Purpose: We performed a phase I trial to investigate the safety, clinical responses, and Wilms' tumor 1 (WT1)-specific immune responses following treatment with dendritic cells (DC) pulsed with a mixture of three types of WT1 peptides, including both MHC class I and II-restricted epitopes, in combination with chemotherapy.

Calicheamicin-conjugated humanized anti-CD33 mouse monoclonal antibody, CMA-676, has recently been introduced to clinics as a promising drug to treat patients with acute myeloid leukemia (AML) in relapse. However, the mechanism of action of CMA-676 has not been well elucidated. The cytotoxic effect of CMA-676 on HL60, NOMO-1, NB4, NKM-1, K562, Daudi, and the multidrug-resistant sublines, NOMO-1/ADR and NB4/MDR, was investigated by cell cycle distribution and morphology. These studies were done by a video-microscopic system, DNA fragmentation, dye exclusion and 3H-thymidine uptake after analysis of CD33, CD34, P-glycoprotein (P-gp), multidrug resistance (MDR)-associated protein and lung-related protein on these cells. A dose-dependent, selective cytotoxic effect of CMA-676 was observed in cell lines that expressed CD33, and was dependent on the amount of CD33 and the proliferative speed of the cells. Sensitive cells were temporally arrested at the G2/M phase before undergoing morphological changes. CMA-676 is not effective on P-gp-expressing multidrug-resistant sublines compared with parental cell lines. MDR modifiers, MS209 and PSC833, restored the cytotoxic effect of CMA-676 in P-gp-expressing sublines. CMA-676 is a promising agent in the treatment of patients with AML that expresses CD33. The combined use of CMA-676 and MDR modifiers may increase the selective cytotoxic effect in multidrug-resistant AML.

Vpr, a HIV-1 accessory protein, was believed to be present in the plasma of HIV-1-positive patients, and our previous work demonstrated the presence of plasma Vpr in 20 out of 52 patients. Interestingly, our data revealed that patients' viral titer was correlated with the level of Vpr detected in their plasma. Here, we first show that rVpr, when incubated with human monocytes or MDMs, caused viral production from latently infected cells, and IL-6 was identified as a responsible factor. The induction of IL-6 by rVpr was dependent on signaling through TLR4 and its adaptor molecule, MyD88. We next provide evidence that rVpr induced the formation of OxPC and that a mAb against OxPC blocked rVpr-induced IL-6 production with the concomitant attenuation of MAPK activation. Moreover, the addition of NAC, a scavenger of ROS, abrogated the rVpr-induced formation of OxPC, the phosphorylation of C/EBP-beta, a substrate of MAPK, and IL-6 production. As rIL-6 reactivated viral replication in latently infected cells, our data indicate that rVpr-induced oxidative stress triggers cell-based innate immune responses and reactivates viral production in latently infected cells via IL-6 production. Our results suggest that Vpr should be monitored based on the viral titer, and they provide the rationale for the development of novel, anti-AIDS therapeutics targeting Vpr.

Runx2 is essential for osteoblast differentiation and chondrocyte maturation. The expression of Runx2 is the first requisite step for the lineage determination from mesenchymal stem cells to osteoblasts. Although the transcript from Runx2 distal promoter is majorly expressed in osteoblasts, the promoter failed to direct green fluorescent protein (GFP) expression to osteoblasts. To find the regulatory region, we generated GFP reporter mice driven by a bacterial artificial chromosome (BAC) of Runx2 locus, and succeeded in the reproduction of endogenous Runx2 expression. By serially deleting it, we identified a 343-bp enhancer, which directed GFP expression specifically to osteoblasts, about 30 kb upstream of the distal promoter. The sequence of the 343-bp enhancer was highly conserved among mouse, human, dog, horse, opossum, and chicken. Dlx5, Mef2c, Tcf7, Ctnnb1, Sp7, Smad1, and Sox6, which localized on the enhancer region in primary osteoblasts, synergistically upregulated the enhancer activity, whereas Msx2 downregulated the activity in mouse osteoblastic MC3T3-E1 cells. Msx2 was predominantly bound to the enhancer in mouse multipotent mesenchymal C3H10T1/2 cells, whereas Dlx5 was predominantly bound to the enhancer in MC3T3-E1 cells. Dlx5 and Mef2 directly bound to the enhancer, and the binding sites were required for the osteoblast-specific expression in mice, whereas the other factors bound to the enhancer by protein-protein interaction. The enhancer was characterized by the presence of the histone variant H2A.Z, the enrichment of histone H3 mono-and dimethylated at Lys4 and acetylated at Lys18 and Lys27, but the depletion of histone H3 trimethylated at Lys4 in primary osteoblasts. These findings indicated that the enhancer, which had typical histone modifications for enhancers, contains sufficient elements to direct Runx2 expression to osteoblasts, and that Dlx5 and Mef2, which formed an enhanceosome with Tcf7, Ctnnb1, Sp7, Smad1, and Sox6, play an essential role in the osteoblast-specific activation of the enhancer.

RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx2 −/− calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter.