MED1/TRAP220, a subunit of the transcriptional Mediator/TRAP complex, is crucial for various biological events through its interaction with distinct activators, such as nuclear receptors and GATA family activators. In hematopoiesis, MED1 plays a pivotal role in optimal nuclear receptor-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we present evidence that MED1 in stromal cells is involved in supporting hematopoietic stem and/or progenitor cells (HSPCs) through osteopontin (OPN) expression. We found that the proliferation of bone marrow ( The specialized microenvironmental niches in the bone marrow (BM), namely, the osteoblastic (or endosteal) and vascular niches, host and interface with hematopoietic stem cells (HSCs) and are the sites where their size and fate are strictly regulated (15, 29; reviewed in references 1, 16, 28, 30, and 34). HSCs and their niches produce diverse molecules, whose interactions control HSC self-renewal and differentiation. In the osteoblastic niche, almost 75% of the HSCs are in a quiescent (slowly cycling or G 0 ) state. In a physiological condition, HSCs migrate from the osteoblastic niche toward the vascular niche, enter the cell cycle, and undergo symmetric cell division or asymmetric division, accompanied by differentiation and final maturation. In this manner, a defined set of mature differentiated progeny is continuously produced without HSC depletion.The transcriptional Mediator complex, originally isolated as a thyroid hormone receptor-associated protein (TRAP) complex and subsequently identified as a mammalian counterpart of the yeast Mediator complex (i.e., a subcomplex of the RNA polymerase II holoenzyme), appears to serve as a bridge between diverse activators and the general transcriptional machinery (reviewed in references 4, 13, 18, and 21). This complex contains approximately 25 polypeptides, among which the MED1/TRAP220 subunit is responsible for specific binding of the complex to several activators, which include nuclear receptors (13), GATA family members (22, 27), C/EBP␤ (20), and BRCA1 (33). Mediator conveys the specific signals of the activators to the recruited general transcriptional machinery to activate transcription by direct communication between MED1 and the activators (5).Through the interaction with MED1, nuclear receptors are involved in various hematocytic differentiations. For example, the vitamin D receptor (VDR) and retinoic acid receptor (RAR) are members of the nuclear hormone receptor superfamily, whose interaction with MED1 is crucial for liganddependent monopoiesis and granulopoiesis, respectively, as well as for peroxisome proliferator-activated receptor ␥ (PPAR␥)-mediated adipogenesis (7, 31). GATA-1, for which MED1 was recently shown to be a specific coactivator, mediates erythropoiesis through its interaction with MED1 (27). However, as Med1 null mice die early during embryogenesis (12,22), it is difficult to determine the physiological role of MED1 in BM hematopoiesis in vivo.Osteopontin (OPN), an acidic glyc...

The mean myeloperoxidase index (MPXI) is calculated during the routine complete blood count performed using the autoanalyzer ADVIA120/2120. The pattern of changes in the neutrophil myeloperoxidase levels in patients with specific infectious diseases was analyzed by assessing the MPXI levels. In patients with bacterial sepsis, identified by positive blood-culture tests, with (n = 29) and without (n = 51) systemic inflammatory response syndrome, the mean MPXI significantly reduced to -3.18 and -2.06, respectively. In contrast, among patients with nontuberculous nonseptic bacterial infections (n = 40), the mean MPXI significantly elevated to 5.51, while tuberculosis patients (n = 37) and patients with viral infection (n = 60) showed an unchanged MPXI (mean values, -0.46 and -1.06, respectively). Among the parameters of inflammation, only the C-reactive protein values showed a weak correlation with the MPXI levels. [Conclusion] These results indicate that MPXI is correlated with some specific infectious states, i.e. MPXI is low in bacterial sepsis and high in nontuberculous nonseptic bacterial infections. MPXI appears to be an independent and useful biomarker for the diagnosis and follow-up of infectious diseases, especially when the MPXI values are obtained at regular intervals during the disease courses of the patients.

The role of proteinase inhibitor (PI)-9 in hematopoietic cells remains unclear. To clarify the role of PI-9 in these cells, we compared the expressions of PI-9 mRNA and antigen with those of granzyme B (GrB). While the strongest expression of PI-9 mRNA was observed in a NK cell line YT-N10, it was also expressed in a B-acute lymphoblastic leukemia cell line U-Tree02, an EpsteinBarr Virus (EBV)-transformed B cell clone, a CD8 + T lymphocyte clone and a megakaryocytic cell line CMK, but not in a T cell line Jurkat. Phorbol 12-myristate 13 acetate (PMA) enhanced PI-9 mRNA expression in the CD8 + T lymphocyte clone and YT-N10 cells prior to GrB mRNA expression. IL-2 and IL-12 also had similar effects. PMA increased PI-9 mRNA expression in the EBV-transformed B cell clone and CMK cells, but IL-6 showed no effect. No changes were noted in PI-9 and GrB antigens after the addition of these agonists. Patients with graft-versus-host disease (GVHD) may have activated CTLs and NK cells. We therefore examined the expression of PI-9 and GrB mRNAs in eight patients after allogeneic hematopoietic stem cell transplantation with GVHD (n = 4) or without chronic GVHD (n = 4). Expression of GrB mRNA was significantly increased in three patients with GVHD and one patient without GVHD. Surprisingly, PI-9 mRNA expression was decreased in the eight patients. These results indicate that earlier synthesis of PI-9 may be essential for the prevention of autolysis of immunocompetent cells, and that the expression of PI-9 and GrB mRNAs may be controlled through different pathways.

250 The multi-protein complex TRAP/Mediator is a subcomplex of RNA polymerase II holoenzyme. Mediator acts as the end-point integrator of a variety of activators and intracellular signaling, and conveys these signals to the general transcription machinery. Among circa 25 subunits, MED1/TRAP220 subunit is crucial for many biological events through the interaction with distinct activators such as nuclear receptors (NRs) and GATA family activators. In hematopoiesis, MED1 plays an important role for optimal NR-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we analyzed the role of MED1 in the niche. We used mouse embryonic fibroblasts (MEFs) as an in vitro niche model, since MEFs have a nuance of the osteoblastic precursor and support long-term culture-initiating cells (LTC-ICs). When syngenic normal bone marrow (BM) cells were cocultured with mitomycin C-treated Med1−/− or Med1+/+ MEFs (in p53−/− background), the number of live cells (cell counts, DNA contents and MTT assays) and DNA synthesis (BrdU incorporation) was significantly suppressed on Med1−/− MEFs compared with the control during the two-week period. However, they recovered to the control level when cocultured on Med1−/− MEFs into which MED1 was stably introduced (Rev-Med1−/− MEFs). Furthermore, when LTC-ICs were assayed in complete methylcellulose media (Methocult M3434, StemCell Technologies) after the 8-week coculture, the number of LTC-ICs was attenuated for BM cells cocultured on Med1−/− MEFs compared to the control, but recovered to the control level when cocultured on Rev-Med1−/− MEFs. In order to identify the direct target(s) of MED1 that was responsible for these phenotypes, a microarray analysis of mRNA comparing Med1+/+ and Med1−/− MEFs was performed, and disclosed approximately 15 genes whose expressions were profoundly attenuated in Med1−/− MEFs. Among molecules encoded by these genes we focused on osteopontin (OPN) because solely OPN was known to have a role in niche to support hematopoietic stem/precursor cells (HSPCs). Angiopoietin-1 and Jagged-1 expressions were comparable. The expression of Opn mRNA was distinctly downregulated in Med1−/− MEFs but recovered to the control level in Rev-Med1−/− MEFs. The Opn expression in Med1+/+ MEFs was also similar to those in MC3T3-E1 mouse osteoblastic, and OP-9 BM stromal, cells. Western blot analysis of Med1+/+ MEFs using the polyclonal antibody that recognized the N-terminus of mouse OPN disclosed the abundant expression of the full-length form of OPN (ca. 70kDa), which was much less in Med1−/− MEFs. In contrast, the N-terminal cleaved form of OPN was barely visible in both MEFs. Next a chromatin immunoprecipitation (ChIP) assay was performed to know if Mediator complex was actually recruited to the Opn promoter. As expected, when the sheared chromatin was immunopurified with the antibody against MED10 subunit, Mediator was proved to be recruited to the Opn promoter more abundantly in Med1+/+ than Med1−/− MEFs, and the recruitment in Rev−Med1−/− MEFs recovered to the level of the former. Transient transfection and luciferase reporter assays disclosed that the basal level transcription as well as vitamin D receptor (VDR)- and Runx2-mediated transcriptional activation of Opn was specifically attenuated in Med1−/− MEFs, and that the basal transcription and ligand-dependent activation were dependent on the N-terminal domain (amino acids 1 to 602) and the two NR-recognition motifs of MED1, respectively. If OPN was the direct target of MED1, OPN might be responsible for, and the addition or depletion of OPN might alter, the BM cell growth and LTC-ICs support. Indeed, the addition of recombinant full-length OPN to Med1−/− MEFs restored, and the addition of the anti-OPN (N-terminus) polyclonal antibody to Med1+/+ MEFs attenuated, both the growth of cocultured BM cells during the two-week coculture and the numbers of LTC-ICs after the long-term coculture. Finally, to exclude the possibility that these phenotypes might have been restricted to MEFs, OP-9 cells, which had widely-accepted niche function, were used for similar experiments. Indeed, the addition of the anti-OPN antibody to OP-9 cells attenuated both the growth of cocultured BM cells and the numbers of LTC-ICs. Taken together, these data suggest that MED1 in niche cells, through upregulating VDR- and Runx2-mediated transcription on the Opn promoter, plays an important role in HSPCs support. Disclosures: No relevant conflicts of interest to declare.

The effects of clinical grade serine protease inhibitors on natural killer (NK) activity were compared. Cytotoxicity was measured with the Calcein-AM release method, using K562, Raji as a target. There is a significant correlation between measurements of NK activity by the Calcein-AM method and the 51Cr release assay. Cytotoxicity was inhibited with a calcium chelating agent or a perform inhibitor. Although up to 65% of cytotoxicity was inhibited by nafamostat mesilate with an E/T ratio of 10:1, and by 55% by ulinastatin, neither gabexate mesilate nor antithrombin III inhibited any cytotoxicity. None of these agents inhibited lymphokine-activated killer cell activity. In clinical applications, it should be noted that some protease inhibitors have been proven to have immunosuppressive effects.