Despite the implementation of new antifungal drugs, invasive aspergillosis (IA) still remains a considerable challenge in pediatric oncology with a severe mortality. Prophylactic and therapeutic measurement have to be evaluated in these rare but poor prognostic patients. Therefore the entire group of patients at risk of developing IA has to be defined before cooperative prospective trials. In a retrospective analysis including all our patients with malignancies we looked for patients with proven/probable IA. Cases of the period from 2003 to 2008 were analyzed in detail.In the period between 2003 to 2008 24 of 755 patients were affected by proven/ probable IA. Compared to former studies incidence increased from 1.3%in 1980 to 3.4% in 2008. AML patients with or without allogeneic/haploidentical stem cell transplantation were at highest risk (24% and 25% respectively, in comparison to 1% in ALL-patients). Survival after 2 years was 50% for patients with AML and IA. In patients with high risk to develop IA the effect of intensified, intravenous antimycotic prophylaxis has to be proven prospectively in a cooperative and randomized setting.

BACKGROUND:For children with hemato-oncologic diseases, especially after hematopoietic stem cell transplantation (HSCT), the risk for developing complications related to pandemic influenza A (H1N1) 2009 (pH1N1) infection is largely unknown.METHODS:A retrospective chart study was performed of pH1N1 cases diagnosed between October 2009 to January 2010 in the hemato-oncologic unit of the University Children's Hospital of Düsseldorf, Germany.FINDINGS:In total, 21 children were diagnosed with laboratory-confirmed pH1N1; in 16 patients with malignancies (acute leukemia 7, lymphoma 4, solid tumors 2, others 3) and in 5 with benign hematologic disorders. Five patients had undergone prior HSCT, although 1 patient was diagnosed during conditioning therapy with high-dose chemotherapy in preparation for haploidentical HSCT. Most frequent symptoms were fever (>38.5°C) and cough (in 100%), and rhinorrhea (57%). The 2 patients acquiring pH1N1 infection under high-dose or intensive chemotherapy did not require intensive care or mechanical ventilation, and both recovered under antiviral therapy. Oseltamivir was administered to 11 patients; in 1 patient, therapy was switched, on a compassionate-use basis, to intravenous zanamivir because of lack of clinical improvement after oseltamivir therapy. Complications were hospitalization (19%), demand of oxygen supplementation, delay/interruption of antineoplastic therapy, and prolonged administration of antibiotics and antipyretics.CONCLUSION:In the investigated patient population, pH1N1 was mild in most cases, but was associated with substantial morbidity in a proportion of patients and led to interruption and delay in anticancer treatment.

Background: CD8 cells are key to antiviral immunity and can be divided by phenotype into early (CD28 + CD27 +), intermediate (CD28-CD27+) and terminally differentiated subsets (CD28 CD27 -). Despite effective HAART there is an unexplained expansion of CD8+CD28-CD27- T cells in HIV-infected children. The cytokine production and specificity of this terminally differentiated CD8 T cell subset in chronic virus infection is unclear.Patients, Methods & Results: In a cohort of 26 HIV-infected children the cytokine production of terminally differentiated CD8 cells was analyzed by intracellular staining and FACS analysis and was compared to children with chronic hepatitis B infection and to healthy children. The specificity of CD8 subsets was analyzed by staining with Gag/Pol tetramers in a cohort of 13 patients. We show that an increased production of interferon-gamma in terminally and early/intermediate differentiated CD8 cell subsets after stimulation is specific for HIV-infection. The expanded population of terminally differentiated CD8+CD28-CD27- T cells does include HIV Gag/Pol specific T cells in adults but not in children.Conclusion: The expansion of terminally differentiated CD8 cells might be important for immunomodulation but in children it does not appear to play a role in HIV Gag and Pol specific immunity.

MHC class II deficiency is a rare and fatal form of primary combined immunodeficiency caused by a lack of T-cell-dependent humoral and cellular immune response to foreign antigens, which can only be cured by allogenic stem cell transplantation. In the literature search, we identified 68 cases of HSCT in MHC class II deficiency in the last 14 yr. Pre- and post-transplant MHC class II deficiency is complicated by overwhelming viral infections, a high incidence of GvHD, and graft failure with a poor overall survival rate below 50%. We report an eight-month-old boy presenting with severe respiratory infections and chronic diarrhea, whose sister died at the age of four yr from septicemia. MHC II deficiency was caused by an RFXANK-mutation and treated successfully by 4/6 mismatched unrelated CBT after a myeloablative conditioning regimen based on anti-thymocyte globulin, busulfane, fludarabine, and cyclophosphamide. At present, our patient is well with full immune reconstitution 3(4/12) yr after CBT. CB may represent an alternative source of stem cells for children with MHC class II deficiency without a suitable donor.

In addition to their multilineage differentiation potential mesenchymal stem cells (MSCs) support hematopoiesis and modulate the adaptive immune system. Based on these properties human MSCs are currently under intensive clinical investigation in different therapeutic applications including tissue repair and immune-mediated disorders such as graft rejection and graft-versus-host disease after haematopoietic stem cell transplantation. However, there is still an ongoing debate on the molecular mechanisms underlying MSC-mediated immunosuppression. Here we show for the first time, that adenosine generated via the CD39 (Ecto-ATPase)/CD73(Ecto-5′-nucleotidase) axis is involved in MSC-mediated T cell-suppression in the human setting. We first demonstrate that in MSC/T cell co-cultures CD39 expression is upregulated on activated T cells which is capable of converting adenosine triphosphate (ATP) to adenosine monophosphate (AMP) via its Ecto-ATPase activity. CD73(Ecto-5′-nucleotidase) constitutively expressed by MSCs subsequently degrades AMP to adenosine thus mediating potent T cell inhibition in cell-to-cell contact conditions as well as in a transwell system. The specific role of CD73-mediated adenosine production was confirmed by addition of the CD73(Ecto-5′-nucleotidase) inhibitor α,β-methylene-ADP (Sigma-Aldrich) which significantly restored T cell proliferation (p=0.015, n=4). This observation may be of significant therapeutic significance as, in addition to its T cell inhibitory function, adenosine has recently been shown to exert major anti-inflammatory and anti-fibrotic effects.

Background: Severe hemophilia A (HA) in females is a very rare phenomenon. Ignoring HA as a possible diagnose can result in fatal complications. Patients: We report a 3-month old girl suffering from severe hemophilia A, presenting with intracranial hemorrhage three weeks after drop down from an infant carrier. Recurrent bleeding after neurosurgery led to the diagnosis of a HA by findings of low levels of factor VIII coagulation activity (F8:C) below 1 % and normal levels of factor von Willebrand activity. Methods: Diagnosis of hemophilia A by one stage clotting test and proof by molecular Studies via long - range - PCR. Chromosome analysis in metaphases from peripheral blood lymphocytes. Results: Molecular analysis showed inversion of intron 22 as the result of a maternally inherited, distal, F8 gene inversion and chromosome analyses a 45,X karyotype indicative of Turner syndrome in our patient. Diagnosis was hampered by the female sex and the presence of neither a family history of bleeding disorders nor clinical signs of Turner syndrome. Conclusion: Our case shows that, although uncommon in female infants, x-linked genetic bleeding disorders like HA are a possible diagnosis by very different reasons. Rare bleeding disorders, although not expected, might be present and the combined clinical, laboratory and genetic analysis are needed to establish the final diagnosis. Repetitive prolonged aPTT and clinical bleeding signs should lead to further hemostasiological investigations. An algorithm for hemostasiological investigations in case of unexplained clinical bleeding is given.

Background: Survivin, a member of the inhibitor of apoptosis protein (IAP) family is transiently expressed at low levels during normal hematopoesis but profoundly overexpressed in adult leukemia potentially contributing to leukemogenesis due to deregulated apoptosis and defective cell cycle control. Alternative splicing results in four different mRNA variants survivin, survivin2B, survivin-Delta Exon3 and survivin-3B, with distinct cellular localization patterns and anti-apoptotic potential. Due to co-localization of survivin and survivin-2B in the cytoplasm survivin-2B may permit interactive fine-tuning of survivin actions and moreover play an attenuating role in its anti-apoptotic function. Lack of survivin-213 is associated with disease progression of malignomas suggesting a differential role of these isolforms in tumorigenesis.Patients and methods: We therefore determined the expression of the functional survivin splice variants performing RT- and real-time PCR in a purely pediatric cohort of 20 patients suffering from precursor B-ALL (BCP-ALL).Results: Here, we demonstrate for the first time in pediatric patients with precursor B-ALL an association between lower survivin-2B expression and affiliation to the high risk group.Conclusion: The idea that survivin-2B may act as natural antagonist of survivin could potentially be used in novel approaches of anti-cancer treatment by influencing the proportional expression of the different splice variants.

Authors' contributions: A.T. and I.S designed research and performed research, analyzed data, and wrote the paper; M.S. and L.G. performed research and analyzed data; U.G. performed research; G.E.J.S. provided vital materials and wrote the paper; K.S.O. wrote the paper; D.D. designed research, analyzed data and wrote the paper. Blood First Edition Paper, prepublished online March 21, 2007; DOI 10.1182/blood-2006-08-038398

The influence of the microenvironment on the activation status and behaviour of ALL blasts is critical for interactions with the immune system in vivo . The capacity of B cells to respond to CD40-ligand (CD40L) stimulation is critical for their sensitisation to immunological control mechanisms and susceptibility to apoptotic signals. Primary precursor B-ALL blasts (BCP-ALL; n=32) lack CD95-expression (mean±SE; 4.2±0.6% positive cells) and are resistant to apoptosis while significant up-regulation of CD95 is apparent upon CD40-stimulation in BCP-ALL blasts that reaches a plateau after 72 h. Yet, in spite of equivalent CD95-upregulation in ALL blasts (58.3±6.5%; n=17) and normal B cells (59.3±13.1%) specific apoptosis is markedly lower in ALL compared to mature B cells (19.1±3% vs 36.7±5.5%). Resistance to apoptosis in ALL blasts and its reversibility after cycloheximid treatment suggest that anti-apoptotic mechanisms prevent induction of cell death via CD95 ligation in CD40 activated blasts. In accordance, in CD40-activated ALL blasts caspase 8 and 3 activity is not enhanced upon CD95 ligation in contrast to an 1.8±0.3 and 1.7±0.3 fold increase in caspase activity in stimulated normal B cells (n=7), suggesting a block of the apoptotic cascade in BCP-ALLrelatively close to the receptor level. CD40L-activated ALL blasts and normal B cells were submitted to western blot analysis with respect to the molecules associated to the death-inducing signalling complex (DISC). FADD and the zymogen form of caspase-8 are constitutively expressed in both malignant and non malignant B cells with no modulation following CD40 ligation. In contrast, the anti-apoptotic short isoform of the c-FLICE inhibitory protein FLIP S is weakly expressed in naive blasts and B cells, but strongly up-regulated upon 72h CD40-ligation in ALL with only barely detectable levels in CD40-activated normal B cells. We therefore propose, that prolonged induction of the FLIP S expression inhibits the onset of apoptosis despite high CD95 surface expression levels in BCP-ALL blasts. As an additional anti-apoptotic mechanism inhibiting the downstream effector caspases we demonstrated significant upregulation of the inhibitor of apoptosis protein (IAP) survivin in CD40-activated BCP-ALL (n=6) compared to the unstimulated control (632pg/ml±200pg/ml vs 180pg/ml±52pg/ml). Thus, we identified FLIP S as a CD40-regulated upstream anti-apoptotic element and concomitant downstream upregulation of survivin protein expression as critical mechanisms contributing to blast cell resistance to apoptosis.