Prevalence of antibodies against influenza A and B viruses in children in Germany, 2008 to 2010.

The prevalence of influenza A and B virus-specific IgG was determined in sera taken between 2008 and 2010 from 1,665 children aged 0-17 years and 400 blood donors in Germany. ELISA on the basis of whole virus antigens was applied. Nearly all children aged nine years and older had antibodies against influenza A. In contrast, 40% of children aged 0-4 years did not have any influenza A virus-specific IgG antibodies. Eightysix percent of 0-6 year-olds, 47% of 7-12 year-olds and 20% of 13-17 year-olds were serologically naïve to influenza B viruses. By the age of 18 years, influenza B seroprevalence reached approximately 90%. There were obvious regional differences in the seroprevalence of influenza B in Germany. In conclusion, seroprevalences of influenza A and influenza B increase gradually during childhood. The majority of children older than eight years have basal immunity to influenza A, while comparable immunity against influenza B is only acquired at the age of 18 years. Children aged 0-6 years, showing an overall seroprevalence of 67% for influenza A and of 14% for influenza B, are especially at risk for primary infections during influenza B seasons.

Prevalence of antibodies against hepatitis A virus among children and adolescents in Germany.

Since hepatitis A virus (HAV) infection during childhood is mostly asymptomatic, only seroprevalence studies can provide reliable information on incidence of HAV infection in children. The prevalence of anti-HAV antibodies was determined in sera taken in 2008 to 2010 from 1,645 children aged 0-17 years and in sera taken in 2010-2011 from 400 adult blood donors in Germany. For examination of trend over time, 715 sera collected between 1999 and 2006 from children at the age of 0-17 years within the federal state Thuringia were included. Antibody testing was carried out using the test kits ETI-AB-HAVK PLUS and ETI-HA-IGMK PLUS from DiaSorin. In children, the overall prevalence of antibodies was 10.8 %. After the seroprevalence declined from 8.8 % among the 0-2 year-olds to 2.4 % among the 3-4 year-olds, there was a significant increase to 20.5 % in the group of the 15-17 year-olds. Boys had with 12.7 % a significantly higher seroprevalence of anti-HAV antibodies compared to 8.8 % among girls. In adult blood donors, there was a HAV seroprevalence of 19.3 %. The likelihood of past infection or immunization within the age groups of children from 0 to 12 years differed significantly from that of adults. In conclusion, in Germany, only a small number of HAV infections occur in children, especially up to the age of 12 years. The proportion of susceptible children is greater than the proportion of susceptible adults. Thus, during outbreaks, the rate of infection among children would usually be higher than the rate among adults.

Response of children with stage IV soft tissue sarcoma to topotecan and carboplatin: a phase II window trial of the cooperative soft tissue sarcoma group.

To investigate antitumor activity and toxicity associated with combined topotecan and carboplatin treatment in children and adolescents with metastasized, untreated soft tissue sarcoma (STS).Patients (n=34) less than 21 years old and untreated, stage IV STS. Patients were treated with topotecan (1 mg/m²/d for 4 days) and carboplatin (150 mg/m²/d for 4 days) (TC course) during week 1 and 4 of a chemotherapy window trial, which was followed by chemotherapy and local therapy from week 6 on. We evaluated the side effects, toxicity and tumor response (using RECIST criteria) 6 weeks after starting the 2 TC chemotherapy courses.The objective response rate (ORR) was 38% (n=13 patients with a partial response (PR)), and a stable disease (SD) was reached in 11 cases. No patient showed a complete response (CR) of all metastatic lesions, although 1 patient showed a CR of the target lesion. 2 patients died of progress of disease (PD). Toxicity was mainly hematological (grade III/IV toxicity 79%), and nonhematological toxicities mainly included infection, fever, nausea,and vomiting. Regarding adverse events, 4 probable and 8 possible events related to study medication occurred among the 66 courses of TC.In conclusion, TC was potent against high-risk STS, but results and toxicity data were not superior to former published monotherapeutic topotecan therapies.

[GATA1-mutation associated leukemia in children with trisomy 21 mosaic]. / GATA1-Mutations-assoziierte Leukämien bei Kindern mit Trisomie 21-Mosaik.

Mutations of the hematopoietic transcription factor GATA1 (GATA1s) are pathognomonic in newborn with transient leukemia and children with Down syndrome and myeloid leukemia (ML-DS). Both TL and ML-DS can also occur in children with trisomy 21 mosaic.Between 2002 and 2011, 15 newborns and infants were diagnosed with DS mosaic. 9 of them presented with TL and 8 children suffered from ML-DS; 2 of them with a history of TL. In children without stigmata the special morphology and immunophenotype of blasts triggered the screening for GATA1 mutation and trisomy 21 mosaic.All newborns with TL achieved complete remission (CR). Due to clinical symptoms caused by the leukemic blasts, in 3 children low-dose cytarabine was applied. 1 patient died due to cardiac defect. In all patients GATA 1 s was confirmed. 6 children with ML-DS were initially treated according the AML-BFM protocol. After ML-DS was confirmed, therapy was continued with the intensity reduced schedule according to the ML-DS 2006 protocol. All children are still in CR (follow-up 1.8-7 years, median 2.7 yrs). 2 children with unknown trisomy 21 mosaic were diagnosed as acute megakaryoblastic leukemia (AMKL) and treated according the high risk arm of the AML-BFM 2004 including allogeneic stem cell transplantation in one child). GATA1 mutation was identified retrospectively. Both children are alive in CR.GATA1s associated leukemia has to be excluded in all young children with AMKL (<5 years old) to prevent overtreatment. Treatment with reduced intensity seems sufficient in children trisomy 21 mosaic and ML-DS.

Redirected behavior in learning tasks: the commercial laying hen (Gallus gallus domesticus) as model.

Redirected behaviors occur when some course of action is thwarted or inhibited (frustration). They also occur as adjunctive behaviors in operant conditioning tasks, where they might reflect frustration about unrewarded responses. Because frustration is associated with stress, which could interfere with learning and memory, we studied whether the occurrence of redirected behavior is correlated with learning success in a series of visual-cue discrimination tasks. Eleven hens, aged 34 wk, were tested on acquisition, reversal, extinction, and relearning of a simple visual discrimination task. The experimenters randomly assigned red and blue cardboard discs as discriminative stimuli. A correct response was recorded when a hen pecked at the correct disc. The learning criterion was 90% correct responses in 20 trials in 2 consecutive task sessions. The following data were documented: number of pecks needed to achieve the learning criterion, latency in choosing, pecks at the experimenter, and pecks at the surroundings. The behavioral responses were analyzed using linear mixed model ANOVA. Redirected pecking at the surroundings was a significant indicator of learning failure in that the more the hens performed this behavior, the more trials they needed to complete the discrimination tasks (P = 0.012). The number of pecks at the experimenter during the tasks significantly influenced learning success (P = 0.020), with hens directing more pecks at the experimenter during reversal, reaching the learning criterion in fewer trials (P = 0.027). The more the hens pecked at the experimenter during acquisition and extinction, however, the more trials they needed to meet the learning criteria (acquisition: P = 0.048; extinction: P = 0.003). Thus, laying hens are susceptible to the effects of frustration as measured in terms of redirected pecking elicited by operant procedures in visual discrimination tasks. In general, any situation in which a desirable goal is obstructed or an expected reward is omitted may lead to frustration-related activities, such as redirected behavior, which could in turn lead to abnormal behavior and welfare issues for the animals.

[Desmopressin testing in children with von Willebrand syndrome in haemostaseologic centers of Saxonia, Saxonia-Anhalt and Thuringia]. / Desmopressin-Tests bei Kindern mit von-Willebrand-Syndrom in den Gerinnungszentren Sachsens, Sachsen-Anhalts und Thüringens.

The influence of desmopressin on hemostasis is mediated by the release of von Willebrand factor and of coagulation factor VIII from vascular endothelium. The necessity of testing desmopressin effectiveness on hemostasis is a matter of controversy and the performance of the test is not yet standardized. For this reason the desmopressin tests in 114 children with von Willebrand syndrome (type 1, n=98; type 2A, n=12; type 2M, n=2; type 2N, n=2) carried out in 7 paediatric haemostaseologic centers were retrospectively analyzed. The effectiveness of desmopressin was assessed using defined response criteria. As expected, the test performance showed a wide variation among the centers. In 99 children desmopressin was given intravenously as a short infusion at a dosage ranging from 0.25 to 0.41 microg/kg and in 15 intranasally at an absolute dose of 40 to 300 microg. The points of time for blood taking after desmopressin application ranged from 0.5 to 12 h. The absent desmopressin response in 7 patients (6%) and the partial response in 15 indicate the necessity of testing desmopressin effectiveness before the first therapeutic use. The application of desmopressin was well tolerated by the patients.

BCRP gene expression is associated with a poor response to remission induction therapy in childhood acute myeloid leukemia.

Breast cancer resistance protein (BCRP), also known as mitoxantrone resistance protein (MRX) or placenta ABC protein (ABC-P), is the second member of the ABCG subfamily of ABC transport proteins (gene symbol ABCG2). Transfection and enforced expression of BCRP in drug-sensitive cells confers resistance to mitoxantrone, doxorubicin, daunorubicin and topotecan. In this study the expression of BCRP gene was measured using TaqMan real-time PCR in 59 children with newly diagnosed AML. Nine patients were also analyzed in relapse. The median of BCRP gene expression was more than 10 times higher in patients who did not achieve remission after the first phase of chemotherapy (n = 24) as compared to patients who did achieve remission at this stage (n = 21; P = 0.012). In first relapse the expression of the BCRP gene was higher than at diagnosis (P = 0.038). Although high levels of BCRP gene expression were more frequent in subtypes of AML with a favorable prognosis, we found that within both risk groups (high and low risk), patients who expressed high levels of BCRP had a worse prognosis (P = 0.023). Our results strongly suggest that the expression of the BCRP gene reduces the response to chemotherapy in AML and that BCRP expression is higher at the time of relapse.

Vascular endothelial growth factor in newly diagnosed and recurrent childhood acute lymphoblastic leukemia as measured by real-time quantitative polymerase chain reaction.

PURPOSE: Overexpression of vascular endothelial growth factor (VEGF) is associated with increased angiogenesis, growth, and metastasis in solid tumors, but to date the significance of VEGF in leukemia has received only limited attention. Therefore, this study examined the cellular VEGF levels in 31 newly diagnosed and 22 recurrent cases of childhood acute lymphoblastic leukemia (ALL). EXPERIMENTAL DESIGN: VEGF was determined with real-time quantitative PCR methods. Kaplan-Meier statistical analyses were conducted for the relapse-free intervals and the overall survival times. The groups were compared by log-rank and rank-sum tests. RESULTS: The VEGF levels were significantly higher in recurrent ALL compared with newly diagnosed ALL (28.0 versus 3.1 units; P = 0.001). Kaplan-Meier estimates were conducted to analyze the prognostic value of VEGF levels in newly diagnosed ALL with regard to the relapse-free intervals and the overall survival times. In this analysis, the median relapse-free interval of patients with low VEGF levels was more than 10 years, whereas the relapse-free interval of patients with high VEGF expression was only 1.2 years. The median overall survival time for the collective with low VEGF levels was >10 years, whereas the survival of the group of patients with high VEGF levels was 3.9 years. This difference was not statistically significant. This may be attributable to the small number of patients involved. CONCLUSION: Our data suggest that VEGF may play an important role in the pathophysiology of ALL. The expression of VEGF raises the possibility of using angiogenesis inhibitors as a novel therapeutic strategy in childhood ALL.

Expression of a novel double-mutant dihydrofolate reductase-cytidine deaminase fusion gene confers resistance to both methotrexate and cytosine arabinoside.

A novel fusion gene consisting of the open reading frame of a double-mutant (Phe22-Ser31) dihydrofolate reductase (dmDHFR) cDNA fused to the open reading frame of cytidine deaminase (CD) was constructed and characterized for the purpose of conferring simultaneous resistance to methotrexate (MTX) and cytosine arabinoside (ara-C). The kinetic properties of purified recombinant dmDHFR-CD fusion protein were compared with those of purified CD and dmDHFR. The fusion protein was found to retain enzymatic properties of both dmDHFR and CD, in that the Km and Kcat values of purified dmDHFR-CD protein were found to be virtually identical to those of CD and dmDHFR alone. Retrovirus-mediated expression of dmDHFR-CD in NIH 3T3 cells conferred significant resistance (10- to 12-fold) against MTX and ara-C, compared with mock- and single gene-infected cells and the level of resistance obtained was similar to that of cells expressing both CD and dmDHFR from a retroviral bicistronic vector. Infection of mouse bone marrow cells with the dmDHFR-CD construct also showed high levels of resistance to MTX and ara-C in a CFU-GM assay. This fusion protein confers resistance to two antineoplastic agents that differ in their mechanism of action, and may be useful in the design of gene transfer strategies for protection of target cells against multiple drugs. Since high-dose ara-C and MTX are used in the treatment of lymphomas, this vector may be of value in protecting human hematopoietic progenitor cells from the toxicity of these antimetabolites.

Determination of DNA topoisomerase II in newly diagnosed childhood acute lymphoblastic leukemia by immunocytochemistry and RT-PCR.

The expression of DNA-topoisomerase II was analysed at the protein level in newly diagnosed cases of acute lymphoblastic leukemia (ALL) in children. Blast cells obtained from 81 children with untreated ALL were determined by means of immunocytochemistry. Of the ALL, 49 (60%) were positive for topoisomerase II and 32 (40%) negative. No significant correlation was found between the expression of topoisomerase II and the relapse rate or relapse-free intervals. These results were substantiated by determining the topoisomerase II mRNA expression in a collective of 21 patients by semiquantitative PCR. The PCR-assay and immunocytochemistry corresponded in 13 of 21 cases (62%).

Messenger RNA expression of resistance factors and their correlation to the proliferative activity in childhood acute lymphoblastic leukemia.

In this report we analyzed the mRNA expression of the resistance-related enzymes DNA topoisomerase II (Topo II), thymidylate synthase (TS), glutathione S-transferase-pi (GST-pi) and glutathione peroxidase (GP) in childhood acute lymphoblastic leukemia (ALL) and their correlation to the proliferative activity, determined by Ki-67. RNA of blast cells from 54 children with untreated ALL were examined by dot blot hybridization. We found a significant positive correlation between Topo II and TS and cell proliferation. No significant correlation was detected between the mRNA expression of the glutathione-dependent enzymes GST-pi or GP and Ki-67. The results were substantiated by a semiquantitative RT-PCR-assay and by immunocytochemistry.

Resistance factors and proliferative activity in childhood acute nonlymphoblastic leukemia.

Thirty-four children with newly diagnosed acute nonlymphoblastic leukemia were analysed for the expression of P-glycoprotein (P-170), glutathione S-transferase-pi, (GST-pi) topoisomerase II (Topo II) and the proliferation antigen Ki-67 by the streptavidin-biotin-peroxidase method. Overexpression of P-170 was present in 26 cases (76%) and GST-pi overexpression was seen in 11 patients (32%). Down regulation of Topo II was found in 16 patients (47%) and Ki-67 positive cells (>5%) were detectable in 9 patients (26%). The remission rate was not influenced by P-170 or GST-pi expression, whereas patients with down regulation of Topo II or low Ki-67 expression had lower remission rates. The data were not significant. The probability of continuous first remission (CR) was lower in patients with P-170 expression (p=0.09). A significantly lower probability of CR was also seen in patients with low proliferative activity (p=0.03, log-rank test). The expression of the resistance proteins and the proliferative activity were found to be independent of the clinical parameters age, sex, FAB-type, and initial white blood cell count.

Prognostic-significance of protein-kinase-C in newly-diagnosed childhood acute lymphoblastic-leukemia.

The expression of protein kinase C (PKC) was analysed in newly diagnosed cases of non-B acute lymphoblastic leukemia (NB-ALL) in children. Blast cells obtained from 104 children with untreated NB-ALL were analysed using the streptavidin-biotin-peroxidase method and the antibody MC5. Of the 104 patients with NB-ALL, 48 (=46%) showed positive staining for PKC. The relapse rate was significantly higher in NB-ALL with expression of PKC than in leukemia without expression of PKC (p=0.008). The relapse-free intervals in the PKC-negative leukemias were significantly longer than in cases of PKC-positive leukemias (p=0.004). The overall survival times were also longer in NB-ALL without expression of PKC than in ALL with PKC-expression (p=0.03). The results of univariate and multivariate analyses demonstrate that in addition to the clinical prognostic indicators PKC expression is a significant prognostic factor for relapse rate, relapse-free intervals, and overall survival times in NB-ALL of children.

Resistance-related proteins in initial and relapsed childhood acute lymphoblastic leukemia.

One hundred and eleven children with initial ALL and 28 children with relapsed ALL were analyzed immunohistochemically for expression of resistance-related proteins. P-glycoprotein (P-170) was found in 35% (initial ALL) vs 54% (relapsed ALL; p=0.07), glutathione S-transferase-pi (GST) in 49% vs 68% (p=0.07), thymidylate-synthase (TS) in 42% vs 64% (p=0.03), dihydrofolate-reductase (DHFR) in 20% vs 32% and metallothionein (MT) in 33% vs 32% of the cases. In initial ALL, the resistance proteins P-170 and GST were expressed more frequently in patients who relapsed under therapy and the frequency was similar to relapsed ALL. These results indicate that P-170 and GST-pi were already present before treatment. In contrast, expression of TS increased during treatment.

Detection of fos, jun and ras in newly-diagnosed childhood acute lymphoblastic-leukemia by immunocytochemistry and PCR.

The expression of the oncogenes c-fos, c-jun and c-pan-ras was analyzed at the protein level in newly diagnosed cases of acute lymphoblastic leukemia (ALL) in children. Blast cells obtained from 104 children with untreated ALL were determined by the streptavidin-biotin-peroxidase method and specific antibodies. Of the ALL 52 cases were positive for Fos (50%), 65 for Jun (63%), and 22 for Ras (21%). Fos-positive ALL had a significantly higher relapse frequency (p=0.0002). A similar trend was found for Jun-positive ALL (p=0.073). In contrast, the expression of Ras showed no significant correlation with the relapse rate (p=0.98). Corresponding results were obtained for the relapse-free intervals. The relapse-free intervals were higher in patients with Fos- and Jun-positive leukemic cells than in patients with negative tumor cells (Fos: p<0.001; Jun: p=0.09; Ras: p=NS, log-rank test). In order to substantiate the significant correlation of c-fos-protein expression with the relapse rate the c-fos-mRNA expression was investigated in a collective of 20 patients by semi-quantitative PCR. The PCR-assay and immunocytochemistry corresponded in 14 of 20 cases (70%). The results of the PCR-assay demonstrated also a trend for increased c-fos-mRNA expression and higher relapse rate.

The anti-malarial artesunate is also active against cancer.

Artesunate (ART) is a semi-synthetic derivative of artemisinin, the active principle of the Chinese herb Artemisia annua. ART reveals remarkable activity against otherwise multidrug-resistant Plasmodium falciparum and P. vivax malaria. ART has now been analyzed for its anti-cancer activity against 55 cell lines of the Developmental Therapeutics Program of the National Cancer Institute, USA. ART was most active against leukemia and colon cancer cell lines (mean GI50 values: 1.11+/-0.56 microM and 2.13+/-0.74 microM , respectively). Non-small cell lung cancer cell lines showed the highest mean GI50 value (25.62+/-14.95 microM) indicating the lowest sensitivity towards ART in this test panel. Intermediate GI50 values were obtained for melanomas, breast, ovarian, prostate, CNS, and renal cancer cell lines. Importantly, a comparison of ART's cytotoxicity with those of other standard cytostatic drugs showed that ART was active in molar ranges comparable to those of established anti-tumor drugs. Furthermore, we tested CEM leukemia sub-lines resistant to either doxorubicin, vincristine, methotrexate, or hydroxyurea which do not belong to the N.C.I. screening panel. None of these drug-resistant cell lines showed cross resistance to ART. To gain insight into the molecular mechanisms of ART's cytotoxicity, we used a panel of isogenic Saccaromyces cerevisiae strains with defined genetic mutations in DNA repair, DNA checkpoint and cell proliferation genes. A yeast strain with a defective mitosis regulating BUB3 gene showed increased ART sensitivity and another strain with a defective proliferation-regulating CLN2 gene showed increased ART resistance over the wild-type strain, wt644. None of the other DNA repair or DNA check-point deficient isogenic strains were different from the wild-type. These results and the known low toxicity of ART are clues that ART may be a promising novel candidate for cancer chemotherapy.

High-dose chemotherapy (HDC) and autologous hematopoietic stem cell transplantation (ASCT) as salvage therapy for relapsed osteosarcoma.

In this report, we describe our experience with high-dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT) in 15 children with relapsed osteosarcoma who were treated by members of the Cooperative Osteosarcoma Study Group. Eight patients received HDC after the first relapse, six patients after the second relapse and one after the sixth relapse. Thirteen patients underwent HDC and ASCT in complete remission and two patients had macroscopic tumor residues. Seven patients received HDC based on melphalan and etoposide. Four of these patients were treated with additional carboplatinum. Two patients received carboplatinum, etoposide, and thiotepa or cyclophosphamide. In six patients double HDC was performed. In all six of these, the first HDC consisted of thiotepa/ cyclophosphamide. The second regimens included melphalan/etposide (two patients), melphalan/etposide/ carboplatinum (one patient), and melphalan/busulfan (one patient). Three of the 15 patients died of toxic complications. Eight patients developed further relapses, two patients showed persistent disease, and two patients are presently in continuous complete remission. The probability of relapse-free survival was 0.20 +/- 0.12 within a median follow-up (MFU) of 8 months and the probability of overall survival was 0.29 +/- 0.12 after a MFU of 16 months. In conclusion, utilization of HDC and ASCT in this patient group did not significantly improve the treatment outcome compared to conventional relapse therapy.

Expression of the retinoblastoma tumor suppressor gene (RB-1) in acute leukemia.

In this report we review current studies concerning the RB-1 gene expression in acute leukemias. The RB-1 gene was analyzed in several studies by protein-, RNA and DNA-techniques in acute lymphoblastic leukemia (ALL) as well as in acute myelogenous leukemia (AML). The frequency of RB-1 inactivation in ALL-patients ranged between 30% and 64% in several studies. Structural abnormalities of the RB-1 gene were reported in 18% of ALL-patients and in 27% of Philadelphia chromosome-positive ALL, respectively. The proportion of AML-patients with absent RB-1 protein expression ranged between 19% and 55%. Structural RB-1-abnormalities in AML were predominantly reported in leukemias with monocytic differentiation. Furthermore, the prognostic value of an abnormal RB-1 gene expression was also estimated in some studies. In childhood ALL RB-1 inactivation was reported to have prognostic significance while in contrast, in another study on adults no prognostic value of RB-1 was found. In 4 out of 5 documented studies AML-patients with RB-1 inactivation generally had a poorer prognosis. In conclusion, RB-1 inactivation is frequently observed in acute leukemia. The prognostic value of low RB-1 expression is controversial but the majority of published studies found low RB-1 expression to be a negative prognostic predictor, in acute leukemia.

Protein expression profile of newly diagnosed acute lymphoblastic leukemia in children developing relapses.

The purpose of this investigation was to evaluate the expression profile of proteins involved in children with newly diagnosed acute lymphoblastic leukemia (ALL) children who are developing relapses. For this reason, the expressions of 10 proteins including proto-oncogene and tumor suppressor gene products, proliferative factors and resistance parameters in 104 initial cases of childhood ALL were analyzed and the proteins correlated with ALL patients who experienced relapses. Applying immunocytochemical assays, we found that 4 out of the 10 parameters revealed a relationship to developing relapses (Fisher's exact tests). These were the oncogene product Fos (p=0.002), the drug resistance proteins glutathione S-transferase (p=0.008) and P-glycoprotein (P-pg/MDR1) (p=0.07) and protein kinase C (p=0.01). By means of hierarchical cluster analysis, we were able to show that the patients could be separated according to their protein expression profile into clusters consisting of patients whose ALL relapsed later and of patients who did not show relapses in the future.