Differentiation and characteristics of undifferentiated mesenchymal stem cells originating from adult premolar periodontal ligaments / 대한치과교정학회지

OBJECTIVE: The purpose of this study was to investigate the isolation and characterization of multipotent human periodontal ligament (PDL) stem cells and to assess their ability to differentiate into bone, cartilage, and adipose tissue. METHODS: PDL stem cells were isolated from 7 extracted human premolar teeth. Human PDL cells were expanded in culture, stained using anti-CD29, -CD34, -CD44, and -STRO-1 antibodies, and sorted by fluorescent activated cell sorting (FACS). Gingival fibroblasts (GFs) served as a positive control. PDL stem cells and GFs were cultured using standard conditions conducive for osteogenic, chondrogenic, or adipogenic differentiation. RESULTS: An average of 152.8 +/- 27.6 colony-forming units was present at day 7 in cultures of PDL stem cells. At day 4, PDL stem cells exhibited a significant increase in proliferation (p < 0.05), reaching nearly double the proliferation rate of GFs. About 5.6 +/- 4.5% of cells in human PDL tissues were strongly STRO-1-positive. In osteogenic cultures, calcium nodules were observed by day 21 in PDL stem cells, which showed more intense calcium staining than GF cultures. In adipogenic cultures, both cell populations showed positive Oil Red O staining by day 21. Additionally, in chondrogenic cultures, PDL stem cells expressed collagen type II by day 21. CONCLUSIONS: The PDL contains multipotent stem cells that have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes. This adult PDL stem cell population can be utilized as potential sources of PDL in tissue engineering applications.

The effect of cortical punching on the expression of OPG, RANK, and RANKL in the periodontal tissue during tooth movement in rats / 대한치과교정학회지

OBJECTIVE: The purpose of this study was to investigate whether cortical punching could stimulate the expression of OPG, RANK, and RANKL during tooth movement by immunohistochemistry. METHODS: 34 sprague-dawley rats (15 weeks old) were allocated into 3 groups: TMC group (experimental group; Tooth Movement with Corticotomy, n = 16), TM group (control group; Tooth Movement only group, n = 16), and non-treatment group (n = 2). 20 gm of orthodontic force was applied to rat incisors by inserting elastic bands. The duration of force application was 1, 4, 7 and 14 days. A microscrew (diameter 1.2 mm) was used for cortical punching of the palatal side of the upper incisors in the TMC group. RESULTS: Distributions of OPG, RANK, and RANKL were evaluated by immunohistochemistry. OPG, RANK and RANKL were observed on experimental and control groups. On the compression side, the degree of the expression of OPG decreased in both groups. The expression of RANK was most prominent in the experimental group of day 4. The expression of RANKL was most intensive and extensive in the experimental group of day 7. However, the expression of OPG was decreased in the experimental and control groups compared to the non treatment group. The expression of OPG, RANK and RANKL after force application were decreased at day 14. CONCLUSIONS: These findings suggested that cortical punching might stimulate remodeling of alveolar bone during a 2 week period of tooth movement without any pathologic change.

The expression of MMP-1, -8, and -13 mRNA in the periodontal ligament of rats during tooth movement with cortical punching / 대한치과교정학회지

OBJECTIVE: The aim of this study was to determine whether cortical punching stimulates the expression of matrix metalloproteinase-1, -8, and -13 in orthodontic tooth movement in rats. METHODS: A total of 32 male sprague-dawley rats at 15 weeks old were divided into two groups of 16 rats each, to form the tooth movement with cortical punching (TMC) group and tooth movement only (TM) group. A total of 20 gm of orthodontic force was applied to rat incisors to cause experimental tooth movement. Cortical punching was performed on the palatal side near the central incisor with a 1.0 mm width microscrew in the TMC group. The duration of tooth movement was 1, 4, 7, and 14 days. RESULTS: Measurements of the mRNA expression were selected as the means to determine the identification of expression of MMP-1, -8, and -13. In the TMC group, the expression of collagen type I was greater than that of the TM group from day 4 to day 14. Expression of TIMP-1 in the TM group was greater than that of the TMC group in the pressure side of PDL and alveolar bone cell at day 4. In the TMC group, TIMP-1 was expressed at the osteoclast, but not at the tooth surface of the TM group at day 14. Maximum induction of the mRNA of MMP-1 was observed on day 4 in the TMC group, but it was observed on day 7 in the TM group. MMP-8 mRNA of the TMC group was twice greater than that of the TM group at 4 days. In the TMC group, maximum induction of MMP-13 mRNA was observed on day 1. CONCLUSIONS: These findings suggested that cortical punching can stimulate remodeling of PDL and alveolar bone connective tissues during experimental orthodontic tooth movement in rats.

Change of the Expression of Human Telomerase Reverse Transcriptase mRNA and Human Telomerase RNA after Cisplatin and 5-Fluorouracil Exposure in Head and Neck Squamous Cell Carcinoma Cell Lines

Telomerase activity appears to be associated with cell immortalization and malignant progression. Understanding how telomerase activity is regulated in vivo is important not only for understanding the molecular biology of telomerase but also for the potential clinical application of anticancer drugs. This study evaluated telomerase activity and quantified the expression of human telomerase reverse transcriptase (hTERT) mRNA and human telomerase RNA (hTR) using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method before and after the exposure of cisplatin and 5-fluorouracil (5-FU) in two head and neck squamous cell carcinoma (HNSCC) cell lines. Two human HNSCC cell lines (PNUH-12 and SNU-899) were studied. Cell cytotoxicity, the change of telomerase activity, and hTERT mRNA and hTR expression by 5-FU and cisplatin exposure were assessed by MTT assay, TRAP assay, and real-time RT-PCR, respectively. In two cell lines, after cisplatin exposure, the telomerase activity and hTERT mRNA expression decreased, but hTR expression in- creased according to the concentration of drug. However, in both cell lines, the telomerase activity and hTR did not show any significant change after 5-FU treatment, but the expression of hTERT mRNA decreased. These results suggest that there may be other important regulating mechanism except hTERT mRNA as the regulation factor of telomerase activity in HNSCC cell lines.

Changes of periodontopathogens and clinical parameters of periodontal tissue after debanding / 대한치과교정학회지

OBJECTIVE: The purpose of this study was to evaluate clinical and microbiological changes in periodontal tissue around the banded molars after debanding. METHODS: This study included 17 young adult patients treated with fixed orthodontic appliances including bands on the last molars more than 1 years. Probing depth and bleeding frequency were measured and plaque samples were collected from the last banded molars in all quadrants of each patient. All the data were collected immediately after debanding and 4 weeks after debanding. RESULTS: Using polymerase chain reaction based on 16S rDNA, the presence of Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola was detected. After debanding, probing depth, bleeding frequency, and prevalance of periodontopathogens were reduced. Probing depth and bleeding frequency were most decreased in the buccal site of the mandibular left molar and were least decreased in the lingual site of the maxillary right molar. CONCLUSION: The results of this study indicated that proper management of oral hygiene after debanding can recuperate unfavorable periodontal condition caused by orthodontic treatment.

Gender Differences in Alcohol Metabolizing Hepatic Enzyme Genotypes in Korean Patients with Alcohol Dependence / 신경정신의학

OBJECTIVES: There are a number of preceding epidemiological studies reporting gender differences in the genetic etiology of alcohol dependence. The author investigated gender difference in the frequencies of ADH2 and ALDH2 genoypes between the patients with alcohol dependence and normal control. METHODS: The subjects were 141 alcohol dependent patients (104 males, 37 females) and 138 normal control (79 males, 59 females). The frequencies of 1/1 and 1/2+2/2 (2+ afterward) genotypes for ADH2 and ALDH2 were investigated in male and female between alcohol dependence and normal control group. DNA was extracted from WBC in peripheral venous blood and PCR-RFLP method was used out for genotyping. RESULTS: First, the frequency of ADH2 1/1 genotype was significantly higher in alcohol dependent patients than normal control in both genders. Second, while there was no gender difference in the frequency of ADH2 1/1 genotype in normal controls, in the patient group however, the frequency was significantly higher in females than males. Third, in male subjects with alcohol dependence, the frequency of ALDH2 1/1 genotype was significantly higher than in male normal control subjects. On the other hand, in female subjects with alcohol dependence, the frequency of ALDH2 2+ genotype was significantly higher than in female normal control subjects. CONCLUSION: These results suggest that while the risk of alcohol dependence is predominantly affected by ALDH2 1/1 genotype in male, the female ADH2 1/1 genotype is mainly associated with the risk of alcohol dependence. This means that there are gender differences in the genetic etiology of alcohol dependence.

IL-1beta and IL-1RN polymorphisms in gastroduodenal diseases / 대한내과학회지

BACKGROUND: IL-1beta and IL-1 receptor antagonist (IL-1RN) genetic polymorphisms have been associated with development of gastric atrophy and increased risk of gastric carcinoma. This study aimed to determine the effects of these polymorphisms in gastroduodenal diseases. METHODS: This study population was comprised of 297 patients and they were grouped into gastritis, gastric ulcer, duodenal ulcer, and gastric cancer. We determined IL-1beta-511/-31/+3954 and IL-1RN genotype by polymerase chain reaction using gastric biopsy specimens. RESULTS: The genotype of IL-1beta-511 C/T, -31 T/C, +3954 C/C, and IL-1RN *1/*1 was predominant in all four groups. Allelic and genotypic frequencies of IL-1beta-511/-31/+3954 and IL-1RN showed no significant difference in four groups. IL-1beta-511 T/T, -31 C/C, +3954 C/T, and IL-1RN *2 carriers did not show increased risk of gastric ulcer, duodenal ulcer and gastric cancer. Classification of gastric cancer into intestinal and diffuse type also showed no significant difference of IL-1beta-511/-31/+3954 and IL-1RN genotypic frequencies. CONCLSUION: There was no significant difference of IL-1beta and IL-1RN polymorphisms between patients with gastritis, gastric ulcer, duodenal ulcer and gastric cancer. Therefore, other endogenous or exogenous factors will play more important role in the development of gastroduodenal diseases in Korean.

Relationship between Clinicopathological Characteristics and Telomerase Activity of Renal Cell Carcinoma / 대한비뇨기과학회지

PURPOSE: Telomerase is a ribonucleoprotein involved in maintaining the telomere length in stem, and immortal or actively dividing, cells. There is controversy relating to the correlation between the telomerase activity and the clinicopathological characteristics of renal cell carcinomas. The relationships between the telomerase activity and the clinicopathological characteristics of renal cell carcinomas were evaluated. MATERIALS AND METHODS: Twenty-seven renal cell carcinoma tissues, and 22 normal renal tissues around renal cell carcinoma tissues, were aseptically obtained from a radical or partial nephrectomy in 27 cases with a renal cell carcinoma. The telomerase activity was analyzed using a PCR-based telomeric repeat amplification protocol (TRAP)-ELISA method. The telomerase activity was compared with the clinicopathological characteristics, such as sex, age of patient, histologic type, stage, grade, size and metastasis, of the renal cell carcinomas. RESULTS: Telomerase activity was detected in 24 of the 27 renal cell carcinoma tissues (88.9%), but not in all of the normal renal tissues. There was no statistical correlation between the telomerase activity and the clinicopathological features. CONCLUSIONS: These findings indicate that the telomerase activity may play a role in the carcinogenesis of renal cell carcinomas, but there is no relationship between the telomerase activity and the clinicopathological characteristics of renal cell carcinomas.

Telomerase Activity in Voided Urine: a Potential Marker of Human Bladder Carcinoma / 대한비뇨기과학회지

PURPOSE: Telomerase is an enzyme that immortalizes cells by maintaining a constant telomere length, and is thought to be active in the transformation of normal somatic cells into immortal human tumor cells. In an attempt to get a more valuable, noninvasive assay for the detection of bladder carcinomas, exfoliated cells from the voided urine of patients for the presence of telomerase were assayed. MATERIALS AND METHODS: Voided urine samples were obtained from 37 patients with known, but untreated, bladder carcinomas and 20 healthy volunteers to determine the presence of telomerase activity. Telomerase activity was analyzed using a PCR-based telomeric repeat amplification protocol (TRAP)-ELISA method. RESULTS: Of the 37 bladder carcinoma samples, 30 (81.1%) and 16 (43.2%) tested positive for the presence of telomerase activity and cytology, respectively (p=0.009). However, for the 20 healthy volunteers samples, no telomerase activity was found. 83.3% (5 of 6) of the grade 1 tumors, 75.0% (18 of 24) of the grade 2 tumors and 100% (7 of 7) of the grade 3 tumors were positive for telomerase activity. Only 33.3% (2 of 6) of the grade 1 tumors, 45.8% (11 of 24) of the grade 2 tumors and 42.9% (3 of 7) of the grade 3 tumors were diagnosed by cytology. For the low (Tis, Ta, T1) and high (T2, T3) stages, the sensitivities were 77.8 and 100, and 37.0 and 60.0% for telomerase activity and cytology, respectively. All of T1 and T2 patients, with carcinoma in situ, and Ta (100%) were also positive for telomerase activity. There was no significant correlation between the urinary telomerase activity and clinicopathological characteristics of bladder carcinomas, with the exception of the multiplicity of the tumor. CONCLUSIONS: These findings suggest that urinary telomerase activity may be a potential marker for detecting bladder carcinomas, especially in low grade or stage tumors.

Aberrant Methylation of p16 Tumor Suppressor Gene and Death-Associated Protein Kinase in Non-Small Cell Lung Carcinoma / 결핵

BACKGROUND: The p16INK4a (p16) tumor suppressor gene is frequently inactivated in human non-small cell lung cancers (NSCLCs), predominantly through homozygous deletion or in association with aberrant promotor hypermethylation. Death-associated protein kinase (DAPK) gene influences interferon γ-induced apoptotic cell death and has important role in metastasis of lung cancer in animal model. Hypermethylation of promoter region of DAP kinase gene may suppress the expression of this gene. METHODS: This study was performed to investigate the aberrant methylation of p16 or DAP kinase in 35 resected primary NSCLCs by methylation-specific PCR (MSP), and demonstrated frequency, diagnostic value and clinical implication of aberrant methylation of two genes. RESULTS: Thirty-two cases were male patients, and 3 cases were female patients with an average age was 57.8±10.5 years. The histologic types of lung cancer were 22 of squamous cell carcinoma, 12 of adenocarcinoma, 1 of large cell carcinoma. Pathologic stages were 11 cases of stage I(1 IA,10 IB), 13 cases of stage II (1 IIA, 12 IIB), and 11 cases of stage III(9 IIIA, 2 IIIB). Regarding for the cancer tissue, p16 aberrant methylation was noted in 13 case of 33 cases (39.4%), DAP kinase in 21 cases of 35 cases (60%). Age over 55 year was associated with p16 aberrant methylation significantly (p<0.05). Methylation status of two genes was not different by smoking history, histologic type, size of tumor, lymph node metastasis and disease progression of lung cancer. There was no correlation between p16 and DAP kinase hypermethylation. CONCLUSION: This investigation demonstrates that aberrant methylation of p16 tumor suppressor gene or DAP kinase showed relatively high frequency (74.3%) in NSCLCs, and that these genes could be a biologic marker for early detection of lung cancer.

Detection of human herpesvirus 8 DNA in pemphigus and chronic blistering skin diseases

Increased incidences of Kaposi's sarcoma and lymphoid malignancies have been observed in patients with pemphigus, and the human herpesvirus 8 (HHV-8) is very strongly associated with these tumors. Because the virus may be one of the triggering factors of pemphigus, we undertook this study to screen for the presence of HHV-8 in chronic blistering skin diseases including pemphigus. A total of 45 paraffin-embedded specimens were studied using nested polymerase chain reaction (PCR) with primers to amplify a 160-base pair HHV-8 fragment. HHV-8 DNA could be detected in 7 of 9 patients with pemphigus vulagris, and 1 of 2 with pemphigus foliaceus. All specimens of other blistering skin diseases were negative for HHV-8. On sequencing PCR products, the sequences were almost identical with the prototypic sequence for HHV-8, and a few base- pair substitutions at 1086C-T and 1139A-C were detected. The results of our study suggests that HHV-8 might have trophism for pemphigus lesions. Further studies including comparison of HHV-8 DNA load in both lesional and normal skin in the same patient, serological and animal studies would be helpful to study the relationship between HHV-8 and pemphigus.

Detection of human herpesvirus 8 DNA in pemphigus and chronic blistering skin diseases

Increased incidences of Kaposi's sarcoma and lymphoid malignancies have been observed in patients with pemphigus, and the human herpesvirus 8 (HHV-8) is very strongly associated with these tumors. Because the virus may be one of the triggering factors of pemphigus, we undertook this study to screen for the presence of HHV-8 in chronic blistering skin diseases including pemphigus. A total of 45 paraffin-embedded specimens were studied using nested polymerase chain reaction (PCR) with primers to amplify a 160-base pair HHV-8 fragment. HHV-8 DNA could be detected in 7 of 9 patients with pemphigus vulagris, and 1 of 2 with pemphigus foliaceus. All specimens of other blistering skin diseases were negative for HHV-8. On sequencing PCR products, the sequences were almost identical with the prototypic sequence for HHV-8, and a few base- pair substitutions at 1086C-T and 1139A-C were detected. The results of our study suggests that HHV-8 might have trophism for pemphigus lesions. Further studies including comparison of HHV-8 DNA load in both lesional and normal skin in the same patient, serological and animal studies would be helpful to study the relationship between HHV-8 and pemphigus.

Detection of Long and Short isoforms of PML-RARA mRNA by RT-PCR in Acute Promyelocytic Leukemia / 대한혈액학회지

BACKGROUND: Chromosomal translocation t (15 ; 17), the breakpoints of which are in the PML gene on chromosome 15 and RARA gene on chromosome 17, is specifically found in acute promyelocytic leukemia (APL). According to the site of breakpoint on PML gene, two major isoforms (Long or Short) of PML-RARA mRNA are produced. METHODS: To detect long (L) and short (S) isoforms, we extracted RNA and amplified PML-RARA mRNA by RT-PCR from leukemic cells of 20 cases of APL. We compared the result of cytogenetic study and the clinical response after chemotherapy or ATRA therapy for remission induction with the isoforms of PML-RARA mRNA. RESULTS: In 19 cases (94%) among 20 cases with APL, PML-RARA mRNA was positive, and negative in a case who showed only i (17q) without t (15;17). In 12 cases (63.2%), L isoform of PML-RARA mRNA was detected, and S isoform (36.8%) in 7 cases of APL. All the cases with t (15;17) were positive for PML-RARA mRNA. In a case of trisomy 8 without t (15;17), PML-RARA mRNA of L isoform was detected. There was no significant difference between L and S isoform in laboratory findings and clinical response after chemotherapy or ATRA treatment. Excluding 6 cases with death before or within 10 days of ATRA treatment or chemotherapy, among 13 patients of positive PML-RARA mRNA, 11 cases (84.6%) reached to complete remission, but a case of negative PML-RARA mRNAwas resistent to ATRA treatment. CONCLUSION: This study suggests that detection of PML-RARA mRNA with two major isofroms using RT-PCR is more sensitive to diagnose APL and to detect minimal residual disease than cytogenetic study and that further study with more cases may be substantiated the types of PML-RARA mRNA isoform as a prognostic marker.

Detection of Major bcr/abl mRNA from Stored Bone Marrow Aspirate Smears Using the Reverse Transcriptase Polymerase Chain Reaction / 대한임상병리학회지

BACKGROUND: The verse transcriptase polymerase chain reaction (RT-PCR) has been widely used to analyze the bcr/abl fusion mRNA in chronic myelogenous leukemia (CML). Fresh or cryopreserved cells may not always be available for molecular diagnosis. So we investigated the value of stored bone marrow aspirate smears as the sources of material for the detection of bcr/abl mRNA. METHODS: We extracted RNA using modified Chomczynski method, and amplified bcr/abl mRNA by RT-PCR from the 70 cases of bone marrow smear slides stored from 7 days to 7 years, which were comprised of 49 CML, 11 other chronic myeloproliferative disorders (CMPD) and 10 acute lymphoblastic leukemia (ALL). Sensitivity of RT-PGR was tested using the slide smears prepared with 10(0)-10(6) K562 cells, and RT-PCR results losing each fresh bone marrow cellular suspension and slide smears in 24 patients were compacted. For major bcr/abl rearrangement, RT-PCR was performed by nested PGR afters GDNA synthesis losing downward primer and beta2-microglobulin was used as RNA controls. RESULTS: The sensitivity of RT-PCR for detecting bcr/abl mRNA was l02 cells per slide. Sixty one cases (86%) of 70 bone marrow aspirate smears showed positive results of beta2-micyoglobulin cDNA as an indicator of intact RNA. Thirty nine cases of 42 beta2-microglobulin cDNA positive CML bone marrow aspirate smears showed 29 b3a2 type mRNA and 10 b2a2 type mRNA. Nine cases of 11 bone marrow aspirate smear with other CMPD showed negative results of bcr/abl mRNA. Two cases of 10 ALL bone mallow aspirate smears had b2a2 type mRNA and b3a2 type mRNA, respectively. The results for detection of bcr/abl mRNA with fresh cell suspensions of 24 patients were same as the bone marrow aspirate smears storied for 7 days to 1 year. CONCLUSIONS: These data indicated that RNA obtained from bone marrow smears storied for less than 1 year was valuable as the source of RT-PGR for the detection of bcr/abl mRNA in CML and the bone marrow smears stored for much longer period ould be assailable as the specimens for retrospective analysis of specific gene alter-ation in other hematologic malignancy.

Rapid Extraction of DNA using Ion Exchange Resin for Early Detection ofMycobacterium tuberculosis by the Polymerase Chain Reaction / 결핵

BACKGROUND: The extraction methods of DNA from clinical samples are the major obstacle to use the PCR(Polymerase Chain Reaction) in routine labortary for early detection of M. tuberculosis. We tried to improve the extraction method of DNA from sputum for establishment of the PCR in routine labortary by reducing the possibility of cross contamination and performing it easily and safely. METHODS: We used the InstaGene(TM) DNA extraction kit(BioRad Co.) using Chelex 100 ion exchange resin for preparation of DNA. We compared InstaGene method in 100 cases of sputum from proteinase K method which is known as the most commonly used method for DNA purification(Experiment 1). And we compared InstaGene method in 98 cases of sputum from Microwave method developed by a company in Korea(Experiment 2). In experiment 1, 245bps of IS6110 were amplified and then 188bps were amplified by nested PCR. In experiment 2, 536bps in primary PCR and 276bps in nested PCR were amplified and analysed by agarose gel electrophoresis and EtBr staining. RESULTS: When we chose AFB smear, culture, or AFB smear and culture as a standard test, PCR had low specificity and positive predictive value in both experiments. The InstaGene method has higher value in sensitivity and negative predictive value significantly than proteinase K method. The InstaGene method and the Microwave methods were similar in sensitivity, specificity, positive predictive value and negative predictive value.. CONCLUSION: Even though both methods had lower possibility of cross contamination, shorter time requrirement, simplicity, and economic advantages than Proteinase K method, the InstaGene method was a little simpler than the Microwave method. Therefore, in terms of usfulness in clinical application, the Instagene method seems to be the most useful method in DNA extraction for detection of M. tuberculosis using PCR. The reliability of this method will be clarified by further studies with enough clinical samples.