TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells.

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Here we identified T cell immunoglobulin mucin-3 (TIM-3) as a surface molecule expressed on LSCs in most types of AML except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). TIM-3(+) but not TIM-3⁻ AML cells reconstituted human AML in immunodeficient mice, suggesting that the TIM-3(+) population contains most, if not all, of functional LSCs. We established an anti-human TIM-3 mouse IgG2a antibody having complement-dependent and antibody-dependent cellular cytotoxic activities. This antibody did not harm reconstitution of normal human HSCs, but blocked engraftment of AML after xenotransplantation. Furthermore, when it is administered into mice grafted with human AML, this treatment dramatically diminished their leukemic burden and eliminated LSCs capable of reconstituting human AML in secondary recipients. These data suggest that TIM-3 is one of the promising targets to eradicate AML LSCs.

Craniofacial malformation in R-spondin2 knockout mice.

In vertebrates, craniofacial formation is accomplished by synergistic interaction of many small elements which are generated independently from distinct germ layers. Because of its complexity, the imbalance of one signaling cascade such as Wnt/beta-catenin pathway easily leads to craniofacial malformation, which is the most frequent birth defect in humans. To investigate the developmental role of a newly identified activator of Wnt/beta-catenin signaling, Rspo2, we generated and characterized Rspo2(-/-) mice. We found CLP with mild facial skeletal defects in Rspo2(-/-) mice. Additionally, Rspo2(-/-) mice also exhibited distal limb loss and lung hypoplasia, and died immediately after birth with respiratory failure. We showed the apparent reduction of Wnt/beta-catenin signaling activity at the branchial arch and the apical ectodermal ridge in Rspo2(-/-) mice. These findings indicate that Rspo2 regulates midfacial, limb, and lung morphogenesis during development through the Wnt/beta-catenin signaling.

In vivo efficacy of anti-MPL agonist antibody in promoting primary human hematopoietic cells.

In a previous study, we generated novel antithrombopoietin receptor agonist antibodies as therapeutic candidates. In this report, we investigated the in vivo effects of one of these antibodies, MA01G4344U, on primary human hematopoietic cells using xenotransplantation. NOD/Shi-scid, IL-2Rgamma(null) (NOG) mice were pretreated by total-body irradiation and received a transplant of human cord blood-derived CD34(+) cells. Weekly intraperitoneal injection of MA01G4344U (100 microg/mouse per week) or Peg-rhMGDF (5 microg/mouse per week) or phosphate-buffered saline (PBS) was performed. Human cells in peripheral blood were analyzed by flow cytometry and bone marrow cells were analyzed by flow cytometry and colony assay. MA01G4344U successfully increased the number of human CD41(+) platelets and human CD45(+) cells in peripheral blood. In the bone marrow, MA01G4344U increased the number of human CD45(+)/CD34(+) cells, which resulted in more multilineage progenitor cells. The efficacy of MA01G4344U in promoting primary human hematopoietic cells in vivo suggests its therapeutic potential for thrombocytopenic and pancytopenic disorders.

Expression profile analysis of aorta-gonad-mesonephros region-derived stromal cells reveals genes that regulate hematopoiesis.

The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis.

Notch ligand Delta-1 differentially modulates the effects of gp130 activation on interleukin-6 receptor alpha-positive and -negative human hematopoietic progenitors.

Interleukin (IL)-6 plays pleiotropic roles in human hematopoiesis and immune responses by acting on not only the IL-6 receptor-alpha subunit (IL-6Ralpha)(+) but also IL-6Ralpha(-) hematopoietic progenitors via soluble IL-6R. The Notch ligand Delta-1 has been identified as an important modulator of the differentiation and proliferation of human hematopoietic progenitors. Here, it was investigated whether these actions of IL-6 are influenced by Delta-1. When CD34(+)CD38(-) hematopoietic progenitors were cultured with stem cell factor, flt3 ligand, thrombopoietin and IL-3, Delta-1, in combination with the IL-6R/IL-6 fusion protein FP6, increased the generation of glycophorin A(+) erythroid cells but counteracted the effects of IL-6 and FP6 on the generation of CD14(+) monocytic and CD15(+) granulocytic cells. Although freshly isolated CD34(+)CD38(-) cells expressed no or only low levels of IL-6Ralpha, its expression was increased in myeloid progenitors after culture but remained negative in erythroid progenitors. It was found that Delta-1 acted in synergy with FP6 to enhance the generation of erythroid cells from the IL-6Ralpha(-) erythroid progenitors. In contrast, Delta-1 antagonized the effects of IL-6 and FP6 on the development of monocytic and granulocytic cells, as well as CD14(-)CD1a(+) dendritic cells, from the IL-6Ralpha(+) myeloid progenitors. These results indicate that Delta-1 interacts differentially with gp130 activation in IL-6Ralpha(-) erythroid and IL-6Ralpha(+) myeloid progenitors. The present data suggest a divergent interaction between Delta-1 and gp130 activation in human hematopoiesis.

Bmi1 cooperates with Dnmt1-associated protein 1 in gene silencing.

Polycomb group (PcG) proteins are involved in gene silencing through chromatin modifications. Among polycomb repressive complexes (PRCs), PRC1 exhibits H2A-K119 ubiquitin E3 ligase activity. However, the molecular mechanisms underlying PRC1-mediated gene silencing remain largely obscure. In this study, we found that Bmi1 directly interacts with Dnmt-associated protein 1 (Dmap1), which has been characterized to associate with the maintenance DNA methyltransferase, Dnmt1. Bmi1 was demonstrated to form a ternary complex with Dmap1 and Dnmt1 with Dmap1 in the central position. Chromatin immunoprecipitations confirmed the ternary complex formation within the context of the PRC1 at the Bmi1 target loci. Loss of Dmap1 binding to the Bmi1 target loci was tightly associated with derepressed gene expression in Bmi1-/- cells. Dmap1 knockdown exhibited the same impact as Bmi1 knockout did on the expression of Bmi1 targets, including Hox genes. Collectively, our findings suggest that Bmi1 incorporates Dmap1 in polycomb gene silencing.

Oxidized galectin-1 advances the functional recovery after peripheral nerve injury.

Oxidized galectin-1 has been shown to promote axonal regeneration from transected-nerve sites in an in vitro dorsal root ganglion (DRG) explant model as well as in in vivo peripheral nerve axotomy models. The present study provides evidence that oxidized galectin-1 advances the restoration of nerve function after peripheral nerve injury. The sciatic nerve of adult rats was transected and the distal nerve was frozen after being sutured into a proximal site with four epineurial stitches. An osmotic pump delivered oxidized galectin-1 peripherally to the surgical site. Functional recovery was assessed by measurement of the degree of toe spread of the hind paw for 3 months after the sciatic nerve lesion. The recovery curves of toe spread in the test group showed a statistically significant improvement of functional recovery after day 21 by the application of oxidized recombinant human galectin-1 (rhGAL-1/Ox) compared to the control group. This functional recovery was supported by histological analysis performed by light microscopic examination. The regenerating myelinated fibers at the site 21 mm distal to the nerve-transected site were quantitatively examined at 100 days after the operation. The frequency distribution of myelinated fiber diameters showed that exogenous rhGAL-1/Ox increased the number and diameter of regenerating myelinated fibers; the number of medium-sized (6-11 microm in diameter) fibers increased significantly (P<0.05). These results indicate that oxidized galectin-1 promotes the restoration of nerve function after peripheral nerve injury. Thus, rhGAL-1/Ox may be a factor for functional restoration of injured peripheral nerves.

Plasmacytoid dendritic cells: from specific surface markers to specific cellular functions.

We have recently described a panel of monoclonal antibodies (mAb), that recognize two novel leukocyte surface antigens, BDCA-2 and BDCA-4. BDCA-2 is a novel type II C-type lectin specifically expressed by plasmacytoid dendritic cells (PDCs) that can internalize antigen for presentation to T cells. Furthermore, signaling via BDCA-2 may play a role in switching from interferon (IFN)-alpha/beta-controlled to interleukin (IL)-12-controlled immune response pathways, as triggering of BDCA-2 potently inhibits secretion of IFN-alpha/beta by PDCs and thereby promotes IL-12 p70 production in PDCs and other cells. Viruses may exploit this switch to escape innate antiviral immunity, but it may be beneficial for patients with systemic lupus erythematosus (SLE) if induced, for instance by anti BDCA-2 mAb treatment. BDCA-4 is shown here to be identical to neuropilin-1 (NP-1), a neuronal receptor for the axon guidance factors belonging to the class-3 semaphorin subfamily, and a receptor on endothelial and tumor cells for vascular endothelial growth factor (VEGF-A). In blood and bone marrow, BDCA-4/NP-1 is exclusively expressed on PDCs, but in tonsils also on a few other cells, primarily follicular B helper memory T cells (T(FH)).