Mesenchymal stem cells (MSC) were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the action of cytokines secreted by these cells. To enhance such cytokine effects, we previously transfected the telomerized human MSC with the BDNF gene using a fiber-mutant adenovirus vector and reported that such treatment contributed to improved ischemic recovery in a rat transient middle cerebral artery occlusion (MCAO) model. In the present study, we investigated whether other cytokines in addition to BDNF, i.e., GDNF, CNTF, or NT3, might have a similar or greater effect in this model. Rats that received MSC-BDNF (P < 0.05) or MSC-GDNF (P < 0.05) showed significantly more functional recovery as demonstrated by improved behavioral test results and reduced ischemic damage on MRI than did control rats 7 and 14 days following MCAO. On the other hand, rats that received MSC-CNTF or MSC-NT3 showed neither functional recovery nor ischemic damage reduction compared to control rats. Thus, MSC transfected with the BDNF or GDNF gene resulted in improved function and reduced ischemic damage in a rat model of MCAO. These data suggest that gene-modified cell therapy may be a useful approach for the treatment of stroke.

Examination of the clinical therapeutic efficacy of using bone marrow stromal cells, including mesenchymal stem cells (MSC), has recently been the focus of much investigation. MSC were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the action of cytokines secreted by these cells. To enhance such cytokine effects, we transfected telomerized human MSC with the BDNF gene using a fiber-mutant F/RGD adenovirus vector and investigated whether these cells contributed to improved functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model. BDNF production by MSC-BDNF cells was 23-fold greater than that seen in uninfected MSC. Rats that received MSC-BDNF showed significantly more functional recovery than did control rats following MCAO. Specifically, MRI analysis revealed that the rats in the MSC-BDNF group exhibited more significant recovery from ischemia after 7 and 14 days. The number of TUNEL-positive cells in the ischemic boundary zone was significantly smaller in animals treated with MSC-BDNF compared to animals in the control group. These data suggest that MSC transfected with the BDNF gene may be useful in the treatment of cerebral ischemia and may represent a new strategy for the treatment of stroke.

The morphology formed in a crystalline-amorphous diblock copolymer, e-caprolactone-blockbutadiene (PCL-fe-PB), has been investigated by small-angle X-ray scattering (SAXS) at various temperatures. The process of the morphology formation is also observed by time-resolved SAXS employing synchrotron radiation. Crystallization of the PCL block brings about a dramatic change in the shape of the SAXS pattern; a diffuse intensity maximum arising from the correlation hole effect of disordered block copolymers could be observed at the melt, while it was replaced by a strong intensity peak at a smaller angle for temperatures below Tm (melting temperature of the PCL block). The time-resolved SAXS curves revealed that the copolymer, which was quenched from the melt into a temperature below both Tm and Ts (microphaseseparation temperature of the copolymer), immediately showed a sharp diffraction due to the microstructure of the copolymer, followed by a strong intensity peak at a smaller angle owing to the crystallization of the PCL block. This indicates that an energetic gain in crystallization overwhelms that in microphase separation, so that the microstructure is completely destroyed by the following crystallization of the PCL block.

CD40-CD40-ligand (CD154) interactions play a critical role in immune activation. Using replication defective adenovirus encoding mouse CD154 (Ad-CD154), we modified human chronic lymphocytic leukemia B cells to express a functional ligand for CD40. This not only induces expression of immune accessory molecules on the infected cell, but also allows it to trans-activate noninfected bystander leukemia B cells. Also, factors that impair the antigen-presenting capacity of leukemia B cells are downmodulated. Ad-CD154- infected leukemia cells are highly effective stimulators in mixed lymphocyte reactions and can induce generation of cytotoxic T lymphocytes specific for autologous nonmodified leukemia cells. As such, Ad-CD154 can induce a host antileukemia response that may have therapeutic potential.

We developed human mesenchymal stem cell (MSC) lines that could differentiate into various tissue cells including bone, neural cells, bone marrow (BM) stromal cells supporting the growth of hematopoietic stem cell (HSC), and so-called 'tumor stromal cells' mixing with tumor cells. We investigated the applicability of MSC as therapeutic cell transplanting reagents (cytoreagents). Telomerized human BM derived stromal cells exhibited a prolonged lifespan and supported the growth of hematopoietic clonogenic cells. The gene transfer of Indian hedgehog (Ihh) remarkably enhanced the HSC expansion supported by the human BM stromal cells. Gene-modified MSC are useful as therapeutic tools for brain tissue damage (e.g. brain infarction) and malignant brain neoplasms. MSC transplantation protected the brain tissue from acute ischemic damage in the midcerebral artery occlusion (MCAO) animal model. Brain-derived neurotrophic factor (BDNF)-gene transduction further enhanced the protective efficacy against the ischemic damage. MSC possessed excellent migratory ability and exerted inhibitory effects on the proliferation of glioma cells. Gene-modification of MSC with therapeutic cytokines clearly augmented the antitumor effect and prolonged the survival of tumor-bearing animals. Gene therapy employing MSC as a tissue-protecting and targeting cytoreagent would be a promising approach.

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SummaryCD2 is an intercellular adhesion molecule that has been implicated in T cell activation and differentiation both in humans and mice. Although the ligand for human CD2 has been defined as LFA-3, that for murine CD2 has not been identified yet . To identify the ligand for mouse CD2, we generated a chimeric molecule consisting of the extracellular domain of mouse CD2 and human immunoglobulin (Ig)G1 Fc (mCD2Rg) . A hamster monoclonal antibody (mAb), HM48-1, was established by screening mAbs that could block the binding of mCD2Rg to T cell lines at the ligand site. The putative mouse CD2 ligand recognized by this mAb was a glycosyl phosphatidylinositol-anchored glycoprotein with an apparent molecular mass of 45 W, which were shared characteristics with human LFA-3. However, its expression was predominantly restricted to hematopoietic cells, unlike human LFA 3. Protein microsequencing analysis for the NH2-terminal 18 amino acid residues of the affinity-purified HM48-1 antigen revealed that it is almost identical with mouse CD48. This identity was further confirmed by the reactivity of HM48-1 with a soluble recombinant CD48 (sCD48) protein and the molecule recognized by a rat mAb raised against sCD48 . A rat anti-CD48 mAb blocked the mCD2Rg binding as well as HM48-1 . Moreover, sCD48 also inhibited the mCD2Rg binding to the cellular ligand . Finally, like anti-CD2 mAb, HM48-1 inhibited the phytohemagglutinin response and, when crosslinked, augmented the anti-CD3 response of splenic T cells . These results indicate that CD48 is a ligand for mouse CD2 and is involved in regulating T cell activation . C D2 has been implicated in T cell adhesion and activation (1, 2). A natural ligand for CD2 has been identified as LFA-3, which is expressed on a variety of cells, including nonhematopoietic cells in humans (3-5). CD2 and LFA-3 molecules have a structural similarity (6) and a close genetic linkage (7), suggesting that these molecules have evolved from a common precursor, which has a homotypic adhesion function-like neural cell adhesion molecule, by a gene duplication (8) . It has been proposed that the intercellular interaction between CD2 and LFA3 leads to bidirectional signaling via each molecule, resulting in activation ofboth T cells and APC (9-11) . However, it has been demonstrated that the binding of LFA-3 was not sufficient for triggering T cell activation via CD2 since the addition of anti-CD2R mAbs was required (12)(13)(14). This suggests the existence of an additional ligand corresponding to anti-CD2R mAb, but it has not been identified yet.CD48, which is known as Blast-1 in humans (15), OX-45 in rats (16), and BCM1 in mice (17), is a glycosyl phosphatidylinositol (GPI)t-anchored glycoprotein expressed on almost all T and B cells, whose function remains unknown . Molecular cloning studies revealed a high structural homology of human CD48 molecule to LFA-3 (18), suggesting that it would also function as an adhesion molecule. We describe here that CD48 is a ligand for mouse CD2 and is involved in regulating T...

Gene targeting using short interfering RNA (siRNA) has become a common strategy to explore gene function because of its prominent efficacy and specificity. For the application of siRNA technology to gene therapy, however, still more efficient transduction of siRNA into target cells is needed. In this study, we developed an adenoviral vector harboring a tandem-type siRNA expression unit, in which sense and antisense strands composing the siRNA duplex were separately transcribed by two human U6 promoters. Targeting survivin, an antiapoptotic molecule widely overexpressed in malignancies but not detected in terminally differentiated adult tissues, this type of adenoviral vector (Adv-siSurv) successfully exerted a gene knockdown effect and induced apoptosis in HeLa, U251, and MCF-7 cells. These cancer cells, once infected with Adv-siSurv, displayed remarkably attenuated growth potential, both in vitro and in vivo. Moreover, intratumoral injection of Adv-siSurv significantly suppressed tumor growth in a xenograft model using U251 glioma cells. This novel modality may be a promising tool for cancer therapy.