Eur J Pediatr (2009) 168:375–386 DOI 10.1007/s00431-008-0912-x

ABSTRACTS

45. ARBEITSTAGUNG FÜR PÄDIATRISCHE FORSCHUNG GÖTTINGEN 19.- 20. Februar 2009

Leitung: Prof. Dr. Klaus-Michael Debatin Universitätsklinik für Kinder- und Jugendmedizin Ulm

376 20 years of Diabetes Incidence Registry (DIARY) Baden-Wuerttemberg, Germany — facts and trends S. Ehehalt1, A.M. Willasch2, A. Neu1, for the DIARY-study Group Baden-Wuerttemberg 1 University Children’s Hospital, Tuebingen, Germany, 2 University Children’s Hospital, Frankfurt, Germany To assess the frequency and the trend in incidence of type 1 diabetes among children and adolescents under the age of 15 in BadenWuerttemberg (BW), Germany. All 31 pediatric departments in BW and one diabetes center participated in the study. Case registration was done according to the EURODIAB-criteria. The current number of datasets exceeds 5,000 cases, which makes this registry one of the largest of its kind in the world. The degree of ascertainment was 97.9%. (1) From 1987 to 2006, the age- and sex-standardised incidence rate was 15.3/100,000/year (95% CI 14.9–15.7, n=5,108). The annual increase in incidence was 4.4% (95% CI 3.9–4.9). (2) We observed no significant differences between the sex-specific incidence rates (p=0.6). (3) On average, the highest rise in incidence occurred in the youngest age group of 0- to 4-year-old patients (7.2%, 95% CI 5.9–8.6), followed by the age groups 5 to 9 (4.2%; 95% CI 3.8–4.7) and 10 to 14 (2.9%; 95% CI 2.7–3.1), respectively. Assuming a quadratic regression model, an analysis of trends estimated that in the year 2025, the incidence will be 37.1/100,000/year.The number of new onsets of T1D among children and adolescents in Germany has been rising at a faster pace than expected. A distinct shift to the left has been observed in terms of the age of manifestation (left-shift). Antimicrobial effector function of human mesenchymal stem cells — a novel feature with significant implications for their clinical use R. Meisel1, K. Heseler2, A. Borkhardt1, D. Dilloo1 and W. Däubener2 1 Clinic for Pediatric Oncology, Hematology & Clinical Immunology and 2Institute for Medical Microbiology & Hospital Hygiene, Heinrich-Heine-University, Düsseldorf, Germany In addition to their multilineage differentiation potential mesenchymal stem cells (MSC) modulate the adaptive immune system. With regard to the clinical use in immune-mediated disorders, it is a matter of concern that application of MSC might inhibit antimicrobial immune responses with an increased risk of infection. We recently demonstrated, that IFNγ-induced expression of the tryptophan catabolizing enzyme indoleamine 2,3dioxygenase (IDO) acts as a T cell inhibitory effector mechanism in MSC (Meisel et al, Blood, 2004). Now we show, that the growth of various bacteria (Staph. aureus, KNS, Enterococci), intracellular parasites (T. gondii) and viruses (HSV, CMV) is up to 10,000-fold reduced when these pathogens are cultured in the presence of MSC stimulated with inflammatory cytokines (p<.05). Addition of the IDO-specific inhibitor 1-MT or excess amounts of tryptophan restored microbial growth thus identifying IDO-mediated tryptophan starvation as the underlying mechanism. Collectively, our data identify human MSC as the first cellular immunosuppressant that at the same time exhibit an antimicrobial effector function. Thus, MSC represent a promising novel tool for the treatment of a broad range of immune-mediated pediatric disorders. Mutations in the putative lysosomal cobalamin exporter LMBD1 cause the cblF inborn error of vitamin B12 metabolism S. Gailus1, I. R. Miousse2, W. Höhne3, B. Gasnier4, D. Rosenblatt2, B. Fowler5, P. Nürnberg6, F. Rutsch1 1 University Children’s Hospital, Münster, Germany; 2McGill University Health Centre, Montreal, Canada; 3Charité University Medicine, Berlin, Germany; 4Centre National de la Recherche Scientifique, Paris, France; 5 University Children’s Hospital Basel, Switzerland; 6Cologne Center for Genomics, Cologne, Germany

Eur J Pediatr (2009) 168:375–386 Vitamin B12 (cobalamin) is essential in animals and humans for the metabolism of branched chain amino acids and odd chain fatty acids, and for the remethylation of homocysteine to methionine. In the cblF inborn error of vitamin B12 metabolism, free vitamin accumulates in lysosomes, thus hindering its conversion to cofactors. Homozygosity mapping in 12 unrelated cblF patients identified on chromosome 6q13, LMBRD1, encoding LMBD1, a lysosomal membrane protein with homology to the lipocalin membrane receptor LIMR, as a candidate gene. Five different frameshift mutations, embedded in a common haplotype of 1.34 Mb, result in loss-of-function of LMBD1. Transfection of cblF patient fibroblasts with the LMBD1 wild type construct rescues cobalamin coenzyme synthesis and function. This work identifies LMBRD1 as the gene for the cblF defect of cobalamin metabolism and suggests that LMBD1 is a lysosomal membrane exporter for cobalamin. Functional analysis of novel GLI2-mutations in patients suffering from Multiple Pituitary Hormone Deficiency (MPHD) GMC Flemming1, J Klammt1, W Kiess1, WF Blum1,2 and RW Pfaeffle1 1 Hospital for Children and Adolescents, University of Leipzig, Leipzig, Saxonia, Germany, 04317 and 2Lilly Research Laboratories, Eli Lilly and Company, Bad Homburg, Hessen, Germany, 61350 Human GLI2 mutations have been shown to lead to a spectrum of midline defects ranging from discrete cranio-facial abnormalities to holo-prosencephaly. We screened 147 patients (pts) with multiple pituitary hormone deficiency (MPHD) by denaturing HPLC after PCR amplification of all coding exons followed by sequencing of suspicious PCR fragments. The impact of identified mutations was tested in luciferase reporter assay. GLI2 mutations were identified in 5 pts. In addition to MPHD, patient 4 showed poly-dactyly and an ectopic posterior pituitary. In the luciferase co-transfection assays the mutations at the C-terminal end of GLI2 (pts 1, 2 and 3) showed a decrease in reporter activity by 20–30%. In contrast, the N-terminal mutation (R188P) caused the most pronounced loss in activity by 70%. We conclude that the pheno-types of pts with GLI2 mutations are highly variable and MPHD may be the only symptom recognized. Our data reinforce the im-portance of the DNA-binding zinc finger domains in transcriptional activation and point to a functional involvement of the C-terminal part of the GLI2 protein in pituitary morphogenesis and function. A critical role of MAdCAM-1 for B cell localization and function in the gut A. Schippers1, C. Leuker2, O. Pabst3, A. Kochhut4, B. Prochnow4, A.D. Gruber5, W. Müller6, N. Wagner1 Department of Pediatrics, University Hospital Aachen, Pauwelsstr. 30, 52074 Aachen In order to elucidate the in vivo function of mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the principal ligand of β7integrin, we generated a MAdCAM-1 deficient mouse strain. MAdCAM-1 is constitutively expressed in the GALT and intestinal venules. Lack of MAdCAM-1 results in reduced size and cellularity of Peyer’s patches (PP). The earliest differences in the size of the PP anlagen become visible at day 3 after birth, indicating that the β7-integrin/MAdCAM-1 interaction is dispensable for embryonic PP development, but affects the recruitment of mature lymphocytes into the organ. Moreover, the number of IgA secreting plasma cells in the small intestinal lamina propria is drastically diminished. These reduced numbers of intestinal

Eur J Pediatr (2009) 168:375–386 effector B cells correlate with impaired humoral responses after oral immunization in MAdCAM-1 deficient mice, suggesting that also plasma cell homing into the intestine requires MAdCAM-1. In conclusion, MAdCAM-1 directs the seeding of Peyer’s patches with lymphocytes, it is critical for localization of plasma cells into the intestinal lamina propria and for the humoral immune response in the gut. (Supported by the DFG) Synthetic lethality siRNA screening to identify novel therapeutic targets in Ewing tumours Jenny Potratz1,2, Poul Sorensen1, Heribert Jürgens2 1 Molecular Oncology, BC Cancer Research Centre, Vancouver, Canada; 2Department of Haematology and Oncology, University Children’s Hospital, Münster, Germany In addition to conventional chemotherapy strategies novel therapeutic approaches are needed to improve survival of sarcoma patients. Single-target-inhibition strategies however are often insufficient to sustainably inhibit tumour growth; hence the search is to be focussed on combination inhibitory strategies. We developed a high-throughput siRNA screening assay in Ewing tumour cell lines and screened a tyrosine kinase (88) and insulin pathway (31) siRNA library. This identified a set of siRNAs that significantly impair cell survival and thus represent potential therapeutic targets. The screening assay was performed in a synthetic lethality approach in the presence and absence of an IGF1R inhibitor, a class of compounds promising in clinical studies. This revealed siRNAs that induce cell death specifically in the presence of otherwise sub-lethal doses of an IGF-1R inhibitor, e.g. the Met related tyrosine kinase Ron, warranting further investigation as potential targets for combination strategies. This same assay identified siRNAs that confer resistance to IGF-1R inhibition. Synthetic lethality siRNA screening can provide a molecular basis for the rational development of targeted (combination) treatment strategies. At the same time it can contribute to the understanding of novel compounds, mechanisms of action and resistance, as well as their targeted signalling pathways. Characterization of a Novel Human Anti-GD2 Anti-Idiotypic Antibody GK8 DNA Vaccine for Immunotherapy in Neuroblastoma S.Weixler, 1S.Fest, Y. Zeng, N. Huebener, G. Gaedicke, J. Krüger, H. N. Lode Charité Universtiy Medicine, Dept. of General Pediatrics and Bone Marrow Transplantation, Augustenburger Platz 1, 13353 Berlin; 1 Ottovon-Guericke-Universität Magdeburg, Pädiatrische Immuntherapie, Leipziger Straße 44, 39120 Magdeburg Disialoganglioside GD2 is an important target for immunotherapy against neuroblastoma. One active immunization strategy in clinical trials is based on a murine anti-GD2 anti-idiotype antibody 1A7 which functionally mimics GD2. Here we describe the identification and characterization of a DNA vaccine encoding for a novel human antiidiotypic antibody GK8 identified from a patient previously treated with ch14.18 at our centre. The vaccine encoding for single-chain variable fragments of GK8 was constructed by sequential PCR. GD2 mimicry was demonstrated and found to best for the GK8 DNA vaccine construct in contrast to the 1A7 control. Importantly, the efficacy of these DNA vaccines was shown in the syngeneic NXS2 neuroblastoma model using attenuated salmonella typhimurium (SL7207) as an oral DNA vaccine

377 carrier. Only mice receiving anti-idiotypic antibody DNA vaccines revealed a reduction of spontaneous liver metastasis. In summary these data provide a baseline for clinical application after mainstream therapy. Allergic airway inflammation is regulated by the composition of the classical antigen-binding site Sebastian Kerzel, Tobias Rogosch, Julia Wagner, Kathrin Preißer, Benjamin Strücker, Larisa Sikula, Harry W. Schroeder Jr.1, Michael Zemlin, and Rolf F. Maier Department of Pediatrics, Philipps-University Marburg, Germany; 1 Developmental & Clinical Immunology, University of Alabama at Birmingham, USA. IgE is a central effector molecule in allergic immune responses. Bound to mast cells, IgE serves as antigen-receptor, permitting specific identification of the allergen. It is unknown if the allergen acts as a superantigen or as a classical antigen. Therefore, we sought to evaluate the contribution of the classical antigen-binding site to the development of an asthma phenotype. Using a murine model of allergic asthma, we characterized a gene targeted mouse strain expressing a modified repertoire of classical antigen-binding sites (ΔD-iD mice). Compared to wildtype, ΔD-iD mice showed (i) a decreased rise in allergen-specific IgE, (ii) an alleviated eosinophilic airway inflammation, and (iii) reduced levels of TH2 cytokines in bronchoalveolar lavage fluids. Moreover, in a model of chronic allergic airway inflammation, the airway hyperreactivity to methacholine was completely abolished in ΔD-iD mice. We conclude that the IgE levels and the asthma phenotype in a murine model of allergic airway inflammation depend on the composition of the predominant classical antigen-binding sites. Our data further emphasize a central role for IgE, not only in mediating but also regulating the allergic immune response. Quantitative sensory testing (QST) in children M Blankenburg, T Hechler, C Maier, F Aksu, B Zernikow Vodafone Foundation Institute for Children’s Pain Therapy and Paediatric Palliative Care, Children’s Hospital Datteln, University Witten/Herdecke, Dr.-Friedrich Steiner Street, 45711 Datteln QST detects functioning of different nerve fibers (Aβ, Aδ, C), pathways (spin thalamic, lemniscal) and central mechanisms to obtained neurobiological pain classification. In the present work we adapted the QST protocol of the German research network on neuropathic pain (DFNS) for children and provided a reference database consisting of 13 parameters obtained for 6 body regions in 176 children aged 6–17 years. Four-way ANOVAs for the withinsubjects factors region and body side and the between subjects factors age and gender confirm that each body region, age and gender groups need their own reference data. Thresholds for thermal and mechanical detection and pain were region-specific with lower thresholds in the face than in the foot. While young children (6– 9 years) were more sensitive to all pain thresholds (p<0.01), they were less sensitive to all none pain detection thresholds than adults (10–17 years; p<0.01). Girls were more sensitive to thermal detection and pain thresholds than boys but not to mechanical stimuli and mechanical pain stimuli. Comparison of left and right body sides within subjects indicates that right-left difference were 5 times more sensitive than absolute reference data. Our results demonstrate that the QST-protocol is feasible for paediatric pain patients but reference data from adults are not transferable to children.

378 The role of NOD1 as major pattern recognition receptor for detection of Campylobacter jejuni by the intestinal epithelia 1,4 M. Zilbauer, 2N. Dorrell, 3G. Núňez, 1N. J. Klein, 2B. W. Wren, 4 S. Wirth, 1M. Bajaj-Elliott 1 Department of Infectious Diseases and Microbiology, Institute of Child Health, London, UK. 2Department of Infections and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK. 3Department of Pathology and Comprehensive Cancer Center, the University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. 4Zentrum für Kinder- und Jugendmedizin, HELIOS Klinikum Wuppertal, Universität Witten Herdecke, Wuppertal, Germany. The aim of our study was to investigate the role of NOD1 and NOD2 as potential pattern recognition receptors (PRR) for C. jejuni in mediating host innate defence. Small interfering (si) RNA targeting NOD1 and NOD2 over-expressing plasmid were transiently transfected into Caco-2 cells prior to bacterial co-culture. IL-8 and human beta defensin 2 (hBD2) gene expression and promoter function was followed by RT-PCR and luciferase reporter gene assay respectively. The effect of NOD1 siRNA on bacterial invasion was analyzed by gentamicin protection assay. Specific knock down of NOD1 in Caco-2 cells lead to significant reduction in C. jejuni mediated IL-8 and hBD2 gene expression. Additionally, an increase in intracellular C. jejuni in cells deficient in NOD1 suggests a critical role for this intracellular PRR in limiting infection by mediating antimicrobial host defence. JNK is a putative target for adjunctive therapy in group B streptococcal sepsis Sybille Kenzel, and Philipp Henneke Centre for Pediatric and Adolescent Medicine, University of Freiburg, Mathildenstrasse 1, 79106 Freiburg, Germany Group B streptococcus (GBS) is the most frequent pathogen causing neonatal sepsis and meningitis, two life-threatening diseases. In vitro, GBS cell wall strongly induces inflammatory cytokines through a pathway that is unique to professional phagocytes and that is dependent on the Toll-like receptor (TLR) adapter protein MyD88. The aim of our study was to identify signaling intermediates that might serve as targets for adjunctive therapy in group B streptococcal sepsis. On the TLR adapter level, we found TNF-α formation to be dependent on MyD88 alone while deletion of other adapter proteins (TRIF, TRAM, MAL) remained without effect. Downstream of MyD88, the MAP-kinases JNK and p38 were both essential for the transcriptional activation of inflammatory cytokines. Inhibition of either p38 or JNK abolished protein phosphorylation, transcription factor activation and proinflammatory cytokine induction. In addition to its central position in the inflammatory response to GBS, p38 -but not JNK- was essential for antimicrobial properties such as phagocytosis and oxygen radical formation, which are beneficial for the host response in sepsis. Thus, we tested JNK as a target for adjunctive sepsis therapy in a neonatal mouse model. We found that inhibition of JNK by the specific inhibitor SP600125 reduced serum TNF-α levels and mortality in these mice without interfering with bacterial clearance. Thus, JNK appears to be a promising target for adjunctive therapy in GBS sepsis. Retention of lysosomal protein CLN5 in the endoplasmic reticulum causes neuronal ceroid lipofuscinosis in Asian sibship Anne-Hélène Lebrun#, Stephan Storch#, Aija Kyttällä*, Sara E. Mole§, Alfried Kohlschütter#, Kurt Ullrich#, Thomas Braulke#, Angela Schulz# #Children’s Hospital, University Hospital Hamburg Eppendorf, Hamburg, Germany; *Institute for Molecular Medicine Finland, Helsinki, Finland; §MRC Laboratory for Molecular Cell Biology, University College London, UK

Eur J Pediatr (2009) 168:375–386 The neuronal ceroid lipofuscinoses (NCLs) form a group of autosomal recessively inherited neurodegenerative disorders that mainly affect children. We have identified the first reported CLN5 patients originating in Asian sibships: In a Pakistani family, two children with symptoms of early juvenile NC were homozygous for a novel homozygous mutation c.1072-1073delTT (p.Leu358AlafsX4) CLN5. In the second Afghan family, two children with symptoms of a late infantile NCL were homozygous for the novel mutation c.1137G>T (p.Trp379Cys) in CLN5. Expression analysis showed that mutant p. Leu358AlafsX4 CLN5 is truncated and lacks a used N-glycosylation at site Asn401. The missense mutation p.Trp379Cys affected neither the size nor glyco-sylation of the CLN5 protein. Double immunofluorescence microscopy showed that while the wild-type CLN5 protein is localized in lysosomes, both mutant CLN5 proteins are retained in the endoplasmic reticulum rather than reaching the lyosome. Toll like receptor (TLR)-independent regulation of cytokine network in juvenile idiopathic arthritis (JIA) V. Umlauf, M. Kirchner, P. Schmidtke, W. Mannhardt-Laakmann Center for Pediatric and Adolescent Medicine, Hospital of the Johannes Gutenberg-University, 55101 Mainz, Germany In rheumatic diseases “damage associated molecular patterns” (DAMP) stimulate TLRs to activate macrophages and secondly T cells as major cytokine producers. While TLR-2 expression is de-creased in JIA, TLR-4 is elevated in certain JIA subgroups. The subgroups can be characterized through specific pro-/anti-inflammatory key cytokines. The current study’s aim was to investigate the pathogenetic association between TLR expression and cytokine levels in JIA subgroups. Methods: Monocytic TLRs and pro-/ antiinflammatory plasma cytokines (150 patients, 32 healthy) were measured by flowcytometry after antibody staining resp. multiplex bead immunoassaying. Results were statistically analysed (non-parametic testing, correlation). Results: Surprisingly, no correlations between TLR-2/-4 expression and cytokine levels were found. Instead, pro- and antiinflammatory cytokines showed regulatory feedback patterns specific for JIA subgroups (systemic JIA: IL6 vs. IL-5; persistent Oligoarthritis: IL-6/IL-2 vs. IL-5; sero-negative polyarthritis: TNF-α vs. IL-4; enthesitis-related arthritis: IL-6/IL-2 vs. IL-5). Conclusion: TLR expression and cytokine production seem to be independent events; pro- and antiinflammatory cytokines apparently regulate themselves within a disease specific cytokine network. However, the regulatory link between TLRs and inflammatory response in JIA is still under investigation. Disease-causing missense-mutations affect enzymatic activity, stability, and oligomerization of mutant glutaryl-CoA dehydrogenase (GCDH) Chris Mühlhausen1, Britta Keyser1, Achim Dickmanns2, Ernst Christensen3, Kurt Ullrich1, and Thomas Braulke1 1 Department of Biochemistry, Children’s Hospital, University Medical Center, Martinistr. 52, 20246 Hamburg; 2Department of Molecular Structural Biology, GCMB, Göttingen, Germany; 3Department of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark Glutaric aciduria type 1 (GA1) is caused by mutations in the glutarylCoA dehydrogenase gene (GCDH). The clinical phenotype is highly variable with no correlation to the genotype. We report on mammalian expression studies of missense mutations identified in GA1 patients. Enzymatically, most mutants were inactive. Western blot and pulsechase analyses demonstrated that the amount of expressed p. Arg402Trp protein was significantly reduced due to rapid intramitochondrial degradation. Upon cross linkage, the formation of

Eur J Pediatr (2009) 168:375–386 homotetrameric GCDH was strongly impaired in p.Met263Val and p. Arg402Trp mutants. In addition, GCDH appears to interact with distinct hetero-polypeptides to form novel 97, 130 and 200 kDa GCDH complexes. Molecular modeling of mutant GCDH suggests that Met263 at GCDH protein surface might be part of the contact interface to interacting proteins. These results indicate that reduced intramitochondrial stability as well as impaired formation of homoand heteromeric GCDH complexes can underly GA1. Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation D. Frommhold, A. Ludwig, M.G. Bixel, A. Zarbock, I. Babushkina, M. Weissinger, B. Lange-Sperandio, L. G. Ellies, J. D. Marth, A. G. Beck-Sickinger, A. Zernecke, C. Weber, D. Vestweber, K. Ley, and M. Sperandio Dept. of Neonatology, University Children’s Hospital, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand. This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of α2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands KC or IL-8. In addition, extravasation of ST3Gal-IV−/− neutrophils into thioglycollate pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that IL-8-binding to isolated ST3Gal-IV−/− neutrophils was markedly impaired. Furthermore, KC-mediated adhesion of ST3Gal-IV−/− leukocytes at physiological flow conditions as well as transendothelial migration of ST3Gal-IV−/− leukocytes in response to KC was significantly reduced. In human neutrophils, enzymatic desialylation led to decreased binding of CXCR2 ligands to CXCR2 and diminished neutrophil degranulation in response to these chemokines. Taken together, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte recruitment during inflammation in vivo. Histological characterisation of cryolesions and results of intracoronary ultrasound and coronary angiography after energy application at growing myocardium Kriebel T, Schneider H, Kroll M, Selle J, Quentin T, Paul T Department of Pediatric Cardiology and Pediatric Intensive Care Medicine, Georg-August-University Göttingen Cryoenergy has emerged as an effective alternative to radiofrequency (RF) in pediatric patients. Up to now, there is lack of data concerning the histopathology of cryolesions and coronary artery obstruction, a possible side effect of RF. In 10 piglets cryoenergy was delivered in the standard fashion at the valve annulus in the right and left atrium and left ventricle. 6 cryolesions were performed in each piglet. 5 animals were restudied after 48 h by coronary angiography and intracoronary ultrasound and sacrificed. The remaining 5 piglets were restudied after 3 and 6 months. Selective coronary angiography and intracoronary ultrasound studies did not demonstrate any coronary artery obstruction after 48 h, 3 and 6 months after cryoablation. 22/30 cryolesions of the first 5 piglets could be used for histological workup. The sharp demarcated hemorrhagic cryolesions were bordered by of neutrophile leucocytes. All atrial lesions exhibited a transmural extension. Thrombus formation was evident in 15/22 lesions. Coronary artery involvement was not noted. In our experimental setting, cryolesions, in contrast to RF lesions, were well demarcated with intact endocardium and without affection of the coronary arteries. Therefore, this energy mode seems to be preferable to RF energy in children.

379 Consequences of lesion-induced language reorganization on cognitive development Lidzba, K Department of Developmental Neurology, University Children’s Hospital, Tübingen Pre- and perinatally acquired focal left-hemispheric (LH) brain lesions often induce right-hemispheric (RH) language representation, leading to good language proficiency. In visuospatial functions, usually represented in the RH, patients with LH brain lesions often have deficits. Two hypotheses have tried to explain this paradox finding. A) “crowding”-hypothesis: The RH is overburdened by the mediation of two normally dissociated functions, leading to deficits in one of the functions. B) “reversed laterality”: The language-shift induces reorganisation of visuospatial functions to the lesioned RH. We examined 13 patients with congenital LH brain lesions with a neuropsychological test-battery and with a word-production task in fMRI. Six of the patients additionally performed a visuospatial task in fMRI. Language lateralization and neuropsychological test results were correlated. A conjunction analysis of verbal and nonverbal tasks in fMRI depicted the topographical relation between the two domains. Results: Verbal and nonverbal functions largely use the same RH network in patients with RH language representation. RH language representation correlates strongly with deficits in visuospatial skills. This corroborates the “crowding”-hypothesis. TBX15 mutations cause craniofacial dysmorphism, hypoplasia of scapula and pelvis, and short stature in cousin syndrome E. Lausch, P. Hermanns, H. Farin, Y. Alanay, S. Unger, S. Nikkel, C. Steinwender, G. Scherer, J. Spranger, B. Zabel, A. Kispert, and A. Superti-Furga Centre for Pediatrics and Adolescent Medicine, Department of Pediatrics, University of Freiburg, 79106 Freiburg, Germany Members of the evolutionarily conserved T-box family of transcription factors are important players in developmental processes that include mesoderm formation and patterning and organogenesis both in vertebrates and invertebrates. The importance of T-box genes for human development is illustrated by the association between mutations in several of the 17 human family members and congenital errors of morphogenesis. We identified two unrelated individuals with a complex cranial, cervical, auricular, and skeletal malformation syndrome with scapular and pelvic hypoplasia (Cousin syndrome) that recapitulates the dysmorphic phenotype seen in the Tbx15-deficient mice, droopy ear. Both affected individuals were homozygous for genomic TBX15 mutations that resulted in truncation of the protein and addition of a stretch of missense amino acids. Although the mutant proteins had an intact T-box and were able to bind to their target DNA sequence in vitro, the missense amino acid sequence directed them to early degradation, and cellular levels were markedly reduced. We conclude that Cousin syndrome is caused by TBX15 insufficiency and is thus the human counterpart of the droopy ear mouse. The structure of tripeptidyl peptidase I (TPP1) provides insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis Robert Steinfeld, Aritra Pal, Tim Grüne, Ralph Krätzner, Jutta Gärtner, Georg Sheldrick Departments of Pediatrics and Structural Chemistry, University of Göttingen, 37077 Götingen, Germany Late infantile neuronal ceroid lipofuscinosis (LINCL) is an autosomal recessive disease caused by mutations in the TPP1 gene resulting in

380 functional defect of the gene product. Clinical symptoms usually begin after two years of age and comprise developmental stagnation, ataxia and epilepsy, finally leading to a complete loss of motor function, vision and speech by the age of ten. More than 50% of the LINCL associated genetic alterations are missense mutations that result in conformational distrubances. To elucidate the structural basis for these TPP1 mutants, we crystallized fully glycosylated TPP1. Xray crystallographic analysis at 2.35Å resolution reveals a globular structure with a subtilisin-like fold, a S475-E272-D360 catalytic triad and an octahedrally coordinated Ca2+ binding site that are characteristic features of the S53 sedolisin family of serine proteases. On the basis of the resolved TPP1 structure we characterized the molecular effects of 28 missense mutations. These mutations either disrupt protein folding leading to instability or compromise the active center resulting in loss of proteolytic activity. Our results extend the understanding of the molecular mechanisms underlying LINCL and offer novel therapeutic options. Preconceptional oral vaccination prevents experimental biliary atresia in newborn mice Carmen Turowski, Johannes Leonhardt, Birgit Teichmann, Albert Heim, Ulrich Baumann, Joachim F. Kübler, Claus Petersen Department of Pediatric Surgery, Hannover Medical School, CarlNeuberg-Str. 1, 30625 Hannover, Germany Rotavirus (RV) induced biliary atresia (BA) in newborn Balb/c-mice endorses the hypothesis of a virally triggered pathomechanism of this devasting disease in humans. In the mouse model, susceptibility to BA depends on a temporary gap in the newborn’s innate immune system. We compared 2 oral rotavirus vaccines. Female dams were orally vaccinated. Overall, 243 newborns were intraperitoneally infected with RRV. Clinical follow-up was performed daily, and viral load of harvested livers was measured by RT-PCR and Bioassay. 14 days p.i., the incidence of BA was 82% in the control group, but only 11% in V1 and 3% in V2 pups. However, at day 7 p.i. the viral load of the liver was high in all tested pups. Continuous virus elimination occurred over the next 2 weeks, but no significant differences occurred between the groups. In the mouse model, oral vaccination of dams against rotavirus prevents most of the pups from developing BA. However, this immunological mechanism is less effective in terms of hepatopism of rotavirus infection and the kinetics of virus elemination. Thus, the findings of this animal study show that development of BA is closely related to a gap in the maternal immunological protection and gives, therefore, reason to reflect also about prophylactic approaches to BA. Identification of a novel primary immunodeficiency syndrome leading to an XLP-like phenotype in girls Kirsten Huck, Oliver Feyen, Franz Rüschendorf, Stefan Knapp, Arndt Borkhardt Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich Heine University, Düsseldorf; Max Delbrück Centre for Molecular Medicine, Berlin-Buch, Germany; Structural Genomics Consortium, University of Oxford, UK X-linked lymphoproliferative disease (XLP), a fatal immuno-deficiency to EBV in boys, is caused by mutations in SH2D1A and XIAP. We examined a consanguineous Turkish family in which two girls died from an EBV-associated lymphoproliferative disorder clinically resembling XLP. SNP array based linkage analysis revealed a candidate gene, involved in thymic selection of conventional and innate T-cells, on chromosome 5. In both girls we found a homozygous missense

Eur J Pediatr (2009) 168:375–386 mutation of a highly conserved residue (R335W) in the SH2 domain of the identified gene product. The patients showed low protein expression levels and, in accordance with corresponding mouse models, a lack of NKT cells and a characteristic phenotype of CD8 + T-cells. In silico modeling and lack of detection of the mutant construct expressed in 293 T-cells by western blot provided further evidence, that R335W causes a profound instability of the protein. This is the first molecular cause of an autosomal EBVassociated lymphoproliferative disease. Antigen-specific recognition of glioblastoma cells by Melan-A-specific T-cells Matthias Wölfl, Matthias Eyrich, Katharina Merker and Paul G. Schlegel University Children’s Hospital, Pediatric Oncology, Würzburg Despite intensive therapy, patients suffering from glioblastoma have a dismal prognosis. Immunotherapy is an additional therapeutic strategy to reduce tumor burden, especially in the setting of surgical resection, where residual microscopic lesions always lead to relapse. Ex vivo induction and expansion of antigen-specific T-cells offers a highly selective theapeutic option against tumor cells being recognized by the specific T-cell receptor complex. Melan-A/MART1 has been extensively studied as a tumor antigen pre-clinically and clinically. Adoptively transferred Melan-A-specific T-cells have lead to some remarkable remissions in melanoma patients. We established a protocol that allows rapid expansion of Melan-A-specific T-cells from the naïve T-cell repertoire, requiring a single stimulation and a reduced culture time of 10 days. This method gives rise to a robust population (mean: 40%–50%) of antigen-specific T-cells, expressing favorable markers such as homing receptors (CD62L) as well as costimulatory molecules (CD27/CD28). Most importantly Melan-A-specific T-cells recognize glioblastoma cells in an HLArestricted manner, producing IFN-gamma and IL-2 and inducing tumor cell lysis in vitro. In conclusion these data suggest that glioblastoma cells are immunogenic and may be targeted by antigen-specific T-cells either in the autologous or in the allogeneic setting. Maternal pro-inflammatory T effector cells pass the placenta and exert recall responses Katrin Hebel, Janine Wienecke, Dirk Reinhold, Annette Pethe, Thomas Brune, Hardy Krause, Michael Steiner, Kolja Hegel, and Monika. C. Brunner-Weinzierl Department of Pediatrics, University Hospital Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany Persistence of maternal cells within the fetus is known, but what kind of differentiated, mature cells are able to pass the placenta and remain functional are unknown. Here, we analyzed the transfer of proinflammatory Th1 and Th17 cells across the placenta by radioactive labeling of T effector cells prior to i.v. injection into pregnant mice 2 days before delivery. Radioactivity appeared in all fetuses when Th17 cells were injected into the mother and in 90% of the fetuses when Th1 cells were injected. Concomitant application of Pertussis toxin enhanced uptake of radioactive labeled T effector cells per fetus implicating that infections of the mother are able to enhance maternofetal cell transfer. In addition, efficacy of the transfer was different at mid gestation and prior to delivery. Prenatally transferred T effector cells were detected upon birth of mice and exerted recall responses suggesting that surrogate immunity of maternal T cells might play a role in the immune response of the offspring.

Eur J Pediatr (2009) 168:375–386 Single-cell phospho-profiling in pediatric acute lymphoblastic leukemia (ALL) reveals constitutive and cytokine induced specific signatures involving Stat5 M. Queudeville, H. Kunze, S. M. Eckhoff, K.-M. Debatin and L. H. Meyer Department of Pediatrics and Adolescent Medicine, University of Ulm, Germany Oncogenesis and tumor progression are supported by alterations in cellular signaling. We used phospho-specific antibodies in flow cytometry to analyze specific signaling profiles of leukemia cells in 7 B cell precursor (BCP)- ALL leukemia cell lines and 10 primary pediatric BCP- ALL xenograft samples. Cells were stimulated by different cytokines and activation of various phosphoepitopes was analyzed and compared to unstimulated samples. The BCP-leukemia cell lines and the BCP xenograft samples both displayed high levels of constitutive phosphorylation in general reducing their ability to react to a given stimulus compared to normal B-lymphocytes. With the most important exception of Stat5: we consistently found that Stat5 phosphorylation is increased in ALL cell lines and primary xenografts after stimulation with IL-7 compared to normal B-lymphocytes. Stat5 is known to enhance proliferation and protect from apoptosis and our data now strongly suggest that Stat5 and Stat5 dependent pathways are critically involved in pathogenesis of pediatric ALL. Early relapse in paediatric BCP- ALL is identified by Time To Leukaemia (TTL) in NOD/SCID mice and characterised by a gene signature involving mTOR S.M. Eckhoff, M. Queudeville, K.-M. Debatin and L.H. Meyer Dept. of Paediatrics and Adolescent Medicine, University of Ulm, Germany Despite increasingly successful treatment of children with acute lymphoblastic leukaemia (ALL), patients who encounter relapse exhibit a poor prognosis, particularly if it occurs early during therapy. In a recent study we transplanted primary leukaemia samples (N=50) into NOD/SCID mice and demonstrated a clearly inferior survival for patients whose cells led to manifestation of disease in the recipients within 10 weeks (TTLshort) in contrast to patient samples with prolonged in vivo growth (TTLlong). The biological properties of the 2 groups were further analysed using a whole genome array approach (Affymetrix U133 Plus 2.0). Specific signatures of differentially expressed genes discriminating TTLshort and TTLlong phenotypes were identified. Applying T-test and Significance Analysis of Microarrays (SAM) 2 genes involved in regulation of cell growth were identified and demonstrated prognostic impact in an independent cohort of paediatric BCP-ALL patients (N=197). In this study we demonstrated the prognostic impact of in vivo proliferation of transplanted leukaemia samples for patient outcome and identified a gene signature associated with relapse pointing to pathways including mTOR, which might serve as potential therapeutic targets. Clinopathological investigation of carcinoid tumors of the appendix in children and adolescent: Is extended resection necessary? Christine Böger, Peter Vorwerk1, Ivo Leuschner Department of Paediatric Pathology, Institute of Pathology, Universitätsklinikum Schleswig-Holstein, Campus Kiel, ArnoldHeller-Str. 3, Haus 14, 24105 Kiel, Germany 1 GPOH-MET97-Study, University Childrens Hospital Magdeburg Dept. Paediatric Oncology, Leipziger Str. 44, 39120 Magdeburg

381 Carcinoid tumors of the appendix in children and adolescent are rare and have caused difficulties in prediction of prognosis and clinical management. In the present work we examinated histopathological and immunhistochemical parameters and correlated them to clinical outcome. Mean age of the 101 involved patients (41 males, 60 females) was 13,2 years (range 8,3–20,3). Mean tumor diameter was 7,3 mm (range 1–30 mm). Right hemicolectomy was performed in 16 cases, ileocoecal pole resection/extended ileocoecal pole resection was performed in 10 cases. Metastases were found in three cases. They were all located in lymph nodes of the mesenteriolum or the ileocoecal pole. All children were cured after surgical treatment. We developed a scoring sytem which includes several histopathological features and which might be helpful in developing new guidelines for the indication of extended surgery. The advantage of hemicolectomy versus ileocoecal pole resection is disputable. The data suggests that carcinoid tumors of the appendix show a good prognosis and that extended surgery might not be necessary in most cases. Evidence for B cell antigen receptor (BCR)-driven, BAFF-facilitated positive selection within a unique population of late transitional B cells Almut Meyer-Bahlburg1,2, Sarah F Andrews2, Karl OA Yu2, Steven A Porcelli2, David J Rawlings2 1 Department of Pediatric Pnemology and Neonatology, Hannover Medical School, Carl-Nebuerg-Str.1, Hannover, Germany; 2Seattle Children’s Hospital Research, Seattle, WA 98101, USA In addition to negative selection, several lines of evidence suggest that positive selection may also play an important role in shaping the naïve mature B cell repertoire. We have characterized a late transitional (T2) B cell subset with a distinct phenotypic, functional, and gene expression profile. Intriguingly a high percentage of T2 B cells are actively cycling as demonstrated by cell cycle analysis, measurement of kappa-deleting recombination excision circles (KRECS) and in vivo BrdU labeling. Proliferation of this subset is BAFF-dependent, consistent with the highest levels of BAFF-R expression among all splenic B cells. Finally, M167 BCR idiotypic-specific B cells are first selected by self-antigen within this subset leading, ultimately, to preferential enrichment within the MZ compartment. In summary, our data indicate that late transitional B cells represent a bipotent precursor for mature splenic B cells; and can function as a target for BCRdriven, BAFF-facilitated homeostatic signaling. These combined findings suggest that this small pool of cells may operate at a previously unknown selection point important for establishment of the mature B repertoire. Defining novel therapeutic strategies in MLL-rearranged leukemias J. Faber 1,2, A. Krivtsov2, M. Stubbs2, S. Armstrong2 1 Universitätskinderklinik Mainz and 2Children’s Hospital Boston MLL-rearranged infant leukemias possess unique characteristics and often have an unfavorable prognosis. Introduction of the MLLAF9fusion oncogene into granulocyte-macrophage progenitors (GMPs) resulted in the generation of leukemia stem cells (LSCs), which reactivate a limited stem cell gene expression program. Select Homeobox genes including HOXA9 were among the most prominent up-regulated genes. Furthermore, gene expression analysis in primary MLL-rearranged leukemias demonstrates high-level HOXA9 expression. Here, HOXA9 suppression by RNAi in murine MLL-rearranged LSCs and primary human MLL-rearranged leukemias lead to a progressive induction of apoptosis in vitro and a significantly lower

382 leukemia burden and prolonged overall survival in vivo. Gene expression profiling further demonstrated co-downregulation of a program highly expressed in human MLL-AML including the transcription factor MEF2C and ChIP analysis identified MEF2C as a direct transcriptional HOXA9 target. Direct RNAi mediated targeting of MEF2C revealed progressive induction of apoptosis in murine MLL-LSC and primary MLL-rearranged leukemias in vitro and a prolonged overall survival in vivo. Our data indicates an important role for HOXA9 and its direct downstream target MEF2C for leukemia maintenance and survival in MLL-rearranged leukemias. Slowly progressive clinical phenotype of Mucopolysaccharidosis IIIA patients exhibiting the mutation p.S298P Ann Meyer, Kai Kossow, Andreas Gal, Chris Mühlhausen, Kurt Ullrich, Thomas Braulke, Nicole Muschol Department of Pediatrics/Institute of Genetics, University Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg Mucopolysaccharidosis IIIA is caused by lysosomal sulfamidase deficiency. The onset and progression of the disease is highly variable. Seventy-five mutations in the SGSH gene have been described. Any relationship between the gene mutation and the onset and clinical course of the disease is unknown. The natural course of the disease was assessed in 71 patients using a questionnaire and “Four-Point Scoring System”. In 54 of these an analysis of the underlying mutations was conducted. By assessing the degree of developmental regression over time a group of 7 patients with a slowly progressive course of the disease were identified. In these 7 and 3 other mildly affected patients the missense mutation p.S298P was found on one allele. These patients showed a lower frequency and later onset of typical symptoms. The onset of regression in speech abilities and cognitive functions were delayed by 0.7 and 0.8 years, respectively, and the onset of regression of motor functions occurred 6.1 years later. Severe regression in speech, cognitive and motor functions were delayed by 5, 5.9, and 11.2 years, respectively. These data suggest a slowly progressive phenotype in patients carrying the mutation p.S298P. This may have an important impact on parental counselling and therapeutic interventions. KCNQ potassium channels are critical determinants of neuronal activity in neonatal mouse brain Axel Neu1,2, Quyen Le2, Iliana Hanganu2, Michel Le van Quyen3, Dirk Isbrandt2 1 Department of Pediatrics & 2ZMNH, UKE, Hamburg 3 LENA-CNRS, Hopital de La Salpetriere, Paris The mechanisms of synchronization and epileptogenesis in hyperexcitable neonatal neuronal networks are only partially understood. To investigate pathophysiological mechanisms linked to KCNQ potassium channel deficiency that is associated with a neonatal epilepsy syndrome (BFNC), we generated Tet-Off system-mediated conditional transgenic mice resulting in a functional knockout. Suppression of KCNQ channels during the neonatal period resulted in a severe phenotype that included neurodegeneration, spontaneous epilepsy, and marked behavioral changes in adult mice, whereas absence of transgene expression during the first two neonatal weeks prevented this phenotype. Acute treatment with the NKCC1 blocker bumetanide that lowers [Cl-] in immature neurons reduced cortical and hippocampal network activities in neonates. Chronic bumetanide treatment during the first two neonatal weeks prevented morphological hippocampal changes and improved the behavioral phenotype to comparable levels as did suppressed transgene expression. Our data suggest that the control of network oscillations by cortical KCNQ channels is critical in the neonatal brain, and bumetanide provides an effective treatment option.

Eur J Pediatr (2009) 168:375–386 Defective membrane tubulation in dysferlin-deficient muscular dystrophy Lars Klinge, Sven Thoms, Irmgard Cierny, Volker Straub, Kate Bushby, Jutta Gärtner Department of Paediatrics and Paediatric Neurology University Medical Centre Göttingen, Germany The muscular dystrophies (MD) are a group of inherited disorders causing progressive muscle weakness and wasting. Many of these diseases have a childhood onset, and so far no curative treatment exists. Recently a novel pathomechanism, defective repair of the plasma membrane, was demonstrated in dysferlin deficient myofibres, but the exact function of dysferlin remains unknown. Dysferlin is the protein involved in limb-girdle muscular dystrophy type 2B, and Miyoshi myopathy. The clinical course of these disorders is distinct from other muscle diseases as patients show good muscle strength in early childhood before onset of overt weakness. In contrast to adult muscle with a predominant sarcolemmal localisation, we identified an association of dysferlin with the T-tubule system in developing muscle. At this stage of development, membrane damage was followed by a specific and Ca2+-dependent translocation of dysferlin to the wounding site identifying the T-tubule system as a compartment involved in membrane repair. Ultrastructural analysis of pre-dystrophic, dysferlin-deficient muscle demonstrated morphological abnormalities of the T-tubule system indicating a functional role of dysferlin in T-tubule formation. These results indicate a role of dysferlin in membrane tubulation and may ultimately help to define treatment strategies for these patients. Metabolic programming of fetal rats: About the need for proper controls Kai-Dietrich Nüsken1, Jörg Dötsch1, Christian Plank1, Manfred Rauh1, Wolfgang Rascher1, Holm Schneider1,2 1 Department of Pediatrics, University of Erlangen-Nuernberg, Erlangen, Germany; 2Experimental Neonatology, Department of Pediatrics, Medical University of Innsbruck, Austria There is accumulating evidence that susceptibility to disease is set already in utero, e.g. by insufficient supply of nutrients and/or oxygen during a sensitive window of development. Rodent models of low protein intake (LP) or ligation of the uterine arteries (LIG) serve as established model systems to investigate the concept of fetal programming. However, these studies critically depend on the suitability of the experimental controls. We compared glucose and lipid metabolism in the offspring of dams which underwent either LIG or sham operation (SOP) with that of untreated controls (C). Placental gene expression of IGF1, IGF2, IGFBP1 and leptin was quantified by RT-PCR. Blood parameters, body weight and food intake were recorded for 30 weeks and glucose tolerance tests were performed. Only LIG offspring showed reduced IGF1 expression, impaired glucose tolerance and elevated glycosylated hemoglobin levels, whereas both LIG and SOP revealed reduced placental leptin expression and increased body fat content relative to C (p<0.001), elevated triglyceride, total cholesterol and leptin levels and reduced HDL cholesterol. Thus, use of LIG models requires both SOP and C controls. Moreover, LP and LIG had opposed effects on leptin and IGF1 expression and metabolic sequelae. Effect of intraamniotic endotoxin induced chorioamnionitis (CA) on development of regulatory T-lymphocytes (Treg) in fetal thymus S. Kunzmann1, B. Kramer1,3, S. Kallapur2, A. Jobe2, C. Speer1 1 Univ. Children’s Hospital Würzburg 2Cincinnati Children’s Hospital, USA 3Univ. Children`s Hospital Maastricht

Eur J Pediatr (2009) 168:375–386 The thymus is essential for the development of T-lymphocytes in fetal and postnatal life. The thymus is the home of Treg mediate homeostasis of the immune system and differentiate under the control of the transcription factor FoxP3. The effects of CA on thymus function are not well understood. CA was induced by a single intraamniotic injection of 10 mg LPS 5 h, 1 d, 2 d or 5 d before delivery of preterm lambs at 123 d GA. Lymphocytes, plasma cortisol and thymus weight were quantified. NF-κB- and FoxP3-positive cells in thymus were evaluated by immunohistochemistry. The number of circulating lymphocytes in cord blood showing a decrease after 1 d and 2 d. Thymus-to-body weight ratios were reduced in the LPS groups, with a maximum at 5 d with a 40% decrease. LPS modestly increased the plasma cortisol concentration with a maximum of 2.6-fold after 2 d. LPS increased NF-κB signalling and reduced FoxP3 positive cells to a minimum at 1 d of 60% in controls. These changes in lymphocytes, cortisol, NF-κB signalling and FoxP3 expression were no longer evident after 5 d. This pathophysical sequence induced by antenatal inflammation during fetal life underlines CA induces changes in fetal thymus, which may have implication on the development of immune function in preterm infants later in life. Post-transplant lymphoproliferative disorders (PTLD) in pediatric solid organ transplant recipients Britta Maecker-Kolhoff, Barbara Meissner, Christian Kebelmann-Betzing, Günther Henze, Christoph Klein Departments of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, and Charite Campus Virchow, Berlin, Germany Epstein-Barr Virus associated post-transplant lymphoproliferative disorders (PTLD) have been recognized as severe side effects of immunosuppressive therapy after organ transplantation. So far, no standard diagnostic and therapeutic approaches have been defined. Here, we report an interim result of the prospective multicenter trial Ped-PTLD 2005 Pilot. Pediatric patients with histologically confirmed CD20 + PTLD were subjected to a standardized diagnostic procedure and treated by weekly infusions of anti-CD20-antibody Rituximab. Initial treatment response was assessed after 3 weeks. Patients who achieved at least a partial remission received 3 further Rituximab infusions on a protracted schedule. All others were stratified to receive a moderate chemotherapy regimen (mCOMP; vincristin, cyclophosphamide, low dose methotrexate and prednisone). 22 Patients have been enrolled so far. 16 patients were treated with CD20-antibody alone, one of whom relapsed. 6 patients received mCOMP chemotherapy, which led to complete remission in 4 patients. 2 patients were non responsive and went to receive standard chemotherapy. One patient died of bowel perforation during treatment, while treatment was well tolerated in all other patients. Overall survival is 90% at 2 years with an event-free survival of 75%. These results support the need for standardized, interdisciplinary approaches for transplant complications. SKAR: a novel target of the mTOR/S6K1 pathway Kristina Jülich, Celeste J. Richardson, Xiaoju Max Ma, John Blenis Dpt. of Neuropediatrics, Charité University Medical Center, Berlin, and Dpt. of Cell Biology, Harvard Medical School, Boston One of the pathways that is frequently affected in human diseases ranging from diabetes to cancer, is the rapamycin-sensitive mTOR/S6K1 signaling pathway. We identified the novel protein SKAR (S6K1 Aly/REF-like target) as a target of the mTOR pathway and characterized it in terms of localization, phosphorylation, interaction and function. SKAR is a specific binding partner and substrate of the cell size regulator S6K1 (S6 kinase 1). SKAR phosphorylation occurs upon insulin stimulation and is sensitive to RNAi-mediated S6K1 reduction in vivo, and RNAi-

383 mediated reduction of SKAR decreases cell size. In addition, SKAR is a nuclear protein with homology to the Aly/REF family of RNA binding proteins which couple splicing and mRNA export. Splicing labels mRNA with a subset of proteins that facilitate later events such as nuclear export and translation, ultimately regulating protein expression. Phosphorylation of other targets such as 4E-BP1 already suggested an involvement of the mTOR pathway in translation regulation. Later work by our group demonstrated that, when activated, S6K1 is recruited to the newly synthesized mRNA by SKAR, and that SKAR and S6K1 increase the translation efficiency of spliced mRNA. Genetic characterization and genotype-phenotype correlation of a large cohort of patients with hypophosphatemic rickets Marius Schumacher, Merle Thieme, Stefan Nissen, Ralf Werner, Harald Jueppner 2, Egbert Herting, Olaf Hiort Deparment of Pediatric and Adolescent Medicine, University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany 2 Endocrine Unit, Harvard Medical School, Boston, USA Hypophosphatemic rickets (HR) are caused by renal phosphate wasting leading to hypophosphatemia, rickets and bone disease, as well as growth retardation. HR is due to mutations in the Phex-, FGF23-, SLC34A3/NaPi-IIc -, and DMP1-gene. We investigated a large HR cohort for their underlying genetic disorder and their clinical presentation. In 80 families (106 individuals) we performed molecular investigations. Moreover, we correlated laboratory values for phosphate, parathormone and bone markers (alkaline phosphatase, urinary desoxypyridinoline (DPD) excretion) with growth data in 168 patients with HR. We detected mutations in 69 families (45 previously not described mutations) in the Phex gene, none in the FGF23 gene, two in the DMP1 gene and one in the SLC34A3/NaPi-IIc gene. In the cohort with Phex gene mutations we demonstrated a strong correlation towards poor growth in patients with disruptive mutations. We also showed a strong correlation of early treatment with good growth. The urinary DPD/Crea ratio seems to be the best parameter to monitor early growth rate. In some patients there seems to be another yet unknown genetic cause for HR. Treatment of Duchenne muscular dystrophy with cyclosporin A — a randomized, double-blind, placebo controlled trial Janbernd Kirschner, Joachim Schessl, Gabriele Ihorst, Rudolf Korinthenberg, and the Muscular Dystrophy Network MD-NET Division of Neuropediatrics and Muscle Disorders, Department of Pediatric and Adolescent Medicine, Mathildenstr. 1, 79106 Freiburg, Germany Treatment with steroids has shown to slow down progression of muscle weakness in patients with Duchenne muscular dystrophy (DMD) but its use is limited by side effects. In a randomized, multicentre, double-blind placebo-controlled trial we investigated the effect of cyclosporin A (CSA) in DMD. 153 patients were randomized to receive either placebo or 4 mg/kg CSA. After three months both groups received additional treatment with intermittent prednisone for another 12 months. In each group 73 patients were available for intention to treat analysis. Baseline characteristics were comparable in both groups. There was no significant difference between the two groups concerning primary (manual muscle strength measure) and secondary (myometry, loss of ambulation, side effects) outcome measures. Peak CSA values were measured blindly and ranged from 12–658 ng/ml (mean 210 ng/ml) in the verum group. In conclusion, CSA does not improve muscle strength as a monotherapy and does not improve the efficacy of intermittent prednisone in DMD. Networks of

384 rare diseases such as the BMBF-funded MD-NET facilitate the conduction of meaningful multicentre trials even in rare diseases. Assessment of growth hormone therapy in the risk of tumor recurrence after treatment of brain tumors Tilman R. Rohrera, Thorsten Langerb, Helmuth G. Dörrb a Department of Paediatrics; University Hospital, Homburg, b University of Erlangen, Hospital for Children and Adolescents To assess the effect of human growth hormone (hGH) treatment and other factors contributing to tumor recurrence after therapy of CNS tumors in children. We retrospectively analyzed the clinical data of 103 children with brain tumors (craniopharyngioma 28.2%, medulloblastoma 45.6% and ependymoma 26.2%) diagnosed from 1973 to 1999. A multifactorial regression analysis was made for possible risk factors (radiotherapy, completeness of tumor removal, age, gender, chemotherapy, and recurrence-free survival time). The incidence of tumor recurrence was compared in patients who did or did not receive hGH. Tumor recurrences occurred in 38 patients, second malignant neoplasms (SMN) in 4 patients. Significant correlations with tumor recurrence were found for completeness of tumor removal (P=.001) and tumor free survival time (P<.001). Thirteen of 44 hGH-treated children (29.5%) and 28 (63.6%) of 59 non-hGH-treated children had tumor recurrence (P=.017). Four children developed SMN,three having received hGH therapy. With a regression model based on completeness of tumor removal and recurrence-free survival, within-sample prediction of recurrence was accurate in 90% of children. Growth hormone treatment after treatment of brain tumors does not increase the risk of tumor recurrence. The major factors correlated with tumor recurrence are completeness of tumor removal and recurrence-free survival time. Allergy prevention starts before conception: Materno-fetal transfer of tolerance protects from the development of asthma Christian Hennig, Tobias Polte, Gesine Hansen Department of Paediatric Pneumology and Neonatology, Hannover Medical School, Hannover, Germany Allergy prevention must start early since epidemiology shows that exposure during pregnancy and shortly after birth determine the disease risk. We asked the question whether induction of tolerance in the mothers before conception can be somehow transferred to the offspring. Therefore, Balb/c mice were tolerized with oral ovalbumin (OVA) before conception. The offspring was subsequently immunized with OVA and analyzed in our murine asthma model. Interestingly enough, the offspring of tolerized mice was completely protected, even when immunized as late as 8 months after birth, whilst the offspring of naïve mothers developed an asthma-like phenotype. Allergen-specific IgG was critically involved in the tolerance trancefer, since FcRn−/− mice, that cannot transport IgG via the placenta, transferred tolerance to the offspring only when the missing diaplacental IgG transfer was compensated by IgG transfer via breast milk from tolerant mothers but not when weaned by naïve wet-nurses. Inhibition of IFN-y, produced by allergen-specific memory T cells in the offspring, abrogated the effect of maternal tolerance transfer demonstrating a crucial role for IFN-y in maintaining allergen-specific tolerance. Our data show that maternal immunologic memory has significant and persistent impact on allergy prevention in the offspring. The transcriptional inferno of acute monosodium urate crystal inflammation 1,3 Pessler, F., 2Mayer, C.T., 3Tobias, J., 4Menetski, J.P., and 3 Schumacher, H.R. 1 Klinik und Poliklinik für Kinder- und Jugendmedizin, TU Dresden; 2 TU München; 3University of Pennsylvania, 4Merck Research Labs.

Eur J Pediatr (2009) 168:375–386 Monosodium urate (MSU) crystals elicit an acute inflammatory response driven by TLRs 2 and 4 and the Nalp3 inflammasome, i.e. factors that relate pathogenically to juvenile idiopathic arthritis and various autoinflammatory disorders. Injecting MSU crystals into the murine air pouch causes inflammation that resembles acute synovitis in humans. Using dissected air pouch membranes and custom lowdensity PCR arrays for 96 target genes, we have begun to define major transcriptional events in a 50 h time course of MSU crystal inflammation in a synovium-like tissue. Averaged over all mRNAs, transcriptional induction was 100-fold, peaked 4 h after crystal injection, and gradually returned to near base-line by 50 h. Euklidian distance similarity calculation confirmed that transcriptional reprogramming was most intense at 4 h. Leukocyte accumulation in the pouch exudate differed kinetically in that it began to rise later and peaked at 9 h. Hierarchical clustering analysis revealed 3 clusters of temporally coregulated mRNAs: an early burst that included major proinflammatory cytokines (IL-1β, IL-6, TNFα), a more sustained second wave including matrix metalloproteases and cell surface receptor s involved in prostaglandin synthesis and TLR signaling, and a prolonged resolution phase characterized by increased expression of CD68 and the anti-inflammatory factors PPAR-γ, h-prostaglandin D synthase and TGF-β. Premature aging of the immune system in children with juvenile idiopathic arthritis Martina Prelog, Nora Schwarzenbrunner, Lothar Bernd Zimmerhackl Department of Pediatrics I, Medical University Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria Juvenile idiopathic arthritis (JIA) is an autoimmune disease of the young with unknown pathogenesis. Premature aging, associated thymic involution and compensatory autoproliferation could play an important role in the pathogenesis of autoimmunity. To evaluate whether patients with JIA demonstrate premature immunosenescence characteristic indicators of immunological aging were measured: quantities of peripheral blood naive T cells, the frequency of T cell receptor excision circles (TRECs) in naive T cells, telomeric erosion and Ki67 expression as estimate of replicative history and proliferation. JIA patients (n=22) demonstrated an accelerated loss of naive (CD4 + CD45RA+) T cells with advancing age and a compensatory increase of memory (CD4 + CD45RO+) T cells. JIA patients showed significantly decreased TREC numbers in naive T cells compared to age-matched healthy donors (HD) (n= 37) (p< 0.01). Telomeric erosion in naive T cells was increased in JIA patients (p<0.01). Disease duration was an independent factor for telomeric erosion and proliferation (p<0.05). Our findings provide evidence for a premature immunosenescence in JIA patients and suggest that the age-inappropriate T cell senescence and the disturbed T cell homeostasis may contribute to the development of JIA. Genetic basis of hyper-IgE syndromes ED Renner 1 , L Schimke 1 , J Reichenbach 3 , M Borte 4 , J Roesler 5 , E Maaß 6 , R Cremer 7 , M Hönig 8 , P Lohse 9 , TR Torgerson 2 , BH Belohradsky 1 , and HD Ochs 2 1 Haunersches Kinderspital, LMU, Munich; 2University of Washington, Seattle, WA (USA); 3University Children’s Hospital, Zurich (Switzerland) 4 St. Georges Hospital, Leipzig; 5University Children’s Hospital, Dresden; 6 Olgahospital Stuttgart; 7Children’s Hospital, Cologne; 8University Children’s Hospital, Ulm; 9Clinical Chemistry, LMU, Munich Hyper-IgE syndromes (HIES) are immunodeficiencies of autosomal dominant or recessive inheritance characterized by eczema, Staphylococcus aureus skin abscesses, recurrent episodes of pneumonia, and

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elevated serum IgE. The identification of a homozygous Tyk2 mutation was instrumental to link autosomal dominant HIES to heterozygous STAT3 mutations. To determine the prevalence and the nature of STAT3 mutations, we sequenced the STAT3 gene in a cohort of 76 patients with clinical findings suggestive of HIES. STAT3 mutations were identified in patient with the complete HIES phenotype. Only missense mutations and in-frame deletions allowing expression of mutant STAT3 protein were found. Mutations were located in three distinct domains. STAT3 plays an important role in cell development, inflammation, neutrophil mobilization, osteoclastogenesis, and the generation of TH17 cells. TH17 cell numbers were found to be reduced not only in STAT3-HIES but also autosomal recessive HIES not due to Tyk2 mutations, shedding light on these entities. In conclusion, progress has been made to define distinct entities of HIES on a molecular basis, but many functional aspects remain unsolved and need further investigation.

7 secondary malignancies in 6/26 patients (23.1%) occurred. Conclusions: To the best of our knowledge this is the first study with a follow up period of more than 25 years in paediatric patients with Hodgkin’s disease. As our findings are to be attributed as long term side effects of the initial therapy and as cardiac complications implicate the third most common reason for death in this formerly treated population our study emphasizes the urgent need of standardized follow up regimen for our patients as well as an early interdisciplinary covering of the patients between the paediatric and adult oncologists.

Neurological and renal outcome in isolated methylmalonic acidurias: prediction by biochemical and clinical parameters Friederike Hörster, Sven F Garbade, Tamaris Zwickler, Martin Lindner, Stefan Kölker Zentrum für Kinderheilkunde und Jugendmedizin, Abteilung I Allgemeine Pädiatrie: Sektion für angeborene Stoffwechselerkrankungen, INF 430, 69120 Heidelberg, Germany

For organprotection during corrective cardiac surgery in children, patients are cooled to a minimal temperature of 17°C during cardiopulmonary bypass (CPB).The aim of our studies was to investigate effects of deep hypothermia and rewarming on the inflammatory response of endothelial cells and furthermore analyse the impact of pre-treatment with methylpredisolone (MP) and tacrolimus (TAC). Primary human umbilical endothelial cells (HUVECs) were stimulated with TNF-α, pre-treated with MP and/or TAC and exposed to dynamic temperature changes analogues to clinical settings during paediatric cardiac surgery: deep hypothermia (17°C), slow rewarming (2 h up to 37°C) and normothermia (20 h at 37°C). Deep hypothermia suppressed inflammation but, interestingly, 2 hours after rewarming the IL-6 concentration was significantly higher than in control cells (37°C). Moreover dynamic temperature changes lead to increased ERK 1/2 and p38 phosphorylation. The combined pre-treatment with MP and TAC served to inhibit this intracellular inflammation via MAP-kinases pathway. Application of combined drugs that affect multiple targets in activated endothelial cells may therefore be considered as a potential new therapeutic strategy to inhibit inflammation after hypothermia and rewarming.

Isolated methylmalonic acidurias (MMAurias) are caused by deficiency of methylmalonyl-CoA mutase or by defects in the synthesis of its cofactor 5′-deoxyadenosylcobalamin. The aim of our studies was to evaluate which parameters predicted the long-term outcome. 273 patients from 20 European Metabolic centres were included into a cross-sectional study based on standardised questionnaires and complex statistical models were applied for data analysis. In a second study 83 patients were enzymatically characterised and prospectively followed (median follow-up period: 18 years) Neonatal onset of the disease was associated with high mortality, high incidence of developmental delay and severe handicap. Cobalamin non-responsive patients with neonatal onset born in the 70s and 80s had a particularly poor outcome. A more favourable outcome was found in patients with late onset of symptoms, especially when cobalamin responsive or classified as mut-. Prevention of neonatal crises in pre-symptomatically diagnosed newborns was identified as protective factor concerning occurrence of handicap. Chronic renal failure was found most frequently in mut0 and cblB patients and was predicted by the urinary excretion of methylmalonic acid prior to renal failure. Cardiac late effects 25 years after treatment for Hodgkin’s disease -A long term follow up studyBeate B. Nenning1, Karl-Friedrich Kreitner2, Peter Gutjahr1 Departments of 1Paediatrics and 2Radiology, Johannes GutenbergUniversity, Mainz, Germany Background: With increasing numbers of childhood cancer survivors clinical research and follow up studies have to focus on therapy related late effects. Aim of the current study was to analyse cardiac late effects 25 years after initial treatment of childhood Hodgkin’s disease. Methods/Patients: 26 long term survivors treated by mediastinal irradiation (mean 33.9 Gy) and poly-chemo therapy were included. Patients underwent a detailed history and physical examination. Subsequently a Cardio Magnet Resonance Imaging, cMRI was performed. Results: 11 patients (42.3%) gave a positive history of cardiac symptoms. In 16 patients (61.5%) clinical examination exhibited a cardiac murmur. MRI-scans revealed cardiac pathology in 18 (69.2%) of the patients. Thyroid sequelae were found in 19 patients (73.1%),

Methylprednisolone and Tacrolimus prevent IL-6 inflammation after hypothermia and rewarming in endothelial cells via ERK 1/2 and p38 pathway Katharina Schmitt, Antje Diestel, Joerg Roessler, Felix Berger Dep. of congenital heart disease and pediatric cardiology, Deutsches Herzzentrum Berlin, Germany

A combined immundeficiency disease with hypomorphic RAG mutations and granulomas C. Schütz, K. Huck, S. Gudowius, U. Pannicke, R. Willemze, U. Göbel, A. Schulz, S. Ehl, KM Debatin, W. Friedrich, K. Schwarz and T. Niehues Department of Pediatrics & Institute for Transfusion Medicine and Immunogenetics (CS, AS, KMD, WF, UP, KS), Ulm University Department of Pediatric Oncology, Hematology and Immunology (KH, SG, UG, TN), Heinrich Heine University, Düsseldorf Department of Dermatology (RW), Leiden Univ. Medical Center Department of Pediatric Immunology (SE), Freiburg University We describe four unrelated children with a combined immunodeficiency and granulomas in the skin, mucous membranes and internal organs. All patients had severe complications after viral infections, including varicella and EBV associated B-cell lymphoma. One patient suffered from severe haemolytic anemia and immune thrombocytopenia. Immuno-logical findings were hypogammaglobulinemia, diminished numbers of T and B cells, and sparse thymic tissue. On molecular analysis three patients were compound heterozygotes and one was homozygous for mutations in recombination activating gene 1 or 2. The mutations were associated with reduced function of RAG in vitro (3 to 30% of normal). In each case, both parents were heterozygous carriers of a RAG mutation. RAG deficiency should be considered in older patients with evidence of combined immunodeficiency, signs of immune dysregulation and otherwise unexplained granulomatous lesions.

386 Polyalanine expansions in hand-foot-genital syndrome Boris Utsch, Michael Ludwig, Wolfgang Rascher, Jeff W. Innis Children’s Hospital, Inselspital, University of Berne, CH-3010 Berne, Switzerland Hand-foot-genital syndrome is a rare autosomal-dominant inherited syndrome characterized by limb and urogenital malformations. Most of the underlying HOXA13 mutations seem to be polyalanine expansions. HOXA13 encodes for a transcription factor. Our HOXA13 polyalanine expansion mouse model showed normal spatial HOXA13 RNA expression in the mutant limb buds but reduced levels of the protein. Since the in vitro translation efficiency of the expanded HOXA13 protein was normal, the reduced in vivo abundance of the expanded protein is likely due to degradation. To address the paradox why HOXA13 polyalanine expansions lead to a loss of function, whereas the paralogous HOXD13 polyalanine expansions found in synpolydactyly type 1 act in a dominant-negative manner, we performed cell culture experiments. Using transfection and immunohistochemistry we showed that HOXA13 polyalanine expanded proteins form cytoplasmic aggregates, and the distribution between cytoplasmic aggregates or the nucleus is polyalanine tract sizedependent. We also found that wt HOXA13 or HOXD13 polyalanine expansions are sequestered in HOXA13 polyalanine expansion cytoplasmic aggregates. Thus, the difference between HOXA13 polyalanine expansion loss of function and HOXD13 polyalanine expansion dominant negative effect is not the ability to aggregate wt group 13 paralogs but perhaps to variation in activities associated with refolding, aggregation or degradation of the proteins.

Eur J Pediatr (2009) 168:375–386 Application of whole genome SNP arrays for molecular karyotyping in acquired bone marrow failure disorders. M.Wlodarski1, J Huh2, J.Maciejewski2, C.Flotho1, C.Niemeyer1 1 Pediatric Hematology and Oncology, University of Freiburg, Mathildenstr. 1, 79106 Freiburg. 2Experimental Hematology, Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195 Hypocellular bone marrow failure (hBMF), including aplastic anemia (AA) and refractory cytopenia (RC) is characterized by pancytopenia due to contraction of the vital stem cell pool. Clonal malignant evolution occurs in a significant proportion of patients. The inability to detect patients at risk early constitutes a significant clinical problem. Metaphase cytogenetics (MC) is often non-informative in hBMF. We hypothesized that karyotyping using SNP-arrays (SNPA) that allows for whole genome analysis from archived DNA samples, will improve detection of genomic lesions in hBMF. We applied Affymetrix 250K/6.0 chips to study whole genomes of hBMF patients (AA, N=106; RC, N=35) and detected genomic gains, losses and copy number neutral LOH. Clonal evolution was observed in 13 AA patients, and remarkably in 38% of evolving AA patients, numerical aberrations were detected earlier by SNPA than MC. Lesions of acquired uniparental disomy were detected in 4 patients and were not present in nonmyeloid lineage. Interestingly, these changes disappeared in 2 patients after therapy and nearly 2 years later newly recruited clones with mono7 dominated as a sole abnormality by SNPA and MC. Our study demonstrates that SNPA is a powerful tool for molecular karyotyping and for early detection of patients harbouring clonal defects previously undetected using traditional MC.