Mutations of hepcidin (HAMP)and IntroductionDuring the past few years, a number of new genes participating in iron metabolism have been identified. Mutations in 2 genes, hepcidin (HAMP) 1 and hemojuvelin (HJV) 2,3 have been shown to result in juvenile hemochromatosis. Hepcidin, a small peptide synthesized predominantly in hepatocytes, is emerging as an important regulator of iron homeostasis, which inhibits iron absorption from the intestine and iron release from macrophages. Hepcidin expression is controlled by iron status and erythropoietic activity, as well as by inflammatory stimuli 4 ; inappropriate expression of hepcidin probably plays a role in the pathophysiology of hereditary hemochromatosis and anemia of inflammation. 5 On the other hand, the function and regulation of hemojuvelin are at present unknown. Prior to identification of the HJV gene, it was speculated that its product could function in the hepcidin signaling pathway, possibly as a hepcidin receptor, 5 whereas a current concept proposes that hemojuvelin could modulate hepcidin expression. 2,6 Orthologs of the HJV gene have been identified in zebrafish, mice, and rats 2 ; the mouse HJV ortholog Rgmc is, like HJV, expressed mainly in skeletal muscle, heart, and liver. 7 The aim of the present study was to examine whether experimental conditions known to influence hepatic Hamp expression in mice will also change hepatic Rgmc mRNA levels and to compare possible similarities or discrepancies in the regulation of these 2 genes. Study designAll animal experiments were approved by the Animal Care Committee of the First Faculty of Medicine. Male C57BL/6N mice (Charles River, Sulzfeld, Germany) were treated with lipopolysaccharide (LPS, serotype 0111:B4, 1 mg/kg intraperitoneally; Sigma Aldrich, Prague, Czech Republic) and humanely killed by cervical dislocation after 90 minutes or 6 hours. Iron overload (600 mg/kg) was induced by a single subcutaneous injection of iron polyisomaltosate (Ferrum Lek; Lek, Ljubljana, Slovenia); mice were humanely killed 1 week or 3 weeks after application. Erythropoietin (EPREX 10 000, Cilag AG, Schaffhausen, Switzerland) was administered at 50 U/mouse for 4 days, and mice were killed on day 5.Liver RNA was extracted using RNABlue (Top-Bio, Prague, Czech Republic), treated with DNase I (Gibco, Life Technologies, Gaithersburg, MD), and 1 g total RNA was reverse transcribed by the RevertAid First-Strand cDNA synthesis kit (Fermentas, Vilnius, Lithuania).Levels of Hamp and Rgmc mRNA were determined by real-time polymerase chain reaction (PCR) on a Roche LightCycler instrument, using LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Mannheim, Germany). Primer sequences were: ␤-actin forward 5Ј-GACATGGAGAAGATCTGGCA-3Ј, reverse 5Ј-GGTCTTTACGGATGT-CAACG-3Ј; Hamp forward 5Ј-CTGAGCAGCACCACCTATCTC-3Ј, Hamp reverse 5Ј-TGGCTCTAGGCTATGTTTTGC-3Ј; Rgmc forward 5-CCCA-GATCCCTGTGACTATGA -3, Rgmc reverse 5-CAGGAAGATTGTCCAC-CTCAG -3. Rgmc primers were designed to amplify a sequence from exons 3 and 4 of Rgmc DNA. 2 Beca...

M2‐polarized macrophages, on one hand, can promote tumour vascularization by producing proangiogenic factors, such as vascular endothelial growth factor (VEGF). On the other hand, the expression of VEGF receptors (VEGFR) in this cell lineage was also reported. Although the function of VEGF/VEGFR axis plays a pivotal role in macrophages infiltration and angiogenesis, however, there is still lack of the direct evidence to show the role of VEGF as an autocrine operating in M2 macrophages, particularly for immunomodulation. In our study, we surprisingly discovered that M2 macrophages polarized by baicalin can simultaneously express VEGF and its receptors. Taking advantage of this unique culture system, we were able to investigate the biological activity of M2 macrophages in response to the autocrine VEGF milieu. Our results showed that the expression of programmed death‐ligand 1 (PD‐L1) on M2 macrophages was significantly up‐regulated in autocrine VEGF milieu. Through the blockade of autocrine VEGF signalling, PD‐L1 expression on M2 macrophages was dramatically down‐regulated. Furthermore, transplantation of PD‐L1+ M2 macrophage stimulated by autocrine VEGF into allogeneic mice significantly suppressed host CD4+/CD8+ T cells in the peripheral blood and increased CD4+ CD25+ regulatory T cells in the bone marrow. In conclusion, our findings provide a novel biological basis to support the current successful strategy using combined VEGF/PD‐1 signalling blockade in cancer therapy.

Extramedullary hematopoiesis (EMH) is a pathological process secondary to underlying bone marrow (BM) insufficiency in adults. It is characterized by the emergence of multipotent hematopoietic progenitors scattered around the affected tissue, most likely in the spleen, liver, and lymph node, etc. EMH in patients frequently receives less medical attention and is neglected unless a compressive or obstructive hematopoietic mass appears to endanger the patient's life. However, on a biological basis, EMH reflects the alteration of relationships among hematopoietic stem and progenitor cells (HSPCs) and their original and new microenvironments. The ability of hematopoietic stem cells (HSCs) to mobilize from the bone marrow and to accommodate and function in extramedullary tissues is rather complicated and far from our current understanding. Fortunately, many reports from the studies of drugs and genetics using animals have incidentally found EMH to be involved. Thereby, the molecular basis of EMH could further be elucidated from those animals after cross-comparison. A deeper understanding of the extramedullary hematopoietic niche could help expand stem cells in vitro and establish a better treatment in patients for stem cell transplantation.

Objectives-A G>T transversion in a tyrosine kinase JAK2 (V617F) was reported in over 80% of patients with polycythemia vera (PV). Current evidence suggests that JAK2 V617F somatic mutation is involved in the pathogenesis of PV, since it confers erythropoietin independent proliferation to erythroid progenitor cells. However, several unanswered questions regarding the essential role of JAK2 V617F arose as a) it is not a dominant mutation, b) it is not PV specific since it is found in several myeloproliferative disorders and c) some (~20%) PV patients lack the JAK2 V617F mutation. We set up to investigate the relative frequency of JAK2 V617F in in vitro expanded PV progenitors.Methods-In vitro expansion of erythroid progenitors from mononuclear cells was optimized. Frequency of JAK2 V617F allele was measured by using allele-specific real-time PCR. Clonality was performed using established procedure.Results-In vitro expansion of PV erythroid progenitors and differentiated dendritic cells resulted in a decrease of the frequency of JAK2 V617F allele compared to granulocytes or CD235 + erythroid progenitors. Clonality analysis demonstrated that while granulocytes of these PV patients were clonal, expanded erythroid cells were polyclonal. However, in vitro expanded PV erythroid progenitors still had ~two fold increased proliferative capacity in comparison to erythroid progenitors from healthy individuals. Erythropoietin favors the cells without JAK2 V617F allele. Dendritic cells in one out of three patients remained clonal. Conclusion-JAK2V617F mutation does not provide a proliferative/survival advantage to the PV clone during in vitro expansion. These data suggest that the JAK2 V617F mutation plays an important role in the biology of PV, yet it may not be the PV-initiating event.

Baicalin is the main active ingredient primary isolated from the Chinese herb, Scutellaria baicalensis Georgi. Although baicalin can induce M2 macrophage polarization, we still do not know the subtype of macrophages polarized by baicalin. In this study, we characterized that murine bone marrow derived macrophages induced by M-CSF can be further polarized into M2C phenotype by baicalin. The signatures of M2C macrophages for mRNA expression like interferon regulatory factor 4 (IRF4), interleukin-10 (IL-10), MERTK and PTX3 were up-regulated. Moreover, we observed the concomitantly decreasing of tumor necrosis factor alpha (TNF-[Formula: see text]), interferon regulatory factor 5 (IRF5), IL-6. In contrast, M2 macrophages polarized by IL-4 increased gene transcript of arginase-1 (Arg-1) and surface marker of CD206 indicates that their identity as M2A rather than M2C subtypes. Interestingly, the phagocytosis as well as efferocytosis activity were significantly enhanced in M2C macrophage polarized by baicalin and these capacities were associated with the expression of MERTK receptor. Finally, we conclude that baicalin induced M2C macrophages polarization with both elevations of efferocytosis and anti-inflammatory activity.