FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells

FLT3 ligand is a hematopoietic growth factor that plays a key role in growth of primitive hematopoietic cells. FLT3 receptor mRNA is found in early hematopoietic progenitors and in human myeloid leukemia blasts. Much less is known about the surface expression of FLT3 receptor on human hematopoietic cells. Using human 125I-FLT3 ligand, we have identified and characterized surface FLT3 receptors on normal and malignant human hematopoietic cells and cell lines. Our results showed that surface display of FLT3 receptor was greatest in fresh myeloid leukemia blast cells and myeloid leukemia cell lines. Erythroleukemic and megakaryocytic leukemia cell lines (n = 5) bound little to no 125I- FLT3 ligand. Scatchard analysis of 125I-FLT3 ligand binding data shows that three myeloid leukemia cell lines, ML-1, AML-193, and HL-60, as well as normal human marrow mononuclear cells, exhibit high affinity FLT3 receptors. Crosslinking of 125I-FLT3 ligand to FLT3 receptors on the surface of ML-1 myeloid leukemia cells indicates that the FLT3 ligand. The rates of FLT3 ligand internalization and degradation were determined by binding 125I-FLT3 ligand to ML-1 cells and acid stripping to distinguish surface bound from internalized ligand. Internalized 125I-FLT3 ligand was detected within 5 minutes after binding to ML-1 cells. In addition, we evaluated the effect of FLT3 ligand on megakaryocytic colony growth and nuclear endoreduplication, alone or in the presence of thrombopoietin. FLT3 ligand did not promote colony forming unit megakaryocyte (CFU-Meg) colony growth or megakaryocyte nuclear maturation, nor did FLT3 ligand augment the effects of thrombopoietin on these measures of megakaryopoiesis. These data indicate that the FLT3 receptor shares several characteristics with the c-kit receptor including dimerization and rapid internalization. However, the more restricted cellular distribution of the FLT3 receptor may target the effects of FLT3 ligand to primitive hematopoietic cells and to myeloid and lymphoid progenitor cells, in contrast to the pleiotropic effects of the c-kit receptor ligand, stem cell factor.     

Screening for co-existence of α-thalassemia in β-thalassemia and in HbE heterozygotes via an enzyme-linked immunosorbent assay for Hb Bart’s and embryonic ζ-globin chain

We sought to demonstrate the ability of levels of Hb Bart’s and ζ-globin chain quantified by enzyme-linked immunosorbent assay (ELISA) in detecting α-thalassemia in β-thalassemia and HbE heterozygotes. We developed an in-house sandwich ELISA method using monoclonal antibodies (mAbs) to Hb Bart’s and ζ-globin chain, and quantified levels of Hb Bart’s and ζ-globin chain in 172 and 223 anonymous blood samples of β-thalassemia and HbE heterozygotes, respectively. Genotypes of α-thalassemia 1, β-thalassemia were identified, and HbE allele was confirmed using a newly developed multiplex allele-specific PCR. The in-house sandwich ELISA method detected Hb Bart’s in 6.4% of β-thalassemia heterozygotes, of which 5.2% showed detectable amounts of the ζ-globin chain. 15.2% of individuals heterozygous for HbE showed a detectable amount of Hb Bart’s, and the ζ-globin chain was detected in 11.2% of this cohort. All samples having detectable amounts of Hb Bart’s and the ζ-globin chain were verified to be SEA-type α-thalassemia 1. ELISA-quantified Hb Bart’s and ζ-globin chain levels can be used to detect double heterozygosity of α- and β-thalassemia and of α-thalassemia and HbE. This strategy may be useful in screening for co-existence of α-thalassemia in β-thalassemia and in HbE heterozygotes, particularly in countries where α-, β-thalassemia and HbE are endemic.     

Oxidative stress indicated by elevated expression of Nrf2 and 8-OHdG promotes hepatocellular carcinoma progression

Reactive oxygen species (ROS) is excessively generated in tumors creating an oxidative stress in tumor microenvironment. We investigated hepatic expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and 8-hydroxydeoxyguanosine (8-OHdG) in hepatocellular carcinoma (HCC) patients, and asked if ROS epigenetically upregulated Nrf2 and enhanced aggressiveness in HCC cells. Expression of Nrf2 (n = 100) and 8-OHdG (n = 53) was remarkably increased in HCC tissues compared with the noncancerous hepatic tissues. Elevated expression of 8-OHdG was associated with poor survival in HCC patients. H₂O₂, as ROS representative, provoked oxidative stress in HepG2 cells, indicated by increased protein carbonyl content and decreased total antioxidant capacity. Nrf2 expression and 8-OHdG formation were markedly increased in the H₂O₂-treated cells compared with the untreated control. Co-treatment with antioxidants, tocopheryl acetate (TA) and S-adenosylmethionine (SAM) effectively attenuated expression of Nrf2 and 8-OHdG in H₂O₂-treated cells. HepG2 cells treated with H₂O₂ had significantly higher migration and invasion capabilities than the untreated control cells, and this aggressiveness was significantly inhibited by TA and SAM. Bisulfite sequencing revealed that CpG dinucleotides in Nrf2 promoter were unmethylated in the H₂O₂-treated cells similar to the untreated control. In conclusion, robust histological evidence of increased antioxidative response and oxidative DNA damage in human HCC tissues was demonstrated. Elevated oxidative DNA lesion 8-OHdG was associated with shorter survival. Experimentally, ROS enhanced Nrf2 expression, 8-OHdG formation and tumor progression in HCC cells. These effects were inhibited by antioxidants. Therefore, oxidative stress-reducing regimens might be beneficial to diminish the ROS-induced HCC progression.     

Robust decentralized design of power system stabilizers taking into consideration system uncertainties

In complex power systems, changes in network configurations, various loading conditions, etc., cause system uncertainties. Without considering such uncertainties in the design, the conventional power system stabilizers (PSSs) may deteriorate the system robust stability. To overcome this problem, the proposed design incorporates the uncertainty model in the system representation. Then, the PSSs in a multi‐machine power system are arranged as the decentralized controller in a multi‐input multi‐output (MIMO) system. The robust stability margin of the closed‐loop control system is guaranteed in terms of the MIMO gain margin and phase margin. Control parameters of PSSs are optimized by a tabu search. Non‐linear simulation studies confirm the robustness of the designed PSSs against various uncertainties. Copyright © 2005 John Wiley & Sons, Ltd.     

Cells with hemopoietic potential residing in muscle are itinerant bone marrow-derived cells

OBJECTIVE: The nature of cells residing in muscle giving rise to hemopoietic colonies in vitro or hemopoietic reconstitution in vivo has been unclear. The goal of the present study was to characterize these cells and uncover their potential site of origin. MATERIALS AND METHODS: Cells prepared from muscle were characterized for surface antigens (CD45, CD34, c-kit, Sca-1, CD31, VCAM-1), for their in vitro clonogenic capacity and in vivo repopulation potential either as unpurified cells or sorted subsets (CD45(+), CD45(-)). The presence of bone marrow (BM)-derived cells in muscle of mice reconstituted with marked BM cells before and after cytokine-induced mobilization was also examined. RESULTS: Our data show: 1) The yield of CD45(+) cells is higher in muscle of neonates and young animals. Their composite phenotype does not favor contamination by blood. 2) The capacity of fresh muscle cell explants to give rise to colonies in vitro and hemopoietic reconstitution in vivo is associated with CD45(+) cells. 3) Irradiated recipients reconstituted with marked BM cells harbor marked BM-derived cells (CD45(+) or CD45(-)) in their muscle several months after transplant. 4) Cytokine-induced mobilization of transplanted animals modestly increases the yield of BM-derived cells recovered from muscle, unlike the yields from spleen, liver, or peripheral blood (PB). CONCLUSIONS: Our data suggest a reinterpretation of previously published conclusions: hemopoietic colonies derived from fresh muscle explants do not originate from transdifferentiated muscle cells, but from BM-derived cells residing in muscle; the hemopoietic reconstituting potential of muscle cells is likewise attributed to these cells.     

Bone marrow-derived CD45+ and CD45- cells reside in skeletal muscle

In this report we examined the kinetics of accumulation in muscle tissue of donor-derived CD45+ and CD45- cells from one to several weeks post-bone marrow transplantation. Only after hemopoiesis is fully reconstituted are CD45+ cells recovered from muscle tissue in proportions similar to ones we reported earlier in non-transplanted mice. Furthermore, using marked donor bone marrow cells, we document that in addition to CD45+ cells, donor-derived CD45- cells with certain phenotypic characteristics progressively accumulate in muscle post-transplantation. The turnover rate and physiologic roles of these populations in muscle are unclear and require further study.