MERTK+/hi M2c Macrophages Induced by Baicalin Alleviate Non-Alcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. An accumulation of fat, followed by inflammation, is the major cause of NAFLD progression. During inflammation, macrophages are the most abundant immune cells recruited to the site of injury. Macrophages are classified into "proinflammatory" M1 macrophages, and "anti-inflammatory" M2 macrophages. In NAFLD, M1 macrophages are the most prominent macrophages that lead to an excessive inflammatory response. Previously, we found that baicalin could polarize macrophages into anti-inflammatory M2c subtype macrophages with an increased level of MERTK expression. Several studies have also shown a strong correlation between MERTK expression and cholesterol efflux, efferocytosis, as well as phagocytosis capability. Therefore, in this study, we aim to elucidate the potential and efficacy of mononuclear-cell (MNC)-derived MERTK+/hi M2c macrophages induced by baicalin as a cell-based therapy for NAFLD treatment. In our results, we have demonstrated that a MERTK+/hi M2c macrophage injection to NAFLD mice contributes to an increased level of serum HDL secretion in the liver, a decline in the circulating CD4+CD25- and CD8+CD25- T cells and lowers the total NAFLD pathological score by lessening the inflammation, necrosis, and fibrosis. In the liver, profibrotic COL1A1 and FN, proinflammation TNFα, as well as the regulator of lipid metabolism PPARÉ£ expression, were also downregulated after injection. In parallel, the transcriptomic profiles of the injected MERTK+/hi M2c macrophages showed that the various genes directly or indirectly involved in NAFLD progression (e.g., SERPINE1, FADS2) were also suppressed. Downregulation of cytokines and inflammation-associated genes, such as CCR5, may promote a pro-resolving milieu in the NAFLD liver. Altogether, cell-based therapy using MERTK+/hi M2c macrophages is promising, as it ameliorates NAFLD in mice.

Withholding of M-CSF Supplement Reprograms Macrophages to M2-Like via Endogenous CSF-1 Activation.

Macrophage colony-stimulating factor (M-CSF or CSF-1) is known to have a broad range of actions on myeloid cells maturation, including the regulation of macrophage differentiation, proliferation and survival. Macrophages generated by M-CSF stimulus have been proposed to be alternatively activated or M2 phenotype. M-CSF is commonly overexpressed by tumors and is also known to enhance tumor growth and aggressiveness via stimulating pro-tumor activities of tumor-associated macrophages (TAMs). Currently, inhibition of CSF-1/CSF-1R interaction by therapeutic antibody to deplete TAMs and their pro-tumor functions is becoming a prevalent strategy in cancer therapy. However, its antitumor activity shows a limited single-agent effect. Therefore, macrophages in response to M-CSF interruption are pending for further investigation. To achieve this study, bone marrow derived macrophages were generated in vitro by M-CSF stimulation for 7 days and then continuously grown until day 21 in M-CSF absence. A selective pressure for cell survival was initiated after withdrawal of M-CSF. The surviving cells were more prone to M2-like phenotype, even after receiving interleukin-4 (IL-4) stimulation. The transcriptome analysis unveiled that endogenous CSF-1 level was dramatically up-regulated and numerous genes downstream to CSF-1 covering tumor necrosis factor (TNF), ras-related protein 1 (Rap1) and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway were significantly modulated, especially for proliferation, migration and adhesion. Moreover, the phenomenal increase of miR-21-5p and genes related to pro-tumor activity were observed in parallel. In summary, withholding of CSF-1/CSF-1R interaction would rather augment than suspend the M-CSF-driven pro-tumor activities of M2 macrophages in a long run.

Autocrine VEGF signalling on M2 macrophages regulates PD-L1 expression for immunomodulation of T cells.

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.

Low c-Kit Expression Level Induced by Stem Cell Factor Does Not Compromise Transplantation of Hematopoietic Stem Cells.

The c-Kit expression level is decreased in regenerating bone marrow, and such bone marrow performs poorly when co-transplanted with normal bone marrow. We asked whether diminished numbers of c-Kit receptors on hematopoietic stem and progenitor cells (HSPCs) after their internalization induced by the binding of the cytokine stem cell factor (SCF) would jeopardize transplantability of HSPCs. We used a battery of functional assays to evaluate the capacity of HSPCs with markedly different c-Kit expression levels to be transplanted. Surprisingly, our experiments testing the homing of transplanted HSPCs to bone marrow of recipient mice and their short-term and long-term engraftment did not reveal any defects in HSPCs with severely reduced numbers of c-Kit receptor molecules. This unexpected result can be ascribed to the fact that HSPCs exposed to SCF replace the consumed c-Kit receptors rapidly. This article demonstrates that exposure of HSPCs to SCF and diminished number of c-Kit receptors in their cell membranes do not compromise the capacity of HSPCs to reconstitute damaged hematopoietic tissue.

Ginsenoside Rg1 improves bone marrow haematopoietic activity via extramedullary haematopoiesis of the spleen.

Cyclophosphamide (CY) is a chemotherapeutic agent used for cancer and immunological diseases. It induces cytotoxicity of bone marrow and causes myelosuppression and extramedullary haematopoiesis (EMH) in treated patients. EMH is characterized with the emergence of multipotent haematopoietic progenitors most likely in the spleen and liver. Previous studies indicated that a Chinese medicine, ginsenoside Rg1, confers a significant effect to elevate the number of lineage (Lin(-) ) Sca-1(+) c-Kit(+) haematopoietic stem and progenitor cells (HSPCs) and restore the function of bone marrow in CY-treated myelosuppressed mice. However, whether the amelioration of bone marrow by Rg1 accompanies an alleviation of EMH in the spleen was still unknown. In our study, the cellularity and weight of the spleen were significantly reduced after Rg1 treatment in CY-treated mice. Moreover, the number of c-Kit(+) HSPCs was significantly decreased but not as a result of apoptosis, indicating that Rg1 alleviated EMH of the spleen induced by CY. Unexpectedly, the proliferation activity of c-Kit(+) HSPCs was only up-regulated in the spleen, but not in the bone marrow, after Rg1 treatment in CY-treated mice. We also found that a fraction of c-Kit(+) /CD45(+) HSPCs was simultaneously increased in the circulation after Rg1 treatment. Interestingly, the effects of Rg1 on the elevation of HSPCs in bone marrow and in the peripheral blood were suppressed in CY-treated splenectomized mice. These results demonstrated that Rg1 improves myelosuppression induced by CY through its action on the proliferation of HSPCs in EMH of the spleen and migration of HSPCs from the spleen to the bone marrow.

Multichannel lens-free CMOS sensors for real-time monitoring of cell growth.

A low-cost platform is proposed for the growth and real-time monitoring of biological cells. The main components of the platform include a PMMA cell culture microchip and a multichannel lens-free CMOS (complementary metal-oxide-semiconductor) / LED imaging system. The PMMA microchip comprises a three-layer structure and is fabricated using a low-cost CO2 laser ablation technique. The CMOS / LED monitoring system is controlled using a self-written LabVIEW program. The platform has overall dimensions of just 130 × 104 × 115 mm(3) and can therefore be placed within a commercial incubator. The feasibility of the proposed system is demonstrated using HepG2 cancer cell samples with concentrations of 5000, 10 000, 20 000, and 40 000 cells/mL. In addition, cell cytotoxicity tests are performed using 8, 16, and 32 mM cyclophosphamide. For all of the experiments, the cell growth is observed over a period of 48 h. The cell growth rate is found to vary in the range of 44∼52% under normal conditions and from 17.4∼34.5% under cyclophosphamide-treated conditions. In general, the results confirm the long-term cell growth and real-time monitoring ability of the proposed system. Moreover, the magnification provided by the lens-free CMOS / LED observation system is around 40× that provided by a traditional microscope. Consequently, the proposed system has significant potential for long-term cell proliferation and cytotoxicity evaluation investigations.

Toll-like Receptor-Mediated Immunomodulation of Th1-Type Response Stimulated by Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2).

PRRSV infects CD163-positive macrophages and skews their polarization toward an M2 phenotype, followed by T-cell inactivation. In our previous study, we found that recombinant protein A1 antigen derived from PRRSV-2 was a potential vaccine or adjuvant for immunization against PRRSV-2 infection due to its ability to repolarize macrophages into M1 subtype, thereby reducing CD163 expression for viral entry and promoting immunomodulation for Th1-type responses, except for stimulating Toll-like receptor (TLR) activation. The aim of our current study was to evaluate the effects of another two recombinant antigens, A3 (ORF6L5) and A4 (NLNsp10L11), for their ability to trigger innate immune responses including TLR activation. We isolated pulmonary alveolar macrophages (PAMs) from 8- to 12-week-old specific pathogen free (SPF) piglets and stimulated them with PRRSV (0.01 MOI and 0.05 MOI) or antigens. We also investigated the T-cell differentiation by immunological synapse activation of PAMs and CD4+ T-cells in the cocultured system. To confirm the infection of PRRSV in PAMs, we checked the expression of TLR3, 7, 8, and 9. Our results showed that the expression of TLR3, 7, and 9 were significantly upregulated in PAMs by A3 antigen induction, similar to the extent of PRRSV infection. Gene profile results showed that A3 repolarizes macrophages into the M1 subtype potently, in parallel with A1, as indicated by significant upregulation of proinflammatory genes (TNF-α, IL-6, IL-1ß and IL-12). Upon immunological synapse activation, A3 potentially differentiated CD4 T cells into Th1 cells, determined by the expression of IL-12 and IFN-γ secretion. On the contrary, antigen A4 promoted regulatory T cell (T-reg) differentiation by significant upregulation of IL-10 expression. Finally, we concluded that the PRRSV-2 recombinant protein A3 provided better protection against PRRSV infection, suggested by its capability to reeducate immunosuppressive M2 macrophages into proinflammatory M1 cells. As M1 macrophages are prone to be functional antigen-presenting cells (APCs), they can call for TLR activation and Th1-type immune response within the immunological synapse.

High levels estradiol affect blastocyst implantation and post-implantation development directly in mice.

BACKGROUND: Previous studies have demonstrated that high levels of estradiol (E2) impair blastocyst implantation through effects on the endometrium; however, whether high E2 directly affects blastocysts is not well established. The present study sought to clarify the direct impacts of high E2 levels on blastocysts in vitro. METHODS: ICR virgin albino mice were used. Using an in-vitro 8-day blastocyst culture model, immunofluorescence staining for the estrogen receptor (ER), blastocyst outgrowth assays, differential staining and TUNEL assays of blastocysts, and embryo transfer, we investigated the main outcomes of exposure to different E2 concentrations (10-7 to 10-4 M) in vitro and in vivo. RESULTS: ERα and ERß expression were detected in pre-implantation stage embryos. In vitro exposure of blastocysts to 10-4 M E2 for 24 h followed by 7 days culture in the absence of E2 caused severe inhibition of implantation and post-implantation development. The late adverse effects of E2 on post-implantation development still occurred at concentrations of 10-7 to 10-5 M. In addition, blastocyst proliferation was reduced and apoptotic cells were increased following exposure to 10-4 M E2. Using an in vivo embryo-transfer model, we also showed that treatment with high E2 resulted in fewer implantation sites (38% vs. 72% in control) and greater resorption of implanted blastocysts (81% vs. 38% in control). CONCLUSION: Exposure to high E2 concentrations in vitro is deleterious to blastocyst implantation and early post-implantation development, mainly owing to direct impacts of E2 on implanting blastocysts. In clinical assisted reproductive technique (ART), high serum E2 concentrations not only affects the endometrium, but also affects blastocysts directly at the period of implantation.

Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) Promotes M1 Repolarization of Porcine Alveolar Macrophages and Th1 Type Response.

The polarization status of porcine alveolar macrophages (PAMs) determines the infectivity of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV infection skews macrophage polarization toward an M2 phenotype, followed by T-cells inactivation. CD163, one of the scavenger receptors of M2 macrophages, has been described as a putative receptor for PRRSV. In this study, we examined two types of PRRSV-2-derived recombinant antigens, A1 (g6Ld10T) and A2 (lipo-M5Nt), for their ability to mediate PAM polarization and T helper (Th1) response. A1 and A2 were composed of different combination of ORF5, ORF6, and ORF7 in full or partial length. To enhance the adaptive immunity, they were conjugated with T cells epitopes or lipidated elements, respectively. Our results showed that CD163+ expression on PAMs significantly decreased after being challenged with A1 but not A2, followed by a significant increase in pro-inflammatory genes (TNF-α, IL-6, and IL-12). In addition, next generation sequencing (NGS) data show an increase in T-cell receptor signaling in PAMs challenged with A1. Using a co-culture system, PAMs challenged with A1 can induce Th1 activation by boosting IFN-γ and IL-12 secretion and TNF-α expression. In terms of innate and T-cell-mediated immunity, we conclude that A1 is regarded as a potential vaccine for immunization against PRRSV infection due to its ability to reverse the polarization status of PAMs toward pro-inflammatory phenotypes, which in turn reduces CD163 expression for viral entry and increases immunomodulation for Th1-type response.

Erythropoietin receptor signaling regulates both erythropoiesis and megakaryopoiesis in vivo.

Transgenic expression of a gain-of-function truncated mouse erythropoietin receptor gene (EpoR) leads to expansion of the HSC pool in response to human erythropoietin (Epo). We have re-examined this observation using a knock-in mouse model, wherein the mouse EpoR gene was replaced in its proper genetic locus by a single copy of either a wild-type human or a polycythemia-inducing truncated human EPOR gene. Bone marrow cells obtained from knock-in mice were transplanted together with competitor bone marrow cells in a model that allows tracking of erythroid, platelet, and leukocyte contributions by each genotype. Secondary transplants were also performed. Stem/progenitor cells were identified phenotypically and isolated for colony-forming assays to evaluate cytokine responsiveness by cells with the wild-type human or truncated human EPOR gene. Augmented Epo signaling increased erythroid repopulation post-transplant as expected, but had no effect on short-term or long-term leukocyte repopulation. However, the wild-type human EPOR knock-in mouse showed decreases in both erythroid and platelet repopulation compared to marrow cells from the mutant human EPOR knock-in mouse or normal B6 animals. These results provide evidence supporting a role for Epo signaling in megakaryopoiesis in vivo and suggest a role for Epo signaling early in hematopoietic development.

Adult Stem Cells in Hibernation: Future Perspectives of Space Travel.

Space traveling is imperative for mankind in the future. Expectedly, hibernation will become an option for space traveler to overcome the endless voyage. With regard to some of the studies pointed out that during hibernation, muscle will undergo atrophy and meantime neurogenesis will reduce, these obstacles were frequently related with stem cell regeneration. Thus, investigation on whether hibernation will lead to dysfunction of stem cell becomes an important issue. By going through four main systems in this article, such as, hematopoietic system, skeletal muscle system, central nervous system and orthopedic system, we are expecting that stem cells regeneration capacity will be affected by hibernation. To date, these researches are majorly the read-out from short term or seasonal hibernating mammals. Proposing and creating a simulated long-term hibernation animal model is turning essential for the further investigation on the effect of longer period of hibernation to human stem cells.

Imatinib effect on growth and signal transduction in polycythemia vera.

OBJECTIVE: An activating mutation of Janus kinase 2 (JAK2) in majority of polycythemia vera (PV) and other myeloproliferative disorders was reported. As imatinib inhibits several tyrosine kinases, we studied its effect in PV. PATIENTS AND METHODS: We employed FDCP reporter cells expressing wild-type JAK2 and mutant JAK2(V617F) to study the efficacy of imatinib by cell proliferation assay and its effect on several cell-signaling events. Imatinib's efficacy was also examined on in vitro expanded native human erythroid progenitors. In addition, analysis of the percent JAK2 T-allele and phospho-signal transducer and activator of transcription-5 (STAT5) in granulocytes of PV patients following imatinib therapy was assessed. RESULTS: Imatinib showed a specific time- and dose-dependent growth inhibitory effect on FDCP cells expressing JAK2(V617F), wherein we observed imatinib's inactivation of JAK2, STAT5 and cKIT proteins. In vitro expanded human PV erythroid progenitors were more sensitive to imatinib than normal erythroid progenitors and FDCP cells expressing JAK2(V617F), with growth inhibition at concentrations attainable in vivo. In an ongoing clinical study, a PV patient showed strong correlation between the percent JAK2 T-allele and his responsiveness to imatinib therapy. CONCLUSION: Our data elucidate the therapeutic benefit of imatinib seen in some PV patients. Our data suggest that JAK2/STAT5 and cKIT activation may be integrated. To our knowledge, this is the first report demonstrating imatinib's effect on PV erythroid progenitors. These studies underscore the limitation of experiments using cell lines expressing the gene of interest.

M2C Polarization by Baicalin Enhances Efferocytosis via Upregulation of MERTK Receptor.

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- α ), 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.

Congenital polycythemia with homozygous and heterozygous mutations of von Hippel-Lindau gene: five new Caucasian patients.

We report on five Caucasian patients with congenital polycythemia and mutations of the von Hipple-Lindau (VHL) gene: a compound heterozygote for the novel exon 1 (VHL 235C->T) and previously reported VHL 562C->G mutations; three homozygotes for Chuvash VHL 598C->T mutation; and a heterozygote for VHL 523->G mutation who also has ataxia-telangiectasia; a rare autosomal disease of childhood onset.

Extramedullary hematopoiesis (EMH) in laboratory animals: offering an insight into stem cell research.

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.

S100P expression is a novel prognostic factor in hepatocellular carcinoma and predicts survival in patients with high tumor stage or early recurrent tumors.

The calcium-binding protein S100P is expressed in a variety of human cancer cells and is important in cancer cell growth and invasion. Using differential display, we found S100P is overexpressed in human hepatocellular carcinoma (HCC). We examined the expression of 305 unifocal, primary HCC tumors using immunohistochemistry. The S100P protein was expressed in 173 of the 305 (56.7%) HCC tumors. The expression of S100P correlated with female sex (P = 0.0162), high serum α-fetoprotein level (P = 0.0001), high tumor grade (P = 0.0029), high tumor stage (P = 0.0319), the presence of the p53 mutation (P = 0.0032), and the absence of the ß-catenin mutation (P = 0.0489). Patients with HCC tumors that expressed S100P were more likely to have early tumor recurrence (ETR) (P = 0.0189) and lower 5-year survival (P = 0.0023). The multivariate analysis confirmed that S100P expression was an independent prognostic factor in HCC. The combinatorial analysis showed an additive unfavorable prognostic interaction between S100P expression and the p53 mutation. In contrast, the ß-catenin mutation was associated with better prognosis in both S100P-positive and -negative HCCs. Furthermore, S100P expression was a predictor of survival in HCC patients with high tumor stage or ETR (P = 0.0026 and P = 0.0002, respectively). Our study indicates the expression of the S100P protein is a novel independent predictor for poor prognosis in HCC, and it is also an unfavorable prognostic predictor in HCC patients with high tumor stage or ETR.

B-lymphopoiesis gains sensitivity to subsequent inhibition by estrogens during final phase of fetal development.

Adult B-lymphopoiesis is suppressed by the inhibitory effects of elevated estrogens during pregnancy. At the same time, hematopoietic cells in the fetal liver are resistant to this suppression by estrogens and ensure active production of B-cells. We investigated whether this unresponsiveness to estrogens of fetal cells also applies to cells obtained from a newborn liver and projects into the adult hematopoiesis when fetal liver cells are transplanted to adult mice. Mixtures of fetal liver (E14.5), neonatal liver (P0.5) and adult bone marrow (BM) cells were co-transplanted into adult primary and secondary recipients treated with high doses of estrogen in the Ly5.1/Ly5.2 congenic mouse model. Total chimerism as a proportion of all nucleated blood cells, chimerism as a proportion of B220+ B-cells, and of other blood cell lineages as well, were determined by flow cytometry. B-lymphopoiesis derived from fetal liver (E14.5) stem cells remained resistant to estrogen after transplantation into both primary and secondary adult recipients, for up to 280 days. In contrast, B-lymphopoiesis derived from neonatal liver (P0.5) stem cells was resistant to estrogen only for approximately 50 days after the primary transplantation to the adult BM microenvironment. These results provide further evidence for a critical developmental period of B-lymphopoiesis during its fetal liver stage. In the mouse, critical developmental events that allow for the subsequent expressed sensitivity of B-lymphopoiesis for suppression by estrogens after sexual maturation appear to occur during the period of late-stage fetal liver hematopoiesis before its migration to the bone marrow.

Early fetal liver readily repopulates B lymphopoiesis in adult bone marrow.

Fetal liver (FL) becomes a major organ of hematopoiesis at mouse embryonic day (E) 11 and E12, when definitive hematopoietic stem cells, originating from the aorta-gonads-mesonephros region, colonize the hepatic tissue. Unipotent B-cell progenitors are very rare in FL by day 12, whereas erythropoiesis prevails. We have studied hematopoiesis in FL from different gestational ages, with special emphasis on B lymphopoiesis. The mRNA levels of selected liver-specific genes, hematopoietic lineage-specific genes, and genes for selected cytokines/hormones as well as for their receptors were evaluated by real-time polymerase chain reaction in FL from E12.5, E14.5, and E17.5, adult liver and adult bone marrow (BM). The level of B lineage-related gene expression in FL was very low at E12.5. There was also a significantly lower fraction of B220+ and CD19+ B cells in E12.5 FL compared with E17.5 FL. To analyze whether these differences reflect different stem cell potentials occurring during FL development, 10(6) or 5 x 10(6) of FL cells collected from embryos at E12.5 or E17.5 and those from adult BM were transplanted into sublethally irradiated (3- or 6-Gy) congenic mice. Short-term and long-term repopulation of B and T cells and granulocyte/macrophage lineages from donor FL or adult BM cells were evaluated in competition to adult hematopoiesis of sublethally irradiated recipients. In short-term repopulation, the transplantation of E12.5 FL cells resulted in a lower blood chimerism compared with that of E17.5 FL cells. However, the proportion of B lymphopoiesis exerted by E12.5 FL cells was not different from that of E17.5 FL or adult BM. This study demonstrates that E12.5 FL contains hematopoietic stem cells with fully developed B-cell repopulating capacity and that the developmental period of fetal hematopoiesis between E12.5 and E17.5 is not an obligatory phase for the adult B lymphopoiesis.

Expression of Rgmc, the murine ortholog of hemojuvelin gene, is modulated by development and inflammation, but not by iron status or erythropoietin.

Mutations of hepcidin (HAMP) and hemo-juvelin (HJV) genes have been recently demonstrated to result in juvenile hemochromatosis. Expression of HAMP is regulated by iron status or infection, whereas regulation of HJV is yet unknown. Using quantitative real-time polymerase chain reaction, we compared expression of Hamp and Rgmc (the murine ortholog of HJV) in livers of mice treated with iron, erythropoietin, or lipopolysaccharide (LPS), as well as during fetal and postnatal development. Iron overload increased Hamp expression without effect on Rgmc mRNA. Erythropoietin decreased Hamp mRNA, but Rgmc expression was unchanged. Hamp mRNA level decreased after birth by 4 orders of magnitude, without significant changes in Rgmc expression. Administration of LPS elevated Hamp mRNA levels, while markedly decreasing hepatic Rgmc mRNA levels (to approximately 5% after 6 hours). The responses of Hamp and Rgmc were quite different and suggested that human HJV expression could be modulated by inflammation.