Chulhun L. Chang

Cancer‐Derived Lysophosphatidic Acid Stimulates Differentiation of Human Mesenchymal Stem Cells to Myofibroblast‐Like Cells

Lysophosphatidic acid (LPA) is enriched in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests cancer‐associated myofibroblasts play a pivotal role in tumorigenesis through secreting stromal cell‐derived factor‐1 (SDF‐1). In the present study, we demonstrate that LPA induces expression of α‐smooth muscle actin (α‐SMA), a marker for myofibroblasts, in human adipose tissue‐derived mesenchymal stem cells (hADSCs). The LPA‐induced expression of α‐SMA was completely abrogated by pretreatment of the cells with Ki16425, an antagonist of LPA receptors, or by silencing LPA1 or LPA2 isoform expression with small interference RNA (siRNA). LPA elicited phosphorylation of Smad2/3, and siRNA‐mediated depletion of endogenous Smad2/3 or adenoviral expression of Smad7, an inhibitory Smad, abrogated the LPA induced expression of α‐SMA and phosphorylation of Smad2/3. LPA‐induced secretion of transforming growth factor (TGF)‐β1 in hADSCs, and pretreatment of the cells with SB431542, a TGF‐β type I receptor kinase inhibitor, or anti‐TGF‐β1 neutralizing antibody inhibited the LPA‐induced expression of α‐SMA and phosphorylation of Smad2. Furthermore, ascites from ovarian cancer patients or conditioned medium from ovarian cancer cells induced expression of α‐SMA and phosphorylation of Smad2, and pretreatment of the cells with Ki16425 or SB431542 abrogated the expression of α‐SMA and phosphorylation of Smad2. In addition, LPA increased the expression of SDF‐1 in hADSCs, and pretreatment of the cells with Ki16425 or SB431562 attenuated the LPA‐stimulated expression of SDF‐1. These results suggest that cancer‐derived LPA stimulates differentiation of hADSCs to myofibroblast‐like cells and increases SDF‐1 expression through activating autocrine TGF‐β1‐Smad signaling pathway.

Evaluation of an Immunochromatographic Assay Kit for Rapid Identification of Mycobacterium tuberculosis Complex in Clinical Isolates

ABSTRACT We evaluated a new immunochromatographic assay (ICA) using mouse monoclonal anti-MPT64 antibody for rapid discrimination between Mycobacterium tuberculosis and nontuberculous mycobacteria in clinical isolates. A study with mycobacteria and other organisms showed excellent sensitivity (≅99%) and specificity (100%) and an appropriate detection limit (105 CFU/ml) when tested with M. tuberculosis H37Rv. This ICA can simplify the identification of M. tuberculosis in clinical laboratories.

Characterization of IncF plasmids carrying the blaCTX-M-14 gene in clinical isolates of Escherichia coli from Korea

OBJECTIVES The purpose of this study was to investigate the molecular epidemiology of CTX-M-14-producing Escherichia coli clinical isolates from Korea. METHODS A total of 138 non-duplicate E. coli clinical isolates showing reduced susceptibility or resistance to ceftazidime and/or cefotaxime were included in the study. Resistance genes, genetic environment, R plasmid size and replicon type, sequence type (ST) and XbaI-macrorestriction patterns were determined. RESULTS Among 138 isolates, 35 were found to carry the bla(CTX-M-14) gene. The ISEcp1 element was identified in the upstream region of the bla(CTX-M-14) gene in 32 isolates. The bla(CTX-M-14) gene was located on an IncF plasmid in 21 isolates, on an IncA/C plasmid in 1 isolate, on the chromosome in 8 isolates and on both the chromosome and an IncF plasmid in 5 isolates. The most prevalent ST was ST405 (n = 8), followed by ST354 (n = 4), ST38 (n = 3), ST69 (n = 3) and the intercontinental ST, ST131 (n = 3). PFGE and multilocus sequence typing experiments demonstrated no major clonal relationship among the CTX-M-14-producing isolates. CONCLUSIONS The bla(CTX-M-14) gene was probably mobilized by IncF plasmids, which can readily spread in E. coli, causing horizontal dissemination of the resistance gene in Korea.

Molecular Epidemiological Profile of Rotavirus in South Korea, July 2002 through June 2003: Emergence of G4P[6] and G9P[8] Strains

To determine the distribution of rotavirus strain genotypes in South Korea, rotavirus-positive stool specimens were collected from July 2002 through June 2003 at 8 hospitals in the Korean Rotavirus Strain Surveillance Network, and they were genotyped by means of reverse-transcription polymerase chain reaction. The globally uncommon G4P[6] type was the most prevalent type identified among strains (27% of strains), the newly emerging G9P[8] strain accounted for 11% of strains, and the globally common genotypes (i.e., G1P[8], G2P[4], G3P[8], and G4P[8]) constituted 55% of the strains characterized. Ninety percent of G4P[6] strains were detected in specimens obtained from neonates. Common genotypes were responsible for the rotavirus epidemic that began in January 2003 and ended in May 2003; however, an early peak in infections with the G4P[6] strain occurred from August through October 2002, and infections with this strain were detected throughout the remaining study period. G4P[6] strains were most commonly identified at 6 urban health care centers, but they were absent from 2 rural health care centers. The newly emerging strain G9P[8] represented a relatively greater proportion of strains identified at a hospital in the central region of Korea and at 2 hospitals in the southern region. The identification of novel rotavirus genotypes in this laboratory-based surveillance study underscores the importance to public health of continued strain surveillance among children for whom prevention of rotavirus infection by vaccination might be considered.

Species Distribution and Susceptibility to Azole Antifungals of Candida Bloodstream Isolates from Eight University Hospitals in Korea

Purpose The incidence of Candida bloodstream infections (BSI) has increased over the past two decades. The rank order of occurrence and the susceptibility to antifungals of the various Candida species causing BSI are important factors driving the establishment of empirical treatment protocols; however, very limited multi-institutional data are available on Candida bloodstream isolates in Korea. Materials and Methods We investigated the susceptibility to azole antifungals and species distribution of 143 Candida bloodstream isolates recovered from eight university hospitals over a six-month period. Minimal inhibitory concentrations (MICs) of fluconazole, itraconazole, and voriconazole for each isolate were determined by the broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI). Results The Candida species recovered most frequently from the blood cultures was C. albicans (49%), followed by C. parapsilosis (22%), C. tropicalis (14%), and C. glabrata (11%). The MIC ranges for the Candida isolates were 0.125 to 64 µg/mL for fluconazole, 0.03 to 2 µg/mL for itraconazole, and 0.03 to 1 µg/mL for voriconazole. Overall, resistance to fluconazole was found in only 2% of the Candida isolates (3/143), while the dose-dependent susceptibility was found in 6% (8/143). The resistance and dose-dependent susceptibility of itraconazole were found in 4% (6/143) and 14% (20/143) of the isolates, respectively. All bloodstream isolates were susceptible to voriconazole (MIC, ≤ 1 µg/mL). Conclusion Our findings show that C. albicans is the most common cause of Candida-related BSI, followed by C. parapsilosis, and that the rates of resistance to azole antifungals are still low among bloodstream isolates in Korea.

Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea

The katG, inhA and ahpC genes, in 71 isoniazid (INH)-resistant and 26 INH-susceptible Mycobacterium tuberculosis isolates, from South Korea were examined by sequencing and MspI restriction enzyme analysis. Mutations in the katG 315 alone, katG 315 plus inhA, katG 315 plus ahpC, katG 309 alone, katG 309 plus inhA, inhA alone, and ahpC alone, were detected in 54.9, 2.8, 1.4, 1.4, 1.4, 19.7, and 5.6% of the 71 INH-resistant isolates, respectively. There was no statistically significant difference (p > 0.05) in the frequencies of these mutations for the INH-monoresistant compared with the multidrug-resistant isolates. Mutations in the katG codon 315 were associated with the high-level of INH resistance (MIC, >1 microg/ml), whereas the mutation in the inhA promoter region was associated with the low-level of INH resistance (MIC, >0.2 to 1 microg/ml). The previously undescribed GGT-->GAT (Gly-->Asp) mutation in the katG codon 309 was found in two rifampin, including-multidrug-resistant isolates, but we cannot assess if this is predictive of INH resistance. The sensitivity and specificity of molecular analysis of the katG codon 315 and/or the inhA promoter region were 80.3 and 100%, respectively. Therefore, mutations in these regions are highly predictive of INH resistance in South Korea.

Lysophosphatidic acid in malignant ascites stimulates migration of human mesenchymal stem cells

Lysophosphatidic acid (LPA) is elevated in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests a pivotal role of mesenchymal stem cells (MSCs) or stromal cells in tumorigenesis. In the present study, we demonstrated that ascites from ovarian cancer patients and LPA increased migration of human MSCs. The migration of MSCs induced by LPA and malignant ascites was completely abrogated by pretreatment with Ki16425, an antagonist of LPA receptors, and by silencing of endogenous LPA1, but not LPA2, with small interference RNA, suggesting a key role of LPA played in the malignant ascites‐induced migration. LPA induced activation of ERK through pertussis toxin‐sensitive manner, and pretreatment of MSCs with U0126, a MEK inhibitor, or pertussis toxin attenuated the LPA‐induced migration. Moreover, LPA induced activation of RhoA in MSCs, and pretreatment of the cells with Y27632, a Rho kinase inhibitor, markedly inhibited the LPA‐induced migration. In addition, LPA and malignant ascites increased intracellular concentration of calcium in MSCs, and Ki16425 completely inhibited the elevation of intracellular calcium. These results suggest that LPA is a crucial component of the malignant ascites which induce the migration of MSCs and elevation of intracellular calcium. J. Cell. Biochem. 104: 499–510, 2008.

In Vitro Antimicrobial Susceptibility of Mycobacterium abscessus in Korea

Mycobacterium abscessus is the second most common etiology of pulmonary disease caused by nontuberculous mycobacteria in Korea. Although antimicrobial susceptibility tests are important for appropriate patient management in M. abscessus lung disease, the tests have never been investigated in Korea. Seventy-four isolates of M. abscessus recovered from patient respiratory samples were tested against eight antimicrobial agents following the guidelines set forth by the National Committee for Clinical Laboratory Standards. Of the parenteral antibiotics, amikacin (99%, 73/74) and cefoxitin (99%, 73/74) were active against most isolates. Imipenem (55%, 36/66) and tobramycin (36%, 27/74) had activity against moderate number of isolates. Of the oral antibiotics, clarithromycin (91%, 67/74) was active against the majority of isolates. Moxifloxacin (73%, 54/74) and ciprofloxacin (57%, 42/74) had activity against a moderate number of isolates. Doxycycline was the least active, inhibiting only 7% (5/74) of isolates. In conclusion, the variations in susceptibility within M. abscessus isolates to currently available antimicrobials suggest that the antimicrobial susceptibilities of any clinically significant M. abscessus isolate be needed individually.

Comparison of a Conventional Antimicrobial Susceptibility Assay to an Oligonucleotide Chip System for Detection of Drug Resistance in Mycobacterium tuberculosis Isolates

ABSTRACT An oligonucleotide chip (Combichip Mycobacteria chip) detecting specific mutations in the rpoB, katG, and inhA genes of Mycobacterium tuberculosis was compared with conventional antimicrobial susceptibility results. The probes detecting drug resistance were as follows: 7 wild-type and 13 mutant probes for rifampin and 2 wild-type and 3 mutant probes for isoniazid. Target DNA of M. tuberculosis was amplified by PCR, followed by hybridization and scanning. Direct sequencing was performed to verify the results of the oligonucleotide chip. One-hundred seven of 115 rifampin-resistant strains (93%) had mutations in the rpoB gene. Eighty-five of 119 isoniazid-resistant strains (71%) had mutations in the katG gene or inhA gene. The diagnostic oligonucleotide chip with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of resistance against rifampin and isoniazid in M. tuberculosis isolates.

pncA mutations in clinical Mycobacterium tuberculosis isolates from Korea

BackgroundPyrazinamide (PZA) is among the first-line drugs for the treatment of tuberculosis. In vitro, it kills semidormant mycobacteria only at low pH. The purpose of this study was to compare PZA resistance with pyrazinamidase (PZase) activity and the genotype to better understand the molecular basis of PZA resistance and to expand the profile of pncA mutations worldwide.ResultsOf the 28 tested strains of Mycobacterium tuberculosis, 6 were susceptible to PZA and positive for PZase activity and had no pncA mutations. Twenty-one strains were resistant to PZA and negative for PZase activity and had mutations in the pncA gene, including 15 point mutations, 5 insertions, and 2 deletions. One strain had no mutation in the pncA gene, even though it was resistant to PZA and negative for PZase activity. Three isolates had adenine to guanine point mutations in the -11 upstream region, making this the most common type of pncA mutations in this study, with at least two different RFLP patterns.ConclusionThese data help in the understanding of the molecular basis of PZA resistance. An adenine to guanine point mutation in the -11 upstream region was the most common type of pncA mutation in our isolates. The results of pncA mutation analyses should be carefully interpreted for epidemiologic purposes.