Preclinical and dose-ranging assessment of hESC-derived dopaminergic progenitors for a clinical trial on Parkinson's disease.

Human embryonic stem cell (hESC)-derived midbrain dopaminergic (mDA) cell transplantation is a promising therapeutic strategy for Parkinson's disease (PD). Here, we present the derivation of high-purity mDA progenitors from clinical-grade hESCs on a large scale under rigorous good manufacturing practice (GMP) conditions. We also assessed the toxicity, biodistribution, and tumorigenicity of these cells in immunodeficient rats in good laboratory practice (GLP)-compliant facilities. Various doses of mDA progenitors were transplanted into hemi-parkinsonian rats, and a significant dose-dependent behavioral improvement was observed with a minimal effective dose range of 5,000-10,000 mDA progenitor cells. These results provided insights into determining a low cell dosage (3.15 million cells) for human clinical trials. Based on these results, approval for a phase 1/2a clinical trial for PD cell therapy was obtained from the Ministry of Food and Drug Safety in Korea, and a clinical trial for treating patients with PD has commenced.

Trophoblast glycoprotein is a marker for efficient sorting of ventral mesencephalic dopaminergic precursors derived from human pluripotent stem cells.

Successful cell therapy for Parkinson's disease (PD) requires large numbers of homogeneous ventral mesencephalic dopaminergic (vmDA) precursors. Enrichment of vmDA precursors via cell sorting is required to ensure high safety and efficacy of the cell therapy. Here, using LMX1A-eGFP knock-in reporter human embryonic stem cells, we discovered a novel surface antigen, trophoblast glycoprotein (TPBG), which was preferentially expressed in vmDA precursors. TPBG-targeted cell sorting enriched FOXA2+LMX1A+ vmDA precursors and helped attain efficient behavioral recovery of rodent PD models with increased numbers of TH+, NURR1+, and PITX3+ vmDA neurons in the grafts. Additionally, fewer proliferating cells were detected in TPBG+ cell-derived grafts than in TPBG- cell-derived grafts. Our approach is an efficient way to obtain enriched bona fide vmDA precursors, which could open a new avenue for effective PD treatment.

Nonconjugated anionic polyelectrolyte as an interfacial layer for the organic optoelectronic devices.

A nonconjugated anionic polyelectrolyte, poly(sodium 4-styrenesulfonate) (PSS-Na), was applied to the optoelectronic devices as an interfacial layer (IFL) at the semiconducting layer/cathode interface. The ultraviolet photoelectron spectroscopy and the Kelvin probe microscopy studies support the formation of a favorable interface dipole at the organic/cathode interface. For polymer light-emitting diodes (PLEDs), the maximum luminance efficiency (LEmax) and the turn-on voltage (Von) of the device with a layer of PSS-Na spin-coated from the concentration of 0.5 mg/mL were 3.00 cd/A and 5.5 V, which are dramatically improved than those of the device without an IFL (LEmax = 0.316 cd/A, Von = 9.5 V). This suggests that the PSS-Na film at the emissive layer/cathode interface improves the electron injection ability. As for polymer solar cells (PSCs), the power conversion efficiency (PCE) of the device with a layer of PSS-Na spin-coated from the concentration of 0.5 mg/mL was 2.83%, which is a 16% increase compared to that of the PSC without PSS-Na. The PCE improvement is mainly due to the enhancement of the short-circuit current (12% increase). The results support that the electron collection and transporting increase by the introduction of the PSS-Na film at the photoactive layer/cathode interface. The improvement of the efficiency of the PLED and PSC is due to the reduction of the Schottky barrier by the formation of a favorable interface as well as the better Ohmic contact at the cathode interface.

Gene silencing of c-Met leads to brain metastasis inhibitory effects.

An unfortunate consequence of improvements in the treatments of advanced primary cancers is the concurrent increase of metastatic brain tumors. Despite of unfavorable clinical prognosis, radiation therapy is still the only viable treatment option for brain metastases. Expression of c-Met induces cell migration and invasion in many cancers, which are indispensable steps for metastasis. Accordingly, we examined the effects of gene silencing of c-Met on brain metastasis to evaluate the possibility of c-Met as a potential target. MDA-MB-435 cells were transfected with c-Met targeting short hairpin RNAs (shRNAs). Effects of c-Met shRNAs on the expression of epithelial mesenchymal transition (EMT) related proteins, in vitro migration, and in vivo brain metastasis were examined. Expression of mesenchymal markers and in vitro migration of MDA-MB-435 cells were significantly inhibited by introduction of c-Met shRNAs. When c-Met-silenced MDA-MB-435 cells were stereotactically implanted into the brains of immune-compromised mice or injected into the right internal carotid arteries, c-Met-silenced MDA-MB-435 cells produced significantly smaller tumor masses or survival time was significantly prolonged, respectively, compared with MDA-MB-435 cells transfected with control shRNA. The data reveal the novel function of c-Met in the process of brain metastasis and its potential as a preventive and/or therapeutic target in this disease.

Occurrence of norovirus infections in asymptomatic food handlers in South Korea.

Prevalence of asymptomatic norovirus infection was investigated in food handlers in South Korea. Among 6,441 subjects, 66 (1.02%) had norovirus infections confirmed by reverse transcription (RT)-PCR (real time and nested). GII-12 and GII-4 were the prevalent genotypes. Our data suggest that infection of asymptomatic food handlers is an important transmission source in norovirus outbreaks.

Combined therapy of temozolomide and ZD6474 (vandetanib) effectively reduces glioblastoma tumor volume through anti-angiogenic and anti-proliferative mechanisms.

Currently, clinically available options for treating glioblastoma (GBM) are quite limited, and there is a clear need to develop novel treatment strategies that can more effectively manage tumors. Here, we present a combination treatment of temozolomide (TMZ), a blood-brain barrier penetrating DNA alkylating agent, and ZD6474 (vandetanib), a VEGFR2 and EGFR dual-targeting anti-angiogenic agent, as a novel treatment strategy for GBM. In a U-87MG orthotopic xenograft model, the combination treatment provided a marked 94% tumor volume reduction. This reduction was greater than that achieved by monotherapy of either agent, and was correlated with a statistically significant reduction in microvessel density (CD31+ cells) and proliferation (PCNA+ cells). These results confirm the necessity to target angiogenesis in addition to utilizing cytotoxic approaches, and provide the rationale for application of TMZ + ZD6474 combination therapy for treating GBM patients in the clinical setting.

Role of coronary artery calcification score on the decrease in GFR among subjects with CT coronary angiography.

OBJECTIVE: Higher levels of coronary artery calcification score (CACS) are associated not only with an increased risk for cardiovascular death, but also with lower glomerular filtration rates (GFRs). However, its role in renal disease progression in patients has not been elucidated. MATERIALS AND METHODS: We evaluated the change of estimated GFR in 279 nondialytic outpatients, who had undergone computed tomographic coronary angiography and follow-up over a period of 3 months. RESULTS: The mean age of the participants was 57.7 ± 10.5 years, and the mean GFR was 88.2 ± 15.7 mL/min/1.73 m(2). Although there was no difference in baseline GFR between the CACS ≤ 200 AU group (n = 240) and the CACS > 200 AU group (n = 39), the latter group had a lower level of final GFR and higher annual reduction rate of GFR than the former group after an observation period of 13.1 ± 5.97 months. After adjusting for confounding variables, including age, gender, baseline GFR, albumin, and proteinuria, high levels of CACS showed an independent association with an annual reduction rate of GFR (r = -0.142, P = .048). CONCLUSIONS: The results suggest that CACS was related to an annual decrease in GFR and may predict the faster decline in GFR in patients with symptoms requiring computed tomographic coronary angiography.

Synergistic therapeutic effects of cytokine-induced killer cells and temozolomide against glioblastoma.

The presence of active immunity within the brain supports the possibility of effective immunotherapy for glioblastoma (GBM). To provide a clinically-relevant adoptive immunotherapy for GBM using ex vivo expanded cytokine-induced killer (CIK) cells, the treatment capability of CIK cells, either alone or in combination with temozolomide (TMZ) were evaluated. Human CIK (hCIK) cells were cultured from PBMC using activating anti-CD3 antibody and IL-2, which were 99% CD3+, 91% CD3+CD8+ and 29% CD3+CD56+. In vitro, hCIK cells showed tumor-specific cytotoxicity against U-87MG human GBM cells. When hCIK cells were injected into tail veins of immune-compromised mice bearing U-87MG tumors in their brains, numerous CIK cells infiltrated into the brain tumors. CIK treatments (1x10(5), 1x10(6) or 1x10(7), once a week for four weeks) inhibited the tumor growth significantly in a dose-dependent manner; 44, 54 and 72% tumor volume reduction, respectively, compared with the control group (P<0.05). Moreover, hCIK cells (1x10(7), once a week for four weeks) and TMZ (2.5 mg/kg, daily for 5 days) combination treatment further increased tumor cell apoptosis and decreased tumor cell proliferation and vessel density (P<0.05), creating a more potent therapeutic effect (95% reduction in tumor volume) compared with either hCIK cells or TMZ single therapy (72% for both, P<0.05). Taken together, CIK cell-immunotherapy and TMZ chemotherapy have synergistic therapeutic effects and could be combined for a successful treatment of GBM.

Combined treatment of tumor-tropic human neural stem cells containing the CD suicide gene effectively targets brain tumors provoking a mild immune response.

Previous studies showed promise of coupling genetically engineered neural stem cells (NSCs) with blood-brain barrier permeable prodrugs as an effective anti-brain tumor therapy. Here, we further advance those findings by testing the suicide gene therapeutic system in syngenic glioblastoma immunocompetent mice. After intracranial injection of HB1.F3.CD, an immortalized human NSC cell line engineered to constitutively produce cytosine deaminase (CD), the prodrug 5-fluorocytosine (5-FC) was administered for five days, q.d., via intraperitoneal injection. The HB1. F3.CD hNSCs migrated specifically to the brain tumor site via the corpus callosum and significantly reduced the tumor volume (67%) by converting 5-FC into the cytotoxic 5-fluorouracil. A corresponding increase in F4/80-positive population was observed in the treatment group, although CD3-positive population remained unchanged compared to control. No toxic effects or morphological changes were observed in the spleen and the lymph nodes. The data suggest that the NSC-enzyme/prodrug treatment is an effective anti-tumor therapeutic strategy that specifically targets only the tumor site with little or no systemic side effects. In addition, the treatment modeled here successfully elicited a macrophagic immune response which seemed to have a synergistic role in reducing tumor volume, thus showing promise for treatment-mediated enhancement of inherent immune responses against brain tumors.

Schedule-dependent synergistic effect of rituximab on methotrexate chemotherapy against lymphoma of the central nervous system.

We hypothesized that methotrexate (MTX) normalizes the increased permeability of the blood-tumor barrier and thus reduces the accessibility of rituximab (RTX) to central nervous system (CNS) lymphoma. Here, we evaluated the combinational treatment capability of RTX and MTX using an alternative treatment schedule against CNS lymphoma. We developed a CNS lymphoma animal model that closely mimics the morphological and molecular characteristics of human CNS lymphoma by injecting Raji human Burkitt lymphoma cells into the brains of immune-compromised mice and tested a novel combinational treatment schedule by which penetration of RTX was not influenced by MTX administration. RTX was conjugated with Alexa Fluor 680, and its distribution in the brain was analyzed by in vivo imaging. When MTX treatment was followed by a 3-day post RTX administration, RTX was scarcely distributed in the brain, and there were only modest statistically insignificant therapeutic effects compared with the control mice which received sham injections. In contrast, RTX administration followed by a 3-day post MTX treatment showed significantly increased distribution of RTX and significantly reduced tumor volume in the brain. Collectively, our data demonstrate that RTX can be successfully combined with MTX using an alternative treatment schedule that allows increased distribution of RTX in CNS lymphoma.

Human neural stem cells can target and deliver therapeutic genes to breast cancer brain metastases.

The tumor-tropic properties of neural stem cells (NSCs) led to the development of a novel strategy for delivering therapeutic genes to tumors in the brain. To apply this strategy to the treatment of brain metastases, we made a human NSC line expressing cytosine deaminase (F3.CD), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil, an anticancer agent. In vitro, the F3.CD cells significantly inhibited the growth of tumor cell lines in the presence of the prodrug 5-FC. In vivo, MDA-MB-435 human breast cancer cells were implanted into the brain of immune-deficient mouse stereotactically, and F3.CD cells were injected into the contralateral hemisphere followed by systemic 5-FC administration. The F3.CD cells migrated selectively into the brain metastases located in the opposite hemisphere and resulted in significantly reduced volumes. The F3.CD and 5-FC treatment also decreased both tumor volume and number of tumor mass significantly, when immune-deficient mouse had MDA-MB-435 cells injected into the internal carotid artery and F3.CD cells were transplanted into the contralateral brain hemisphere stereotactically. Taken together, brain transplantation of human NSCs, encoding the suicide enzyme CD, combined with systemic administration of the prodrug 5-FC, is an effective treatment regimen for brain metastases of tumors.