25- Study design Cell separation and cultureDCs and MACs were derived from human blood monocytes or CD34 ϩ as described previously. 15,16 Terminal maturation was induced with 10 ng/mL tumor necrosis factor (TNF) or 10 ng/mL lipopolysaccharide (LPS) or a mixture containing 10 ng/mL TNF-␣, 10 ng/mL interleukin-1␤ (IL-1␤), 1000 U/mL IL-6, and 1 g/mL prostaglandin E 2 . 17 Northern blot analysisA cDNA fragment of 25-hydroxyvitamin D 3 -1␣-hydroxylase (25(OH)D 3 -1␣-hydroxylase) complementary to the nucleic acids ϩ931 base pair (bp) to ϩ2073 bp (GenBank/European Molecular Biology Laboratory [EMBL] accession no. AB005989) was used for hybridization. As a loading control, membranes were rehybridized with an 18S rRNA-oligonucleotide (5Ј-ACG GTA TCT GAT CGT CTT CGA ACC-3Ј). ImmunohistochemistryDCs were fixed with glutaraldehyde and a standard alkaline phosphatase antialkaline phosphatase (APAAP) staining was performed with a polyclonal sheep antibody against mouse and human 25(OH)D 3 -1␣-hydroxylase (The Binding Site, Birmingham, United Kingdom), a secondary rabbit antisheep antibody (Abcam, Cambridge, United Kingdom), APAAP reagent (Dianova, Hamburg, Germany), and Fast Red as a detection substrate (BioGenex, San Ramon, CA). For personal use only. on May 9, 2018. by guest www.bloodjournal.org From 1␣,25-Dihydroxyvitamin D 3 ELISA Cells were seeded (10 6 cells/well/mL) in RPMI medium in a 6-well plate. After incubation for 24 hours with 5 ϫ 10 Ϫ8 M 25(OH)D 3 (Sigma, Deisenhofen, Germany) with or without 100 ng/mL LPS (Salmonella abortus equi, kindly provided by Dr Chris Galanos, MPI, Freiburg, Germany), the supernatant and the cells were harvested. 1,25(OH) 2 D 3 was separated from other vitamin D metabolites by extraction columns (Immundiagnostik, Bensheim, Germany) and determined with a commercially available enzyme-linked immunosorbent assay (ELISA; Immundiagnostik). This ELISA is specific for 1,25(OH) 2 D 3 and does not recognize other vitamin D metabolites. Mixed lymphocyte reaction (MLR)T cells (10 5 ) were incubated with different amounts of immature and mature allogenic DCs in RPMI containing 5% T-cell autologous plasma. On day 6 of coculture, 1 Ci (0.037 MBq) of 3 H-methyl-thymidine/well was added, and incorporated radioactivity was determined after 20 hours. All samples were performed in triplicates and values represent mean Ϯ SEM. Results and discussion25(OH)D 3 -1␣-hydroxylase, the mitochondrial cytochrome P450 enzyme that catalyzes the conversion of 25(OH)D 3 , was detectable only in the late stages of DC differentiation, and the level of expression was low independent of the culture condition (granulocyte-macrophage colony-stimulating factor [GM-CSF] plus IL-4 vs interferon ␣ [IFN␣]). 16 Terminal differentiation of DCs with LPS clearly up-regulated the expression ( Figure 1A). In contrast, MACs cultured either in human AB-group serum or with fetal calf serum (FCS) plus GM-CSF showed a strong expression of 25(OH)D 3 -1␣-hydroxylase mRNA. CD34 ϩ -derived DCs 15 cultured with stem cell factor (SCF), GM-CSF, a...

The differentiation of macrophages from their progenitors is controlled by macrophage colony-stimulating factor (CSF-1), which binds to a receptor (CSF-1R) encoded by the c-fms proto-oncogene. We have previously used the promoter region of the CSF-1R gene to direct expression of an enhanced green fluorescent protein (EGFP) reporter gene to resident macrophage populations in transgenic mice. In this paper, we show that the EGFP reporter is also expressed in all granulocytes detected with the Gr-1 antibody, which binds to Ly-6C and Ly-6G or with a Ly-6G-specific antibody. Transgene expression reflects the presence of CSF-1R mRNA but not CSF-1R protein. The same pattern is observed with the macrophage-specific F4/80 marker. Based on these findings, we performed a comparative array profiling of highly purified granulocytes and macrophages. The patterns of mRNA expression differed predominantly through granulocyte-specific expression of a small subset of transcription factors (Egr1, HoxB7, STAT3), known abundant granulocyte proteins (e.g., S100A8, S100A9, neutrophil elastase), and specific receptors (fMLP, G-CSF). These findings suggested that appropriate stimuli might mediate rapid interconversion of the major myeloid cell types, for example, in inflammation. In keeping with this hypothesis, we showed that purified Ly-6G-positive granulocytes express CSF-1R after overnight culture and can subsequently differentiate to form F4/80-positive macrophages in response to CSF-1.

Clinical research has resulted in an improvement of treatment options for patients with immune thrombocytopenia (ITP) over the last years. However, only few data exist on the real-life management of patients with ITP. To expand the knowledge, a multicenter, national survey was undertaken in 26 hematology practices distributed all over Germany. All patients with a diagnosis of ITP were documented using questionnaires, irrespective of the diagnosis date over a period of 2 years. Overall, data of 1023 patients were evaluated with 56% of patients being older than 60 years. Seventy-nine percent of the patients had chronic (> 12 months), 16% persistent (> 3–12 months), and 5% newly diagnosed (0–3 months) ITP. In 61% of cases, the disease lasted 3 or more years before survey documentation started. Main strategies applied as first-line therapy consisted of steroids in 45% and a “watch and wait” approach in 41% of patients. During second- and third-line strategies, treatment with steroids decreased (36% and 28%, respectively), while treatment modalities such as TPO-RAs increased (19% and 26%, respectively). As expected, patients with a low platelet count and thus a higher risk for bleeding and mortality received treatment (esp. steroids) more frequently during first line than those with a higher platelet count. Up to a third of patients were treated with steroids for more than a year. Overall, our study provides a cross-section overview about the current therapeutic treatment landscape in German ITP patients. The results will help to improve therapeutic management of ITP patients.

No abstract

MADDAM (Metalloprotease And Disintegrin Dendritic Cell Antigen Marker, ADAM19), a human metalloprotease belonging to the ADAM-family, is strongly expressed during in vitro differentiation of monocytes into dendritic cells (DC), whereas differentiation of monocytes into macrophages (MAC) is associated with a loss of MADDAM transcription. To investigate the mechanisms underlying this cell-type specific expression pattern we defined the transcriptional start site and the proximal promoter of the MADDAM gene. Gene bank analysis of the CpG island promoter and first intron revealed putative binding sites for several transcription factors, including VDR, NF-kB and Sp1-family factors. EMSA demonstrated binding of Sp1, Sp3, NF-kB and VDR to their putative binding sites in the proximal promoter region and mutation of these elements led to a decreased reporter activity of the proximal promoter in luciferase assays. A minimal promoter construct of 150-bp showed weak reporter activity in primary monocyte-derived MAC and a threefold higher activity in monocyte-derived DC, indicating that differential binding of transcription factors contributes to the cell-type specific regulation of MADDAM. Transfection of monocytic THP-1 cells with the 150-bp fragment also resulted in significant reporter activity, despite the lack of endogenous MADDAM expression. Interestingly, Trichostatin A (TSA), a known inhibitor of histone deacetylation, lead to a dose dependent induction of MADDAM mRNA in THP-1 cells. Chromatin immunoprecipitation (ChIP) assays demonstrate increased levels of acetylated histones H3 and H4 in DC as compared to MAC, indicating an important role of histone acetylation in the cell-type specific regulation of the MADDAM gene.