The striatum plays important motor, associative and cognitive roles in brain functions. However, the rodent dorsolateral (the primate putamen) and dorsomedial (the primate caudate nucleus) striatum ...are not anatomically separated, making it difficult to distinguish their functions. By contrast, anatomical separation exists between the caudate nucleus and putamen in primates. Here, we successfully decreased dopamine D1 receptor (D1R) or D2R mRNA expression levels selectively in the marmoset caudate using shRNA knockdown techniques, as determined using positron emission tomography imaging with specific D1R and D2R ligands and postmortem in situ hybridization analysis. We then conducted a voxel-based correlation analysis between binding potential values of PET imaging and visual discrimination learning task performance in these genetically modified marmosets to find a critical role for the caudate D2R but no apparent role for the caudate D1R. This latter finding challenges the current understanding of the mechanisms underlying D1R activation in the caudate.
Peritoneal fibrosis (PF), a serious pathophysiology of peritoneal dialysis (PD), is implicated in various types of chronic inflammation. In the present study, we examined the benefits of interleukin ...(IL)-10, which exerts anti-inflammatory effects, in an experimental rat model of methylglyoxal (MGO)-induced PF. We injected an adeno-associated virus (AAV) vector encoding rat IL-10 or enhanced green fluorescent protein (GFP) into male Sprague-Dawley rats at 6 weeks of age. Four weeks later, the rats received continuous peritoneal injections of conventional PD fluid (PDF) with MGO for 3 weeks. Then, the peritoneal histology and the expression levels of fibrogenic mediators and proinflammatory cytokines were analyzed. The rats demonstrating persistent IL-10 expression showed significantly reduced fibrous peritoneal thickening compared with those with GFP expression. The infiltration of macrophages, the expression of tumor necrosis factor-α, IL-1β, IL-6, transforming growth factor-β1, Snail, and matrix metalloproteinase 2 genes as well as the proliferation of mesenchymal-like mesothelial cells augmented by MGO were all significantly suppressed by IL-10 expression. IL-10 also abrogated the extent of MGO-induced bowel adhesions mimicking a cocoon-like mass. Our findings provide valuable insight into the potential benefit of immunomodulation with IL-10 as one potentially effective therapeutic strategy for preventing the onset of peritoneal injury resulting in PF.
In vivo gene transduction with adeno-associated virus (AAV)-based vectors depends on laborious procedures for the production of high-titer vector stocks. Purification steps for efficient clearance of ...impurities such as host cell proteins and empty vector particles are required to meet end-product specifications. Therefore, the development of alternative, realistic methods to facilitate a scalable virus recovery procedure is critical to promote in vivo investigations. However, the conventional purification procedure with resin-based packed-bed chromatography suffers from a number of limitations, including variations in pressure, slow pore diffusion, and large bed volumes. Here we have employed disposable high-performance anion- and cation-exchange membrane adsorbers to effectively purify recombinant viruses. As a result of isoelectric focusing analysis, the isoelectric point of empty particles was found to be significantly higher than that of packaged virions. Therefore, AAV vector purification with the membrane adsorbers was successful and allowed higher levels of gene transfer in vivo without remarkable signs of toxicity or inflammation. Electron microscopy of the AAV vector stocks obtained revealed highly purified virions with as few as 0.8% empty particles. Furthermore, the membrane adsorbers enabled recovery of AAV vectors in the transduced culture supernatant. Also, the ion-exchange enrichment of retroviral vectors bearing the amphotropic envelope was successful. This rapid and scalable viral purification protocol using disposable membrane adsorbers is particularly promising for in vivo experimentation and clinical investigations.
Azacitidine, an inhibitor of DNA methyltransferase, is reported to have antileukemic efficacy and is approved for the treatment of myelodysplastic syndromes in Western countries. We have conducted a ...Phase I/II study of azacitidine in Japanese patients with myelodysplastic syndromes to evaluate its pharmacokinetics, efficacy, and safety. In all, 53 patients received 75 mg/m2 azacitidine subcutaneously or intravenously once daily for seven consecutive days on a 28‐day cycle. The Cmax following intravenous administration was approximately 3.7‐fold higher than that following subcutaneous administration, whereas the area under the plasma concentration–time curve from time zero to infinity was comparable for subcutaneous and intravenous administration. The bioavailability of azacitidine following subcutaneous administration was 91.1%, indicating that azacitidine is nearly completely absorbed after subcutaneous administration. The hematologic improvement and hematologic response rates were 54.9% (28/51) and 28.3% (15/53), respectively, and there were no differences between the two routes of administration. Azacitidine was generally well tolerated and clinically manageable in Japanese patients with myelodysplastic syndromes. Adverse events occurred in ≥20% of patients included hematologic toxicity, gastrointestinal events, and general disorders, such as malaise. Grade 3/4 adverse events that occurred in ≥50% of patients were all due to hematologic toxicity. The safety profile of azacitidine was generally similar for both routes of administration, with the exception of injection site reactions observed following subcutaneous administration. These results indicate that azacitidine can be expected to be a useful therapeutic agent in Japanese patients with myelodysplastic syndromes. (Cancer Sci 2011; 102: 1680–1686)
Autophagy, a cellular degradation system has been demonstrated in some hematopoietic malignant cell lines, but there is much still remaining to be known about its role and the mechanisms. We observed ...the excessive autophagy in chronic myelogenous leukemia (CML) cell line, K562, associated with treatment of 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), which can induce K562 cells to differentiate into megakaryocytic lineage. Confocal microscopic analysis demonstrated that autophagic cells did not express a megakaryocyte marker, the CD41 molecule, indicating that the autophagy was independent of megakaryocytic differentiation. After remarkable autophagic degradation, the cells finally underwent autophagic cell death (APCD). On the other hand, a block of TPA‐induced autophagy by chloroquine rapidly promoted cell death that was not APCD. This result suggested that autophagy regulated two mechanisms in K562 cells: both the cell survival system and APCD. To confirm that autophagy regulates the cell survival system in K562 cells, imatinib was used to induce cell death in K562 cells. Autophagy has not been considered during imatinib treatment; nonetheless, co‐treatment with imatinib and chloroquine markedly enhanced imatinib‐induced cell death, compared to K562 cells treated only with imatinib. Furthermore, imatinib‐resistant cell lines, BaF3/T315I and BaF3/E255K, also underwent cell death by co‐treatment with imatinib and chloroquine. From these data, we concluded that autophagy is deeply related to the cell survival system and that inhibition of autophagy accelerates TPA‐ or imatinib‐induced cell death. The block of autophagy could be a new strategy in the treatment of CML. (Cancer Sci 2008; 99: 2200–2208)
Abstract Mesenchymal stem cells (MSCs) are considered to be a promising platform for cell and gene therapy for a variety of diseases. First, in the field of hematopoietic stem cell transplantation, ...there are two applications of MSCs: 1) the improvement of stem cell engrafting and the acceleration of hematopoietic reconstitution based on the hematopoiesis-supporting ability; and 2) the treatment of severe graft-versus-host disease (GVHD) based on the immunomodulatory ability. Regarding the immunosuppressive ability, we found that nitric oxide (NO) is involved in the MSC-mediated suppression of T cell proliferation. Second, tumor-bearing nude mice were injected with luciferase-expressing MSCs. An in vivo imaging analysis showed the significant accumulation of the MSCs at the site of tumors. The findings suggest that MSCs can be utilized to target metastatic tumors and to deliver anti-cancer molecules locally. As the third application, MSCs may be utilized as a cellular vehicle for protein-supplement gene therapy. When long-term transgene expression is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS1 locus on the chromosome 19 (19q13.4) by using the integration machinery of adeno-associated virus (AAV) would be particularly valuable. There will be wide-ranging applications of MSCs to frontier medical treatments in the near future.
Bortezomib is a novel proteasome inhibitor with significant antimyeloma activity. Its frequent adverse effects are manageable, including gastrointestinal symptoms, peripheral neuropathy, and ...thrombocytopenia. Severe lung toxicity has not previously been reported. Between June 2004 and September 2005, 13 Japanese patients with multiple myeloma were treated with bortezomib in Toranomon Hospital, Juntendo University School of Medicine, and Jichi Medical School. Four of them developed severe pulmonary complications, and 2 died of respiratory failure without progression of underlying disease. To our knowledge, this is the first report on life-threatening pulmonary adverse effects after bortezomib therapy. Previous clinical studies on bortezomib, mostly in the United States and Europe, have shown low incidences of pulmonary adverse effects. Our study suggests that bortezomib can cause serious lung injury, and that its incidence might vary among different ethnicities. Clinicians need to be alert to the possibility.
Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in ...GALR1‐expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin‐dependent kinase inhibitors, whereas, in GALR2‐expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp‐2 and KB cell lines using a recombinant adeno‐associated virus (rAAV)‐green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (>90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40–60% after 72 h in both cell lines. Additionally, the annexin V‐positive rate and sub‐G0/G1 phase population were significantly elevated in HEp‐2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp‐2 cells, GALR2‐mediated apoptosis was caspase‐independent, involving downregulation of ERK1/2, followed by induction of the pro‐apoptotic Bcl‐2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC.
Transduced HEp‐2 and KB cell lines using a recombinant adeno‐associated virus (rAAV)‐GFP vector showed a high GFP expression rate. GALR2 express in the presence of galanin suppressed cell viability in both cell lines. The annexin V‐positive rate and sub‐G0/G1 phase population were significantly elevated in the both cells. Furthermore, in HEp‐2 cells, GALR2‐mediated apoptosis was caspase‐independent, involving down‐regulation of ERK1/2, followed by induction of the pro‐apoptotic Bcl‐2 protein, Bim.
Lymph node metastasis is the most important prognostic factor of endometrial cancer. However, effective therapy has not been established against lymph node metastasis. In this study, we explored the ...efficacy of gene therapy targeting lymph node metastasis of endometrial cancer by suppressing the action of vascular endothelial growth factor (VEGF)‐C through soluble VEGF receptor‐3 (sVEGFR‐3) expression. For this purpose, we first conducted a model experiment by introducing sVEGFR‐3 cDNA into an endometrial cancer cell line HEC1A and established HEC1A/sVEGFR‐3 cell line with high sVEGFR‐3 expression. The conditioned medium of HEC1A/sVEGFR‐3 cells inhibited lymphatic endothelial cell growth in vitro, and sVEGFR‐3 expression in HEC1A cells suppressed in vivo lymph node and lung metastases without inhibiting the growth of a subcutaneously inoculated tumor. To validate the therapeutic efficacy, adeno‐associated virus vectors encoding sVEGFR‐3 were injected into the skeletal muscle of mice with lymph node metastasis. Lymph node and lung metastases of HEC1A cells were completely suppressed by the muscle‐mediated expression of sVEGFR‐3 using adeno‐associated virus vectors. These results suggest the possibility of gene therapy against lymph node and lung metastases of endometrial cancer by using muscle‐mediated expression of sVEGFR‐3.
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