric Dept, AUof Thessaloniki,
Thessaloniki, Greece.
The need for the development of growth charts specific for
children with Cerebral palsy (CPsGC) has been emphasized
during the past decades. However, there is some skepticism
over the validity of such growth charts. Those who are
opposed to the use of such charts believe that disease-
specific growth charts should be developed only for children
with genetic abnormalities, in which chromosomal defects
determine growth, and that the use of CPsGC in children
with CP can only mask the inadequate nutritional care
that is provided to these children. The aim of this study is
to evaluate whether the use of the established CPsGC
provides a more accurate assessment of nutritional status
and growth in children with CP than the use of charts used
for healthy children (CDC). This is accomplished by
comparing each patients classification in both charts to
his/her triceps-skinfold thickness classification (t-stc),
which is considered an effective tool for the assessment of
these patients.
Patients: Children with CP: 57, fulfilling the age criteria to
be enrolled in the study (age 110 y, no diseases or drugs
interfering with growth): 33 (22 males, mean age 4.2 y, age
range: 1.18.8 y).
 ESPGHAN 38TH ANNUAL MEETING
Methods: Weight, height percentiles on growth charts of
both types (CDC and CPsGC) and t-stc were assessed. The
correspondence between t-stc and weight and height percen-
tiles according to CDC growth charts or CPsGC was
examined for every child. The 10th percentile for t-stc was
considered as the cut-off point for undernutrition.
Results: Based on the above criterion 39% of the patients
were undernourished. According to the CDC charts 48% fell
below the 5th percentile for weight, whereas none did so
according to the CPsGC. The distribution pattern of t-stc
corresponded to the distribution pattern of weight (73%) and
height (67%) on the CDC charts and not on the CPsGC.
Moreover, it was found that only 9% of the children had their
nutritional assessment better reflected by the use of CPsGC.
Conclusions: Growth charts for healthy children better reflect
the nutritional status of children with CP than CPsGC do.
Krick et al. 1996, Stallings et al. 1993, Fung et al. 2002
PN2-16 DURING GASTROINTESTINAL DIGESTION OF MILK
CASEIN PEPTIDES APPEAR WITH INSULIN MIMICRY,
TRANSITION METAL ION-BINDING AFFINITY AND
AMYLOID PROPENSITY JH Wissler1, G Georgi2,
G Boehm2. 1Arcons Applied Research, Bad Nauheim,
Germany. 2Numico Research, Friedrichsdorf, Germany.
Aim: Investigation of nutritional components of milk
proteins as released during gastrointestinal digestion for
relations to metalloregulated amyloidogenic insulins [Wissler
et al, FASEB J. 16: A980, 2002; Amer. J. Clin. Nutr. 75:
360S, 2002; Mol. Biol. Cell. Suppl. 13: 392a, 2002].
Methods: Peptides derived after tryptic/chymotryptic in vitro
digestion have been separated by gel permeation chromatog-
raphy and fractions were further purified by reverse phase
chromatography. Peptides were characterised by ESI-MS/MS
and MALDI. In bioassays in vitro with SV3T3 mice cell line
or human skin fibroblasts, selected mixtures and individual
peptides (derived from caseins) were tested for effect on
glucose uptake equivalents [redox metabolism and vitality;
methods: Rehder et al, J. Biol. Inorg. Chem. 7:384396:
2002; Meier et al, ALTEX 20:199:2003].
Results: Cryptic metalloregulated structural domains with
epitope and other homologies to insulin chains were found.
Peptides from bovine caseins differ from human and camel
peptides in additional inhomologous transition metal ion-
PN2-18 CDNA MACROARRAY ANALYSIS OF INTESTINAL
binding domains, e.g. -SWxHxxHxx-. Peptide libraries from
digests of de- and phosphorylated nutritional components like
caseins may form stable soluble and aggregating amyloid-
like transition metal [Cu, Zn] ion-peptide complexes, re-
spectively. Several of these peptides (termed casoinsulins)
display insulin-like bioactivity in terms of glucose uptake
equivalents.
Summary: Peptides derived from tryptic/chymotryptic di-
gestion from milk caseins demonstrate insulin mimicry,
transition metal ion-binding affinity and amyloid propensity.
This insulin-like activity which might occur during gastroin-
testinal digestion of milk caseins might have beneficial
physiological effects like gut maturation and/or therapeutic
effects similar to oral application of insulin. However, it might
also suggest a possible nutritional risk for development of
insulin antibodies. Both hypotheses need further evaluation.
PN2-17 SINGLE NUCLEOTIDE POLYMORPHISMS IN THE
APO(A) KRINGLE IV TYPE 8 DOMAIN AND ATHERO-
THROMBOTIC SERUM LIPOPROTEIN (A) CON-
CENTRATION, IN A PORTUGUESE PEDIATRIC
POPULATION H Ferreira. Mansilha1, E Costa2, E Vieira3,
701
R Pimenta4, J Lourencxo. Gomes1, J Barbot5, R Dos. Santos2.
1
Dept Pediatrics - Maria Pia Childrens Hospital, Porto,
Portugal. 2High School of Health of Politec. Inst., Braganc
xa,
Portugal. 3Molecular Genetic Unit of Dr Jacinto de
Magalhaes Inst., Porto, Portugal. 4System Dept of Health
Tecnology High School, Porto, Portugal. 5Dept Hematology -
Maria Pia Childrens Hospital, Porto, Portugal.
Lipoprotein (a) (Lp[a]) is a complex of apolipoprotein (a)
(apo[a]) and low-density lipoprotein (LDL), which is
associated with atherothrombotic disease. Most of the
interindividual variations in plasma levels of Lp(a) can be
attributed to sequence differences linked to the apo(a) gene
locus. Human apo(a) is a glycoprotein containing 10 different
types of kringle (K) IV domains, which share a high degree of
homology with plasminogen K IV. Recently, single nucleo-
tide polymorphisms (SNP) in the exons and intronic
sequences of the apo(a) K IV, have been investigated as to
their effect on Lp(a) particle formation.
The aim of this study was to investigate a possible link between
some SNPs in the apo(a) K IV type 8 domain and athero-
thrombotic serum Lp(a) concentrations (Lp[a] .30 mg/dl).
We studied 97 paediatric patients, 51 with serum Lp(a) con-
centration above and 46 with concentrations below 30 mg/dl.
In all patients direct sequencing of the two exons and flanking
intronic sequences of the apo(a) K IV type 8 domain was
performed. Sequencing was carried out on the primary
amplicons, using the respective forward and reverse primers,
in independent sequencing reactions and subsequently re-
solved on an ABI Prism 310 GeneticAnalyser (Applied
Biosystems, Foster City, USA).
In this population, we found three SNPs, two in exon 1
(c.3888A . C and c.3955G . A) and one in intron 1 of the
apo(a) K IV type 8 domain (c.3982+1G . A). The c.3888A .
C polymorphism is most common with a heterozygosity
frequency of 15.46%. The c.3955G . A and c.3982+1G . A
polymorphisms were found in a frequency of 5.15% and
1.03%, respectively. No statistically significant difference was
found in the genotype distribution between the two groups of
patients, with Lp(a) above and below 30 mg/dl. Our results
suggest that these SNPs in the apo(a) K IV type 8 domain are
not directly associated with atherothrombotic serum Lp(a)
concentrations in our population.
GENE EXPRESSION PROFILES IN PIGLETS DURING
WEANING D Mazurais1, V Rome1, I Le Huerou-Luron1.
1
INRA-SENAH, Saint-Gilles, France.
Aim: During piglet weaning, intestinal function modifica-
tions (digestion and nutrient absorption, water and electrolyte
secretion, barrier properties, metabolism, gut immune
system) are more likely due to quantitative and qualitative
variations of feed intake. The aim of the present study was to
characterize gene expression profiles in the small intestine of
sow-reared, fasted or weaned piglets in order to better
understand molecular effects of weaning-induced abrupt
dietary change.
Methods: At 21 d of age, twenty-four sow-reared piglets
were assigned to 3 dietary treatments. In the first group, eight
sow-reared piglets were slaughtered at 21 d of age. In the
second group, piglets were fasted for 2 d before slaughtering.
In the third group, 2 d fasted piglets were refed a starter diet
for 5 d before sacrifice. The proximal jejunum was collected
for histological, brush-border enzyme activity and genomic
analysis (macroarray).
Results: In agreement with the literature, morphologic and
enzyme activity analysis confirmed that food deprivation
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
702 ESPGHAN 38TH ANNUAL MEETING
affected intestinal functions. Of the 1057 spotted cDNA
clones, 297 were expressed in the jejunum and altogether 128
potential genes were affected by dietary treatments (Gene-
ANOVA, p , 0.05). Clustering analysis revealed subsets of
genes regulated by dietery treatment. Compared to sow-
reared piglets, 69 and 54 genes were up- and down-expressed
in fasted piglets, respectively. Functional clustering of genes
revealed groups of enzymes of intermediary metabolism, of
transcription factors, and of proteins involved in cell
organization, cell proliferation and protein synthesis. Special
attention is now given to analyse the main functions modified
by the dietary treatments.
Conclusion: In the present nutrigenomic study, gene
expression profiling might bring new insights into underlying
molecular mechanisms involved in intestinal perturbation
during food deprivation and refeeding.
PN2-19 THE BILE SALT-STIMULATED LIPASE IS EXPRESSED
IN HUMAN GRANULOCYTES S Lindquist1, C Lundell1,
P Naredi2, O Hernell1. 1Department of Clinical Sciences,
Pediatrics, Umea University, Umea, Sweden. 2Department of
Surgical and Perioperative Sciences, Umea University,
Umea, Sweden.
Background: The bile salt-stimulated lipase (BSSL) is as
a lipolytic enzyme contributing to lipid digestion in the small
intestine. Initially its tissue origin was thought to be confined
to exocrine pancreas, and in some species (including human)
lactating mammary gland. However, BSSL is now considered
an intriguing, multifunctional enzyme with broader tissue
distribution. The BSSL level in serum of healthy individuals
has been suggested to influence serum cholesterol concen-
tration. The origin of circulating BSSL is unknown, but low
expression of the enzyme has been reported in macrophages,
aortic endothelial cells (HAECs), aortic homogenates, small
intestine and liver but data on tissue and cellular origin are
contradictory. We hypthesized that if BSSL is active in the
circulation, or in the arterial wall, the liver or the vascular
system should be the likely origin for the circulating enzyme.
Aim of the Study: To explore the expression of BSSL in
PN2-21 INTESTINAL PERMEABILITY IN HEALTHY TERM
human liver.
Methods: Biopsies from human livers were studied. Immu-
nohistochemistry was used to demonstrate presence and
cellular distribution of BSSL protein. Quantitative real-time
PCR as well as in situ hybridization was used to verify BSSL
mRNA expression.
Results: We could demonstrate BSSL mRNA expression in
human liver. However, immunohistochemistry clearly
showed that BSSL was not associated to hepatocytes, or
other liver-specific cells but to CD15+ granulocytes. The
BSSL mRNA expression was 10-fold increased in a patient
with a typical fatty liver compared to other subjects.
Conclusion: Our novel finding that BSSL is expressed not by
hepatocytes but by granulocytes suggests that BSSL is
involved in inflammatory processes, e.g. liver steatosis and
atherosclerosis. Our observation could lead to a better
understanding of the possible function(s) of BSSL besides
digestion of dietary lipids. It could also help to explain the
contradictory reports regarding BSSL tissue distribution.
PN2-20 QUANTITATIVE AND QUALITATIVE STUDY OF GAS-
TRIC LIPOLYSIS IN PREMATURE NEWBORN
C Roman1, F Carrie`re2, V Millet3, M Leclaire3,
U Simeoni3, J Sarles1. 1Service de Pediatrie Multidisciplin-
aire, Marseille, France. 2CNRS UPR 9025, Marseille,
France. 3Service de Neonatologie, Marseille, France.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
Aim: To determine the role of milk fatty acid (FA)
composition on the intragastric fat digestion in premature
newborn and to analyse the lipolysis products released in the
stomach.
Methods: Premature newborn fed either with fresh human
milk (HM) (10% medium chain triglycerides (MCT)) or
infant formula (IF) (PreGallia, 25% MCT) were included.
Gastric contents were aspirated twice a day during 5 days, just
before a gavage feeding (T0 = T180 min) and at various times
after the end of the same gavage (15, 30, 60, 90, 120 min).
Human gastric lipase (hGL) activity against tributyrin was
measured using a Radiometer pH-stat equipment. Lipids were
extracted with chloroform-methanol (2/1 v/v) and separated
using thin-layer chromatography coupled to a flame ionization
detector (TLC-FID). FA methyl esters were analysed by gas
chromatography.
Results: Eight infants were included in the study (mean age
and weight: 34.3 6 1.8 wks; 1791 6 165 g). Two were fed
with HM and 6 with IF. hGL concentration increased during
digestion, but final levels were 2.5 fold lower than in healthy
adults (43.7 6 28.8 vs 100120 mg/mL). Total hGL secretory
ouputs after 180-min digestion period did not differ in
relation to the type of milk (137.5 6 53 mg for HM vs 113 6
52 mg for IF). The maximum lipolysis level recorded in the
gastric contents (21.4 6 4.3 %) was similar to that recorded in
adults (24.4 6 5.7%). Mean total intragastric lipolysis in the
8 newborn was 6.15 6 2.2%, vs 10 6 1% in healthy adults.
The total amount of free FA released upon gastric lipolysis
was the same with both types of milk. Free FA mainly
released in the stomach were oleic acid (C18:1) and linoleic
acid (C18:2), whatever the type of milk
Conclusion: Intragastric lipolysis is not quantitatively more
important in preterm newborn than in adults and is not
affected by the amount of ingested MCT. The preferential
release of long chain FA might be linked to the fact that they
are essential to optimise the intestinal lipolysis by pancreatic
lipase. MCT supplementation has therefore no apparent
effects on intragastric fat digestion in the newborn and its
use for preterm feeding probably needs to be further
investigated.
NEWBORNS WITH PHYSIOLOGIC JAUNDICE
F Indrio1, ME Baldassarre1, R Francavilla2, V Miniello2,
A Cirillo. Marucco , L Polimeno , F Gatti , F Raimondi4.
1 3 3
1
Dept. of Pediatrics Neonatology Section University of Bari,
Bari, Italy. 2Dept. of Pediatrics University of Bari, Bari,
Italy. 3Dept. of Emergency Organ Transplantation Gastro-
enterology Section, Bari, Italy. 4Dept. of Pediatrics Neo-
natology Section University Federico II, Naples, Italy.
Aim: Previous in vivo studies (1) have shown that un-
conjugated bilirubin may be responsible for an increase of
intestinal permeability (IP). This can be relevant for neonatal
health. Aim of the study is to investigate the effects of
unconjugated bilirubin on intestinal permeability of healthy,
term neonates.
Design/Methods: IP was measured by the sugar absorption
test (SAT) performed at the third day of life in 12 healthy term
jaundiced AGA newborns (total bilirubin .13 mg/dl) and
compared to that of 12 healthy term non-jaundiced AGA
newborns (total bilirubin ,8 mg/dl) matched for sex, GA,
birth weight and Apgar score. In the SAT, the urinary
lactulose/mannitol ratio (La/Ma) was measured after oral
ingestion of a solution containing 86 mg of lactulose (La) and
1.4 mg of mannitol (Ma) per 1 ml water at a dose of 2 ml/kg.
Urine was then collected for five hours, La and Ma
concentrations were measured by HPLC (high liquid phase
chromatography) (mg/l) and La/Ma calculated.
 ESPGHAN 38TH ANNUAL MEETING
Results: Jaundiced newborns have a significantly higher
La/Ma than non-jaundiced [mean 0.31 (sd 6 0.28) vs. mean
0.054 (sd 6 0.04)]. A significant correlation was found
between hyperbilirubinemia and La/Ma (r2 = 0.526 p =
0.008).
Summary: This first pilot clinical study shows that hyper-
bilirubinemia is responsible for an increase of IP in term,
healthy newborns with uncomplicated jaundice.
Conclusion: We speculate that such an increase of IP may
facilitate the passage of food allergens through the intestinal
epithelial barrier. Further studies are needed to detect the
underlying mechanisms of this liaison.
Reference:
(1) Jaerhrig K, Balke EH, Koenig A, Meisel P, Trans-
epithelial electric potential difference in newborns un-
dergoing phototherapy. Ped Res. 1987;21:283284.
PN2-22 GENETIC PREDISPOSITION FOR PRIMARY HYPO-
LACTASIA OF THE ADULT TYPE IN CHILDREN AND
ADOLESCENTS WITH RECURRENT ABDOMINAL
PAIN D Weber-Mzell1, AC Hauer1, M Gugatschka2,
A Fahrleitner-Pammer2, H Dobnig2, A Deutschmann1,
J Deutsch1, BM Obermayer-Pietsch2. 1Div. of General
Paediatrics, Dept. of Paediatrics, Medical University, Graz,
Austria. 2Div. of Endocrinology and Nuclear Medicine, Dept.
of Internal Medicine, Medical University, Graz, Austria.
Aim: Primary hypolactasia of the adult type (HL) presents
clinically as lactose intolerance (LI), a frequent cause of
recurrent abdominal pain (RAP). It bears the risk of impaired
bone growth due to avoidance of dairy products and thus
inadequate calcium intake. Using a molecular genetic assay,
genetic predisposition for HL was found in 24% of female
adults in our region. There are only few data on this
predisposition in children and adolescents as assessed by this
method. We used it to determine genetic HL predisposition in
children and adolescents with RAP to identify individuals at
risk for LI and impaired bone growth.
Methods: From VII-XII04 65/220 patients with self
reported RAP fulfilled the ROME II criteria for RAP (f: 38;
m: 27; 25/65 , 8 yrs. 40/65 . 8 yrs.) and presented clinically
with LI. The LCT genotypes CC, TC and TT were determined
by an allele specific PCR-based assay (defining the LCT-
[13910]-polymorphism near the lactase phlorizin hydrolyse
gene), using 150 microliters of capillary blood or saliva. For
compliance reasons lactose H2 breath tests were performed in
the elder patients only (17/65).
Results: 36/65 patients with RAP and clinical LI were CC
homozygote (55.4%; f: 24, m: 12). 23/65 (35.4%; f: 12, m:
11) were TC heterozygote and 6/65 (9.2%; f: 3, m: 3) TT
homozygote. 9/17 patients undergoing H2 breath tests had
positive results: 7/9 had the CC-, and 2/9 the TC-genotype.
Genotypes of the 8 patients with negative results were as
follows: CC = 3, TC = 2, TT = 3.
Summary: 90.8% of patients with RAP and clinically
suspected LI had the CC or TC genotype for HL. Because
these genotypes are both associated with a higher risk for
osteoporosis, these patients and those with positive H2 breath
tests are warranting close observation. An explanation for the
negative functional test results in the 3 CC patients might be
a higher individual threshold to the oral lactose load.
Conclusion: The PCR-based genotyping for lactose gene
polymorphisms presented might serve as a valuable screening
method for paediatric patients with RAP and at risk for LI and
osteoporosis that needs to be performed only once, is
unexpensive, easy and feasible from minute blood and saliva
volumes respectively.
703
PN2-23 ACIDIC OLIGOSACCHARIDES ENHANCE SYSTEMIC
IMMUNE RESPONSES IN MICE: DOSE-RESPONSE,
INTERACTION AND COMPARATIVE EXPERIMENTS
IN A MURINE VACCINATION MODEL AP Vos1,
B Stahl2, G Boehm2, J Garssen1, L MRabet1. 1Numico
Research, Wageningen, The Netherlands. 2Numico Research,
Friedrichdorf, Germany.
Breast milk contains a high variety of oligosaccharides,
including acidic oligosaccharides (AOS). It has been
suggested that oligosaccharides in breast milk might play
a role in the development of the immune system in infants.
The effect of AOS on the murine systemic immune system
was evaluated in dietary supplementation experiments with
AOS, derived after hydrolysis of pectin. In addition, the
interaction of AOS with a prebiotic mixture of galacto- and
fructo-oligosaccharides (GOS/FOS) was investigated. Earlier
work revealed that GOS/FOS was able to stimulate systemic
immune responses in mice. Furthermore, a combination of
AOS and GOS/FOS was tested in timing experiments.
Finally, the effect of GOS/FOS on the immune response
was compared to other commercially available oligosacchar-
ides. All experiments were performed using an influenza
vaccination model in C57Bl/6 mice, in which the primary
parameter was the T-helper1 dependent delayed-type hyper-
sensitivity (DTH) response.
In the dose-response experiment, AOS induced an increase in
DTH response in dosages between 1 and 5% (w/w in the
pellet diet), that was both significant and dose-dependent. In
interaction experiments, results indicated that a combination
of GOS/FOS and AOS acts synergistically on the immune
response, compared to the single test agents. In timing
experiments, the timing of AOS and GOS/FOS supplemen-
tation was varied in relation to the vaccination schedule. The
results from these experiments suggest that the effect on the
immune response occurs in the priming phase of the response.
Finally, other oligosaccharides were tested and compared to
the effect of GOS/FOS on the immune response. The results
demonstrated that for the oligosaccharides that were tested,
only GOS/FOS enhanced the immune response significantly.
In conclusion, the results indicate that GOS/FOS and AOS
enhance systemic immune responses in a murine vaccination
model synergistically and through specific mechanisms.
PN2-24 INFLUENCE OF FEEDING LACTOBACILLUS F19 DUR-
ING WEANING ON ANTIBODY RESPONSE TO HAE-
MOPHILUS INFLUENZAE TYPE B, DIPHTERIA- AND
TETANUS TOXOIDS GIVEN AT 3, 5 AND 12 MONTHS
OF AGE CE West1, O Hernell1. 1Department of Clinical
Sciences, Pediatrics, Umea, Sweden.
Aim: To investigate the effect of feeding Lactobacillus F19
(F19) during weaning on antibody response to Haemophilus
influenzae type B (Hib), diphteria (DT) - and tetanus toxoid
(TT) vaccine.
Methods: In a placebo controlled double blinded interven-
tion study, healthy, term infants, were randomised to porridge
with (n = 90) or without (n = 90) 1 x 108 CFU of L. F19 per
serving from 4 to 13 months of age. They were immunized
with Infanrix (GlaxoSmithKline) at (3), 5 and 12 months.
Venous blood was drawn before and 4 weeks after the 2nd
and 3rd immunizations. Antibodies to Hib and TT were
measured by Enzyme Immunoassay, and to DT by neutral-
ization test on Vero cells. Stool samples collected before and
3 times during the intervention were analysed for F19 using
a specific 16SrRNA probe and RT-PCR.
Results: 75 infants in the F19 and 79 in the placebo group
remained in the analysis. There was no difference in antibody
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
704 ESPGHAN 38TH ANNUAL MEETING
response between the groups. F19 was detected in faeces in
4/79 and 73/75 infants in the placebo and F19 groups
respectively. In 52/75 infants, F19 was present in all three
samples. Antibody response in these 52 infants and all infants
in the placebo group was evaluated by repeated measures
ANOVA adjusting for breastfeeding (BF) ,6 months or $6
months, total amount of F19 and other lactobacilli in faeces.
There was a significant interaction of intervention and BF. In
the F19 group, infants BF ,6 months had higher antibody
titers to DT and TT at 6, 12 and 13 months of age (p , 0.05 ).
Infants BF ,6 months had higher titers to Hib during
immunization, but there was no effect of F19 (p , 0.05).
Summary: Duration of BF had a strong impact on the
antibody response to Hib. Feeding F19 from 413 months
enhanced the response to DT and TT in infants BF , 6
months but had no effect in infants BF . 6 months. F19 had
no impact on the response to the Hib antigen independent of
duration of BF.
Conclusions: Our results show that the effect of the probiotic
bacterium Lactobacillus F19 on antibody response to
a common vaccine is strongly modulated by the duration of
breastfeeding modulated by breastfeeding.
PND-01 A NOVEL MULTIFACTORIAL/MULTISYSTEMIC AP-
PROACH TO THE MANAGEMENT OF CHILDHOOD
FEEDING DISORDERS C Martin1, S Ghosal2. 1Dept.
of Paediatric Psychology, Mid-Staffordshire NHS Trust,
Stafford, United-Kingdom. 2Dept of Paediatrics, Mid-
Staffordshire NHS Trust, Stafford, United-Kingdom.
Introduction: The transition from being fed to self feeding
(eating) is a crucial learned behaviour in infancy and
childhood. Restoring normal eating behaviour in children
who have feeding disorders is a major challenge and these
children remain dependent on prolonged enteral tube feeding.
We have successfully used the Multi-factorial/Multisystemic
approach in the assessment and treatment of several children
in an in-patient setting and restored adequate eating within
seven days.
Methods: The case of a typical two year old girl with a severe
feeding disorder, weight loss and dependent on nasogastric
tube feeds is described. At assessment, family and ward staff
interviews, video observations and quantitative measures
were carried out. Causal and maintaining factors were
identified and the subsequent intervention used Multisyste-
mic Therapy principles (MST). The Oral Feeding Programme
consisted of a multi-component package of treatment: (i) Bio-
Behavioural approaches, (ii) Parent Training Procedures, (iii)
Play Therapy, (iv) Health Education related to psychosocial
aspects of paediatric care and (v) Systemic intervention at
Mesosystem level.
Results: At admission, the only feeding behaviour observed
was finger licking. Over the next seven days, the number and
variety of feeding behaviours displayed and the amount of
foods taken increased significantly. The nasogastric tube was
removed because she started eating adequately. Behaviours
learned were maintained during the follow up and generalised
into the home environment. An increase in her capacity for
emotional expression was also observed. Follow-up at
community setting confirmed the maintenance of the new
feeding dynamic by the child and the family. No further
readmissions were required and adequate growth was
achieved.
Conclusions: The multi-factorial/Multisystemic approach is
successful in resolving complex childhood feeding disorders.
This is due to effective trandisciplinary collaboration and the
inclusion of the family in all stages of the process.
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
PND-02 THE EFFECTS OF FREEZING TEMPERATURES ON
LIPIDIC PEROXIDATION OF BREAST MILK
I Almansa1, M Miranda1, M Muriach1, FJ Romero1,
E Jareno2, M Perez2, MD1, Silvestre1. 1Universidad
Cardenal Herrera-CEU, Valencia, Spain. 2Centro de Salud
Moncada, Valencia, Spain.
Introduction: Malondialdehyde (MDA) is a good indicator
of lipid peroxidation. Our team has shown that MDA
concentration in breast milk was increased after 10 days of
being maintained under a temperature of 220C, although
this increase was not statistically significant (1). It is
important to establish the optimal conditions for freezing
(time/temperature) breast milk to avoid these alterations and
the possible harmful effects to the healthy infant.
Aim: The objective of this study was to find out the effect
freezing temperatures exercise over the stability of the lipidic
fraction of breast milk, by evaluating MDA concentration
after 15 and 30 days of freezing.
Methods: A total of 10 samples of mature human milk
were collected from 7 healthy well-nourished women using
an electric breast pump (Mamilat S.M. 122) coupled with
a polypropylene container in which the samples were
collected directly. Samples were divided in five parts:
A/was analyzed immediately; B/ was frozen 220C and
was analyzed 15 days after; C/ was frozen 280C and
analyzed 15 days after; D/ was frozen 220C and analyzed
30 days after; E/ was frozen 280C and analyzed 30 days
after. MDA, a lipid peroxidation product, concentration was
measured by liquid chromatography according to a modifica-
tion of the method of Richard et al. (2).
Results: The mean values of MDA for each set of
experiments were: (0.715 6 0.4421), (0.797 6 0.2212),
(0.930 6 0.4551), (1.226 6 0.5913), (0.654 6 0.2916) for the
samples A, B, C, D, E respectively. At 15 days, no significant
variation was detected between either of the two sets of
samples subjected to freezing temperature and fresh milk.
After 30 days, the samples refrigerated at 220C showed
a significant increase in the MDA concentration compaired
with milk frozen at 280C during the same period.
Conclusion: The preservation of breast milk over periods
surpassing 15 days, should take place at temperature of
280C, rather than the usual of 220C, with the objective of
avoiding lipid peroxidation and the consequent increase in
MDA.
References:
(1) Miranda M, Muriach M, Almansa I, Jareno E, Bosch-
Morell F, Romero FJ and Sivestre D. Effect of storage on
oxidative status of human milk. Biofactors (in press)
(2) Richard MJ, Guiraud P, Meo J and Favier A. High
performance liquid chromatography separation of ma-
londialdehyde thiobarbituric acid adduct in biological
materials (plasma and human cell) using a comercially
available reagent. J Chromatogr. 1992;577:918.
PND-03 SELENIUM STATUS AND TOTAL BLOOD ANTIOXI-
DANT ACTIVITY IN RUSSIAN PARTURIENT WOMEN
AND THEIR NEWBORNS N Shilina1, I Gmoshinsky1,
M Gmoshinskaya1, I Kon1. 1Institute of Nutrition of RAMS,
Moscow, Russia.
The aim: To study selenium (Se) status in Russian parturient
women and their newborns as well as total serum blood
antioxidant activity (AOA) as one of the integral indices of
newborn and woman resistance to oxidative stress during
labour.
 ESPGHAN 38TH ANNUAL MEETING
Methods: 81 mother-child pairs were enrolled in the opened,
prospective medical observation. 51 pairs were from Moscow
and 29 - from the other Russian town Ryazan with lower
socio-economic conditions of population life comparatively
to Moscow. Selenium contents in serum blood of women and
cord blood were measured by microfluorimetric method.
Total serum blood AOA was determined by evaluation of
malonic dialdehyde production decrease in egg yolk lip-
oproteins after adding to them of serum blood specimens.
Results: Mean level of Se in serum blood was 75.7 6 2.2 mkg/l
(M 6 m) in Moscow women, and 53.1 6 2.2 mkg/l in
Ryazanian women. Mean Se level in cord blood was 53.8 6
1.4 mkg/l in Moscow and 44.3 6 1.9 mkg/l - in Ryazan. The
difference between cities was significant in both cases. Se
status of Russian parturient women and their newborns was
lower comparatively to normal levels according to the literature
data (95110 mkg/l for pregnant women and 81 mkg/l for
newborns, respectively). Se content of cord blood correlated
significantly with Se content in blood of women. Total serum
cord blood AOA in Moscow as well as at Ryazan was higher
then in parturient women blood. Total AOA depended on Se
cord blood content but only at a range of Se level 5095 mkg/l
of cord blood.
Summary: The Se status of Russian parturient women and
their newborns is not optimal, especially at Ryazan, and
needs in improvement. Total AOA is significantly higher in
cord blood than in womens blood. Total AOA correlated with
Se blood level only in cord blood where Se level was in some
cases very low.
Conclusion: Se status of newborns depends on Se status of
parturient women. The data received demonstrate the
necessity to enrich the pregnant women diet with Se. Low
Se status may be one of the factors limiting an increase of
blood AOA necessary for prevention of oxidative stress in
some physiological conditions that demand significant in-
crease of blood AOA.
PND-04 A SURVEY OF ENTERAL FEEDING PRACTICES IN
PATIENTS WITH SHORT BOWEL SYNDROME IN THE
UNITED KINGDOM E Buchanan1, T Probert1. 1Royal
Hospital for Sick Children,Yorkhill Division, Glasgow,
United-Kingdom.
Aim: There is no consensus amongst clincians who manage
patients with Short Bowel Syndrome (SBS) on the most
appropriate enteral feeds and introduction of solids.
Method: A questionnaire was sent to all dietitians (40) who
are Associate Members of the British Society of Paediatric
Gastroenterology, Hepatology and Nutrition to ascertain their
current practice on the type of enteral feeds used, monitoring
tolerance of feeds, anthropometry, the use of novel substrates
(pectin, glutamine, pre and probiotics), and the introduction
of solids in the management of patients with SBS.
Results: Breast milk was the most commonly used feed
initially. 68% of dietitians then used a hydrolysed protein
formula when tolerance became a problem. Continuous feeds
were given initially in 66% of patients. The most common
reason for changing the type of feed was increased stool
frequency, the presence of fat or reducing substances in stools
and poor weight gain. 62% of dietitians occasionally used
a modular feed. Weight was most commonly measured twice
weekly, length/height and head circumference monthly. 80%
of dietitians did not measure Mid-arm Circumference or
Triceps skinfold thickness. 4 centres followed set protocols
for biochemical monitoring. Investigations in other centers
varied. Novel substrates were infrequently used. Solids were
most commonly introduced at 6 months for patients on
parenteral nutrition and between 56 months for those who
705
were on complete enteral feeds. Exclusion of milk, gluten,
eggs and soya varied between centres.
Conclusion: Practice throughout the United Kingdom varied
immensely on the dietetic management of patients with SBS
with each unit following their own protocols and personal
practice. A more consistent approach to the nutritional
management of SBS patients should be pursued.
PND-05 IMPROVED QUALITY OF LIFE FROM A NURSE LED
CHILDRENS CONSTIPATION SERVICE K Woolliscroft1,
S Ghosal1. 1Dept of Paediatrics, Mid-Staffordshire NHS Trust,
Stafford, United-Kingdom.
Introduction: Constipation is a common problem in
Paediatrics with significant morbidity and impaired quality
of life.
Setting: Patients attending a Advanced Nurse Practitioner
(ANP) led constipation clinic at a medium sized UK District
General Hospital (DGH) with 900 patients per year. Initially
a multidisciplinary service set up on accepted guidelines,
with ANP, Psychologist and Play therapist; it evolved to
become an ANP delivered service after unified training.
Methods and Interventions: We designed a questionnaire to
assess health related quality of life and service delivery for
children with constipation. The questionnaire had 36 items in
6 domains (Demographic data (6), Symptoms including stool
frequency, pain and soiling (8), Social activity (8), Medica-
tion (5), Information (6) and Support (5)). Children and
parents scored their experiences during the previous two
weeks. A pilot with a sample of 18 showed that the form was
simple to fill in and responsive to changes in their clinical
condition. An independent observer carried out a survey of
118 patients selected at random to assess quality of life before
and after clinic treatment. SPSS v11.0 and Wilcoxon signed
rank tests were used for analysis.
Results: Internal reliability was reasonably good (Cronbach
alpha 0.610.74). The study demonstrated: (i) Decreased
frequency of abdominal pain from 81% to 23% (p , 0.0001)
(ii) Increased bowel motions from 0.3 stools/d to 0.7 stools/d
(p , 0.0001), (iii) Increased numbers of children on
medication from 66% to 90%, with combination therapy
increasing from 31% to 84%. (iv) Decreased school absence
from 57% to 12% (p , 0.0001) (v) Absence from social
events declined from 48% to 7% (p , 0.0001) (vi) 90% of
patients found the service informative and very supportive
(vii) 79% of families were confident to manage their
condition at home.
Conclusions: (i) Quality of life for this group of children and
their families had significantly improved. (ii) The service was
cost-effective and is applicable to all DGH settings.
PND-06 EFFECT OF OMEPRAZOLE TREATMENT ON INTRA-
GASTRIC PH IN CHILDREN WITH NEUROLOGICAL
DISEASES B Stephansson-Nystrom1, C Cederfjall2,
L Grahnquist1. 1Unit of Pediatric Gastroenterology and
Nutrition, Karolinska University Hospital, Stockholm,
Sweden. 2Institue of Women and Child Health, Karolinska
Institutet, Stockholm, Sweden.
Aim: To study the effect of omeprazole treatment on
intragastric pH in children with neurological diseases.
Methods: Twelve intragastrical pH registrations (24 hours)
were performed on children between 13 and 217 months of
age with neurological diseases, gastrostomy button and on
current treatment with omeprazole. pH data was captured
intragastrically with a catheter (Zinetics 24 pediatric,
Medtronic), recording advice (Digitrapper pH recorder)
and analysed by the Polygram NET software. Time used for
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
706 ESPGHAN 38TH ANNUAL MEETING
gastrostomy feeding was extracted from the analysis.
Gastrointestinal symptoms were recorded based on an
interview with the parents.
Results: The dose of omeprazole varied between 0.6 and
3.7 mg/kg bw (mean 1.75).The actual registered time varied
between 12 and 24 hours (mean 18.7).The mean percentage
of time with pH ,4 was 64. Three measurements showed pH
,4 less than 50% of the time and these children had a dose of
omeprazole above 2.5 mg/kg bw. The parents of those
children scored much less symptoms than the others.
Summary: Children with neurological diseases, treated with
recommended doses of omeprazole had a low effect on acid
suppression.
Conclusion: The knowledge about the effect of acid
suppressors on children with neurological diseases is scarce.
The possibilities of the children to communicate symptoms
are limited. This first study supports our theory that theses
children are undertreated.
PND-07 A DEPENDENCY SCORING SYSTEM TO PREDICT
TRAINING TIMES FOR HOME PARENTERAL NUTRI-
TION (HPN) FAMILIES C Holden1, C Bunford2, E Sexton3.
1
Birmingham Childrens Hospital NHS Trust, Birmingham,
United-Kingdom. 2Birmingham Childrens Hospital NHS
Trust, Birmingham, United-Kingdom. 3Birmingham Child-
rens Hospital NHS Trust, Birmingham, United-Kingdom.
HPN is a complex therapy that has facilitated improved care
for children with intestinal failure. Successful programmes
can only be achieved by committing a large amount of time to
preparation and education of families. Increasingly, nurses
are required to train parents/carers quickly to prevent
J Pediatr Gastroenterol Nutr, Vol. 40, No. 5, May 2005
catheter-related sepsis and bed shortage issues. Clinicians
require key information about how long training programmes
are likely to take to facilitate a multi-disciplinary review of
the care the child requires.
Aim: The aim of this project was to look at the key factors
which determine training times and identify a scoring system,
which could accurately predict actual training times for the
multi-disciplinary team.
Methods: Key factors were identified independently by three
Nutritional Care nurses and a numerical score was assigned to
each factor. Factors include acceptance of diagnosis, financial
issues/constraints, communication issues, multiple treatments
the child required, the age of the child, travelling distance to
and from the centre, length of time receiving parenteral
nutrition (PN), complications related to PN, existing support
and suitability of housing.
Results: Twenty-six families were identified and training was
evaluated using the dependency scoring system. Training
times were analysed into low, medium and high dependency.
Conclusion: The multi-factorial parameters have informed the
multi-disciplinary team of the expected duration of training
and likely discharge. The tool requires ongoing assessment and
re-evaluation when parameters change. Ongoing review will be
required to ensure validity of this tool.
Patients Scoring system Predicted training times
N=6 Low dependency (919) 23 weeks
N = 18 Medium dependency (2023) 48 weeks
N=2 High dependency (2437) .8 weeks
Predicted scores range from 9 37 with a median of 21.