Hee-Seung Bom

Galactosylated chitosan as a synthetic extracellular matrix for hepatocytes attachment.

Galactose moiety as the hepatocyte anchorage was covalently coupled with chitosan for the development of synthetic extracellular matrix. Hepatocytes adhesion to galactosylated chitosan (GC)-coated polystyrene (PS) dish became as high as 94.7% after 2 h incubation whereas the hepatocytes adhesion to chitosan-coated PS dish was 69.1%, indication of galactose-specific recognition between GC molecules and asialoglycoprotein receptors of hepatocytes. The DNA synthesis of the hepatocytes adhered to GC-coated dish was increased in the presence of epidermal growth factor (EGF) at low concentration of GC (0.05 microg/ml) whereas the DNA synthesis of the hepatocytes adhered to GC-coated dish was decreased in the presence of EGF at high concentration of GC (5 microg/ml). The spreading shapes of the hepatocytes adhered to the surface in the presence of EGF at low concentration of GC (0.05 microg/ml) were enhanced than in the absence of EGF. The hepatocytes adhered to the surface at high concentration of GC (5 microg/ml) showed round shapes and exhibited many spheroid formation after 24 h in the presence of EGF.

Prognostic significance of interim 18F-FDG PET/CT after three or four cycles of R-CHOP chemotherapy in the treatment of diffuse large B-cell lymphoma

PURPOSE (18)F-fluoro-2-dexoy-D-glucose-positron emission tomography (FDG-PET)/computerised tomography (CT) has been used for staging and monitoring responses to treatment in patients with diffuse large B cell lymphoma (DLBCL). The sequential interim PET/CT was prospectively investigated to determine whether it provided additional prognostic information and could be a positive predictable value within patients with the same international prognostic index (IPI) after the use of rituximab in DLBCL. METHODS One hundred and sixty-one patients with newly diagnosed DLBCL were enroled; the assessment of the PET/CT was performed at the time of diagnosis and mid-treatment of rituxibmab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP). RESULTS Sixty-seven patients (41.6%) presented with advanced stage disease and 27 (16.8%) had bulky lesions. Forty-three patients (26.7%) continued to have positive metabolic uptakes with a significantly high relapse rate (62.8%) compared to the patients with a negative interim PET/CT (12.1%) (P<0.01). After a median follow-up of 30.8months, the positivity of interim PET/CT was found to be a prognostic factor for both overall survival (OS) and progression-free survival (PFS), with a hazard ratio of 4.07 (2.62-6.32) and 5.46 (3.49-8.52), respectively. In the low-risk IPI group, the 3-year OS and PFS rates were significantly different in the patients with positive (53.3% and 52.5%) and negative (93.8% and 88.3%) interim PET/CT, respectively (P<0.01). These significant prognostic differences of interim PET/CT responses were consistent with the results of the patients with high-risk IPI group (P<0.01). CONCLUSIONS Interim PET/CT scanning had a significant predictive value for disease progression and survival of DLBCL in post-rituximab treatment; it might be the single most important determinant of clinical outcome in patients with the same IPI risk.

Inhibition of Tumor Growth and Metastasis by a Combination of Escherichia coli–mediated Cytolytic Therapy and Radiotherapy

We have reported that Escherichia coli K-12 colonizes hypoxic and necrotic tumor regions after intravenous injection into tumor-bearing mice. In this study, we established a novel strategy for cancer therapy using engineered bacteria to enhance the therapeutic effects of radiation. E. coli strain K-12 was engineered to produce cytolysin A (ClyA), and its effects on tumor growth in primary and metastatic tumor models were evaluated. A single treatment with E. coli-expressing ClyA significantly decreased tumor growth rates initially (9 days after treatment); however, the tumors tended to grow thereafter. With only radiotherapy (RT; 21 Gy), the tumor growth rates were retarded, but not the tumor sizes. A combination of therapy with E. coli-expressing ClyA and radiation [a total of 5 x 10(7) colony-forming units (CFU) and 21 Gy] resulted in significant tumor shrinkage and even complete disappearance of tumors in mice with tumors derived from murine CT26 colon cancer. Furthermore, treatment with E. coli-expressing ClyA markedly suppressed metastatic tumor growth and prolonged the survival time in mice. The results described here indicate that therapy with engineered E. coli could significantly improve the results of RT, and could exert a striking inhibitory effect on the development of lung metastasis.

Noninvasive Real-time Imaging of Tumors and Metastases Using Tumor-targeting Light-emitting Escherichia coli

PurposeA number of bacteria types are known to preferentially grow in tumors. We have taken advantage of this phenomenon to target luciferase-expressing Escherichia coli to tumors and metastases in mouse models to image them noninvasively.Methods and ResultsAfter intravenous injection of pLux-expressing E. coli (108 CFU), bioluminescence signals from the bacteria were detected exclusively in tumor tissue after 24 hours. The balanced-lethal host–vector system using the gene encoding aspartate β-semialdehyde dehydrogenase (asd) enabled stable maintenance of the pLux in the tumor-targeting E. coli. This phenomenon of selective tumor targeting and proliferation of E. coli was observed in a diverse range of tumors implanted in nude mice. More importantly, E. coli was capable of targeting both primary tumors and metastases, enabling them to be imaged noninvasively in both nude and immunocompetent mice.ConclusionsOur results suggest the potential clinical use of this technology for tumor targeting.

In vivo bioluminescence imaging of cord blood derived mesenchymal stem cell transplantation into rat myocardium

ObjectiveThe conventional method for the analysis of myocardial cell transplantation depends on postmortem histology. Here, we have sought to demonstrate the feasibility of a longitudinal monitoring of transplanted cell survival in living animals, accomplished with optical imaging techniques and pharmacological interventions.MethodsHuman cord blood (50 ml) was donated with parental consent. After getting cord blood derived mesenchymal stem cells (CBMSCs), cells were transfected (MOI = 100) overnight with adenovirus encoding firefly luciferase gene (Ad-CMV-Fluc). Our experimental Sprague-Dawley rats (n = 12) were given intramyocardial injections containing 1 × 106 CBMSCs, which had been made to express the firefly luciferase (Fluc) reporter gene. Optical bioluminescence imaging was then conducted using a cooled charged-coupled device (CCD) camera (Xenogen), beginning on the day after the transplantation (day 1). Groups of mice were intraperitoneally injected with cyclosporine (5 mg/kg) or tacrolimus (1 mg/kg), in an attempt to determine the degree to which cell survival had been prolonged, and these values were then compared with the cell survival values of the negative control group. The presence of transplanted CBMSCs on in vivo images confirmed by in situ hybridization for human specific Alu in the myocardium.ResultsCardiac bioluminescence signals were determined to be present for 6 days after transplantation: day 1 (97000 ± 9100 × 105 p/s/cm2/sr), day 3 (9600 ±1110 p/s/cm2/sr), and day 5 (3200 ± 550 p/s/cm2/sr). The six mice that received either cyclosporine or tacrolimus displayed cardiac bioluminescence signals for a period of 8 days after transplantation. We observed significant differences between the treated group and the non-treated group, beginning on day 3 (tacrolimus; 26500 ± 4340 p/s/cm2/sr, cyclosporine; 27200 ± 3340 p/s/cm2/sr, non-treated; 9630 ± 1180 p/s/cm2/sr, p < 0.01), and persisting until day 7 (tacrolimus; 12500 ± 2946 p/s/cm2/sr, cyclosporine; 7310 ±1258 p/s/cm2/sr, non-treated; 2460 ± 160 p/s/cm2/sr, p < 0.01). The human-derived CBMSCs were detected in the myocardium 3 days after transplantation by in situ hybridization.ConclusionsThe locations, magnitude, and survival duration of the CBMSCs were noninvasively monitored with a bioluminescence optical imaging system. We determined that optical molecular imaging expedites the fast throughput screening of pharmaceutical agents, allowing for the noninvasive tracking of cell survival within animals. In rat cardiac CBMSC transplant models, transient immunosuppressive treatment with tacrolimus or cyclosporine was shown to improve donor cell survival.

Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin

Engineered Salmonella secreting heterologous bacterial flagellin suppress tumor growth by activating intratumoral macrophages. Two bacteria can be better than one In some cases, injecting tumors with specific bacteria can help eradicate the tumors by stimulating inflammation and triggering an antitumor immune response. A classic example of this is injection of bladder cancer with bacillus Calmette-Guérin, but more recent approaches have used bacteria such as Clostridium and Salmonella species. Building on the idea of antitumor bacterial therapy, Zheng et al. engineered a weakened strain of Salmonella typhimurium to produce the flagellin B protein from another bacterium, Vibrio vulnificus. The engineered bacteria induced an effective antitumor immune response, successfully treating tumors in several different mouse models with no evidence of toxicity. We report a method of cancer immunotherapy using an attenuated Salmonella typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (FlaB) in tumor tissues. Engineered FlaB-secreting bacteria effectively suppressed tumor growth and metastasis in mouse models and prolonged survival. By using Toll-like receptor 5 (TLR5)–negative colon cancer cell lines, we provided evidence that the FlaB-mediated tumor suppression upon bacterial colonization is associated with TLR5-mediated host reactions in the tumor microenvironment. These therapeutic effects were completely abrogated in TLR4 and MyD88 knockout mice, and partly in TLR5 knockout mice, indicating that TLR4 signaling is a requisite for tumor suppression mediated by FlaB-secreting bacteria, whereas TLR5 signaling augmented tumor-suppressive host reactions. Tumor microenvironment colonization by engineered Salmonella appeared to induce the infiltration of abundant immune cells such as monocytes/macrophages and neutrophils via TLR4 signaling. Subsequent secretion of FlaB from colonizing Salmonella resulted in phenotypic and functional activation of intratumoral macrophages with M1 phenotypes and a reciprocal reduction in M2-like suppressive activities. Together, these findings provide evidence that nonvirulent tumor-targeting bacteria releasing multiple TLR ligands can be used as cancer immunotherapeutics.

Prognostication of Recovery in Patients With Acute Ischemic Stroke Through the Use of Brain SPECT With Technetium-99m—Labeled Metronidazole

Background and Purpose— We hypothesized that technetium-99m-ethylene dicysteine-metronidazole (99mTc-EC-MN) localizes to brain tissue that is hypoxic but viable. This study prospectively evaluated the relationship between neurological outcome and uptake of 99mTc-EC-MN in peri-infarcted regions of the brain. Methods— Eight patients with acute ischemic stroke in the territory of the left middle cerebral artery underwent 99mTc-EC-MN and 99mTc-ethyl cysteinate dimer (ECD) brain SPECTs on the same day during the subacute stage (10.3±2.5 days). The infarct volumes from 99mTc-ECD images (IVECD), infarct volumes from diffusion-weighted MRI images (IVDW), and hypoxic volume (HV) from 99mTc-EC-MN images were calculated. The net infarct volume (NIVECD) was defined as IVECD minus HV. The National Institutes of Health Stroke Scale scores were measured on admission and days 1, 3, 7, and 30. Results— IVECD was greater than IVDW. The lesion-to-normal count-density ratios of 99mTc-EC-MN ranged from 1.80 to 5.96. HV was 60.2±65.2 cm3, and the mean percent HV was 24.5±28.1% of IVECD. NIVECD was 162.6±133.4 cm3 and was significantly smaller than IVECD. NIVECD was significantly correlated with National Institutes of Health Stroke Scale score at 1 month and was a significant predictor of neurological deficit at 1 month. Conclusions— 99mTc-EC-MN brain SPECT can detect hypoxic tissue after acute ischemic stroke and, in combination with 99mTc-ECD brain SPECT, is useful in predicting neurological outcome in ischemic stroke patients.

Evaluation of a Mitochondrial Voltage Sensor, (18F-Fluoropentyl)Triphenylphosphonium Cation, in a Rat Myocardial Infarction Model

Radiolabeled lipophilic cationic compounds, such as 18F-labeled phosphonium salt, accumulate in the mitochondria through a negative inner transmembrane potential. The purpose of this study was to develop and evaluate (18F-fluoropentyl)triphenylphosphonium salt (18F-FPTP) as a myocardial PET agent. Methods: A reference compound of 18F-FPTP was synthesized via 3-step nucleophilic substitution reactions and was radiolabeled via 2-step nucleophilic substitution reactions of no-carrier-added 18F-fluoride. Accumulations of 18F-FPTP, 3H-tetraphenylphosphonium, and 99mTc-sestamibi were compared in a cultured embryonic cardiomyoblast cell line (H9c2). The biodistribution of 18F-FPTP was assessed using BALB/c mice. The 18F-FPTP small-animal PET study was performed in Sprague–Dawley rats with or without left coronary artery (LCA) ligation. Results: 18F-FPTP was synthesized with a radiochemical yield of 15%–20% and radiochemical purity of greater than 98%. Specific activity was greater than 6.3 TBq/μmol. Cell uptake of 18F-FPTP was more than 15-fold higher in H9c2 than in normal fibroblasts (human normal foreskin fibroblasts). Selective collapse of mitochondrial membrane potential substantially decreased cellular uptake for 18F-FPTP and 3H-tetraphenylphosphonium, compared with that for 99mTc-sestamibi. The biodistribution data in mice (n = 24) showed rapid blood clearance and high accumulation in the heart. Heart-to-blood ratios at 10 and 30 min were 54 and 133, respectively. Heart-to-lung and heart-to-liver ratios at 10, 30, and 60 min were 4, 4, and 7 and 4, 5, and 7, respectively. Dynamic small-animal PET for 60 min after injection of 18F-FPTP showed an initial spike of radioactivity, followed by retention in the myocardium and rapid clearance from the background. 18F-FPTP small-animal PET images in LCA-occluded rats demonstrated sharply defined myocardial defects in the corresponding area of the myocardium. The myocardial defect size measured by 18F-FPTP small-animal PET correlated closely with the hypoperfused area measured by quantitative 2,3,5-triphenyltetrazolium chloride staining (r2 = 0.92, P < 0.001). Conclusion: The excellent pharmacokinetics of 18F-FPTP and its correlation with 2,3,5-triphenyltetrazolium chloride staining in normal and LCA-occluded rats suggest that this molecular probe may have a high potential as a mitochondrial voltage sensor for PET. This probe may also allow high throughput, with multiple daily studies and a wide distribution of PET myocardial imaging in the clinic.

Comparison of 131I whole-body imaging, 131I SPECT/CT, and 18F-FDG PET/CT in the detection of metastatic thyroid cancer

PurposeThe aim of this study was to compare 131I whole-body scintigraphy (WBS), WBS with 131I single photon emission computed tomography/computed tomography (SPECT/CT), and 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT in the detection of distant metastases of differentiated thyroid cancer (DTC).MethodsA total of 140 patients with 258 foci of suspected distant metastases were evaluated. 131I WBS, 131I SPECT/CT, and 18F-FDG PET/CT images were interpreted separately. The final diagnosis was obtained from histopathologic study, serum thyroglobulin level, other imaging modalities, and/or clinical follow-up.ResultsOf the 140 patients with 258 foci, 46 patients with 166 foci were diagnosed as positive for distant metastasis. The sensitivity, specificity, and diagnostic accuracy of each imaging modality were 65, 55, and 59%, respectively, for 131I WBS; 65, 95, and 85% for 131I SPECT/CT, respectively; and 61, 98, and 86%, respectively, for 18F-FDG PET/CT in patient-based analyses. Lesion-based analyses demonstrated that both SPECT/CT and PET/CT were superior to WBS (p<0.001) in all patient groups. SPECT/CT was superior to WBS and PET/CT (p<0.001) in patients who received a single challenge of radioiodine therapy, whereas PET/CT was superior to WBS (p=0.005) and SPECT/CT (p=0.013) in patients who received multiple challenges.ConclusionBoth SPECT/CT and PET/CT demonstrated high diagnostic performance in detecting metastatic thyroid cancer. SPECT/CT was highly accurate in patients who underwent a single challenge of radioiodine therapy. In contrast, 18F-FDG PET/CT presented the highest diagnostic performance in patients who underwent multiple challenges of radioiodine therapy.

Impact of Medication Discontinuation on Increased Intestinal FDG Accumulation in Diabetic Patients Treated With Metformin

OBJECTIVE We evaluated the impact of stopping medication for 2 days on reductions in the high intestinal FDG uptake induced by metformin. SUBJECTS AND METHODS One hundred thirty-eight diabetic patients were divided into two groups: one in which the antihyperglycemic drug regimen included metformin (group A; n = 107) and one in which the regimen did not include metformin (group B; n = 31). Fifty-two patients without diabetes mellitus served as the control group (group C). Group A was divided into two subgroups: 77 patients (group A1) were taking metformin at the time of FDG PET/CT scans, whereas the remaining 30 patients (group A2) were asked to stop taking metformin for 2 days before PET/CT scans. In addition, 10 diabetic patients underwent two consecutive PET/CT scans before and after the discontinuation of metformin. The intestinal FDG uptake and blood glucose levels were compared among the four groups, as well as before and after the discontinuation of metformin. RESULTS The high intestinal FDG uptake in group A1 was significantly reduced after the discontinuation of metformin (p < 0.001 vs group A2); thus, there were no significant differences among group A2, group B, and group C (p = 0.581-0.872). There were also no statistically significant differences in the blood glucose levels among the three groups of diabetic patients (p > 0.9). In 10 patients who underwent serial PET/CT scans, mean intestinal FDG uptake decreased by 64% without significant changes in the blood glucose level. Hidden colorectal malignancies were revealed in two patients after the discontinuation of medication. CONCLUSION The discontinuation of metformin for 2 days is feasible for reducing the high intestinal FDG uptake induced by metformin.