[show abstract][hide abstract] ABSTRACT: The two interferon-γ release assays such as QuantiFERON-TB Gold / In-Tube (QFT-TB) and T-SPOT.TB-are useful tools for the rapid diagnosis of tuberculosis (TB) but can yield indeterminate test results (ITRs). While some studies have identified risk factors for ITRs in the QFT-TB test, there have been few such studies for the T-SPOT.TB test. The aim of this study was to investigate the risk factors associated with ITRs in the T-SPOT.TB test.
From April 2008 to August 2010, all patients with suspected extrapulmonary tuberculosis (E-TB) were enrolled in a tertiary hospital in Korea. ITR was defined as < 20 spots in the positive control well or > 10 spots in the negative control well.
Out of a total of 368 patients, 32 (8.7%, 95% CI, 6.0% to 11.7%) had ITRs in their T-SPOT.TB tests. The ITRs were due to a low mitogen response in 13 (40.6%) patients and to a high nil response in the other 19 (59.4%) patients. Statistical analysis revealed that old age, underlying diseases, immunosuppressive treatment, lymphopenia, and clinical manifestations of E-TB were not significantly associated with ITRs.
Indeterminate results in the T-SPOT.TB test are not affected by age, underlying disease, immunosuppressive treatment, lymphopenia, or clinical manifestations of E-TB, which are known risk factors for indeterminate results in the QFT-TB test.
[show abstract][hide abstract] ABSTRACT: Forty-three patients with miliary tuberculosis (TB) were evaluated for diagnostic usefulness of ELISPOT assay. Among non-invasive rapid tests available within 3-5 days, ELISPOT had the highest sensitivity (93%), compared with acid-fast bacilli stain (sputum 32% and bronchoalveolar lavage 7%), M.TB-PCR (sputum 53% and bronchoalveolar lavage 36%), and tuberculin skin test (22%). In comparison to 44 patients with lymph node TB, the sensitivity of the ELISPOT assay in patients with miliary TB (93%) was as high as in those with lymph node TB (95%, P = 0.63), whereas the sensitivity of the tuberculin skin test was substantially lower in patients with miliary TB (22%) than in those with lymph node TB (73%, P <.001).
[show abstract][hide abstract] ABSTRACT: Uncontrolled oxidative stress impairs bone formation and induces age-related bone loss in humans. The FoxO family is widely accepted to play an important role in protecting diverse cells from reactive oxygen species (ROS). Activation of FoxO1, the main FoxO in bone, stimulates proliferation and differentiation as well as inhibits apoptosis of osteoblast lineage cells. Despite the important role of FoxO1, little is known about how FoxO1 expression in bone is regulated. Meanwhile, several recent studies reported that microRNAs (miRNAs) could play a role in osteoblast differentiation and bone formation by targeting various transcriptional factors. Here, we identified one additional crucial miRNA, miR-182, which regulates osteoblastogenesis by repressing FoxO1 and thereby negatively affecting osteogenesis. Overexpression of miR-182 in osteoblast lineage cells increased cell apoptosis and inhibited osteoblast differentiation, whereas in vivo overexpression of miR-182 in zebrafish impaired bone formation. From in silico analysis and validation experiments, FoxO1 was identified as the target of miR-182, and restoration of FoxO1 expression in miR-182-overexpressing osteoblasts rescued them from the inhibitory effects of miR-182. These results indicate that miR-182 functions as a FoxO1 inhibitor to antagonize osteoblast proliferation and differentiation, with a subsequent negative effect on osteogenesis. To treat bone aging, an antisense approach targeting miR-182 could be of therapeutic value.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 03/2012; 27(8):1669-79. · 6.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine(-) and bromodeoxyuridine [BrdU](+)) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ(0) cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction.
PLoS ONE 01/2012; 7(3):e32778. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The forkhead box C2 (Foxc2) protein, a member of the forkhead/winged helix transcription factor family, plays an important role in regulation of metabolism, arterial specification, and vascular sprouting. Foxc2-null mutants die prenatally or perinatally, and they exhibit hypoplasia of the vertebrae and insufficient chondrification or ossification of medial structures. However, the role of Foxc2 in osteoblastogenesis is not yet fully understood. According to the degree of differentiation of osteoblasts, we found that Foxc2 expression was gradually increased and dose-dependently up-regulated by well-known bone anabolic agents, such as hPTH(1-34) and BMP2. In ex vivo mouse calvarial organ culture, a significant reduction of the basal expression of Foxc2 induced by siFoxc2 remarkably suppressed cell proliferation and differentiation and induced cell death. Knockdown of Foxc2 expression using siFoxc2 in both MC3T3-E1 and primary mouse calvarial cells also resulted in a significant suppression of proliferation and differentiation, and induced cell death, supporting the ex vivo observations. In addition, the resistance to apoptosis induced by serum deprivation and phosphorylation of both Akt and ERK was significantly reduced after siFoxc2 treatment. Conversely, overexpression of Foxc2 increased the proliferation of MC3T3-E1 and primary mouse calvarial cells. Furthermore, we found that Foxc2 enhanced the expression of integrin β1, an important modulator of osteoblastogenesis, by direct binding to a Forkhead-binding element in its promoter. Taken together, these results indicate that Foxc2 plays an important role in osteoblastogenesis by promoting osteoblast proliferation, survival and differentiation through up-regulation of integrin β1 in response to stimuli which induce bone formation.
[show abstract][hide abstract] ABSTRACT: The single photon emission computed tomography (SPECT) based on pixelated Cadmium Telluride (CdTe) detector has been promising for high resolution small animal imaging. For the feasibility of our ultra-high resolution SPECT system with CdTe, we compared the quality of the reconstructed image of our SPECT system to that of a conventional small animal SPECT system with NaI(Tl) detector. The pixel size of CdTe detector was 0.35 mm × 0.35 mm which was available for a real detector (PID350, AJAT, Finland). The detector size was 44.8 × 44.8 mm with 128 × 128 pixels and the thickness of this detector was 1 mm. The intrinsic resolution of CdTe detector and that of NaI(Tl) detector were 0.35 mm and 2.3 mm, respectively. The height parallel hole collimator made of lead was 25 mm and that of a septum was 0.2 mm. The shape of that parallel hole collimator and its radius were hexagonal and 0.5 mm, respectively. A SPECT liver scan was simulated for both SPECT with CdTe and conventional SPECT system using voxelized MOBY phantom. All SPECT images were obtained using 120 projection views acquired from 0° to 360° with a 3°. Slices were reconstructed using an Ordered Subsets Expectation Maximization (OS-EM) and 5 iterations with 4 subsets. We compared the quality of images for two systems in terms of spatial resolution (FWHM), sensitivity, signal-to-noise ratio (SNR), and contrast-to-noise (CNR). Our simulation results indicated that the reconstructed SPECT images obtained with CdTe detector showed higher resolution compared to those with the conventional scintillation detector. These results demonstrated that the SPECT imaging based on pixelated CdTe detector can improve the performance of SPECT system for small animal imaging.
Journal- Korean Physical Society 01/2011; 60(7). · 0.51 Impact Factor
[show abstract][hide abstract] ABSTRACT: Osteoblasts originate from mesenchymal stem cells by the coordinated activities of different signaling pathways that regulate the expression of osteoblast-specific genes. Runt-related transcription factor 2 (Runx2) is the master transcription factor for osteoblast differentiation. Despite the importance of Runx2 in the developing skeleton, how Runx2 expression is regulated remains a pivotal question. Snail, a zinc finger transcription factor, is essential for triggering epithelial-to-mesenchymal transitions (EMTs) during embryonic development and tumor progression. Here, we report that Runx2 expression is significantly up- or down-regulated relative to Snail expression. We demonstrate that Snail binds to the Runx2 promoter and that repression of Runx2 transcription by Snail is dependent on specific E-box sequence within the promoter. With antisense morpholino oligonucleotide (MO)-mediated knockdown of Snail expression in zebrafish, we observed alterations in osteogenic potential. These results indicate that Snail plays a crucial role in osteogenic differentiation by acting as a direct Runx2 repressor.
Bone 03/2010; 46(6):1498-507. · 3.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diagnosing abdominal tuberculosis (TB) remains a challenge. A recently developed RD-1 gene-based assay for diagnosing tuberculosis infection shows promising results. We evaluated the diagnostic usefulness of this assay compared with conventional tests in patients with suspected abdominal TB in clinical practice.
All patients with suspected abdominal TB were prospectively enrolled in a tertiary hospital during a 1-year period. In addition to the conventional tests for diagnosing TB, the IFN-gamma-producing T-cell response to ESAT-6 and CFP-10 by ELISPOT assay using peripheral blood mononuclear cells (PBMC) and peritoneal fluid mononuclear cells (PF-MC) were performed.
Forty eight patients with suspected abdominal TB were enrolled. Of these patients, 30 (63%) were classified as abdominal TB including 14 TB peritonitis (12 confirmed + 1 probable + 1 possible), 6 abdominal TB lymphadenitis (3 confirmed + 3 probable), 4 hepatic TB (3 confirmed + 1 possible), 2 intestinal TB (1 confirmed + 1 probable), 3 renal TB (1 confirmed + 2 probable), and 1 pancreatic TB (1 confirmed). Eighteen (38%) were classified as not TB. ELISPOT assay using PBMC was performed on samples from all 48 subjects. The sensitivity and specificity of the PBMC ELISPOT assay were 89% (95% CI, 71-98%) and 78% (95% CI, 52-94%), respectively. In the 11 patients in whom PF-MC ELISPOT assay was performed, it was positive in 5 of 6 patients with TB peritonitis, and negative in all 5 patients with not TB.
The ELISPOT assay using PBMC and PF-MC is a useful adjunct to the current tests for diagnosing abdominal TB.
The Journal of infection 09/2009; 59(6):409-15. · 4.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fibroblast growth factor 2 (FGF2), the potent bone anabolic agent, regulates the bone development, as well as the growth, remodeling and healing of the fracture. The intracellular signaling of FGF2 leads to activation of genes involved in cell proliferation, migration, differentiation and survival. However, little is known about FGF2-regulated proteins in the osteoblasts. Therefore, in this study, protein profiling in FGF2-treated MC3T3-E1 preosteoblast cells was evaluated using proteomic technologies. Six proteins including asparaginyl-tRNA synthetase (NARS), eukaryotic translation termination factor 1 (ETF1), GDP-forming succinyl-CoA synthetase (SUCLG2), heat shock protein 84 (HSP 84), sorting nexin 9 (SNX9) and alpha glucosidase 2alpha neutral subunit (GANAB) were increased more than 3-fold after the FGF2 treatment. Also, two proteins including beta-tropomyosin and tropomyosin 2 were decreased to 2-folds. Among these proteins, asparaginyl-tRNA synthetase (NARS), a member of aminoacyl-tRNA synthetases (AARS), was strikingly up-regulated more than 900-fold. The overexpression of NARS significantly increased the proliferation of both the MC3T3-E1 and the primary mouse calvarial cells. In contrast, significant reduction of the basal expression of NARS by siNARS remarkably suppressed the proliferation and induced the death of cell. After the siNARS treatment, the resistance to apoptosis induced by serum deprivation was also significantly reduced. The level of p-Akt was also reduced and the activity of caspase 3 significantly enhanced. In addition, NARS-induced protection against apoptosis was abolished by the treatment of PI3K inhibitors, wortmannin and LY294002. In conclusion, we suggest that NARS is one of the important mediators of FGF2 induced survival signaling in osteoblasts through the activation of PI3K/Akt survival pathway.
Bone 08/2009; 45(5):994-1003. · 3.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: The forkhead box C2 (Foxc2) protein is a member of the family of winged helix/forkhead transcription factors. Foxc2-deficient mice display defective formation of the aortic arches, multiple craniofacial bones, and vertebral columns. To investigate the role of Foxc2 in osteoblast differentiation, DNA containing Foxc2 was transfected into the developing cranial suture mesenchymal cells by electroporation. Compared to the controls, alkaline phosphatase (ALP) and bone sialoprotein were expressed strongly in suture mesenchymal cells in the Foxc2 overexpressed calvaria. After Foxc2-siRNA transfection, ALP staining was rarely observed in the suture mesenchyme and adjacent parietal bone of the calvaria. Meanwhile, overexpression of Foxc2 increased protein levels of β-catenin and stimulated TCF/LEF transcriptional activity. The protein kinase A inhibitor H-89 suppressed Foxc2-mediated increases in TCF/LEF transcriptional activity (−40%, P < 0.01). In conclusion, our results demonstrated that Foxc2 stimulated osteoblast differentiation of mesenchymal cells and preosteoblasts. Activation of canonical Wnt-β-catenin signals might be involved in the Foxc2-mediated stimulation of osteoblast differentiation.
Biochemical and Biophysical Research Communications. 01/2009;
[show abstract][hide abstract] ABSTRACT: Brain ischemia activates Ca(2+)-dependent synaptic vesicle exocytosis. The synaptosomal-associated protein 25 (SNAP-25) and syntaxin proteins, located on presynaptic terminals, are components of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex and play a key role in regulating exocytosis. Changes in the expression of SNAREs could affect SNARE complex formation, fusion of vesicles with the presynaptic membrane, and release of neurotransmitters through exocytosis. To investigate the relationship of glucose/oxygen deprivation (GOD)/reperfusion-induced neuronal damage and alteration of presynaptic function, we examined the expression of SNAREs and complexin during GOD and reperfusion using organotypic hippocampal slice cultures. Microtubule-associated protein 2 (MAP-2) staining and transmission electron microscopy showed that neuronal damage increased in a time-dependent manner and both types of neuronal death can occur at different times during GOD and reperfusion. The immunoreactivity of SNAREs such as SNAP-25, vesicle-associated membrane protein and syntaxin and complexin increased in pyramidal cell bodies in the CA1 and CA3 areas in a time-dependent manner following reperfusion. Our data suggest that alteration of presynaptic function may play a partial role in delayed neuronal death during GOD and reperfusion in organotypic hippocampal slice cultures.
[show abstract][hide abstract] ABSTRACT: Enterobacter spp., Serratia marcescens, Citrobacter freundii, and Morganella morganii are characterized by chromosomally encoded AmpC beta-lactamases and possess the ability to develop resistance upon exposure to broad-spectrum cephalosporins. To determine the incidences of the emergence of resistance during antimicrobial therapy for infections caused by these organisms and the effect of the emergence of resistance on patient outcomes, all patients who were admitted to the Asan Medical Center (Seoul, Republic of Korea) from January 2005 to June 2006 and whose clinical specimens yielded Enterobacter spp., S. marcescens, C. freundii, or M. morganii were monitored prospectively. The main end point was the emergence of resistance during antimicrobial therapy. A total of 732 patients with infections were included for analysis. The overall incidence of the emergence of antimicrobial resistance during antimicrobial therapy was 1.9% (14/732). Resistance to broad-spectrum cephalosporins, cefepime, extended-spectrum penicillin, carbapenem, fluoroquinolones, and aminoglycosides emerged during treatment in 5.0% (11/218), 0% (0/20), 2.0% (2/100), 0% (0/226), 0% (0/153), and 1.1% (1/89) of patients, respectively. The emergence of resistance to broad-spectrum cephalosporins occurred more often in Enterobacter spp. (8.3%, 10/121) than in C. freundii (2.6%, 1/39), S. marcescens (0%, 0/37), or M. morganii (0%, 0/21). Biliary tract infection associated with malignant bile duct invasion was significantly associated with the emergence of resistance to broad-spectrum cephalosporins (P = 0.024 at a significance level of 0.042, by use of the Bonferroni correction). Only 1 of the 14 patients whose isolates developed resistance during antimicrobial therapy died. The emergence of resistance was more frequently associated with broad-spectrum cephalosporins than with the other antimicrobial agents tested, especially in Enterobacter spp. However, the emergence of resistance was associated with a low risk of mortality.
Antimicrobial Agents and Chemotherapy 04/2008; 52(3):995-1000. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: The FIGNL1 gene was proven to be a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). In this in vitro study, the AAA proteins inhibited osteoblast proliferation and stimulated osteoblast differentiation. We showed that FIGNL1 may play some regulatory role in osteoblastogenesis.
The fidgetin-like 1 (FIGNL1) gene encodes a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). Although the FIGNL1 protein localizes to both the nucleus and cytoplasm, the function of FIGNL1 remains unknown. In a previous study, we identified several genes that mediate the anabolic effects of basic fibroblast growth factor (bFGF) on bone by using microarray data. FIGNL1 was one of the genes that downregulated >2-fold in MC3T3-E1 cells after treatment with bFGF. Therefore, this study was aimed to identify and confirm the function of FIGNL1 on osteoblastogenesis.
We examined the effect of the FIGNL1 gene on proliferation, differentiation, and apoptosis in mouse osteoblast cells (MC3T3-E1 and mouse primary calvarial cells) using flow cytometry, RT-PCR, cell proliferation assay, and cell death assay. MC3T3-E1 cells and mouse calvarial cells were transfected with small interfering RNA (siRNA) directed against the FIGNL1 or nontargeting control siRNA and examined by cell proliferation and cell death assays. Also, FIGNL1 was fused to enhance green fluorescent protein (EGFP), and the EGFP-fused protein was transiently expressed in MC3T3-E1 cells.
Reduced expression of FIGNL1 by bFGF and TGF-beta1 treatment was verified by RT-PCR analysis. Overexpression of FIGNL1 reduced the proliferation of MC3T3-E1 and calvarial cells, more than the mock transfected control cells did. In contrast, siFIGNL1 transfection significantly increased the proliferation of osteoblasts, whereas overexpression of FIGNL1 did not seem to alter apoptosis in osteoblasts. Meanwhile, overexpression of FIGNL1 enhanced the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in osteoblasts. In contrast, siFIGNL1 decreased the expression of ALP and OCN. A pEGFP-FIGNL1 transfected into MCT3-E1 cells had an initially ubiquitous distribution and rapidly translocated to the nucleus 1 h after bFGF treatment.
From these results, we proposed that FIGNL1, a subfamily member of the AAA family of proteins, might play some regulatory role in osteoblast proliferation and differentiation. Further analyses of FIGNL1 will be needed to better delineate the mechanisms contributing to the inhibition of proliferation and stimulation of osteoblast differentiation.
Journal of Bone and Mineral Research 06/2007; 22(6):889-96. · 6.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: We compared in vitro activities of carbapenems against 264 penicillin-resistant Streptococcus pneumoniae (PRSP) isolates. The MIC(50)/MIC(90) (microg/mL) values of imipenem, meropenem, ertapenem, and panipenem were 1/1, 0.25/0.25, 0.25/0.5, and 0.125/0.25, respectively. The susceptibility rates to imipenem, meropenem, and ertapenem were 0%, 85.2%, and 99.6%, respectively. Compared with imipenem and meropenem, ertapenem and panipenem had better in vitro activities against PRSP.
[show abstract][hide abstract] ABSTRACT: The DesVIII is an auxiliary protein which enhances the transfer of TDP-d-desosamine catalyzed by DesVII glycosyltransferase in the biosynthesis of macrolide antibiotics, neomethymycin, methymycin and pikromycin, in Streptomyces venezuelae ATCC 15439. Homologues of the desVIII gene are present in a number of aminosugar-containing antibiotic biosynthetic gene clusters including eryCII from the erythromycin producer Saccharopolyspora erythraea, oleP1 from the oleandomycin producer Streptomyces antibioticus, dnrQ from the doxorubicin producer Streptomyces peucetius, and tylMIII from the tylosin producer Streptomyces fradiae. In order to gain further insight into the function of these DesVIII homologues, interspecies complementation experiments were carried out by expressing each gene in a desVIII deletion mutant strain of S. venezuelae. Complementation by expressing EryCII, OleP1, and DnrQ in this mutant strain restored the production of glycosylated macrolides to an approximate level of 66%, 26% and 26%, respectively, compared to self-complementation by DesVIII. However, expression of TylMIII did not restore the antibiotic production. These results suggest that the DesVIII homologues (except for TylMIII) can functionally replace the native DesVIII for glycosylation to proceed in vivo and their functions are similar in acting as glycosyltransferase auxiliary proteins. The requirement of glycosyltransferase auxiliary protein seems to be more widespread in polyketide biosynthetic pathways than previously known.
[show abstract][hide abstract] ABSTRACT: Dihydrotestosterone (DHT) is the most potent male hormone that causes androgenetic alopecia. The type II 5alpha-reductase is an enzyme that catalyzes the conversion of testosterone (T) to DHT, therefore it can be expected that specific inhibitors for type II 5alpha-reductase may improve the pathophysiologic status of androgenetic alopecia. In this study, we attempted to establish the reliable and convenient screening model for type II 5alpha-reductase inhibitors. After transfection of human cDNA for type II 5alpha-reductase into HEK293 cells, the type II 5alpha-reductase over-expressing stable cells were selected by G418 treatment. RT-PCR and Western blot analyses confirmed that type II 5alpha-reductase gene was expressed in the stable cells. In in vitro enzymatic assay, 10 microg of stable cell extract completely converted 1 microCi (approximately 0.015 nmol) of T into DHT. The type II 5alpha-reductase activity was inhibited by finasteride in a dose-dependent manner, confirming the reliability of screening system. In cell culture condition, 2 x 10(5) of stable cells completely converted all the input T (approximately 0.03 nmol) into DHT by 4h incubation, demonstrating that the stable cell line can be used as a cell-based assay system. Using this system, we selected the extracts of Curcumae longae rhizoma and Mori ramulus as the potential inhibitors for type II 5alpha-reductase. These results demonstrate that the type II 5alpha-reductase over-expressing stable cell line is a convenient and reliable model for screening and evaluation of inhibitors.
The Journal of Steroid Biochemistry and Molecular Biology 01/2007; 107(3-5):245-52. · 3.98 Impact Factor