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This paper concerns the new results obtained on the characterization of chitins and chitosans. Large series of samples was analyzed covering a wide range of water soluble and insoluble materials. The water soluble polymers were obtained by heterogeneous deacetylation and by homogeneous reacetylation. The calibration of IR spectrum was proposed and shown to be valid in all the range of DA. Application of 15N and 13C solid state NMR was developed to be able to determine DA even in situon insoluble natural materials. All the methods proposed give a very coherent set of results. The molecular weight distribution was established by GPC using cationic porous supports and the good solvent earlier proposed 0.3M AcOH/0.2M AcONa. The role of the distribution of acetyl groups along the chain is also discussed and analyzed by NMR; it is demonstrated clearly the difference between homogeneously acetylated samples and heterogeneous samples coming from different routes of preparation. The dependence of Mark‐Houwink parameters allowing to relate the intrinsic viscosity with the molecular weight is also briefly reported. In good concordance with experimental data, molecular modeling helps in understanding the role of the N‐acetyl group content and distribution on the stiffness of the chains.

The degree of acetylation (DA) of various samples has been evaluated by ¹H liquid-state NMR and ¹³C and ¹⁵N CP−MAS solid-state NMR over the whole range of DA. A good agreement has been found for all the experiments. The ¹³C and ¹⁵N CP−MAS experiments have permitted the evaluation of the chitin DA and content in the structural polysaccharides in a fungus named Aspergillus niger. The fungus structural carbohydrates mainly consist in pure chitin associated with glucans. Comparison of the ¹³C CP−MAS spectra with standard (1→3)-β-d-glucans strongly suggests that chitin and glucans are linked via covalent bonds.

Abstract 5125

No relevant conflicts of interest to declare.

Sickle cell disease (SCD) pathophysiology is associated with a hypercoagulable state that may contribute to the initiation and propagation of vaso-occlusion. Increased platelet activation has been described in SCD and SCD platelets may present augmented adhesion to the vascular endothelium, potentially contributing to vaso-occlusion.This study investigated whether platelets (PLTs) from SCD individuals are able to activate endothelial cells per se.Human umbilical vein endothelial cells (HUVEC) were cultured (1×106cells/well) on 6-well plates (37°C, 5% CO2). Subsequently, HUVEC were co-cultured in direct contact, or not, with washed PLTs (1×108PLTs/well) from healthy control individuals (CON, n=23) or steady-state SCD patients (SCD, n=47; 26 of which were on hydroxyurea therapy; 20mg/Kg/day) for 4h, 37°C, 5%CO2. After incubation, PLTs were removed; supernatants were reserved for cytokine quantification by ELISA, and HUVEC were analyzed by flow cytometry for CD62E (E-Selectin) and CD54 (ICAM-1) surface expression; gene expressions of ICAM1 and NFKBIA were analyzed by qPCR.Basal ICAM-1 expression on the surface of HUVEC (39.6±3.2%, n=15) was significantly increased following their incubation in direct contact with SCD PLTs (46.1±3.1%, n=26, p<0.05, Wilcoxon test), but not CON PLTs (41.3±4.7%, n=12). E-selectin expression was also low level on the surface of HUVEC (0.9±0.2%, n=17), and was slightly but significantly increased following incubation of cells with SCD PLTs (6.1±2.2%, n=26, p<0.001), but not CON PLTs (3.6±1.5%, n=12, p>0.05). Repetition of these assays, but with the placement of transwell inserts in culture plates to separate the PLTs from HUVEC resulted in a 45% decrease in ICAM-1 expression (p<0.05), and 85% decrease in E-selectin (p<0.05) expression on the surface of HUVEC, following their incubation with SCD PLTs. HUVEC produce and release interleukin-8 (IL-8); basal IL-8 production by HUVEC (1×106cells/well) was 1.160±0.187ng/mL (n=21); this production was augmented in the presence of SCD PLTs (1.280±0.149ng/mL, n=42, p<0.01), but not by CON PLTs (1.127±0.157ng/mL, n=23). The influence of PLT IL-8 production on these values was negligible, as shown by data (not shown) demonstrating that PLT IL-8 production is low level and does not differ between CON and SCD PLTs. IL-1β is produced and released by PLTs (CON, 3.4±1.4ρg/mL, n=12; SCD, 6.5±1.5 ρg/mL, n=25, p>0.05), but this production was further increased when PLTs were co-cultured with HUVEC: SCD PLTs (10.3±4.2ρg/mL, n=47; p<0.01) and CON PLTs (5.8±2.4ρg/mL, n=25; p>0.05), compared to HUVEC alone (1.27±0.4ρg/mL, n=24). Gene expression of ICAM1 by HUVEC increased 6.3-fold in the presence of SCD PLTs (n=25, p<0.01), compared to basal expression (n=11), but was not altered in the presence of CON PLTs (n=11, p>0.05). The expression of the gene encoding the NFkB transcription factor, NKBIA, increased 3.4-fold in HUVEC following incubation with SCD PLTs (n=25, p<0.05), compared to basal NFKBIA expression (n=12); however NFKBIA expression in HUVEC was not significantly altered by CON PLTs (n=10, p>0.05).Results indicate that the contact of platelets, or products released from platelets, from patients with SCD may activate endothelial cells, in vitro, increasing adhesion molecule and IL-8 production, associated with an augmented expression of the gene encoding NFkB. Platelets produce IL-1β in greater quantities in the presence of endothelial cells, possibly contributing to endothelial cell activation; however the fact that transwell inserts significantly reduced SCD PLT-mediated endothelial activation indicates that the direct contact of PLTs (possibly via adhesion) is required for this activation. Data indicate that platelets adhered to vessel walls may play an important role in endothelial activation and, therefore, vaso-occlusive mechanisms in SCD.No relevant conflicts of interest to declare.

Abstract 1002

Extensive studies have led to a considerable understanding of the cellular and molecular control of haemoglobin production during red blood cell differentiation, however, identification of the genes expressed as part of the erythroid differentiation programme remains an important goal because of the insights that these data will bring to erythrocyte biology and disease. Previous results using SAGE identified 93 differentially-expressed genes during erythroid development. One of these genes, EYA3, a homologue gene of Eyes Absent 3 in Drosophila, is a transcription cofactor with intrinsic phosphatase activity and its expression was observed to be high at the end of CD34+ cell differentiation and in human bone marrow.To evaluate globin gene, fetal hemoglobin (HbF) expressions and apoptosis levels in the erythroleukemic K562 cell line after EYA3 gene silencing and induction with hemin.Four different cultures from human K562 cells (1×105cells/mL in DMEM, 10% FBS, penicillin/streptomycin, 5% CO2, 37°C) were transfected with control or EYA3 knockdown lentiviruses (MOI=2.5). After proliferation and selection of successfully transfected cells with puromicin (2.0 ug/mL), cells were treated with 30μM hemin and collected after 0, 24, 48, 72 and 96h for gene expression and flow cytometry analyses. EYA3, LXN, α, and g-gene expression was measured by qRT-PCR and normalized using the Genorm program. HbF expression and apoptosis were evaluated by flow cytometry.Analysis of globin gene expression showed that α-globin gene was downregulated in EYA3 silenced K562 culture cells compared with the control culture in 72h after hemin addition (1.791±0.1735; 0.7404±0.1709, respectively, **P<0.001, n=4). g-globin gene expression was found to be downregulated in K562 EYA3 silenced culture after 24h (1.350±0.1296; 0.5285±0.1736, respectively, *P<0.05, n=4) and 72h (1.554±0.1042; 0.6889±0.1535, respectively, **P<0.001, n=4). HbF expression was found to be downregulated in the same culture compared to the control culture at 72h after hemin addition (3568±41.00; 1947±206.50, respectively, *P<0.05, n=4). Apoptosis levels were found increased in EYA3 silenced K562 culture cells compared with the control culture in 72h after hemin addition (4.7±0.10; 8.55±0.55, respectively, *P<0.001, n=4).Our results show that silencing EYA causes modifications in the expression pattern of α- and g-globin gene expression as well as in HbF expression pattern and apoptosis levels in a model of erythroid differentiation. Further studies should be performed in primary erythroid cell cultures using siRNA-based gene silencing and overexpression of these genes to determine how these genes are involved in the mechanisms of globin gene regulation.Support by FAPESP, CNPq and INCTSNo relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 4797

Abstract 3195

No relevant conflicts of interest to declare.

Abstract 2066

Off Label Use: Development of TKI in the treatment of CML.

Erythroid differentiation is a dynamic and complex process in which a pluripotent stem cell undergoes a series of developmental changes that commit it to a specific lineage. These alterations involve changes in gene expression profiles. Extensive studies have led to a considerable understanding of the cellular and molecular control of hemoglobin production during red blood cell differentiation, however, a complete understanding of human erythropoiesis will require a robust description of the entire transcriptome of these cells during differentiation. From a global point of view of cell metabolic regulation, where genomic information could be complemented with gene expression, the use of methods that enable quantification of the entire transcriptome of the red blood cell during differentiation is of great importance. In this study, the gene expression profiles during differentiation of Human erythroid cells of a normal blood donor in a two-phase liquid culture (Fibach & Rachmilewitz, 1993) were evaluated using Serial analysis of gene expression (SAGE). Global gene expression was evaluated in cells collected immediately before the addition of erythropoeitin (0 hour) and 192 and 336 hours after the addition of this hormone. We generated a total of 30512 tags at 0h, 30117 tags at 192h and 30189 tags at 336h, representing 12026, 11709 and 11337 unique tags, respectively. In the 0h library, a high expression of ferritin genes and CD74 antigen gene was observed. As expected, the expression of globin genes started during intermediate stages of differentiation (predominantly basophilic erythroblasts) and were the most expressed genes at the end of the culture (predominantly orthocromatic erythroblasts). Ribosomal genes were the most expressed genes at 192 hours, indicating an increase in protein synthesis. To identify the genes that were differentially expressed between the libraries, a P value < 0.01 and fold ≥ 5 were considered as statistically significant. In the comparison of the 0h and 192h libraries, 179 differentially expressed transcripts were identified. From these genes, in addition to the globin genes, we found an up-regulation of several genes related to protein binding (LXN, GSTM3, and TRIP6), transcription factor (GATA-1), hydrolase activity (TPSAB1), ion transport (SLC12A9) and regulation of apoptosis (PRDX2). Comparing the 192h and 336h libraries, 103 differentially expressed transcripts were identified. The up-regulated genes were generally related to hemoglobin synthesis, such as ALAS2, involved in the biosynthesis of the heme group or related to intracellular transport such as MSCP and NUDT4 and cell differentiation such as GDF15. The transcription of some of these genes, such as SLC12A9, TRB3, EYA3 and TWIST2 are described for the first time during erythroid differentiation. The results indicated that the global aspects of the transcriptome were similar during differentiation for the majority of the genes and that probably a relative small set of genes is involved in the modification of erythroid cells during differentiation. The results of this study amplify the previous published data (Komor et al, 2005) and may contribute to the comprehension of erythroid differentiation and identification of new target genes involved in some erythroid diseases.

The term, hereditary persistence of fetal hemoglobin (HPFH), describes a hereditary benign disease, characterized by an increase in fetal hemoglobin (HbF) during adult life. Non-deletional forms of HPFH are characterized by single-base mutations in the promoter region (most of them between −114 and −202 from the cap site) of either the Gγor Aγ-globin gene, resulting in an increase of HbF ranging from 3 to 20% in heterozygotes. Many point mutations in this region have been described, including the Aγ-195 C →G mutation that causes the Brazilian type of HPFH (HPFH-B). A previous study showed that the -195 mutations alone was not able to increase gene expression in vitro and a modest increase was observed when a LCR element fragment (HS2) was introduced in the construction. This study showed too, that the the mechanism of HbF elevation by the -195 mutation was neither mediated by the Sp-1 transcription factor nor by the creation of a CACCC box, as described for the -198 mutation. Thus, other proteins may be involved in the over-expression of the γ-globin chain and/or may depend on DNA structure and these mechanisms remain to be clarified. To better understand this mechanism we have developed HPFH-B transgenic mice. The promoter with mutation was amplified using the genomic DNA of a HPFH-B patient and cloned in a μLCRAγψβδβ cosmid. This construct, containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into single cell mouse embryos. To detect the differences in developmental regulation of the human gamma-globin gene expression in the transgenic mice, we analyzed the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5 of both mutated and non-mutated transgenic mice by RNAse Protection Assay (RPA) and Real time PCR (RT-PCR). Levels of expression of murine alpha-globin mRNA were used as internal controls in the RPA experiment and levels of murine-GAPDH were used in RT-PCR. mRNA levels of human gamma-globin in fetal liver of transgenic mice containing mutation were clearly higher, as compared with control transgenic mice bearing cosmid construct with wild a type sequence gamma promoter. Additionally a nearly 10-fold increase in human gamma-globin expression was detected in fetal livers of HPFH transgenic mice at E13.5 compared to the yolk sac. Thus, our data indicate that the - 195 mutation is the unique cause of elevation of HbF in Brazilian HPFH, but the exact mechanism needs to be clarified.