No abstract

The present article describes an l-amino acid oxidase from Bothrops atrox snake venom as with antiprotozoal activities in Trypanosoma cruzi and in different species of Leishmania (Leishmania braziliensis, Leishmania donovani and Leishmania major). Leishmanicidal effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Leishmania spp. cause a spectrum of diseases, ranging from self-healing ulcers to disseminated and often fatal infections, depending on the species involved and the host's immune response. BatroxLAAO also displays bactericidal activity against both Gram-positive and Gram-negative bacteria. The apoptosis induced by BatroxLAAO on HL-60 cell lines and PBMC cells was determined by morphological cell evaluation using a mix of fluorescent dyes. As revealed by flow cytometry analysis, suppression of cell proliferation with BatroxLAAO was accompanied by the significant accumulation of cells in the G0/G1 phase boundary in HL-60 cells. BatroxLAAO at 25 μg/mL and 50 μg/mL blocked G0-G1 transition, resulting in G0/G1 phase cell cycle arrest, thereby delaying the progression of cells through S and G2/M phase in HL-60 cells. This was shown by an accentuated decrease in the proportion of cells in S phase, and the almost absence of G2/M phase cell population. BatroxLAAO is an interesting enzyme that provides a better understanding of the ophidian envenomation mechanism, and has biotechnological potential as a model for therapeutic agents.

Despite serological evidences of presence of hepatitis E virus (HEV) in humans or other animals, until this study the virus had not been detected and molecular characterization of the isolate that circulates in Brazil had not been described. Thus, we collected stool samples of young pigs and tested for presence of HEV RNA by RT-PCR, using primers for partial amplification of ORF2 sequence. Phylogenetic analysis with sequence obtained from the amplified products revealed that the HEV isolate identified here was most closely related to HEV isolates of genotype 3, which is commonly detected in HEV infected pigs. Nucleotide sequence analyses carried out with the entire amplified fragment, ORF2/ORF3 overlapping and ORF2 non-overlapping sequences showed highest identities with the US isolate of genotype 3. Similarly, amino acid sequence analyses done with the entire amplified fragment, ORF2 non-overlapping, ORF2 and ORF3 overlapping sequences also showed highest identities with the genotype 3 isolate. Presence, in Brazil, of HEV of genotype 4, which also infects pigs, as well as HEV strains that infect humans still remain to be detected and characterized.

No abstract

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 μg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.

Here, we evaluated whether the overexpression of transcriptionally inactive ΔNp73 cooperates with PML/RARA fusion protein in the induction of an APL-leukemic phenotype, as well as its role in vitro in proliferation, myeloid differentiation, and drug-induced apoptosis. Using lentiviral gene transfer, we showed in vitro that ΔNp73 overexpression resulted in increased proliferation in murine bone marrow (BM) cells from hCG-PML/RARA transgenic mice and their wild-type (WT) counterpart, with no accumulation of cells at G2/M or S phases; instead, ΔNp73-expressing cells had a lower rate of induced apoptosis. Next, we evaluated the effect of ΔNp73 on stem-cell self-renewal and myeloid differentiation. Primary BM cells lentivirally infected with human ΔNp73 were not immortalized in culture and did not present significant changes in the percentage of CD11b. Finally, we assessed the impact of ΔNp73 on leukemogenesis or its possible cooperation with PML/RARA fusion protein in the induction of an APL-leukemic phenotype. After 120 days of follow-up, all transplanted mice were clinically healthy and, no evidence of leukemia/myelodysplasia was apparent. Taken together, our data suggest that ΔNp73 had no leukemic transformation capacity by itself and apparently did not cooperate with the PML/RARA fusion protein to induce a leukemic phenotype in a murine BM transplantation model. In addition, the forced expression of ΔNp73 in murine BM progenitors did not alter the ATRA-induced differentiation rate in vitro or induce aberrant cell proliferation, but exerted an important role in cell survival, providing resistance to drug-induced apoptosis.

In approximately 15% of patients with acute myeloid leukemia (AML), total and phosphorylated EGFR proteins have been reported to be increased compared to healthy CD34+ samples. However, it is unclear if this subset of patients would benefit from EGFR signaling pharmacological inhibition. Pre-clinical studies on AML cells provided evidence on the pro-differentiation benefits of EGFR inhibitors when combined with ATRA or ATO in vitro. Despite the success of ATRA and ATO in the treatment of patients with acute promyelocytic leukemia (APL), therapy-associated resistance is observed in 5-10% of the cases, pointing to a clear need for new therapeutic strategies for those patients. In this context, the functional role of EGFR tyrosine-kinase inhibitors has never been evaluated in APL. Here, we investigated the EGFR pathway in primary samples along with functional in vitro and in vivo studies using several APL models. We observed that total and phosphorylated EGFR (Tyr992) was expressed in 28% and 19% of blast cells from APL patients, respectively, but not in healthy CD34+ samples. Interestingly, the expression of the EGF was lower in APL plasma samples than in healthy controls. The EGFR ligand AREG was detected in 29% of APL patients at diagnosis, but not in control samples. In vitro, treatment with the EGFR inhibitor gefitinib (ZD1839) reduced cell proliferation and survival of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Moreover, the combination of gefitinib with ATRA and ATO promoted myeloid cell differentiation in ATRA- and ATO-resistant APL cells. In vivo, the combination of gefitinib and ATRA prolonged survival compared to gefitinib- or vehicle-treated leukemic mice in a syngeneic transplantation model, while the gain in survival did not reach statistical difference compared to treatment with ATRA alone. Our results suggest that gefitinib is a potential adjuvant agent that can mitigate ATRA and ATO resistance in APL cells. Therefore, our data indicate that repurposing FDA-approved tyrosine-kinase inhibitors could provide new perspectives into combination therapy to overcome drug resistance in APL patients.

3536 ΔNp73 is an alternative TP73 gene transcript lacking the transactivation (TA) domain that is generated via alternative splicing and/or P2 promoter. The encoded protein acts as a potent transdominant inhibitor of wild type TP53 and full-length TAp73. In several human malignancies the unbalanced expression of transcriptionally active (TAp73) and inactive (ΔNp73) variants correlates with treatment outcome. We have previously reported that higher ΔN/TA isoform expression ratio was associated with poorer prognosis and resistance to cytarabine induced apoptosis in patients with acute myeloid leukemia (AML) (Lucena-Araujo et al., 2008). In acute promyelocytic leukemia (APL), both isoforms are expressed, but the clinical significance remains unknown. The aim of this study was to determine whether the ΔN/TA expression ratio was associated with treatment outcome of APL patients and to investigate the mechanisms by which ΔNp73 may contribute to PML-RARa+ cell survival. Using isoform-specific probes for ΔNp73 and TAp73, their expression was analyzed in 166 APL patients by Real-time quantitative polymerase chain reaction (RQ-PCR). Patients were divided into tertiles for ΔN/TA expression ratio (median value=23.62; 33rd/66th percentiles=12.8/42.3) and their clinical and laboratory characteristics were compared. Patients in the highest tertile presented higher white blood cells (WBC) counts than those in intermediate/lower tertiles (p <0.001), but no significant differences were observed for age, gender, PML breakpoint, or platelets count. Higher ΔN/TA expression ratio values were significantly associated with the presence of FLT3-ITD (p =0.001). Treatment outcome was obtained for 131 APL patients enrolled in the APL99 (n=41) and IC-APL (n=90) trials. The mean follow-up was 29.1 months, ranging from 1 to 85.5 months. The mean overall survival (OS) of all patients was of 66.8 months [95%CI; 60.8 to 72.8], whereas it was of 67.1 months [56.5 to 77.7] for patients in the lower and 41.7 months [32 to 51.4] for those in the higher tertile for ΔN/TA expression ratio (p=0.014, Figure 1A). Univariate analysis identified WBC counts above 10,000/μl (p =0.003), FLT3-ITD mutation (p =0.011) and ΔN/TA expression ratio (p =0.014) as predictive factors for OS. However, in multivariate Cox analysis, these three prognostic factors were not independent. Until April 2011, a total of eight relapses (6.1%) were recorded. The disease free survival (DFS) rate at five-years for all patients was of 88.3% ± 4.2% and the mean DFS was of 76.1 months [71.2 to 80.9]. DFS was significantly shorter in patients at the higher tertile ΔN/TA expression ratio compared with patients at the lower tertile (72.1 ± 11.2% vs 97.1 ± 2.8%, respectively; p <0.001; Figure 1B) and was the only variable found to be significant in the univariate analysis. To test the functional significance of the association of PML-RARa with high ΔNp73 gene expression, primary murine bone marrow cells from hCG-PML-RARa transgenic mice were transfected with MSCV-based retroviral vector carrying the ΔNp73 cDNA upstream of IRES-GFP cassette (PML-pMIG-ΔN). Expression of ΔNp73 in PML-RARa+ cells increased cell proliferation rate by 2.5-fold compared to PML-RARa+ transfected with the empty vector (p =0.03). This increase resulted from accumulation of cells at the G2/M phase (5.79 ± 0.08% for PML-pMIG vs 9.8 ± 0.35% for PML-pMIG-ΔN, p <0.001), as well as at S phase of the cell cycle (27.74 ± 0.89% for PML-pMIG vs. 36.78 ± 0.81% for PML-pMIG-ΔN, p =0.001). In addition, transfection of ΔNp73 resulted in resistance to cytarabine-induced apoptosis. After 24h of culture with 50μg/ml of cytarabine (ED-50%), the fractional effect for the drug (% Anexin V-positive in (treated – untreated) cells /100 - % Anexin V-positive in non-treated cells) was 32.1% for PML-pMIG-ΔN and 54.8% for PML-pMIG (p <0.001). In conclusion, ΔN/TA expression ratio was associated with shorter OS and DFS in APL, which may reflect increased cell proliferation and apoptosis resistance due to ΔNp73 activity.Figure 1.Overall (A) and disease-free survival (B) in acute promyelocytic leukemia patients according to ΔN/TA expression ratio.Figure 1. Overall (A) and disease-free survival (B) in acute promyelocytic leukemia patients according to ΔN/TA expression ratio. Disclosures: No relevant conflicts of interest to declare.