Engrafted mesenchymal stem cells from human deciduous dental pulp (SHEDs) support recovery from neural insults via paracrine mechanisms that are poorly understood. Here we show that the conditioned serum-free medium (CM) from SHEDs, administered intrathecally into rat injured spinal cord during the acute postinjury period, caused remarkable functional recovery. The ability of SHED-CM to induce recovery was associated with an immunoregulatory activity that induced anti-inflammatory M2-like macrophages. Secretome analysis of the SHED-CM revealed a previously unrecognized set of inducers for anti-inflammatory M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9). Depleting MCP-1 and ED-Siglec-9 from the SHED-CM prominently reduced its ability to induce M2-like macrophages and to promote functional recovery after spinal cord injury (SCI). The combination of MCP-1 and ED-Siglec-9 synergistically promoted the M2-like differentiation of bone marrow-derived macrophages in vitro, and this effect was abolished by a selective antagonist for CC chemokine receptor 2 (CCR2) or by the genetic knock-out of CCR2. Furthermore, MCP-1 and ED-Siglec-9 administration into the injured spinal cord induced M2-like macrophages and led to a marked recovery of hindlimb locomotor function after SCI. The inhibition of this M2 induction through the inactivation of CCR2 function abolished the therapeutic effects of both SHED-CM and MCP-1/ED-Siglec-9. Macrophages activated by MCP-1 and ED-Siglec-9 extended neurite and suppressed apoptosis of primary cerebellar granule neurons against the neurotoxic effects of chondroitin sulfate proteoglycans. Our data suggest that the unique combination of MCP-1 and ED-Siglec-9 repairs the SCI through anti-inflammatory M2-like macrophage induction.

No abstract

(7,8). On the other hand, oscillatory changes in [IP 3 ] i have been suggested by the observed cyclical translocation of a GFPtagged pleckstrin homology domain of PLC-␦ (GFP-PHD) (9, 10). However, in other experiments using more specific IP 3 biosensors, IP 3 was shown to accumulate gradually with little or no fluctuation during Ca 2ϩ oscillations (11). These discrepant observations may be attributable to differences between various IP 3 biosensors and a lack of quantitation.There are two types of IP 3 biosensors, GFP-PHD and IP 3 Rbased FRET sensors. GFP-PHD binds to both PIP 2 and IP 3 ; thus it has been thought that changes in [IP 3 ] i could be monitored indirectly by the release of membrane-bound GFP-PHD (9). IP 3 R-based FRET biosensors consist of the ligand-binding domain of IP 3 R and a pair of fluorescent proteins, cyan fluorescent protein and yellow fluorescent protein. Since the successful monitoring of IP 3 with LIBRA (12), the first IP 3 R-based FRET biosensor, several different groups have used similar biosensors for IP 3 monitoring (11,13,14). In principle, quantitative measurements of [IP 3 ] i are not possible with GFP-PHD. In addition, it is recognized that GFP-PHD may be released from the plasma membrane by decreases in available PIP 2 (15), which could be attributed to PIP 2 hydrolysis or the occupation by other molecules. IP 3 R-based FRET biosensors offer significant benefits for monitoring IP 3 based on their high selectivity for IP 3 and ratiometric measurement.In this study, we developed a series of improved IP 3 biosensors that exhibit high pH stability and varying IP 3 affinities. They also possess higher selectively and afford a larger dynamic range than that of original LIBRA. In combination with these * This work was supported by Grant-in-aid for Scientific Research 16390532 (to A. T.), by HAITEKU (2007) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Science and Technology Agency. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Interleukin 2 (IL-2) 1 was discovered through its function as a T cell growth factor (TCGF) (1, 2). Indeed, the availability of T cell lines for the quantitative measurement of TCGF activity has been crucial for the characterization of IL-2 (3, 4) and IL-2 receptors on activated T cells (5-7) in different species. Human IL-2, which acts on murine and human T cells (4,8,9), can now rapidly be purified to apparent homogeneity with monoclonal antibodies (8, 9) and its cDNA has been cloned (10, 11). Human I L-2 is a T ceil-generated 15 kilodalton peptide hormone of 133 amino acids.Although it was observed in several studies (3, 4, 12-14) that IL-2-rich T cell supernatants (SN) enhance the generation of plaque-forming cells (PFC) in certain B cell culture systems, it is widely believed that B cells do not directly respond to IL-2. Thus, a variety of other T cell-derived factors (but not IL-2) were reported to enhance the proliferation of B cells activated by either Tindependent antigens (15), dextran (16), lipopolysaccharide (LPS) (17, 18), antiimmunoglobulin antibodies (anti-Ig) (16,(19)(20)(21), or other means (22). In one study (23), showing an effect of immunoaffinity-purified IL-2 on anti-Iginduced B cell proliferation, the interpretation of the results was complicated because it was also observed that T cells participated in the B cell response. LPSactivated murine B cells were not found to bind radiolabeled IL-2 (5) but, in another study (7), such LPS blasts reacted weakly with an anti-IL-2 receptor monoclonal antibody.However, there still exist controversies with regard to the activation signals that B cells require to respond to growth factors (24). A recent study (25) showed in a limit-dilution culture system for murine B cells that, in fact, either a cell contact-dependent T-B cell interaction or LPS was required in conjunction with anti-Ig for optimal induction of growth factor responsiveness. Whereas anti-Ig

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3؉). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3؉ cells than in GD3؊ cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3؉ cells than in GD3؊ cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipidenriched microdomain/rafts in GD3؉ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3؊ cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3؊ cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.