George Th. Tsangaris

Hodgkin's disease in a child with sickle cell disease treated with hydroxyurea.

Hydroxyurea (HU) is an oral drug that ameliorates the clinical course of sickle cell anemia by ¯ increasing the levels of fetal hemoglobin and decreasing the adhesion of red cells to endothelium. Although HU has minimal short-term toxicity, few data are available about the long-term safety and the potential risk for carcinogenesis or leukemogenesis. An 8-year-old child with sickle cell / g 0- thalassemia who received HU treatment for painful crises is described. Six months after the initiation of the HU treatment he developed Hodgkins disease, lymphocyte predominance subtype. Chemotherapy induced a complete remission. After discontinuation of chemotherapy the painful crises recurred and bone marrow transplantation was decided at the age of 12 years. Two years after the bone marrow transplantation, the child is in complete remission without painful crises. Although the authors suggest that the development of Hodgkins disease is a coexisting event, questions arise about the safety of HU treatment in childhood.

Proteomic Analysis of Amniotic Fluid in Pregnancies with Turner Syndrome Fetuses

Turner syndrome, occurring in 1:2500 female births, is caused by the complete or partial absence of one X chromosome. Amniotic fluid supernatant proteins from five second trimester pregnancies with Turner syndrome fetuses and five normal ones were analyzed by 2DE, MALDI-TOF-MS, and Western blot. Serotransferin, lumican, plasma retinol-binding protein, and apolipoprotein A-I were increased in Turner syndrome, while kininogen, prothrombin, and apolipoprotein A-IV were decreased. Since differentially expressed proteins are likely to cross the placenta barrier and be detected in maternal plasma, proteomic analysis may enhance research for noninvasive prenatal diagnosis of Turner syndrome.

Low relapse rate in children with acute lymphoblastic leukemia after risk-directed therapy.

Purpose Even though acute lymphoblastic leukemia (ALL) responds well to chemotherapy, relapse remains the major problem. This study documents relapse and survival rates in 85 consecutive children (33 at good risk, 52 at high risk) with ALL diagnosed in 1991 to 1996. Patients and Methods Until 1993, the New York II protocol for the high-risk group and a combination of UKALL XI (induction) and R blocks of ALL-REZ BFM-87 (intensification) regimens for patients at good risk were used. To reduce toxicity, the protocols were subsequently modified. Consolidation treatment was the same for both groups, consisting of a lower cytarabine dose and methotrexate removal, whereas intensification was changed only for the high-risk group using the BB block of the NHL-BFM-90 protocol. The bone marrow clearance of leukemia was assessed on day 22, and minimal residual disease was detected using polymerase chain reaction analysis of Ig heavy-chain gene rearrangements. Results Seventy patients had common precursor B lineage ALL, six had pre-B-ALL, eight had T-ALL, and one had B-ALL. Two patients never achieved remission and died. Six patients died of consolidation-related complications. Four more patients died, two during induction and two during maintenance therapy. Two other children had relapse (2.3%), both of whom were treated with the earlier protocols and then underwent bone marrow transplantation. Four more children with morphologically complete remission showed minimal residual disease (which reached the levels of 1 leukemic cell among 10 2 –10 4 normal cells) with the use of clone-specific probes at several points of the study intervals, but never had relapse. The 5-year overall and event-free survival rates were 86% and 83%, respectively. The 5-year overall survival rates for good-risk and high-risk groups were 94% and 81%; the corresponding event-free rates were 91% and 78%. The 5-year event-free survival rate in the patients at high risk was significantly higher after the protocol change (90% vs. 65%, P = 0.04). Conclusions The modification proved to be effective in diminishing the therapeutic toxicity and improving the efficacy, mainly for the high-risk group.

Potential biomarkers for Turner in maternal plasma: possibility for noninvasive prenatal diagnosis.

Turner syndrome (TS) is the most common sex chromosome abnormality in females, caused by the complete or partial absence of one X chromosome. To identify biomarkers for TS, we compared the protein composition of maternal plasma samples from pregnant women with normal and TS fetuses, using a proteomic approach consisting of 2D-E separation and MS analysis for the identification of the differentially expressed proteins. Samples were routinely obtained in the second trimester of pregnancy, stored, and used after prenatal determination of the fetal karyotype. Nine proteins (C1S, CO3, CLUS, AFAM, HABP2, IGHA1, HPT, SHBG, and CD5L) were significantly increased in the plasma of women carrying TS fetuses, whereas KNG1, IGJ, and TTHY were decreased. Identified proteins were further evaluated by immunoblot analysis while functional network association was carried out to asses significance. The identification of specific biomarkers may facilitate the development of noninvasive prenatal diagnosis and improve our understanding of the pathology of TS. Nevertheless, testing a larger cohort of pregnant women is necessary to evaluate the relevance of the reported findings.

Proteomics studies of childhood pilocytic astrocytoma.

Childhood pilocytic astrocytoma is the most frequent brain tumor affecting children. Proteomics analysis is currently considered a powerful tool for global evaluation of protein expression and has been widely applied in the field of cancer research. In the present study, a series of proteomics, genomics, and bioinformatics approaches were employed to identify, classify and characterize the proteome content of low-grade brain tumors as it appears in early childhood. Through bioinformatics database construction, protein profiles generated from pathological tissue samples were compared against profiles of normal brain tissues. Additionally, experiments of comparative genomic hybridization arrays were employed to monitor for genetic aberrations and sustain the interpretation and evaluation of the proteomic data. The current study confirms the dominance of MAPK pathway for the childhood pilocytic astrocytoma occurrence and novel findings regarding the ERK-2 expression are reported.

Identification and characterization of Inc766, an inclusion membrane protein in Chlamydophila abortus-infected cells

We have identified the gene product of locus 766 in the transmembrane head region (TMH/Inc-region) in the Chlamydophila abortus genome by using mass spectrometry and a monoclonal antibody that reacted with the inclusion membrane. The identified protein at 32 kDa, termed Inc766, formed highly stable oligomers when solubilized in the absence of beta-mercaptoethanol. These oligomers were resistant to SDS, to heat denaturation and to 8M urea, but very sensitive to beta-mercaptoethanol, consistent with conformations resulting from protein-protein interactions stabilized through disulphide bonds. Mass spectrometry analysis of immunoprecipitated infected cell lysates indicated that a dimer at 56 kDa was the most prominent form in solution. Cross-linking with DSP provided supporting evidence for the formation of oligomers in situ. Inc766 was expressed at 20-24h post infection and its localization pattern in the extra-inclusion space was common in all C. abortus strains tested. Taken together, Inc766 displays unique biochemical and cellular features not encountered in other Incs from other Chlamydiaceae species. Future studies of the particular characteristics especially the interactive properties of Inc766 should contribute to our understanding of the relationship of the different chlamydial species with their respective hosts.

Proteomic studies of pediatric medulloblastoma tumors with 17p deletion.

CNS tumors are the leading cause of cancer-related death in children. Medulloblastoma is the commonest pediatric CNS malignancy, wherein, despite multimodal therapy with surgery, radiation, and chemotherapy, 5 year survival rates merely approach 60%. Until present, gene expression and cytogenetic studies have produced contradicting findings regarding the molecular background of the specific disease. Through integration of genomics, bioinformatics, and proteomics, the current study aims to shed light at the proteomic-related molecular events responsible for MBL pathophysiology, as well as to provide molecular/protein/pathway answers concerning tumor-onset. Experiments were performed on tissues collected at surgery. With 17p loss being the commonest chromosomal aberrance observed in our sample set, array-CGH were employed to first distinguish for 17p-positive cases. 2-DE coupled to mass spectrometry identification exposed the MBL-specific protein profile. Protein profiles of malignant tissues were compared against profiles of normal cerebellar tissues, and quantitative protein differences were determined. Bioinformatics, functional and database analyses, characterization, and subnetwork profiling generated information on MBL protein interactions. Key molecules of the PI3K/mTOR signaling network were identified via the techniques applied herein. Among the findings IGF2, PI3K, Rictor, MAPKAP1, S6K1, 4EBP1, and ELF4A, as part of the IGF network (implicating PI3K/mTOR), were founded to be deregulated.

Serum protein profiling of adults and children with Crohn disease.

Objectives: Crohn disease (CD) and ulcerative colitis (UC), known collectively as inflammatory bowel diseases (IBDs), are chronic immunoinflammatory pathologies of unknown aetiology. Despite the frequent use of biomarkers in medical practice, there is a relative lack of information regarding validated paediatric biomarkers for IBD. Furthermore, biomarkers proved to be efficacious in adults are frequently extrapolated to the paediatric clinical setting without considering that the pathogenesis of many diseases is distinctly different in children. In the present study, proteomics technology was used to monitor differences in protein expression among adult and young patients with CD, identify a panel of candidate protein biomarkers that may be used to improve prognostic–diagnostic accuracy, and advance paediatric medical care. Methods: Male and female serum samples from 12 adults and 12 children with active CD were subjected to 2-dimensional gel electrophoresis. Following the relative quantitation of protein spots exhibiting a differential expression between the 2 groups by densitometry, the spots were further characterized by matrix-assisted laser desorption tandem time-of-flight mass spectrometer. The results were confirmed by Western blot analysis. Results: Clusterin was found to be significantly overexpressed in adults with CD, whereas ceruloplasmin and apolipoprotein B-100 were found to be significantly overexpressed in children, indicating that the expression of these proteins may be implicated in the onset or progression of CD in these 2 subgroups of patients. Conclusions: Interestingly, we found a differential expression of several proteins in adults versus paediatric patients with CD. Undoubtedly, future experiments using a larger cohort of patients with CD are needed to evaluate the relevance of our preliminary findings.

Proteomic analysis of human reproductive fluids.

Fertilization, fetal development, and delivery depend upon a coordinated series of events in the oocyte, the embryo, and the supporting tissues and fluids. Proteomic techniques which are capable of identifying and characterizing multiple proteins simultaneously have added new dimensions to the field of human reproduction. Application of these high throughput methodologies in pregnancy‐related research has begun to provide a novel perspective on the biochemical pathways involved in pregnancy and its related disorders. Most of the existing research on human reproduction and gestation has focused on follicular fluid (FF) and amniotic fluid (AF). Proteome analysis of FF has yielded significant information relevant to oocyte maturation and quality. Studies performed on the protein content of AF cells and supernatant contributed to the comprehension of the underlying pathophysiology, clinical diagnosis of pregnancy‐related disorders and identification of relevant disease biomarkers. Although proteome technologies in reproduction research are not as yet widely applied, characterization of the proteome of reproductive fluids can be expected to significantly improve maternal healthcare in the future.

Targeted Downregulation of s36 Protein Unearths its Cardinal Role in Chorion Biogenesis and Architecture during Drosophila melanogaster Oogenesis

Drosophila chorion represents a model biological system for the in vivo study of gene activity, epithelial development, extracellular-matrix assembly and morphogenetic-patterning control. It is produced during the late stages of oogenesis by epithelial follicle cells and develops into a highly organized multi-layered structure that exhibits regional specialization and radial complexity. Among the six major proteins involved in chorion’s formation, the s36 and s38 ones are synthesized first and regulated in a cell type-specific and developmental stage-dependent manner. In our study, an RNAi-mediated silencing of s36 chorionic-gene expression specifically in the follicle-cell compartment of Drosophila ovary unearths the essential, and far from redundant, role of s36 protein in patterning establishment of chorion’s regional specialization and radial complexity. Without perturbing the developmental courses of follicle- and nurse-cell clusters, the absence of s36 not only promotes chorion’s fragility but also induces severe structural irregularities on chorion’s surface and entirely impairs fly’s fertility. Moreover, we herein unveil a novel function of s36 chorionic protein in the regulation of number and morphogenetic integrity of dorsal appendages in follicles sporadically undergoing aged fly-dependent stress.