Yeonhee Lee

Human amniotic fluid-derived stem cells have characteristics of multipotent stem cells

Abstract.  Objectives: To characterize mesenchymal stem cell‐like cells isolated from human amniotic fluid for a new source of therapeutic cells. Materials: Fibroblastoid‐type cells obtained from amniotic fluid at the time of birth. Methods: The ability of ex vivo expansion was investigated until senescence, and stem cell‐like characteristics were analyzed by examining differentiation potential, messenger RNA expression and immunophenotypes. Results and Conclusions: A morphologically homogenous population of fibroblastoid‐type (HAFFTs) cells, similar to mesenchymal stem cells from bone marrow (BM‐MSCs), was obtained at the third passage. The cells became senescent after 27 passages over a period of 8 months while undergoing 66 population doublings. Under appropriate culture conditions, by the 8th passage they differentiated into adipocytes, osteocytes, chondrocytes and neuronal cells, as revealed by oil red O, von Kossa, Alcian blue and anti‐NeuN antibody staining, respectively. Immunophenotype analyses at the 17th passage demonstrated the presence of TRA‐1–60; SSEA‐3 and‐4; collagen types I, II, III, IV and XII; fibronectin; α‐SMA; vimentin; desmin; CK18; CD44; CD54; CD106; FSP; vWF; CD31; and HLA ABC. Reverse transcriptase–polymerase chain reaction analysis of the HAFFTs from passages 6–20 showed consistent expression of Rex‐1, SCF, GATA‐4, vimentin, CK18, FGF‐5 and HLA ABC genes. Oct‐4 gene expression was observed up to the 19th passage but not at the 20th passage. HAFFTs showed telomerase activity at the 5th passage with a decreased level by the 21st passage. Interestingly, BMP‐4, AFP, nestin and HNF‐4α genes showed differential gene expression during ex vivo expansion. Taken together, these observations suggest that HAFFTs are pluripotent stem cells that are less differentiated than BM‐MSCs, and that their gene expression profiles vary with passage number during ex vivo expansion.

Antiobesity effect of trans‐10,cis‐12‐conjugated linoleic acid‐producing Lactobacillus plantarum PL62 on diet‐induced obese mice

Aims:  To observe the antiobesity activity of trans‐10,cis‐12‐conjugated linoleic acid (CLA)‐producing lactobacillus in mice.

Effect of Weissella confusa strain PL9001 on the adherence and growth of Helicobacter pylori.

ABSTRACT Viable and nonviable Weissella confusa strain PL9001 inhibited the binding of Helicobacter pylori to human gastric-cell line MKN-45 cells by more than 90%. Spent culture supernatant of PL9001 rapidly decreased the viability of H. pylori, rupturing cell walls. The results suggest that PL9001 is a probiotic that can reduce the infectivity and persistence of H. pylori.

Different induction of adaptive response to ionizing radiation in normal and neoplastic cells

Since the beneficial effects of low-dose radiation (0.01 Gy) are usually observed in normal cells, we investigated whether the adaptive response was induced by low-dose radiation in neoplastic cells of different origin as well as in normal cells. Cell lines used in this experiment were as follows: mouse lymphocytes (NL); L929 cells established from mouse connective tissue; primary mouse keratinocytes (PK); line 308 from mouse papilloma; X-ray sensitive lymphoma cells, L5178Y-S and EL-4 cells from mouse lymphoma. The adaptive response was determined by cell survival and apoptosis. The involvement of apoptosis in the adaptive response was examined by ELISA and TUNEL assay. Adaptive response was induced by pretreatment with low-dose radiation of 0.01 Gy in normal cells such as NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. In addition, the reduction of apoptosis by pretreatment with low-dose radiation was observed in NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. These results suggested that the adaptive response could be induced by pretreatment with low-dose radiation and the phenomena were observed in normal cells, not in neoplastic cells. In addition, pretreatment with low-dose radiation reduced apoptosis, suggesting that an anti-apoptotic pathway may be involved in the adaptive response.

The identification of CTX-M-14, TEM-52, and CMY-1 enzymes in Escherichia coli isolated from the Han River in Korea

From water samples collected monthly between 2000 and 2001 from the Han River in Seoul, sixteen strains of Escherichia coli which confer resistance to at least 10 kinds of antimicrobial agents were isolated. From these isolates, 2 kinds of extended-spectrum β-lactamases (ESBLs) and one plasmid-mediated AmpC β-lactamase were detected; CTX-M-14 from 10 isolates, TEM-52 from 5 isolates, and CMY-1 from one isolate. Class 1 integron gene cassettes, such as aadA1, dfr12-orfF-aadA2, and dfr17-aadA5, were also detected and the integrons are the same as those found in E. coli isolated from swine, poultry, and humans in Korea. The result of this study indicated the importance of river water as a reservoir for antimicrobial resistance genes and resistant bacteria.

Adaptive response is differently induced depending on the sensitivity to radiation-induced cell death in mouse epidermal cells.

We investigated the relationship between induction of radio-adaptive response and cell death in mouse normal and neoplastic epidermal cells. Mouse normal primary keratinocytes (PK), cancer-prone cells [v-rasHa-transfected mouse keratinocytes (ras-PK), and line 308 cells (mouse skin papilloma cells which have activatedrasHa gene with A-to-T transversion at codon 61) were primed with a low dose of γ-rays (0.01 Gy), and were challenged with a high dose (4 Gy) after a 4 or 7 h interval. The induction of cell death in PK was 2–10 times higher and was also more rapid in PK than in ras-PK or 308 cells. Low-dose pretreatment with a 4 h interval decreased cell death, and this adaptive response was prominent in PK, whereas it was less obvious in the cases of ras-PK and 308 cells. The response of each protein kinase C (PKC) isozymes to high-dose radiation, especially PKCα, PKCδ, PKCε, and PKCη, were different between the normal andras oncogene-activated neoplastic keratinocytes; translocation of these isozymes to membrane occurred more rapidly in normal than in neoplastic cells. Furthermore, low-dose pretreatment did not induce the translocation of PKCδ in PK significantly more than in ras-PK and 308. Thus, the difference in the induction of radio-adaptive responses between mouse normal and neoplastic epidermal cells reflects difference in the rapidity of cell death, and responsiveness of PKC may affect this adaptive response.

Enhancement in Ex Vivo Phagocytic Capacity of Peritoneal Leukocytes in Mice by Oral Delivery of Various Lactic-Acid-Producing Bacteria

Lactic-acid-producing bacteria (LABs) are known to have immunomodulating activity. In the current study, various LABs were tested for their immunity-enhancing activity, especially the phagocytic activity of leukocytes. Viable but not heat-killed cells of Weissella kimchii strain PL9001, Lactobacillus fermentum strain PL9005, and L. plantarum strain PL9011 significantly increased the ex vivo phagocytic capacity of mouse peritoneal leukocytes to ingest fluorescein isothiocyanate (FITC)-labeled Escherichia coli in a strain-dependent manner. Results of this and previous studies suggest these LABs as candidates for new probiotics. This is the first report of the enhancement of peritoneal leukocyte activity of these species.

Antimicrobial Resistance Patterns and Corresponding Multilocus Sequence Types of the Campylobacter jejuni Isolates from Human Diarrheal Samples

A total of 121 Campylobacter isolates from 4,788 humans with gastroenteritis were identified and characterized by biochemical detection methods, polymerase chain reaction, and multilocus sequence typing (MLST). These samples were obtained during a 3-year period, from January 2007 to December 2009, using the National Notifiable Diseases Surveillance System at the Research Institute of Public Health and Environment in Seoul Metropolitan, Korea. Antimicrobial susceptibilities of the bacterium were also determined with the agar dilution method. All 121 isolates were identified as Campylobacter jejuni, with all (100%) of them having two virulence genes (ceuE and cadF) and a toxin gene (cdtB). Twenty-three different sequence types (STs), including 9 new STs, were determined by MLST. The most prevalent ST and clonal complex (CC) observed in this study were ST-45 (28.9%) and ST-45 CC (53.7%), respectively. Percentages of antimicrobial-resistant isolates were 1.9% for ampicillin, 0.8% for chloramphenicol, 24% for ciprofloxacin, 46.3% for enrofloxacin, 0.8% for erythromycin, 6.6% for gentamicin, and 46.3% for tetracycline. This study demonstrated that the majority of the Campylobacter isolates obtained from human samples in Korea were C. jejuni with ST-45 CC, which has been detected mainly in broilers worldwide, and all strains with new STs were uniformly resistant to enrofloxacin and tetracycline. This study indicates that broilers may be a breeding ground for bacteria as well as an important potential source of human campylobacteriosis.

Immunoenhancing effects of a new probiotic strain, Lactobacillus fermentum PL9005

The immunoenhancing effects of Lactobacillus fermentum PL9005 were assessed via mouse intragastric inoculation. The number of immunoglobulin A-positive cells in the small intestine, CD4+ T lymphocytes in the peripheral blood, and the lymphocyte proliferation response to mitogen stimulation (lipopolysaccharide) increased in mice fed L. fermentum PL9005. The lactic acid concentration also increased dose dependently in the small intestine of mice fed L. fermentum PL9005. No differences were found in body weight, food intake, and clinical signs between mice fed L. fermentum PL9005 and the control group. Results indicated that L. fermentum PL9005 is a probiotic with immunoenhancing properties.

Characterization of erythromycin-resistant porcine isolates of Campylobacter coli.

Erythromycin-resistant Campylobacter organisms were isolated from swine, and their resistance to the antibiotic was characterized. One hundred fourteen Campylobacter organisms were isolated from 572 swine intestinal samples. All isolates were identified as Campylobacter coli by sequence analysis of 16S rRNA gene and polymerase chain reactions with primers specific to hippuricase gene in Campylobacter jejuni and aspartokinase gene in C. coli. Minimal inhibitory concentrations (MICs) of erythromycin were determined by using the agar dilution method, and 80 isolates were found to be resistant to erythromycin (MIC ≥ 4 μg/ml). Of these, 31 isolates had low-level resistance (MIC =4-16 μg/ml), and 49 isolates had high-level resistance (HLR, MIC ≥ 32 μg/ml). The HLR isolates carried a point mutation at position A2075 → G in domain V of the 23S rRNA gene, whereas the low-level resistance isolates carried no mutation. These 49 HLR isolates were characterized by pulsed-field gel electrophoresis and multilocus sequence typing to study their genetic diversity. Pulsed-field gel electrophoresis identified 16 distinct types with 50% genetic similarity as the cutoff. On the other hand, 28 different sequence types (STs), including 10 new STs, were identified with multilocus sequence typing. Forty-six of 49 erythromycin HLR isolates showed crossresistance to 6 macrolide derivatives. The correlation between the inhibitory activity of carbonyl cyanide m-chlorophenylhydrazone and the existence of cmeB, which is responsible for efflux in HLR isolates, was found to be low. Erythromycin resistance was transferred from 38 of the 43 HLR isolates to susceptible C. coli by natural transformation, with a frequency of 1.217 x 10(-8)-4.618 x 10(-5) per recipient cell. All transformants were erythromycin resistant and had A2075 → G mutation in at least one of three copies of the 23S rRNA gene. Results indicate that variable genotypes of HLR C. coli coexist in swine and high-level erythromycin resistance can be transferred to other strains.