IntroductionRecent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.MethodsSkin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.ResultsIn the skin from SSc patients, the number of CD163+ cells or CD204+ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14+ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14+ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163+ cells belong to CD14brightCD204+ population.ConclusionsThis is the first report indicating CD163+ or CD204+ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14brightCD163+CD204+ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163+ or CD204+ macrophages in the skin.

The efficacy and safety of romiplostim, a thrombopoietin-mimetic peptibody, were evaluated in a double-blind, placebo-controlled, randomized trial of Japanese patients with chronic immune thrombocytopenia (ITP). Thirty-four ITP patients received romiplostim (n = 22) or placebo (n = 12) for 12 weeks, with a starting romiplostim dose of 3 μg/kg weekly. The primary end point was the number of weeks with platelet response, defined as a platelet count ≥50 × 10(9)/L (not including the 4 weeks after rescue medication administration). Patients received a median of 4 (range 1-19) prior ITP therapies including splenectomy in 44%. On study, 68% also received concomitant ITP therapy. Weekly responses occurred for a median of 11 weeks with romiplostim as compared to 0 weeks with placebo (p < 0.0001). Most romiplostim-treated patients (95%) achieved platelet responses; two showed extended responses after the treatment period. The use of rescue medication was required in 9% of romiplostim-treated patients as compared with 17% of placebo-treated patients. Both treatment groups had similar incidences of adverse events (91% romiplostim, 92% placebo). Adverse events that occurred more frequently (>10%) in romiplostim-treated patients included nasopharyngitis, headache, peripheral edema, back pain, and extremity pain. In conclusion, romiplostim significantly increased and maintained platelet counts and was well tolerated in Japanese patients with ITP.

Because of their known myelopoietic activities, both interleukin (IL)-3 and IL-1 are often used in combination with other cytokines for in vitro (ex vivo) expansion of stem cells. We have investigated the effects of IL-3 and IL-1 on in vitro expansion of murine hematopoietic stem cells with long-term engraftment capabilities, using a highly purified progenitor population. Lineage-negative, Ly-6A/E+, c-kit+ bone marrow cells from male mice were cultured in suspension in the presence of stem cell factor, IL-6, IL-11, and erythropoietin with or without IL-3 or IL-1. Kinetic 200 units/ml; SCF, 100 ng/ml; IL-6, 100 ng/ml; Ep, 2 units/ml; 20 ng/ml; IL-la, 1 ng/ml;

Recently, the ligand for the Mpl receptor (ML) was identified to be thrombopoietin, the principal regulator of megakaryocytopoiesis and thrombopoiesis. We examined the effects of ML, as a single factor or in combinations with early acting factors such as steel factor (SF), interleukin (IL)-3, IL-1, IL-6, and granulocyte colony-stimulating factor (G-CSF), on colony formation from primitive progenitors of mice. Cells enriched for cell cycle dormant primitive progenitors were isolated from bone marrow cells of 5-fluorouracil (5-FU)-treated mice by a combination of Nycodenz density gradient separation, immunomagnetic selection for lineage-negative cells, and fluorescence- activated cell sorter (FACS) sorting for Ly-6A/E+Kit+ cells. ML, in the presence of erythropoietin, could support the formation of only a few megakaryocyte colonies. However, ML acted synergistically with SF or IL- 3 to support the formation of multiple types of hematopoietic colonies including multilineage colonies. Effects of the combination of ML and SF on multipotential progenitors were not mediated through other cells, as demonstrated by micromanipulation of individual progenitors. In suspension culture, the combination of ML and SF increased the number of multipotential progenitors. ML also acted synergistically with IL- 11, IL-6, or G-CSF to support colony formation in serum-containing, but not in serum-free, cultures. However, the multilineage colony formation seen in serum-containing culture was completely abrogated by addition of ACK2, a neutralizing antibody to Kit protein. Serial observation (mapping studies) of colony development from multipotential progenitors suggested that ML triggers the cell division of dormant progenitors. Based on these observations, we propose that ML can function as an early acting cytokine and stimulate the proliferation of cell cycle dormant progenitors by shortening their G0 period.

We report the results of a randomized study to elucidate whether addition of granulocyte colony-stimulating factor (G-CSF) to immunosuppressive therapy is valuable for the treatment of severe aplastic anemia (SAA) in adults. A total of 101 previously untreated patients (median age, 54 years; range, 19 to 75 years) were randomized to receive antithymocyte globulin (ATG) and cyclosporin A (CyA) (G-CSF؊ group) or ATG, CyA, and G-CSF (G-CSF؉ group). In the G-CSF؉ group, the hematologic response rate at 6 months was higher (77% vs 57%; P ‫؍‬ .03) than in the G-CSF؊ group. No differences were observed between the groups in terms of the incidence of infections and febrile episodes. There were no differences between the G-CSF؊ group and the G-CSF؉ group in terms of survival (88% vs 94% at 4 years), and the development of myelodysplastic syndrome (MDS)/acute leukemia (AL) (1 patient vs 2 patients). However, the relapse rate was lower in the G-CSF؉ group compared with the G-CSF؊ group (42% vs 15% at 4 years; P ‫؍‬ .01). Further follow-up is required to elucidate the role of G-CSF in immunosuppressive therapy for adult SAA. (Blood.