Tae Hyun Choi

Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice.

BACKGROUND & AIMSnMany studies of embryonic stem cells have investigated direct cell replacement of damaged tissues, but little is known about how donor cell-derived signals affect host tissue regeneration. We investigated the direct and indirect roles of human embryonic stem cell-derived cells in liver repair in mice.nnnMETHODSnTo promote the initial differentiation of human embryonic stem cells into mesendoderm, we activated the β-catenin signaling pathway with lithium; cells were then further differentiated into hepatocyte-like cells. The differentiated cells were purified by indocyanine green staining and laser microdissection and characterized by immunostaining, polymerase chain reaction, biochemical function, electron microscopy, and transplantation analyses. To investigate indirect effects of these cells, secreted proteins (secretomes) were analyzed by a label-free quantitative mass spectrometry. Carbon tetrachloride was used to induce acute liver injury in mice; cells or secreted proteins were administered by intrasplenic or intraperitoneal injection, respectively.nnnRESULTSnThe differentiated hepatocyte-like cells had multiple features of normal hepatocytes, engrafted efficiently into mice, and continued to have hepatic features; they promoted proliferation of host hepatocytes and revascularization of injured host liver tissues. Proteomic analysis identified proteins secreted from these cells that might promote host tissue repair. Injection of the secreted proteins into injured livers of mice promoted significant amounts of tissue regeneration without cell grafts.nnnCONCLUSIONSnHepatocyte-like cells derived from human embryonic stem cells contribute to recovery of injured liver tissues in mice, not only by cell replacement but also by delivering trophic factors that support endogenous liver regeneration.

Hepatic siRNA delivery using recombinant human apolipoprotein A-I in mice.

Apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein (HDL), plays a key role in reverse cholesterol transport from peripheral tissues to liver or steroidogenic organs. Class B, type 1 scavenger receptor (SR-BI) is abundantly expressed in these target tissues and recognizes apo A-I of HDL for selective cholesteryl ester uptake. Recently, we reported the liver-targeting potential of plasma-derived apo A-I and the efficient delivery of therapeutic small interfering RNAs (siRNA) assembled with cationic liposome and apo A-I. In this study, we expressed and purified recombinant human apo A-I (rhapo A-I), low endotoxin grade, from an Escherichia coli expression system. The liver-targeting property of rhapo A-I was compared to that of plasma-derived apo A-I. Using a hepatitis C virus mouse model, intravenous administration of virus-specific siRNA with liposome and rhapo A-I significantly inhibited viral protein expression, demonstrating great promise for its use in clinical applications.

Automation of the synthesis of highly concentrated 188Re–MAG3 for intracoronary radiation therapy☆

We have developed an efficient method and an automated synthetic system for the preparation of highly concentrated 188Re-MAG3. Routine production of 188Re-MAG3 for use in intracoronary radiation therapy was performed by compressed air driven semi-automated shielded system. 188Re-MAG3 was prepared with a commercial kit and reducing agents, purified and concentrated by C18 Sep-Pak cartridges to desired radioactivity and volume. Using this automated system, reproducible radiolabeling yields of 80-85% were obtained.

A simple Cu-64 production and its application of Cu-64 ATSM.

One of the positron emission radionuclides, (64)Cu, has been reported to be a particularly effective radioisotope in PET imaging study. This utility of (64)Cu depends on the chemical stability in water with proper energy and half-life as gamma-emitters. Hence, we tried to develop a simple method for producing this isotope using an old cyclotron model in our site (50MeV, Scantronics co.). In particular, we designed the equipments of enrich (64)Ni plating system and radioactive (64)Cu separation using plastic cartridge column; (64)Ni plating system on gold foil which located in the 13 degrees angle target toward beam irradiation. For the nuclear reaction of (64)Cu, it was applied to (64)Ni(p, n) (64)Cu at low energy under the degrader composed of Al and Ta foils. After beam irradiation, (64)Cu was identified by multichannel analyzer installed for a HPGe detector and its utility was certified by the microPET images of (64)Cu-ATSM (CT-26 tumor bearing mouse as reported previously). The image quality of (64)Cu was also very similar to that of (18)F radioisotope in microPET scanner. In conclusion, a method of (64)Cu production and its application was successfully established in old cyclotron having high energy.

Combined radionuclide–chemotherapy and in vivo imaging of hepatocellular carcinoma cells after transfection of a triple-gene construct, NIS, HSV1-sr39tk, and EGFP

The sodium iodine symporter (NIS) or mutant Herpes-simplex virus type1 sr39 thymidine kinase (HSV1-sr39tk) gene is used for in vivo imaging and cancer therapy. Transfection of both NIS and HSV1-sr39tk genes to hepatocellular carcinoma cells (Huh-7/NTG) could enhance intracellular accumulation of therapeutic radionuclides and guanosine nucleoside analogue prodrugs to produce better outcomes than single gene therapy. Non-invasive imaging with I-124, F-18 FHBG and combination therapy with I-131 and GCV were performed in hepatocellular carcinoma cells transfected with NIS, HSV1-sr39tk and GFP. Our results show that: (1) all three genes are stably expressed in Huh-7/NTG cells, (2) I-125 and H3-PCV uptake were markedly increased in the Huh-7/NTG cells in vitro, (3) cellular survival and tumor growth of Huh-7/NTG was inhibited by I-131 or GCV both in vitro and in vivo, and was much prominent with combination therapy, (4) in vivo imaging with I-124 and F-18 FHBG revealed increased uptake in the Huh-7/NTG tumor. Our results demonstrated the potential of combination gene therapy using NIS and HSV1-sr39tk followed by radioiodine treatment and chemotherapy in human hepatocellular carcinoma cells.

Effective microPET imaging of brain 5-HT(1A) receptors in rats with [(18) F]MeFWAY by suppression of radioligand defluorination.

Introduction: [18F]MeFWAY has been developed for imaging the serotonin 1A receptors in the brain. The purpose of this study were to verify the metabolic stability of [18F]MeFWAY, to measure the degree of defluorination of [18F]MeFWAY in vivo, to investigate methods of inhibition of defluorination of [18F]MeFWAY, and to assess the efficacy of [18F]MeFWAY in rat brains in vivo. Methods: MicroPET experiments in rats were conducted to confirm the distribution of radioactivity in the brain. Nondisplaceable binding potential (BPND) in the hippocampus and frontal cortex were also analyzed. Miconazole and fluconazole were tested for the ability to suppress defluorination of [18F]MeFWAY. We conducted a blockade and displacement experiment by treating with WAY‐100635. Results: In vitro stability tests showed that MeFWAY was very stable in serum for 6 h, but PET revealed that authentic [18F]MeFWAY underwent significant defluorination in vivo. In vitro inhibition study against decreasing parent activity in liver microsomes, miconazole and fluconazole suppressed metabolic elimination of MeFWAY. However, in the PET study, fluconazole showed more potent inhibitory activity than miconazole. In the suppression of metabolizing enzymes using fluconazole, radioactivity in skull was dramatically decreased by 81% (compared with 69% with miconazole) and it was coupled with an increase in brain uptake. Moreover, BPND in hippocampus was 5.53 and 2.66 in frontal cortex. The blockade and displacement study showed the specificity of [18F]MeFWAY to 5‐HT1A receptors. Conclusion: In the rat brain, [18F]MeFWAY microPET showed skull uptake due to defluorination in vivo. We can effectively overcome this drawback with fluconazole. Synapse, 2012.

Relationship between dopamine deficit and the expression of depressive behavior resulted from alteration of serotonin system

Depression frequently accompanies in Parkinsons disease (PD). Previous research suggested that dopamine (DA) and serotonin systems are closely linked with depression in PD. However, comprehensive studies about the relationship between these two neurotransmitter systems are limited. Therefore, the purpose of this study is to evaluate the effect of dopaminergic destruction on the serotonin system. The interconnection between motor and depression was also examined. Two PET scans were performed in the 6‐hydroxydopamine (6‐OHDA) lesioned and sham operated rats: [18F]FP‐CIT for DA transporters and [18F]Mefway for serotonin 1A (5‐HT1A) receptors. Here, 6‐OHDA is a neurotoxin for dopaminergic neurons. Behavioral tests were used to evaluate the severity of symptoms: rotational number for motor impairment and immobility time, acquired from the forced swim test for depression. Region‐of‐interests were drawn in the striatum and cerebellum for the DA system and hippocampus and cerebellum for the 5‐HT system. The cerebellum was chosen as a reference region. Nondisplaceable binding potential in the striatum and hippocampus were compared between 6‐OHDA and sham groups. As a result, the degree of DA depletion was negatively correlated with rotational behavior (R2u2009=u20090.79, Pu2009=u20090.003). In 6‐OHDA lesioned rats, binding values for 5‐HT1A receptors was 22% lower than the sham operated group. This decrement of 5‐HT1A receptor binding was also correlated with the severity of depression (R2u2009=u20090.81, Pu2009=u20090.006). Taken together, this research demonstrated that the destruction of dopaminergic system causes the reduction of the serotonergic system resulting in the expression of depressive behavior. The degree of dopaminergic dysfunction was positively correlated with the impairment of the serotonin system. Severity of motor symptoms was also closely related to depressive behavior. Synapse 69:453–460, 2015.

Targeted therapy for Epstein-Barr virus-associated gastric carcinoma using low-dose gemcitabine-induced lytic activation

The constant presence of the viral genome in Epstein-Barr virus (EBV)-associated gastric cancers (EBVaGCs) suggests the applicability of novel EBV-targeted therapies. The antiviral nucleoside drug, ganciclovir (GCV), is effective only in the context of the viral lytic cycle in the presence of EBV-encoded thymidine kinase (TK)/protein kinase (PK) expression. In this study, screening of the Johns Hopkins Drug Library identified gemcitabine as a candidate for combination treatment with GCV. Pharmacological induction of EBV-TK or PK in EBVaGC-originated tumor cells were used to study combination treatment with GCV in vitro and in vivo. Gemcitabine was found to be a lytic inducer via activation of the ataxia telangiectasia-mutated (ATM)/p53 genotoxic stress pathway in EBVaGC. Using an EBVaGC mouse model and a [125I] fialuridine (FIAU)-based lytic activation imaging system, we evaluated gemcitabine-induced lytic activation in an in vivo system and confirmed the efficacy of gemcitabine-GCV combination treatment. This viral enzyme-targeted anti-tumor strategy may provide a new therapeutic approach for EBVaGCs.

Mycobacterium marinum infection in Drosophila melanogaster for antimycobacterial activity assessment

OBJECTIVESnThe major advantages of Drosophila melanogaster are a well-characterized immune system and high degree of susceptibility to tuberculosis caused by Mycobacterium marinum. The D. melanogaster-M. marinum infection model is gaining momentum as a screening tool because it is genetically amenable, low priced, rapid, technically convenient and ethically acceptable. In this context, the aim of this study was to develop a new, effective D. melanogaster-M. marinum in vivo efficacy model for antimycobacterial drug discovery.nnnMETHODSnD. melanogaster were challenged with intra-abdominal injections of M. marinum and infected flies were fed with a fly medium containing isoniazid, rifampicin, ethambutol, pyrazinamide, amikacin, dinitrobenzamide or ampicillin dissolved in DMSO at different concentrations (0, 100 and 500 mg/L). Bacterial dissemination in flies was monitored by fluorescence microscopy/cfu counts and a fly survival curve was plotted.nnnRESULTSnThe D. melanogaster-M. marinum model allowed assessment of the effectiveness of antibiotic treatment not only with conventional drugs, but also with newly discovered antimycobacterial agents. Rifampicin, dinitrobenzamide, amikacin and isoniazid effectively extended the life span of infected flies and ethambutol showed slightly improved survival. However, M. marinum infection was not cured by ampicillin or pyrazinamide.nnnCONCLUSIONSnThis D. melanogaster-M. marinum infection/curing methodology may be valuable in the rapid evaluation of the activity of new antimycobacterial agents in drug discovery.

Effectiveness of [124I]-PET/CT and [18F]-FDG-PET/CT for Localizing Recurrence in Patients with Differentiated Thyroid Carcinoma

Although the prognosis of patients with differentiated thyroid carcinoma (DTC) is generally encouraging, a diagnostic dilemma is posed when an increasing level of serum thyroglobulin (Tg) is noted, without detection of a recurrent tumor using conventional imaging tools such as the iodine-131 whole-body scanning (the [131I] scan) or neck ultrasonography (US). The objective of the present study was to evaluate the diagnostic value of [124I]-PET/CT and [18F]-FDG-PET/CT in terms of accurate detection of both iodine- and non-iodine-avid recurrence, compared with that of conventional imaging such as the [131I] scan or neck ultrasonography (US). Between July 2009 and June 2010, we prospectively studied 19 DTC patients with elevated thyroglobulin levels but who do not show pathological lesions when conventional imaging modalities are used. All involved patients had undergone total thyroidectomy and radioiodine (RI) treatment, and who had been followed-up for a mean of 13 months (range, 6-21 months) after the last RI session. Combined [18F]-FDG-PET/CT and [124I]-PET/CT data were evaluated for detecting recurrent DTC lesions in study patients and compared with those of other radiological and/or cytological investigations. Nine of 19 patients (47.4%) showed pathological [18F]-FDG (5/19, 26.3%) or [124I]-PET (4/19, 21.1%) uptake, and were classed as true-positives. Among such patients, disease management was modified in six (66.7%) and disease was restaged in seven (77.8%). In particular, the use of the described imaging combination optimized planning of surgical resection to deal with locoregional recurrence in 21.1% (4/19) of patients, who were shown to be disease-free during follow-up after surgery. Our results indicate that combination of [18F]-FDG-PET/CT and [124I]-PET/CT affords a valuable diagnostic method that can be used to make therapeutic decisions in patients with DTC who are tumor-free on conventional imaging studies but who have high Tg levels.