Apoptosis induced by synthetic compounds containing a 3,4,5-trimethoxyphenyl fragment against lymphoid immature neoplasms.

T-cell acute lymphoblastic leukemia is an aggressive hematological malignancy originating from the malignant transformation of progenitor T cells at different stages of development. The treatment causes severe adverse effects and is associated with relapses and high morbidity and mortality rates. The present study aimed to evaluate the cytotoxic activity of 28 new compounds containing 3,4,5-trimethoxyphenyl analogues on hematological neoplastic cells lines. Cytotoxicity screening by the MTT method revealed that compound 1d was the most promising. Cell viability of neoplastic cells decreased in a concentration- and time-dependent manner, with compound 1d not causing hemolysis or reducing peripheral blood mononuclear cells viability, suggesting a selective cytotoxicity. We also suggested that compound 1d induced apoptotic-like cell death with mitochondrial involvement in Jurkat cells.

Background levels of trace elements in brown and red seaweeds from Trindade, a remote island in South Atlantic Ocean.

Trace elements in organisms are normally higher in well-developed coastal areas than on oceanic islands. Few studies have used seaweeds as their sentinels on islands. This study established background levels of trace elements (As, Cd, Pb, Zn, Cu and Hg) for four seaweed species (Dictyopteris delicatula and Canistrocarpus cervicornis, brown algae; Ceratodictyon variabile and Palisada perforata, red algae) from Trindade, an oceanic Brazilian island, and verified potential differences associated to distinct environmental conditions. Spatial differences were not detected for As, Hg and Cd in samples, although the highest concentrations of these elements were observed in brown seaweeds. The highest Zn, Pb and Cu concentrations in seaweeds from the only inhabited beach may be a signal of the onset of human footprints on this still pristine, remote island. By comparison with background described in the literature, concentrations of trace elements in seaweeds were low, thus, allowing them to be considered reference levels.

Evaluation of leucocytes from sputum samples of pulmonary tuberculosis patients using flow cytometry.

SETTING: Information about the sputum cells of pulmonary tuberculosis (PTB) patients is scarce. The analysis of sputum cells using optical microscopy (OM) is a well-established method, but it has some serious limitations. OBJECTIVE: To establish a new flow cytometry (FC) protocol for the leucocyte evaluation of sputum samples from PTB patients. DESIGN: A new FC protocol using 0.1% dithiothreitol and 0.5% paraformaldehyde was developed to fluidise sputum samples and kill Mycobacterium tuberculosis, respectively, to allow the analysis of sputum samples collected from TB patients. The protocol was validated by comparing it with OM, and the cellularity of 30 sputum samples from patients with PTB was evaluated. RESULTS: The comparison between leucocyte subsets analysed using OM and FC showed agreement. Immunophenotyping of leucocytes from sputum samples showed that neutrophils (95.7%) comprised the largest proportion of sputum cells, followed by monocytes/macrophages (2.6%) and lymphocytes (1.6%). Among the total T-lymphocytes (100%), 12.3% were T-helper cells, 24.1% were cytotoxic T-cells and 62.9% were gamma/delta T; none of the T lymphocytes had the CD4+/CD8+ phenotype. CONCLUSION: FC is a useful method for evaluating the different subtypes of leucocytes present in the sputum samples of PTB patients.

Normal variation of bone marrow B-cell precursors according to age - reference ranges for studies in myelodysplastic syndromes in Brazil.

BACKGROUND: Normal B lymphoid maturation occurs in bone marrow (BM) throughout life, but immature B-cell progenitors (BCPs) are more numerous in children than in adults. To assess the normal values according to age became important as BCPs are decreased in myelodysplastic syndromes and have been considered an important diagnostic and prognostic feature in these clonal disorders. METHODS: in a multicenter retrospective study from the Brazilian Group of Flow Cytometry we analyzed the variation of BCPs in normal BM according to age and technical peculiarities of each laboratory. We analysed of 45 BM donors and 89 cases examined for elucidation of transitory reactive cytopenias presenting a normal BM immunophenotyping. BCPs were enumerated as CD19+ /CD34+ /CD45dim /CD10+ cells (panel 1) or CD19+ /CD34+ /CD45dim cells (panel 2) among the total nucleated non-erythroid cells and as percentage of CD34+ cells. RESULTS: we included 134 cases. Panel 1 was applied in 88 cases and panel 2 was used in 46. Age range: 10 months to 89 years. In a multiple regression, % BCPs/total nucleated cells was an exponential function of age. Age explained alone 49.4% of the variance, while 'panel used' explained 1.8% and 'laboratory' explained 0.7%. Age explained only 24.9% of the variance of BCPs/CD34+ cells. CONCLUSIONS: in normal individuals, BM B-cell precursors varied mainly according to age, but were also dependent on technical peculiarities of operators and equipments. Analysis by phenotype and as percentage of total nucleated cells was more accurate and less susceptible to variation than evaluating % BCPs/total CD34+ cells. © 2017 International Clinical Cytometry Society.

Screening for the SMIM1*64_80 del Allele in blood donors in a population from Southern Brazil.

BACKGROUND AND OBJECTIVES: Serological screening for the Vel- phenotype is complex given the large individual variation in the levels of expression of the Vel antigen, and the polyclonal anti-human sera of immunised persons, when available, show heterogeneous reactivity levels. Studies of the SMIM1 gene have enabled the development of several molecular methodologies that will be crucially important for the screening of different populations, including Brazilians. To evaluate the deletion of 17 bp in the SMIM1 gene in a population from the south of Brazil, 448 unrelated blood donors from 7 regions comprising the haemotherapy network in the state of Santa Catarina were evaluated between August 2011 and March 2014. MATERIALS AND METHODS: DNA samples from these donors were analysed employing a 5' nuclease real-time polymerase chain reaction (PCR) assay targeting the 17 bp deletion in the SMIM1 gene. RESULTS: Among the 448 samples analysed, 10 (2·23%) harboured the 17 bp deletion of the gene SMIM1, and all were heterozygote for the SMIM1*64_80 del allele. CONCLUSION: The allelic frequency found differed from those observed in other Caucasian populations. This difference can be explained by the ethnic make-up of each Caucasian population. The data obtained are important to characterise the correct phenotype of the donor as the serological assay results are not reliable due to variations in the expression intensity of the Vel antigen in heterozygote donors for the SMIM1*64_80 del allele. Moreover, the tool used in this study is of great value for identifying a donor Vel- phenotype and supplying a possible need for transfusion.

Molecular Cytotoxic Mechanisms of 1-(3,4,5-Trihydroxyphenyl)-dodecylbenzoate in Human Leukemia Cell Lines.

Recent studies have shown that gallic acid and its alkylesters induce apoptosis in different cell lines. Since new compounds with biological activity and less cytotoxicity to normal cells are necessary for cancer therapy, the aim of this study was to evaluate the cytotoxic effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. The cell viability was determined by MTT method. The apoptosis induction was assessed by bromide and acridine orange staining and by Annexin V-FITC Apoptosis Detection kit. The cell cycle analysis was carried out by flow cytometry using propidium iodide. Cytometric analysis was also performed to evaluate the expression of the following proteins: AIF, p53, Bcl-2 and Bax. The mitochondrial potential was also assessed by flow cytometry using MitoView633 kit. The results showed that the compound significantly reduced the cell viability of K562 and Jurkat cells in a concentration and time dependent manner (IC50 of 30 µM). The compound induced cell cycle arrest in G0/G1phase and significantly increased the proportion of cells in the sub-G0/G1phase. Apoptosis was confirmed by the sight of morphological characteristics of apoptosis and by phosphatidylserine externalization (73.47±5.71% of cells expressing annexin). The results also showed that the compound promotes a modification in Bax:Bcl-2 ratio and increases p53 expression. Thus, it is possible to conclude that 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate induces apoptosis by inhibiting the antiapoptotic protein Bcl-2 and by increasing the release of AIF, Bax and p53. In addition, it blocks the cell cycle at G0/G1, stopping cell proliferation. So far, the results suggest that this compound may have a potential therapeutic effect against leukemia cells.

Polymer films with surfaces unmodified and modified by non-thermal plasma as new substrates for cell adhesion.

The surface properties of biomaterials, such as wettability, polar group distribution, and topography, play important roles in the behavior of cell adhesion and proliferation. Gaseous plasma discharges are among the most common means to modify the surface of a polymer without affecting its properties. Herein, we describe the surface modification of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) films using atmospheric pressure plasma processing through exposure to a dielectric barrier discharge (DBD). After treatment the film surface showed significant changes from hydrophobic to hydrophilic as the water contact angle decreasing from 95° to 37°. All plasma-treated films developed more hydrophilic surfaces compared to untreated films, although the reasons for the change in the surface properties of PS and PMMA differed, that is, the PS showed chemical changes and in the case of PMMA they were topographical. Excellent adhesion and cell proliferation were observed in all films. In vitro studies employing flow cytometry showed that the proliferation of L929 cells was higher in the film formed by a 1:1 mixture of PS/PMMA, which is consistent with the results of a previous study. These findings suggest better adhesion of L929 onto the 1:1 PS/PMMA modified film, indicating that this system is a new candidate biomaterial for tissue engineering.

Newly diagnosed adult AML and MPAL patients frequently show clonal residual hematopoiesis.

Adult acute myeloid leukemia (AML) is a highly heterogeneous stem cell malignancy characterized by the clonal expansion of immature myeloid precursors. AML may emerge de novo, following other hematopoietic malignancies or after cytotoxic therapy for other disorders. Here, we investigated the clonal vs reactive nature of residual maturing bone marrow cells in 59 newly diagnosed adult AML and mixed phenotype acute leukemia (MPAL) patients as assessed by interphase fluorescence in situ hybridization analysis of AML and myelodysplastic syndrome-associated cytogenetic alterations and/or the pattern of chromosome X inactivation, in females. In addition, we investigated the potential association between the degree of molecular/genetic involvement of hematopoiesis and coexistence of altered immunophenotypes by flow cytometry. Our results indicate that residual maturing neutrophils, monocytes and nucleated red cell precursors from the great majority of newly diagnosed AML and MPAL cases show a clonal pattern of involvement of residual maturing hematopoietic cells, in association with a greater number of altered immunophenotypes. These findings are consistent with the replacement of normal/reactive hematopoiesis by clonal myelopoiesis and/or erythropoiesis in most newly diagnosed AML and MPAL cases, supporting the notion that in most adults presenting with de novo AML, accumulation of blast cells could occur over a pre-existing clonal hematopoiesis.

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow.

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

Killing of lymphoblastic leukemia cells by nitric oxide and taxol: involvement of NF-kappaB activity.

Nitric oxide (NO) and taxol are cytotoxic towards leukemia and tumor cells and interfere with the transcription factor NF-kappaB activity. NO and taxol inhibited NF-kappaB activity and were cytotoxic to human and murine leukemia cells, but at a different magnitude (30% cell killing and 80% inhibition of NF-kappaB). Sub-effective concentrations of SNAP and taxol synergized in killing L-1210 cells but either alone or in combination completely inhibited NF-kappaB. Pyrrolidine dithiocarbamate (PDTC) was cytotoxic on its own and inhibited NF-kappaB activity. It potentiated NO and taxol killing but again there was no direct relationship between inhibition of NF-kappaB and cell killing. Neither NO nor taxol cytotoxicity was related to the cytoskeleton. Our results show that NO, taxol and PDTC induced apoptosis and NF-kappaB inhibition in leukemic cells but their cytotoxicity either alone or in combination, does not seem to be dependent on the inhibition of NF-KB activity.