Gwang Lee

Toll-like receptor signal transduction

Toll-like receptors (TLRs) are the archetypal pattern recognition receptors in sensing exogenous pathogens. Activation of TLRs is a first line of defense of the immune system, leading to the activation and recruitment of neutrophils and macrophages to sites of infection and enhances antimicrobial activity. The TLR signaling through different intracellular molecules, such as MAP kinases and IκB kinases which are conserved signaling elements for many receptors, leads to a distinct set of proinflammatory gene expressions. However, how these pathways differentially and precisely control the transcription of identical genes remains largely unknown. Our review focuses on the details of up-to- date signaling molecules including negative regulators and their role in controlling innate immune response. We also stress the importance of developing systemic approaches for the global understanding of TLR signaling so that appropriate drug therapeutic targets can be identified for regulating inflammatory diseases.

Cell Stretching Measurement Utilizing Viscoelastic Particle Focusing

We present an efficient method for measuring cell stretching based on three-dimensional viscoelastic particle focusing. We suspended cells in a biocompatible viscoelastic medium [poly(vinylpyrrolidone) solution in phosphate-buffered saline]. The medium viscoelasticity significantly homogenized the trajectories of cells along the centerline of a simple straight channel, which could not be achieved in conventional Newtonian media. More than 95% of red blood cells (RBCs) were successfully delivered to the stagnation point of a cross-slot microchannel and stretched by extensional flow. By computational simulations, we proved that this method prevents inaccuracies due to random lateral distributions of cells and, further, guarantees rotational-free cell stretching along the shear-free channel centerline. As a demonstration, we characterized the differences in RBC deformabilities among various heat treatments. Furthermore, we monitored the decrease of deformability due to nutrient starvation in human mesenchymal stem cells. We envisage that our novel method can be extended to versatile applications such as the detection of pathophysiological evolution in impaired RBCs due to malaria or diabetes and the monitoring of cell quality in stem cell therapeutics.

In vivo tracking of 111In-labeled bone marrow mesenchymal stem cells in acute brain trauma model

INTRODUCTIONnThis study was to evaluate the in vivo distribution of intravenously transplanted bone marrow-derived mesenchymal stem cells (BMSCs) in an acute brain trauma model by (111)In-tropolone labeling.nnnMETHODSnRat BMSCs were labeled with 37 MBq (111)In-tropolone. Their labeling efficiency and in vitro retention rate were measured. The viability and proliferation of labeled BMSCs were evaluated for 14 days after labeling. The biodistribution of (111)In-labeled BMSCs in trauma models was compared with those of sham-operated rats and normal rats on gamma camera images. The migration of (111)In-BMSCs to the traumatic brain was evaluated using confocal microscope.nnnRESULTSnThe labeling efficiency of (111)In-BMSCs was 66+/-5%, and their retention rate was 85.3% at 1 h after labeling. There was no difference in the number of viable cells between (111)In-BMSCs and controls at 48 h after labeling. However, the proliferation of (111)In-BMSCs was inhibited after the third day of labeling, and it did not reach confluency. On gamma camera images, most of the (111)In-BMSCs uptake was observed in the liver and spleen at the second day of injection. The brain uptake of (111)In-BMSCs was detected prominently in trauma models (1.4%) than in sham-operated (0.5%) or normal rats (0.3%). Radiolabeled BMSCs were observed at the traumatic brain on the confocal microscope as they have a homing capacity, although its proliferation capacity was suppressed.nnnCONCLUSIONnAlthough growth inhibition by (111)In-labeling need to be evaluated further prior to use in humans, (111)In-labeled BMSCs are useful for the tracking of intravenously transplanted mesenchymal stem cells in brain disease models.

Cytosine deaminase-producing human mesenchymal stem cells mediate an antitumor effect in a mouse xenograft model

Background and Aim:u2002 Stem cell transplantation offers potential gene therapy for brain tumors. However, this approach has received little attention as a treatment for gastrointestinal tumors. In the present study, we explored the possibility of human bone marrow‐derived mesenchymal stem cells (hMSC) producing cytosine deaminase (CD), followed by systemic 5‐fluorocytosine (5‐FC) administration, showing an antitumor effect on a mouse gastric cancer xenograft.

Early distribution of intravenously injected mesenchymal stem cells in rats with acute brain trauma evaluated by 99m Tc-HMPAO labeling ☆

INTRODUCTIONnStem cell tracking is essential for evaluation of its migration, transplantation and therapeutic response. The aim of this study was to evaluate early distribution of intravenously transplanted rat bone marrow mesenchymal stem cells (BMSCs) in rats with acute cerebral trauma by labeling with (99m)Tc-hexamethylpropyleneamine oxime ((99m)Tc-HMPAO).nnnMETHODSn(99m)Tc-HMPAO-labeled BMSCs were injected intravenously to trauma rats (n=14) and sham-operated controls (n=13). Gamma camera images were acquired at 4 h after injection, and then organs were removed for gamma counting. Confocal microscope was used to confirm the migration of (99m)Tc-BMSCs by co-labeling with PKH26. Cytometric analysis was performed to evaluate apoptotic or necrotic change until the seventh day after labeling.nnnRESULTSn(99m)Tc-BMSCs were distributed mostly to lungs, liver and spleen at 4 h, and uptake of these organs was not significantly different between traumatic rats and controls. Meanwhile, the cerebral uptake of (99m)Tc-BMSCs was significantly higher in the traumatic rats than in controls (0.40% vs. 0.20%; P=.0002). Additionally, (99m)Tc-BMSCs uptake of traumatic hemisphere was significantly higher than that of contralateral ones (0.27% vs. 0.13%; P=.0001) in traumatic rats. Regardless of radiolabeling, BMSCs migrated to traumatic regions, but not to nontraumatic hemispheres. However, gamma camera failed to demonstrate (99m)Tc-BMSCs in traumatic hemispheres. No significant apoptotic or necrotic change was observed until 7 days after radiolabeling.nnnCONCLUSIONSnEarly distribution of BMSCs in traumatic brain disease could be monitored by (99m)Tc-labeling, which does not induce cellular death. However, our data showed that the amount of migrated (99m)Tc-BMSCs was not enough to be demonstrated by clinical gamma camera.

Drugs targeting Toll-like receptors.

Animals and plants are exposed to myriads of potential microbial invaders. In case of animals, Toll-like receptors (TLRs) act as the primary defense against infection by pathogens. Arguably, less is known regarding the activation of TLRs that connect the innate and adaptive immune systems. Some TLR ligands have been used as adjuvants in various vaccines and have gained a great deal of attention due to their ability to elicit an effective immune response. Understanding the intricate relationships between various molecules involved in TLR signaling and their positive or negative regulation is a key focus for the development of effective therapeutics. In this review, recent developments in TLR signaling that will be very important in providing new drug target molecules and a better understanding of molecular regulation of innate immunity are discussed.

Simultaneous clinical monitoring of lactic acid, pyruvic acid and ketone bodies in plasma as methoxime/tert-butyldimethylsilyl derivatives by gas chromatography–mass spectrometry in selected ion monitoring mode

Simultaneous determination of lactic acid, pyruvic acid, 3-hydroxybutyric acid and acetoacetic acid for clinical monitoring of lactic acidosis and ketone body formation in human plasma (20 microL) was performed by gas chromatography-mass spectrometry in selected ion monitoring (SIM) mode after generating methoxime/tert-butyldimethylsilyl derivatives. All of the targeted carboxylic acids were detected by characteristic fragment ions, which permitted sensitive and selective identification in the presence of co-extracted free fatty acids and other acidic metabolites at much higher levels. The method was linear (r>or=0.9991), reproducible (% relative standard deviation=1.2-5.8), and accurate (% relative error=-7.2-7.6), with detection limits of 0.05-1.7 ng/mL. This rapid, accurate and selective method using minimal plasma samples (20 microL) is useful in the clinical monitoring of lactic acidosis and ketone body formation in plasma.

Dichloroacetate attenuates hypoxia-induced resistance to 5-fluorouracil in gastric cancer through the regulation of glucose metabolism

In this study, we investigated whether gastric cancer with hypoxia-induced resistance to 5-fluorouracil (5-FU) could be re-sensitized following treatment with low-dose dichloroacetate (DCA), an inhibitor of the glycolytic pathway. The expression profiles of hypoxia-inducible factor-1α (HIF-1α) and pyruvate dehydrogenase kinase-1 (PDK-1) were analyzed in tissues from 10 patients with gastric cancer who had different responses to adjuvant 5-FU treatment. For the in vitro assays, cell viability and apoptosis were evaluated with and without treatment with 20mM DCA in the AGS and MKN45 cell lines, as well as in PDK1 knockdown cell lines. The expression levels of HIF-1α and PDK-1 were both elevated in the tumor tissues relative to the normal gastric tissues of most patients who showed recurrence after adjuvant 5-FU treatment. Cellular viability tests showed that these cell lines had a lower sensitivity to 5-FU under hypoxic conditions compared to normoxic conditions. Moreover, the addition of 20mM DCA only increased the sensitivity of these cells to 5-FU under hypoxic conditions, and the resistance to 5-FU under hypoxia was also attenuated in PDK1 knockdown cell lines. In conclusion, DCA treatment was able to re-sensitize gastric cancer cells with hypoxia-induced resistance to 5-FU through the alteration of glucose metabolism.

Quantitative measurement of organic acids in tissues from gastric cancer patients indicates increased glucose metabolism in gastric cancer.

The levels of organic acids representing metabolic pathway end products are important indicators of physiological status, and may be associated with metabolic changes in cancer. The aim of this study is to investigate the levels of organic acids in cancerous and normal tissues from gastric cancer patients and to confirm the role of metabolic alterations in gastric carcinogenesis. Organic acids in normal and cancerous tissues from forty-five patients with gastric adenocarcinoma were investigated by gas chromatography-mass spectrometry in selected ion monitoring mode as methoxime/tert-butyldimethylsilyl derivatives. We analysed the significant differences in the levels of organic acids in normal and cancer tissues and investigated the correlation of these levels in cancer tissues with clinicopathological features. The levels of Krebs cycle components, including α-ketoglutaric acid, succinic acid, fumaric acid, malic acid and oxaloacetic acid, were significantly increased in cancer tissues compared to normal tissues. In addition, the levels of glycolytic products, including pyruvic acid and lactic acid, as well as the levels of ketone bodies, including 3-hydroxybutyric acid, were also significantly increased in cancer tissues compared to normal tissues. The levels of ketone bodies in cancer tissues with differentiated histology and in intestinal-type cancer tissues were significantly increased. The organic acid profiling analysis described here may be a generally useful clinical tool for understanding the complexity of metabolic events in gastric adenocarcinoma, and organic acids may have potential as metabolic markers for the future discovery of diagnostic and therapeutic modalities.

Random Networks of Single-Walled Carbon Nanotubes Promote Mesenchymal Stem Cell’s Proliferation and Differentiation

Studies on the interaction of cells with single-walled carbon nanotubes (SWCNTs) have been receiving increasing attention owing to their potential for various cellular applications. In this report, we investigated the interactions between biological cells and nanostructured SWCNTs films and focused on how morphological structures of SWCNT films affected cellular behavior such as cell proliferation and differentiation. One directionally aligned SWCNT Langmuir-Blodgett (LB) film and random network SWCNT film were fabricated by LB and vacuum filteration methods, respectively. We demonstrate that our SWCNT LB and network film based scaffolds do not show any cytotoxicity, while on the other hand, these scaffolds promote differentiation property of rat mesenchymal stem cells (rMSCs) when compared with that on conventional tissue culture polystyrene substrates. Especially, the SWCNT network film with average thickness and roughness values of 95 ± 5 and 9.81 nm, respectively, demonstrated faster growth rate and higher cell thickness for rMSCs. These results suggest that systematic manipulation of the thickness, roughness, and directional alignment of SWCNT films would provide the convenient strategy for controlling the growth and maintenance of the differentiation property of stem cells. The SWCNT film could be an alternative culture substrate for various stem cells, which often require close control of the growth and differentiation properties.