Influence of a Physiologically Formed Blood Clot on Pre-Osteoblastic Cells Grown on a BMP-7-Coated Nanoporous Titanium Surface.

Titanium (Ti) nanotopography modulates the osteogenic response to exogenous bone morphogenetic protein 7 (BMP-7) in vitro, supporting enhanced alkaline phosphatase mRNA expression and activity, as well as higher osteopontin (OPN) mRNA and protein levels. As the biological effects of OPN protein are modulated by its proteolytic cleavage by serum proteases, this in vitro study evaluated the effects on osteogenic cells in the presence of a physiological blood clot previously formed on a BMP-7-coated nanostructured Ti surface obtained by chemical etching (Nano-Ti). Pre-osteoblastic MC3T3-E1 cells were cultured during 5 days on recombinant mouse (rm) BMP-7-coated Nano-Ti after it was implanted in adult female C57BI/6 mouse dorsal dermal tissue for 18 h. Nano-Ti without blood clot or with blood clot at time 0 were used as the controls. The presence of blood clots tended to inhibit the expression of key osteoblast markers, except for Opn, and rmBMP-7 functionalization resulted in a tendency towards relatively greater osteoblastic differentiation, which was corroborated by runt-related transcription factor 2 (RUNX2) amounts. Undetectable levels of OPN and phosphorylated suppressor of mothers against decapentaplegic (SMAD) 1/5/9 were noted in these groups, and the cleaved form of OPN was only detected in the blood clot immediately prior to cell plating. In conclusion, the strategy to mimic in vitro the initial interfacial in vivo events by forming a blood clot on a Ti nanoporous surface resulted in the inhibition of pre-osteoblastic differentiation, which was minimally reverted with an rmBMP-7 coating.

Panobinostat (LBH589) increase survival in adult xenografic model of acute lymphoblastic leukemia with t(4;11) but promotes antagonistic effects in combination with MTX and 6MP.

Patients diagnosed with acute lymphoblastic leukemia (ALL) bearing t(4;11)/MLL-AF4 have aggressive clinical features, poor prognosis and there is an urgent need for new therapies to improve outcomes. Panobinostat (LBH589) has been identified as a potential therapeutic agent for ALL with t(4;11) and studies suggest that the antineoplastic effects are associated with reduced MLL-AF4 fusion protein and reduced expression of HOX genes. Here, we evaluated the in vitro effects of the combination of LBH589 with methotrexate (MTX) or 6-mercaptopurine (6MP) by cell proliferation assays and Calcusyn software in ALL cell line (RS4;11); the in vivo effects of LBH589 in xenotransplanted NOD-scid IL2Rgammanull mice measuring human lymphoblasts by flow cytometry; and the expression of HOX genes by qPCR after treatment in an adult model of ALL with t(4;11). LBH589 combination with MTX or 6MP did not promote synergistic effects in RS4;11 cell line. LBH589 treatment leads to increased overall survival and reduction of blasts in xenotransplanted mice but caused no significant changes in HOXA7, HOXA9, HOXA10, and MEIS1 expression. The LBH589, alone, showed promising antineoplastic effects in vivo and may represent a potential agent for chemotherapy in ALL patients with t(4;11).

NTAL is associated with treatment outcome, cell proliferation and differentiation in acute promyelocytic leukemia.

Non-T cell activation linker (NTAL) is a lipid raft-membrane protein expressed by normal and leukemic cells and involved in cell signaling. In acute promyelocytic leukemia (APL), NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway. The role of NTAL in APL cell processes, and its association with clinical outcome, has not, however, been established. Here, we show that reduced levels of NTAL were associated with increased all-trans retinoic acid (ATRA)-induced differentiation, generation of reactive oxygen species, and mitochondrial dysfunction. Additionally, NTAL-knockdown (NTAL-KD) in APL cell lines led to activation of Ras, inhibition of Akt/mTOR pathways, and increased expression of autophagy markers, leading to an increased apoptosis rate following arsenic trioxide treatment. Furthermore, NTAL-KD in NB4 cells decreased the tumor burden in (NOD scid gamma) NSG mice, suggesting its implication in tumor growth. A retrospective analysis of NTAL expression in a cohort of patients treated with ATRA and anthracyclines, revealed that NTAL overexpression was associated with a high leukocyte count (P = 0.007) and was independently associated with shorter overall survival (Hazard Ratio: 3.6; 95% Confidence Interval: 1.17-11.28; P = 0.026). Taken together, our data highlights the importance of NTAL in APL cell survival and response to treatment.

The lipid raft protein NTAL participates in AKT signaling in mantle cell lymphoma.

Lipid rafts are ordered membrane domains, which provide an environment for the proteins participating in signal transduction. Perifosine is an alkylphospholipid (APL) that inhibits the AKT pathway, cytotoxic to neoplastic cells. We have shown that the lipid raft adaptor protein NTAL is a target of APLs in leukemic cells. Using human mantle cell lymphoma (MCL) Granta-519 cell line we showed here that perifosine decreased NTAL in lipid raft fractions reducing AKT phosphorylation before apoptosis. We also showed that the NTAL-knockdown by shRNA induced a state of reduced AKT activation. Experimental NTAL-knockdown in NSG mouse MCL xenografts reduced AKT activity, increased the basal apoptotic rate by 3-fold (n = 8) and decreased tumor weight by 2.7-fold (n = 5), indicating that NTAL participates in tumor growth. NTAL protein was detected by western blotting in circulating cells of 7 of 8 MCL patients in the leukemic phase, suggesting involvement in the progression of the disease.

CCR2 Plays a Protective Role in Rocio Virus-Induced Encephalitis by Promoting Macrophage Infiltration Into the Brain.

Rocio virus (ROCV) is a highly neuropathogenic mosquito-transmitted flavivirus responsible for an unprecedented outbreak of human encephalitis during 1975-1976 in Sao Paulo State, Brazil. Previous studies have shown an increased number of inflammatory macrophages in the central nervous system (CNS) of ROCV-infected mice, implying a role for macrophages in the pathogenesis of ROCV. Here, we show that ROCV infection results in increased expression of CCL2 in the blood and in infiltration of macrophages into the brain. Moreover, we show, using CCR2 knockout mice, that CCR2 expression is essential for macrophage infiltration in the brain during ROCV infection and that the lack of CCR2 results in increased disease severity and mortality. Thus, our findings show the protective role of CCR2-mediated infiltration of macrophages in the brain during ROCV infection.

Reactive oxygen species production triggers green tea-induced anti-leukaemic effects on acute promyelocytic leukaemia model.

Green tea (GT) has been consumed as a beverage for thousands of years because of its therapeutic properties observed over time. Because there is no sufficient evidence supporting the protective role of tea intake during the development of acute myeloid leukaemia, we herein study GT extract effects on an acute promyelocytic leukaemia model. Our results demonstrated that GT reduces leucocytosis and immature cells (blasts) in peripheral blood, bone marrow (BM), and spleen of leukaemic mice, parallel with an increase of mature cells in the BM. In addition, GT induces apoptosis of cells in the BM and spleen, confirmed by activation of caspase-3, -8 and -9; GT reduces the malignant clones CD34+ and CD117+ in the BM and reduces CD117+ and Gr1+ immature myeloid cells in the spleen; GT increases intracellular reactive oxygen species (ROS) in the BM Gr1+ cells while reducing CD34+ and CD117+ cells; GT reduces CXCR4 expression on CD34+ and CD117+ cells, and reduces the nuclear translocation of HIF-1α. GT has anti-proliferative effects in leukaemia in vivo by inhibiting malignant clone expansion, probably by modulating the intracellular production of ROS.