Severe graft-versus-host disease (GVHD) is an often lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). The safety of clinical-grade mesenchymal stem cells (MSCs) has been validated, but mixed results have been obtained due to heterogeneity of the MSCs. In this phase I study, the safety of bone marrow-derived homogeneous clonal MSCs (cMSCs) isolated by a new subfractionation culturing method was evaluated. cMSCs were produced in a GMP facility and intravenously administered to patients who had refractory GVHD to standard treatment resulting after allogeneic HSCT for hematologic malignancies. After administration of a single dose (1x10(6) cells/kg), 11 patients were evaluated for cMSC treatment safety and efficacy. During the trial, nine patients had 85 total adverse events and the rate of serious adverse events was 27.3% (3/11 patients). The only one adverse drug reaction related to cMSC administration was grade 2 myalgia in one patient. Treatment response was observed in four patients: one with acute GVHD (partial response) and three with chronic GVHD. The other chronic patients maintained stable disease during the observation period. This study demonstrates single cMSC infusion to have an acceptable safety profile and promising efficacy, suggesting that we can proceed with the next stage of the clinical trial.
Bone Marrow ; Drug-Related Side Effects and Adverse Reactions ; Graft vs Host Disease* ; Hematologic Neoplasms ; Hematopoietic Stem Cell Transplantation ; Humans ; Mesenchymal Stromal Cells* ; Myalgia ; Population Characteristics

Mesenchymal stem cells (MSCs) in the bone marrow and other somatic tissues reside in an environment with relative low oxygen tension. Cobalt chloride (CoCl2) can mimic hypoxic conditions through transcriptional changes of some genes including hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF). This study evaluated the potential role of CoCl2 preconditioning on multi-lineage differentiation of C3H/10T1/2, a murine MSC line to understand its possible molecular mechanisms in vitro. CoCl2 treatment of MSCs markedly increased HIF-1alpha and VEGF mRNA, and protein expression of HIF-1alpha. Temporary preconditioning of MSCs with CoCl2 induced up-regulation of osteogenic markers including alkaline phosphatase, osteocalcin, and type I collagen during osteogenic differentiation, followed by enhanced mineralization. CoCl2 also increased chondrogenic markers including aggrecan, sox9, and type II collagen, and promoted chondrocyte differentiation. CoCl2 suppressed the expression of adipogenic markers including PPARgamma, aP2, and C/EBPalpha, and inhibited adipogenesis. Temporary preconditioning with CoCl2 could affect the multi-lineage differentiation of MSCs.
Adipogenesis ; Aggrecans ; Alkaline Phosphatase ; Anoxia ; Bone Marrow ; Chondrocytes ; Cobalt ; Collagen Type I ; Collagen Type II ; Mesenchymal Stromal Cells* ; Osteocalcin ; Oxygen ; PPAR gamma ; RNA, Messenger ; Up-Regulation ; Vascular Endothelial Growth Factor A

Adult hippocampal dentate granule neurons are generated from neural stem cells (NSCs) in the mammalian brain, and the fate specification of adult NSCs is precisely controlled by the local niches and environment, such as the subventricular zone (SVZ), dentate gyrus (DG), and Toll-like receptors (TLRs). Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid in green tea that has neuroprotective activities, but there is no clear understanding of the role of EGCG in adult neurogenesis in the DG after neuroinflammation. Here, we investigate the effect and the mechanism of EGCG on adult neurogenesis impaired by lipopolysaccharides (LPS). LPS-induced neuroinflammation inhibited adult neurogenesis by suppressing the proliferation and differentiation of neural stem cells in the DG, which was indicated by the decreased number of Bromodeoxyuridine (BrdU)-, Doublecortin (DCX)- and Neuronal Nuclei (NeuN)-positive cells. In addition, microglia were recruited with activatingTLR4-NF-kappaB signaling in the adult hippocampus by LPS injection. Treating LPS-injured mice with EGCG restored the proliferation and differentiation of NSCs in the DG, which were decreased by LPS, and EGCG treatment also ameliorated the apoptosis of NSCs. Moreover, pro-inflammatory cytokine production induced by LPS was attenuated by EGCG treatment through modulating the TLR4-NF-kappaB pathway. These results illustrate that EGCG has a beneficial effect on impaired adult neurogenesis caused by LPSinduced neuroinflammation, and it may be applicable as a therapeutic agent against neurodegenerative disorders caused by inflammation.
Adult* ; Animals ; Apoptosis ; Brain ; Bromodeoxyuridine ; Dentate Gyrus ; Hippocampus ; Humans ; Inflammation ; Lipopolysaccharides ; Mice* ; Microglia ; Neural Stem Cells ; Neurodegenerative Diseases ; Neurogenesis* ; Neurons ; Tea ; Toll-Like Receptors

To conduct a kinetic study of paraquat (PQ), we investigated 9 patients with acute PQ intoxication. All of them ingested more than 20 ml of undiluted PQ herbicide to commit suicide and arrived at our hospital early, not later than 7 h after PQ ingestion. The urine dithionite test for PQ in all of the nine patients was strongly positive at emergency room. Blood samples were obtained every 30 min for the first 2~3 h and then every 1 or 2 h, as long as the clinical progression was stable among the patients for 30 h after PQ ingestion. The area under the plasma concentration-time curve (AUCinf), which was extrapolated to infinity, was calculated using the trapezoidal rule. Toxicokinetic parameters, such as the terminal elimination half-life, apparent oral clearance, and apparent volume of distribution (Vd/F) were calculated. The maximum PQ concentration (Cmax) and the time to reach maximum PQ concentration (Tmax) were also obtained. Plasma PQ concentrations in nine patients were well described by a bi-exponential curve with a mean terminal elimination half-life of 13.1+/-6.8 h. Cmax and AUCinf were 20.8+/-25.7 mg/l and 172.5+/-160.3 h.mg/l, respectively. Apparent volume of distribution and apparent oral clearance were 50.9+/-61.3 l/kg and 173.4+/-111.2 l/h, respectively. There were a significant correlation (r =0.84; p<0.05) between the PQ amount ingested and Cmax. AUCinf also showed a significant correlation (r =0.83; p<0.05) with the PQ amount ingested. These correlations provide evidence that PQ has dose-linear toxicokinetic characteristics.
Dithionite ; Eating ; Emergency Service, Hospital ; Half-Life ; Humans ; Paraquat* ; Pharmacokinetics* ; Plasma ; Poisoning* ; Suicide

Ion channels in carcinoma and their roles in cell proliferation are drawing attention. Intracellular Ca2+ ([Ca2+]i)-dependent signaling affects the fate of cancer cells. Here we investigate the role of Ca(2+)-activated K+ channel (SK4) in head and neck squamous cell carcinoma cells (HNSCCs) of different cell lines; SNU-1076, OSC-19 and HN5. Treatment with 1 microM ionomycin induced cell death in all the three cell lines. Whole-cell patch clamp study suggested common expressions of Ca(2+)-activated Cl- channels (Ano-1) and Ca(2+)-activated nonselective cation channels (CAN). 1-EBIO, an activator of SK4, induced outward K+ current (ISK4) in SNU-1076 and OSC-19. In HN5, ISK4 was not observed or negligible. The 1-EBIO-induced current was abolished by TRAM-34, a selective SK4 blocker. Interestingly, the ionomycin-induced cell death was effectively prevented by 1-EBIO in SNU-1076 and OSC-19, and the rescue effect was annihilated by combined TRAM-34. Consistent with the lower level of ISK4, the rescue by 1-EBIO was least effective in HN5. The results newly demonstrate the role of SK4 in the fate of HNSCCs under the Ca2+ overloaded condition. Pharmacological modulation of SK4 might provide an intriguing novel tool for the anti-cancer strategy in HNSCC.
Carcinoma, Squamous Cell* ; Cell Death* ; Cell Line ; Cell Proliferation ; Head* ; Ion Channels ; Ionomycin ; Neck* ; Neoplasms, Squamous Cell

Acetaminophen (APAP) overdose is one of the most common causes of acute liver failure. The study aimed to investigate the protective effect of carnosic acid (CA) on APAP-induced acute hepatotoxicity and its underlying mechanism in mice. To induce hepatotoxicity, APAP solution (400 mg/kg) was administered into mice by intraperitoneal injection. Histological analysis revealed that CA treatment significantly ameliorated APAP-induced hepatic necrosis. The levels of both alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum were reduced by CA treatment. Moreover, CA treatment significantly inhibited APAP-induced hepatocytes necrosis and lactate dehydrogenase (LDH) releasing. Western blot analysis showed that CA abrogated APAP-induced cleaved caspase-3, Bax and phosphorylated JNK protein expression. Further results showed that CA treatment markedly inhibited APAP-induced pro-inflammatory cytokines TNF-alpha, IL-1beta, IL-6 and MCP-1 mRNA expression and the levels of phosphorylated IkappaBalpha and p65 protein in the liver. In addition, CA treatment reduced APAP- induced hepatic malondialdehyde (MDA) contents and reactive oxygen species (ROS) accumulation. Conversely, hepatic glutathione (GSH) level was increased by administration of CA in APAP-treated mice. Mechanistically, CA facilitated Nrf2 translocation into nuclear through blocking the interaction between Nrf2 and Keap1, which, in turn, upregulated anti-oxidant genes mRNA expression. Taken together, our results indicate that CA facilitates Nrf2 nuclear translocation, causing induction of Nrf2-dependent genes, which contributes to protection from acetaminophen hepatotoxicity.
Acetaminophen ; Alanine Transaminase ; Animals ; Aspartate Aminotransferases ; Blotting, Western ; Caspase 3 ; Cytokines ; Glutathione ; Hepatocytes ; Injections, Intraperitoneal ; Interleukin-6 ; L-Lactate Dehydrogenase ; Liver ; Liver Failure, Acute ; Malondialdehyde ; Mice* ; Necrosis ; Reactive Oxygen Species ; RNA, Messenger ; Tumor Necrosis Factor-alpha

Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the Na(+)-K(+)-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain (3.0 micromol/L) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 (3.0 micromol/L), an inhibitor for reverse mode of Na(+)-Ca(2+) exchangers (NCX), but did not by L-type Ca2+ channel blocker nifedipine (1.0 micromol/L) or protein kinase A (PKA) selective inhibitor H-89 (3.0 micromol/L). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline (100.0 micromol/L), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP (0.5 micromol/L) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 (30 micromol/L), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.
8-Bromo Cyclic Adenosine Monophosphate ; Adenosine ; Cardiomegaly ; Colforsin ; Cyclic AMP-Dependent Protein Kinases ; Endothelin-1* ; Heart Diseases ; Nifedipine ; Ouabain* ; Phosphotransferases ; Protein Kinases

Damage in the periphery or spinal cord induces maladaptive plastic changes along the somatosensory nervous system from the periphery to the cortex, often leading to chronic pain. Although the role of neural circuit remodeling and structural synaptic plasticity in the 'pain matrix' cortices in chronic pain has been thought as a secondary epiphenomenon to altered nociceptive signaling in the spinal cord, progress in whole brain imaging studies on human patients and animal models has suggested a possibility that plastic changes in cortical neural circuits may actively contribute to chronic pain symptoms. Furthermore, recent development in two-photon microscopy and fluorescence labeling techniques have enabled us to longitudinally trace the structural and functional changes in local circuits, single neurons and even individual synapses in the brain of living animals. These technical advances has started to reveal that cortical structural remodeling following tissue or nerve damage could rapidly occur within days, which are temporally correlated with functional plasticity of cortical circuits as well as the development and maintenance of chronic pain behavior, thereby modifying the previous concept that it takes much longer periods (e.g. months or years). In this review, we discuss the relation of neural circuit plasticity in the 'pain matrix' cortices, such as the anterior cingulate cortex, prefrontal cortex and primary somatosensory cortex, with chronic pain. We also introduce how to apply long-term in vivo two-photon imaging approaches for the study of pathophysiological mechanisms of chronic pain.
Animals ; Brain ; Chronic Pain* ; Fluorescence ; Gyrus Cinguli ; Humans ; Microscopy ; Models, Animal ; Nervous System ; Neuroimaging ; Neurons ; Plastics* ; Prefrontal Cortex ; Somatosensory Cortex ; Spinal Cord ; Synapses

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.
Humans ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Pertussis Toxin ; Phenotype* ; Phosphorylation ; Protein Kinase C ; Proto-Oncogene Proteins c-raf ; Thrombin

We investigated the combined moisturizing effect of liposomal serine and a cosmeceutical base selected in this study. Serine is a major amino acid consisting of natural moisturizing factors and keratin, and the hydroxyl group of serine can actively interact with water molecules. Therefore, we hypothesized that serine efficiently delivered to the stratum corneum (SC) of the skin would enhance the moisturizing capability of the skin. We prepared four different cosmeceutical bases (hydrogel, oil-in-water (O/W) essence, O/W cream, and water-in-oil (W/O) cream); their moisturizing abilities were then assessed using a Corneometer(R). The hydrogel was selected as the optimum base for skin moisturization based on the area under the moisture content change-time curves (AUMCC) values used as a parameter for the water hold capacity of the skin. Liposomal serine prepared by a reverse-phase evaporation method was then incorporated in the hydrogel. The liposomal serine-incorporated hydrogel (serine level=1%) showed an approximately 1.62~1.77 times greater moisturizing effect on the skin than those of hydrogel, hydrogel with serine (1%), and hydrogel with blank liposome. However, the AUMCC values were not dependent on the level of serine in liposomal serine-loaded hydrogels. Together, the delivery of serine to the SC of the skin is a promising strategy for moisturizing the skin. This study is expected to be an important step in developing highly effective moisturizing cosmeceutical products.
Hyaluronic Acid* ; Hydrogel* ; Hydrogels* ; Liposomes ; Serine* ; Skin* ; Water