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.

L-type amino acid transporter 1 (LAT1) is a neutral amino acid transport system and is a major route for the transport of large neutral amino acids, including methionine, through the plasma membrane. LAT1 requires the heavy chain of 4F2 cell surface antigen (4F2hc) for its functional expression. Positron emission tomography (PET) with L-[methyl-(11)C] methionine (MET) provides information about amino acid metabolism in brain tumors. We conducted a clinicopathologic study to elucidate the correlation of LAT1 and 4F2hc expression with MET uptake in patients with newly diagnosed human gliomas. Thirty-three newly diagnosed glioma patients were enrolled in this study. Uptake of MET in the tumor was evaluated with the maximum standardized uptake value (SUVmax). Expression of the LAT1, 4F2hc, and CD34, and Ki-67 labeling index of the tumor were analyzed by immunohistochemical staining, and the correlation with the SUVmax in the tumors was examined. Expression of LAT1 and 4F2hc was higher in high-grade gliomas than in low-grade gliomas. The grade of LAT1 immunostaining increased with glioma grade. LAT1 was mainly expressed in the tumor cytoplasm and vascular endothelium and 4F2hc was mainly expressed in the tumor cytoplasm and plasma membrane. Expression of LAT1 but not 4F2hc was significantly correlated with MET SUVmax. Expression of LAT1 in the tumor vascular endothelium is significantly correlated with CD34 positive microvessel density. In conclusion, MET SUVmax correlates with LAT1 expression in the tumor in newly diagnosed gliomas. MET transport may be increased by an increased number of microvessels combined with a higher density or activity of LAT1 in the tumor endothelial cells in high-grade gliomas. Use of MET-PET as a molecular target combined with anti-angiogenesis in glioma therapy should be addressed in future studies.

PET studies using MET and FLT are useful for tumor detection in newly diagnosed gliomas. However, there is no complimentary information in tumor detection with simultaneous measurements of MET- and FLT-PET in low grade gliomas. FLT-PET seems to be superior than MET-PET in noninvasive tumor grading and assessment of proliferation activity in gliomas of different grades.

Protein transduction therapy is a newly developing method that allows proteins, peptides, and biologically active compounds to penetrate across the plasma membrane by being fused with cell-penetrating peptides such as polyarginine. Polyarginine-fused p53 protein penetrates across the plasma membrane of cancer cells and inhibits the growth of the cells. However, the protein is often entrapped inside macropinosomes in the cytoplasm. Therefore, high dose concentrations of the protein are needed for it to function effectively. To overcome this problem, in the present study, polyarginine-fused p53 was linked with the NH 2 -terminal domain of influenza virus hemagglutinin-2 subunit (HA2), which is a pH-dependent fusogenic peptide that induces the lysis of membranes at low pH levels. The protein was capable of efficiently translocating into the nucleus of glioma cells and induced p21 WAF1 transcriptional activity more effectively than did polyarginine-fused p53 protein. Moreover, low concentrations of the protein significantly inhibited the growth of cancer cells. These results suggest that protein transduction therapy using polyarginine and HA2 may be useful as a method for cancer therapy.The cellular delivery of various biological compounds such as bioactive protein has been improved recently by conjugating the compounds to short peptides known as cell penetrating peptides (CPPs) 1 or protein transduction domains (PTDs) (1, 2). The PTD of human immunodeficiency virus type-1 TAT protein, which consists of an 11-amino acid polypeptide, is one of the most well known CPPs (3, 4). Despite their broad acceptance as molecular carriers, the mechanism of internalization of CPPs and their cargo are still being discussed. Previous studies (5-7) have demonstrated that the internalization of CPPs and PTD do not involve endocytosis or specific protein transporters. However, recent studies (2,8,9) have shown that the cellular internalization occurs through a temperature-dependent endocytic pathway. A very recent study (10) has shown that TAT-PTD fusion proteins are internalized rapidly by lipid raft-dependent macropinocytosis. After internalization via the macropinocytotic pathway, the proteins are carried to macropinosomes, where most of them are then degraded (10). In order for the molecules delivered by CPPs to function in the cell, they generally must reach the cytosol. Therefore, protein delivery into the cytosol of target cells via macropinosomal escape is an important route of delivery.The CPP, consisting of an 11-mer polyarginine (11R), efficiently delivers peptides and proteins into cells (11,12). The 11R-fused p53 protein (p53-11R) is delivered effectively into cancer cells and has transcriptional regulatory activity there (13). Moreover, p53-11R inhibits the proliferation of the cancer cells (13). However, a high concentration (Ͼ1 M) and repeated administration of p53-11R are needed for transcriptional activation and the growth inhibition of cancer cells (13). Entrapment of the transduced protein in macropinosomes may we...