Eftichia Stiakaki

Altered actin polymerization dynamics in various malignant cell types: evidence for differential sensitivity to cytochalasin B☆

Using the DNase I inhibition assay, fluorimetric measurements, and immunoblot analysis, we studied quantitatively changes in the actin polymerization dynamics in primary cultures of normal and malignant human lymphocytes, normal human endometrial cells, and in various leukemic and endometrial adenocarcinoma cell lines. The G/total-actin ratio of malignant cells was found to be 1.37 to 1.81-fold higher compared to normal cells, indicating that malignant cells express reduced amounts of polymerized actin. The above findings were corroborated by fluorescence measurements of the amounts of rhodamine-phalloidin-labeled F-actin in normal and neoplastic cells, which showed significantly lower F-actin content in malignant cell preparations. Moreover, the total actin content, as quantitated by the DNase I inhibition assay and by immunoblot analysis, was found to be significantly decreased in the primary cultures of malignant human lymphocytes and endometrial cells when compared to the total actin levels in corresponding normal cells. Proliferation and viability measurements of normal and neoplastic cells in culture, treated equally with cytochalasin B (CB), revealed an increased susceptibility of malignant cells to this anticytoskeletal agent. This was not due to increased CB incorporation in neoplastic cells, as indicated by 3H-CB uptake experiments. In addition, fluorescence microscopy, in the presence of graded concentrations of CB, showed destabilization of microfilaments in the poorly differentiated endometrial adenocarcinoma HEC-50 cells, compared to the well-differentiated Ishikawa cells. In conclusion, all investigated malignant cells are characterized by: (a) higher G/total-actin ratio; (b) decreased F- and total-actin content; and (c) lower resistance to CB treatment. These quantitatively determined parameters may represent potential biochemical indicators reflecting malignant transformation. Moreover, it seems worthwhile to explore whether or not the differential sensitivity of malignant cells to anticytoskeletal drugs may provide a valuable approach to the manipulation of malignant cells.

Comparative study of stemness characteristics of mesenchymal cells from bone marrow of children and adults

BACKGROUND AIMS Age-related changes that could affect the biologic features of mesenchymal stromal cells (MSC), such as a decrease in proliferation and osteoblast differentiation capacity and an increase of senescence markers and apoptosis, have been reported recently. The aim of this study was the evaluation of age-related characteristics and the correlation of age with the functional properties of MSC. METHODS The doubling time (DT), colony-forming unit–fibroblast (CFU-F) colonies and surface antigen expression of MSC isolated from bone marrow (BM) of children (C-MSC) were compared with those from adults (A-MSC). The expression of Oct-4 and Nanog transcripts and the relative telomere length were evaluated in both groups. RESULTS DT values were lower in C-MSC compared with A-MSC, and a higher CFU-F count was observed in children. However, the expression of Oct-4 and Nanog did not differ between C-MSC and A-MSC and was not correlated with the proliferative capacity. The telomere length was significantly higher in C-MSC compared with A-MSC. CONCLUSIONS These data suggest that childrens BM-derived MSC could be a more advantageous source of these cells for tissue engineering and cell therapy.

Prophylaxis with urokinase in pediatric oncology patients with central venous catheters.

This study evaluated the effects of urokinase in the prevention of central venous catheter (CVC)-related complications in children with malignancy. Fifteen patients with 16 CVCs (study group A) received an intraluminal application of urokinase (10,000 IU in each catheter lumen for 4 h) once a week. They were monitored prospectively with quantitative blood cultures and ultrasonography (color Doppler ultrasound of the great veins and echocardiography). The rate of complications was compared with that of 15 children with 19 CVCs without thromboprophylaxis, treated the previous year (control group B). The authors found a significantly lower incidence of CVC dysfunction (3/16 versus 13/19), no major thrombosis, fewer CVC-related bacteremias (2/16 versus 8/19), and a higher salvage of CVCs (1/16 versus 5/19 CVC removals due to persistent bacteremia) in the thromboprophylaxis group. Asymptomatic thrombosis rate was also lower (7/16 cases in group A versus 9/11 in group B when sonography was performed). No hemorrhagic complications were noted. Thromboprophylaxis with urokinase seems a safe and effective measure for reducing the rate of CVC-related complications.

Childhood Acute Lymphoblastic Leukemia and Indicators of Early Immune Stimulation: A Childhood Leukemia International Consortium Study

The associations between childhood acute lymphoblastic leukemia (ALL) and several proxies of early stimulation of the immune system, that is, day-care center attendance, birth order, maternally reported common infections in infancy, and breastfeeding, were investigated by using data from 11 case-control studies participating in the Childhood Leukemia International Consortium (enrollment period: 1980-2010). The sample included 7,399 ALL cases and 11,181 controls aged 2-14 years. The data were collected by questionnaires administered to the parents. Pooled odds ratios and 95% confidence intervals were estimated by unconditional logistic regression adjusted for age, sex, study, maternal education, and maternal age. Day-care center attendance in the first year of life was associated with a reduced risk of ALL (odds ratio = 0.77, 95% confidence interval: 0.71, 0.84), with a marked inverse trend with earlier age at start (P < 0.0001). An inverse association was also observed with breastfeeding duration of 6 months or more (odds ratio = 0.86, 95% confidence interval: 0.79, 0.94). No significant relationship with a history of common infections in infancy was observed even though the odds ratio was less than 1 for more than 3 infections. The findings of this large pooled analysis reinforce the hypothesis that day-care center attendance in infancy and prolonged breastfeeding are associated with a decreased risk of ALL.

Ochrobactrum anthropi bacteremia in pediatric oncology patients.

Ochrobactrum anthropi is an emerging pathogen in immunocompromised hosts, particularly in patients with indwelling catheters. We report the characteristics of 14 O. anthropi bacteremic episodes in 11 children with Hickman-type central catheters. Children presented with fever and nonspecific clinical manifestations. Bacteremia was successfully treated with antibiotics, but catheter removal was necessary to achieve cure in four cases.

The Role of the Methylenetetrahydrofolate Reductase 677 and 1298 Polymorphisms in Cretan Children with Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common form of malignancy in children. Recently, many studies have examined factors influencing both the susceptibility to ALL and the metabolism of widely used chemotherapeutic agents. These factors include, among others, single-nucleotide polymorphisms in various genes, such as the gene encoding for methylenetetrahydrofolate reductase (MTHFR), which has been proven polymorphic at the nucleotide positions 677 and 1298. Thirty-five children with ALL and 48 healthy adults of Cretan origin were genotyped for the presence of the MTHFR 677 and 1298 single-nucleotide polymorphisms. The possible correlation of the polymorphisms with the risk for ALL and the presence of methotrexate-induced toxicities were examined. No significant association between the MTHFR genotypes and the susceptibility to ALL was observed. A borderline statistically significant relationship was detected after methotrexate administration, between the C677T genotype (polymorphisms) and leukopenia (p = 0.050) and between the A1298C polymorphism and normal aspartate transaminase and alanine transaminase values (p = 0.065 and p = 0.053, respectively), which was strengthened for aspartate transaminase, after grouping the A1298A and A1298C genotypes together (p = 0.039). In our population the MTHFR C677T and A1298C polymorphisms are related with hematologic toxicity and hepatotoxicity, respectively, and could be suggested as prognostic factors for these adverse events.

Could cord blood be a source of mesenchymal stromal cells for clinical use

BACKGROUND Cord blood (CB) has long been regarded as an easily accessible source of hematopoietic progenitors suitable for transplantation, but its efficiency as a source of mesenchymal stromal cells (MSC) remains controversial. The aim of this study was to assess CB as a potential source of MSC, to determine the optimal culture requirements for CB MSC expansion and to compare their functional and immunophenotypic characteristics with bone marrow (BM) MSC from children. METHODS Mononuclear cells from 18 full-term CB samples and 23 BM samples from children were set in culture under MSC-inducing conditions. Their immunophenotypic characteristics were assessed by flow cytometry and their differentiation potential was evaluated. RESULTS Isolation of CB MSC was achieved in 25% of the samples cultured under optimal conditions: high initial cell concentration, fetal calf serum (FCS) enrichment of the culture medium, high FGF-2 concentration and high sample volume. Isolated CB MSC were morphologically similar to the ones derived from BM, but appeared late in culture. An adherent cell layer was formed and reached confluency in 34 days (passage 1; P1) and needed 55 days subsequently (from P1 to P2). CB MSC retained their characteristics for two successive passages. Immunophenotypic analysis showed no expression of CD34 and varying expression of CD45, ranging from 0% to 17.83%, and CD105, from 49% to 83%. CFU-F colonies developed in one case. DISCUSSION These findings suggest that CB cannot be considered a sufficient source of MSC for clinical use, although easily accessible. Further research should aim for alternative sources.

Ondansetron and tropisetron in the control of nausea and vomiting in children receiving combined cancer chemotherapy.

Ondansetron (Zofron, Glaxo) and tropisetron (Navoban, Sandoz) are selective serotonin (5HT3) antagonists that have proven very effective in the prevention of vomiting and nausea in adults and children receiving cancer chemotherapy. This study compared the efficacy of the two agents in the prevention of vomiting and nausea in children receiving chemotherapy for solid tumors and blood malignancies. A total of 23 children were studied in 205 chemotherapeutic cycles (116 one-day regimens and 89 multiple-day regimens). In 102 chemotherapeutic cycles the children received ondansetron as an antiemetic agent in a dose of 5 mg/m2 30 min before chemotherapy was given and then 4 mg/m2 every 8 h i.v. (group A) and in 103 cycles they received tropisetron in one dose of 0.2 mg/kg 24 h-1 i.v. (max dose 5 mg) 30 min before cytotoxic drugs administration every day they received chemotherapy (group B). The response was defined as complete in the absence of nausea and vomiting per 24 h of chemotherapy, as partial given the presence of 1-4 events of vomiting and/or nausea less than 5 h per 24 h, and as failure if there were more than 4 events of vomiting and/or nausea for more than 5 h per 24 h of chemotherapy. The response of the two groups was studied independently and depending on the degree of emetogenicity of the chemotherapeutic agents, which were divided into mildly, moderately, and highly emetogenic. The comparison of the two groups not taking into consideration the emetogenicity of the chemotherapeutic agents showed that ondansetron was more effective in 1-day regimens (P = .023), whereas the two agents were equally effective in multiple-day regimens (P = .2). The statistical analysis depending on the emetogenicity of the chemotherapeutic agents showed increased efficacy of ondansetron in mild (P = .017) and moderately emetogenic chemotherapeutic agents, whereas there was no difference in the highly emetogenic drug group. Ondansetron is found to be more effective than tropisetron in controlling acute nausea and vomiting in children receiving mild and moderately emetogenic chemotherapeutic drugs, although there is no difference in the efficacy of both antiemetic agents when highly emetogenic drugs are administered.

Properties and potential of bone marrow mesenchymal stromal cells from children with hematologic diseases

BACKGROUND Mesenchymal stromal cells (MSC) have become the focus of cellular therapeutics but little is known regarding bone marrow (BM) MSC derived from children. As MSC constitute part of BM stroma, we examined their properties in children with hematologic diseases. METHODS BM MSC from children with non-malignant hematologic disorders and acute lymphoblastic leukemia (ALL) were isolated and expanded. MSC were immunophenotypically characterized and their functional characteristics were assessed by CFU-F assay and cell doubling time calculation. Their ability for trilineage differentiation was verified by molecular and histochemical methods. Apoptosis was evaluated and clonal analysis was performed. RESULTS MSC were isolated from BM of all groups. They acquired the mesenchymal-related markers from the first passage, with a simultaneous decrease of hematopoietic markers. A very low percentage of apoptotic cells was detected in all passages. The proliferative and clonogenic capacity did not differ among groups, with the exception of ALL at diagnosis, in which they were defective. Histochemical and molecular analysis of differentiated MSC yielded characteristics for adipocytes, osteoblasts and chondrocytes. Clonal analysis in a number of BM samples revealed a highly heterogeneous population of cells within each clone. DISCUSSION MSC from BM of children with hematologic disorders, with the exception of ALL at diagnosis, can be isolated in sufficient number and quality to serve as a potential source for clinical applications.

Enhanced levels of the apoptotic BAX/BCL-2 ratio in children with acute lymphoblastic leukemia and high-risk features

It has been suggested that leukemia is characterized by an impaired balance between the proliferation of blood cells and their capacity to undergo apoptosis. The aim of this study was to examine the expression of key molecules related to apoptosis (BCL-2, BAX, FAS, FAS-L) in children with acute lymphoblastic leukemia (ALL). Measurement of BCL-2 and BAX mRNA was performed by quantitative real-time PCR, and membrane expression of FAS and FAS-L was assessed by flow cytometry in bone marrow mononuclear cells, both at diagnosis and at remission following induction chemotherapy. At diagnosis, increased levels of the apoptotic BAX/BCL-2 ratio were observed in children older than 10 years and with higher white blood cell counts. A DNA index < 1.16 was associated with increased BAX/BCL-2, both at diagnosis and at remission, and the del(9p) chromosome abnormality with increased BAX/BCL-2 at remission. The expression of the apoptotic receptor FAS was significantly higher at remission compared to diagnosis, which might reflect enhanced sensitivity of the leukemic clone to apoptosis and response to treatment. Altogether, our results highlight the association of apoptosis-related genes with clinical and cytogenetic prognostic parameters in pediatric ALL. A better understanding of the mechanisms and regulation of apoptosis should enable the design of novel targeted therapies for these patients.