Normal gingival fibroblasts functioning is fundamental for the maintenance of periodontal connective tissue as well as wound healing. Nicotine have been found to affect DNA synthesis and cell proliferation, which appear to depend on the type of cells. This in vitro study was done to determine the effects of nicotine, a major component of tobacco, on cell proliferation, viability, activity, cell cycle distribution, and expression of cell cycle regulatory proteins in human gingival fibroblasts. Nicotine has been tested for 2 days or 4 days in 5 different concentrations; 0.1 microgram/ml; 1 microgram/ml; 10 microgram/ml; 100 microgram/ml; 1000 microgram/ml. To assess cell proliferation and viability, viable and non-viable cells were counted by hemocytometer; to evaluate cellular activity, MTT assay was employed; to analyze cell cycle distribution, fluorescent propidium iodide-DNA complex were measured using fluorocytometer; to determine the expression of cell cycle regulatory proteins, western blot analysis was performed. After 2 days and 4 days incubation respectively, at concentrations of 1 microgram/ml - 1000 microgram/ml, nicotine significantly inhibited proliferation comparing to non-supplemented controls. The cell viability was significantly decreased after 2 days and 4 days at concentrations of 1 microgram/ml - 1000 microgram/ml and at 10 microgram/ml - 1000 microgram/ml respectively. After 2 days and 4 days, the cellular activity was significantly decreased at concentrations of 10 microgram/ml - 1000 microgram/ml. Treatment with 100 microgram/ml nicotine for 48 hours caused an increase in the proportion of G1-phase cells (from 46.41% to 53.46%) and a decrease in the proportion of S-phase cells (from 17.80% to 14.27%). The levels of cyclin D1 and CDK 4 proteins in nicotine-treated fibroblasts were lower than that of controls, whereas the levels of p16 and pRB were higher than that of controls. These results suggest that the decrease of cell proliferation and lengthened Gap phases (G1) by nicotine may due to the increased expression of p16 and pRB as well as decreased expression of cyclin D1 and CDK 4 in human gingival fibroblasts.
Blotting, Western ; Cell Cycle Proteins* ; Cell Cycle* ; Cell Proliferation ; Cell Survival ; Connective Tissue ; Cyclin D ; Cyclin D1 ; DNA ; Fibroblasts* ; Humans* ; Nicotine* ; Propidium ; Tobacco ; Wound Healing

Epithelial-mesenchymal interaction plays a important role in cell growth and differentiation. This interaction is already well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular microenvironment which provide a epithelial-mesenchymal interaction. Because conventional monolayer culture lacks epithelial-mensenchymal interaction, cultivated cells have an morphologic, biochemical, and functional characteristics differ from in vivo tissue. Moreover, it's condition is not able to induce cellular differention due to submerged culture condition. Therefore, the aims of this study were to develop and evaualte the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted oral keratinocytes by histological and immunohistochemical analysis. The results were as follow; 1. Gingival keratinocytes reconstituted by three-dimensional organotypic culture revealed similar morphologic characteristics to biopsied patient specimen showing stratification, hyperkeratinosis, matutation of epithelial architecture. 2. Connective tissue structure was matured, and there is no difference during stratification period of epithelial 3-dimensional culture. 3. The longer of air-exposure culture on three-dimensionally reconstituted cells, the more epithelial maturation, increased epithelial thickness and surface keratinization 4. In reconstitued mucosa, the whole epidermis was positively stained by anti-involucrin antibody, and there is no difference according to air-exposured culture period. 5. The Hsp was expressed in the epithelial layer of three-dimensionally cultured cells, especially basal layer of epidermis. The change of Hsp expression was not significant by culture stratification. 6. Connexin 43, marker of cell-cell communication was revealed mild immunodeposition in reconstitued epithelium, and there is no significant expression change during stratification. These results suggest that three-dimensional oragnotypic co-culture of normal gingival keratinocytes with dermal equivalent consisting type I collagen and gingival fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. And this culture system seems to provide adequate micro-environment for in vitro tissue reconstitution. Therefore, further study will be focused to study of in vitro gingivitis model, development of novel perioodntal disease therapeutics and epithelial-mensenchymal interaction.
Cells, Cultured ; Cellular Microenvironment ; Coculture Techniques ; Collagen Type I ; Connective Tissue ; Connexin 43 ; Epidermis ; Epithelium ; Fibroblasts* ; Gingivitis ; Humans ; Keratinocytes* ; Models, Theoretical ; Mucous Membrane

Prokaryotic and eukaryotic cells respond to heat stress and other environmental abuses by synthesizing a small set of stress proteins and by inhibiting post-transcription synthesis of normal proteins. The purpose of the present study was to document the stress response produced by inflamed gingival tissue in vivo, and cytokine induced human periodontal ligament cells. Human PDL cells were exposed to TNF-alpha(1ng/ml), INF-gamma(200 U/ml), LPS(100ug/ml), combination of cytokine, and SDS-PAGE gels running and Western blotting analysis was done. In vivo studies, the healthy gingival tissusse of a control group and inflamed gingival tissue of adult periodontitis were studied by immunohistochemistry and histology. The results were as follows 1. HSP 47 was distributed on basal layer in healthy gingiva, but stronger stained in basal, suprabasal, and spinous layer of inflamed gingiva. 2. HSP 47 was rare on endothelial cells and mononuclear cells in healthy gingiva, but stronger expressed in inflamed gingira. 3. HSP 70 expression was rare on epihelium and inflammatory cells in both healthy & inflamed gingiva. 4. HSP 70 was actively expressed on endothelial cells and inflammatory cells of capillary lumen in moderately & mild inflamend gingiva. 5. PDL cells showed low level of HSP 47 protein expression which was significantly induced by cytokine stimulation(LSP only and combination). 6. Maximum HSP 70 protein induction was seen with stimulation by a combination of the cytokine, Combination of TNF-alpha, INF-gamma, LPS have been shown to synergistically effects of HSP 70 expression. On the above findings, HSP is influenced by cytokine and chronic inflammation in vivo, and may be involved in protection of tissue during periodontal inflammatiom.
Blotting, Western ; Capillaries ; Chronic Periodontitis ; Electrophoresis, Polyacrylamide Gel ; Endothelial Cells ; Eukaryotic Cells ; Gels ; Gingiva ; Heat-Shock Proteins* ; Hot Temperature* ; Humans ; Immunohistochemistry ; Inflammation ; Periodontal Ligament ; Running ; Tumor Necrosis Factor-alpha

The significance of intraoperative electrophysiologic monitoring during microvascular decompression was evaluated prospectively in 261 patients with the hemifacial spasm from 1985 to 1995. The patients were divided into a monitored group and a non-monitored group. Identification of the offending vessels was facilitated by the monitoring during the surgical procedure and the complication rate of the monitored group was significantly lower than that of the non-monitored group (p< 0.05). In addition, the abnormal muscle response continued to improve during the follow-up period, thus the electrophysiological status of the hemifacial spasm after the microvascular decompression improved significantly with time (p< 0.05). In conclusion, intraoperative monitoring is useful for identifying the exact offender among multiple vessels, and lowering the complication rate of the microvascular decompression for the hemifacial spasm.
Adult ; *Decompression, Surgical ; *Facial Muscles ; Female ; Follow-Up Studies ; Human ; Male ; Middle Age ; *Monitoring, Intraoperative ; Spasm/physiopathology/*surgery

The efficacy of hydrocolloid occlusive dressing technique was compared with that of the conventional wet-to-dry gauze dressing technique in decubitus ulcer of stage I and II. Forty-four patients were randomly divided into two treatment groups and each received treatment according to the two different protocols. As a result, 80.8% of the hydrocolloid occlusive dressing group (group 1) and 77.8% of the conventional wet-to-dry gauze dressing group (group 2) healed completely with no statistically significant difference between the two groups. However, the time required for complete healing was shorter in group 1 with 18.9 days compared to 24.3 days in group 2. Ulcer healing speed was also slightly faster in group 1 with 9.1 mm2/day compared to 7.9 mm2/day for group 2. Average treatment time spent by a medical staff member was significantly shorter in group 1 with 20.4 minutes/day compared to 2017 minutes/day in group 2. The hospital cost of the ulcer treatment was higher in group 2 compared to group 1 even without taking into consideration the medical personnel's labor cost. These results indicate that the hydrocolloid occlusive dressing technique offers less time consuming and less expensive method of treatment compared to the conventional technique in stage I andII decubitus ulcers.
Adult ; Aged ; Comparative Study ; Decubitus Ulcer/*therapy ; Female ; Human ; Male ; Middle Age ; *Occlusive Dressings

In the present study, we investigated if priming of autoreactive CD8+ T cells would be inhibited by competitive peptides for major histocompatibility complex (MHC) class I binding. We used a mouse model of vitiligo which is induced by immunization of Kb-binding tyrosinase-related protein 2 (TRP2)-180 peptide. Competitive peptides for Kb binding inhibited IFN-gamma production and proliferation of TRP2-180-specific CD8+ T cells upon ex vivo peptide restimulation, while other MHC class I-binding peptides did not. In mice, the capability of inhibition was influenced by T-cell immunogenicity of the competitive peptides. The competitive peptide with a high T-cell immunogenicity efficiently inhibited priming of TRP2-180-specific CD8+ T cells in vivo, whereas the competitive peptide with a low T-cell immunogenicity did not. Taken together, the inhibition of priming of autoreactive CD8+ T cells depends on not only competition of peptides for MHC class I binding but also competitive peptide-specific CD8+ T cells, suggesting that clonal expansion of autoreactive T cells would be affected by expansion of competitive peptide-specific T cells. This result provides new insights into the development of competitive peptides-based therapy for the treatment of autoimmune diseases.
Animals ; Autoimmune Diseases ; Immunization ; Major Histocompatibility Complex ; Mice ; Peptides ; T-Lymphocytes ; Vitiligo

Application of membranes for guided tissue regeneration(GTR) have been confined to the subgingival barrier functions; however, many studies have provided evidence that some drugs, including tetracycline, initially can promote the growth of periodontal ligament or alveolar bone in peridontal therapy. Osseous regeneration in periodontal defects is increased by local administration of tetracycline due to its anti-collagenolytic effect, which enhances bone-forming ability via osteoblast cell chemotaxis and reduced bone resorption. The aim of this study was to evaluate effects of tetracycline loaded poly-L-lactide(PLLA) barrier membranes for guided bone regenerative potential. Tetracycline was incorporated into the PLLA membrane with the ratio 10% to PLLA by weight. Ability to guided bone regeneration of the membranes were tested by measuring new bone in the tibial defects(7x10x5 mm3) of the beagle dog for 4, 5, and 6 weeks. In control, drug-unloaded PLLA membranes were used in same size of defect. In histologic finding of the defect area, a few inflammatory cells were observed in both groups. These membrane were not perforated by connective tissue and maintained their mechanical integrity for the barrier function for 4-6 weeks. New bone formation was greater in defects covered by tetracycline-loaded membrane than in defects covered by drug- unloaded membranes. In bone regeneration guiding potential test, tetracycline-loaded membrane was more effective than drug- unloaded membranes(p<0.05). These results suggest that tetracycline-loaded PLLA membranes potentially enhance guided bone regenerative efficacy and might be a useful barrier for GTR in periodontal treatment.
Animals ; Bone Regeneration* ; Bone Resorption ; Chemotaxis ; Connective Tissue ; Dogs* ; Membranes* ; Osteoblasts ; Osteogenesis ; Periodontal Ligament ; Regeneration ; Tetracycline

Human gingival fibroblasts have proven to useful as a species specific cell culture system in various system on periodontal disease and regeneration. However, their use is limited, since they are hard to obtain and lifespan is short due to replicative senescence. To overcome these disadvantages, we transfected primary human gingival fibroblasts by the E6 and E7 genes of the Human papilloma virus(HPV) 16. The full length of HPV 16 E6 and E7 was cloned from the pBR322 into BamH1 and Sal I of a pBabe vector including hygromycin B resistance. Before pBabeE6/E7 plasmid transfection, peak 8 GFP including G418 resistance was transfected into primary GF to check the transfection efficency. PBabe E6/E7 plasmid was transfected using Lipofectamine plus following manufacter's instruction into primary normal human gingival fibroblasts in 60mm dishes with FBS free DMEM. After 2 days of transfection, the cells were treated with hygromycin for 2 weeks until the transfected control cells died. The resulting hygromycin resistant colonies were pooled, and clonned, and sucessful transfection was established for immortalized gingival fibroblast cell lines. Immoralized GF cells showed stellate shape, that is similar to that of orange grains, and more rapid growth and higher proliferation than that of primary gingival fibroblasts. This cell lines overcame crisis and could be cultured over 30 subcultured, could be use for three dimentional culture, epithelial-mesenchymal interaction study.
Cell Aging ; Cell Culture Techniques ; Cell Line* ; Edible Grain ; Citrus sinensis ; Clone Cells ; Fibroblasts* ; Human papillomavirus 16 ; Humans* ; Hygromycin B ; Papilloma ; Periodontal Diseases ; Plasmids ; Regeneration ; Transfection

PURPOSE: In this study, nosocomial bloodstream infection rate and fatality rate for 774 and 386 patients, who whose blood cultivation were obtained after 48 hours of hospitalization between March 1999 and February 2000 in two university hospitals, were sought. A distribution of etiologic agent and risk factors of the nosocoial bloodstream infection were also investigated. METHODS: This study was carried out through medical record review and a structural questionnaire. Besides registers of microbe cultivation in the department of clinical pathology and medical records of patients were checked. The nosocomial bloodstream infection was also checked through medical records of patients using the standard of CDC. Statistical analysis were performed using SAS 6.12. RESULTS: The nosocomial bloodstream infection rate in hospital K and hospital A were 3.9 and 3.5 per 1,000 discharged patients, respectively. Although the rates were increased accoding to patients' age, they were different by medical departments, showing the highest level in the ICU. The fatality rate from nosocomial bloodstream infection in hospital K and hospital A were 12.5% and 21.8%, respectively. A distribution of etiologic agent of the nosocomial bloodstream infection in hospital K was 17 cases(21.8%) of Coagulase negative staphylococcus(CNS), 12 cases(15.0%) of Staphylococcus aureus and 8 cases(10.0%) of Enterococcus spp. For hospital A, it was 14 cases925.4%) of Coagulase negative taphylococcus(CNS), 9 cases(16.4%) of Staphylococcus aureus and 7 cases(12.7%) of Klebsiella pneumoniae. While risk factors of the nosocomial bloodstream infection edentified in hospital K were ICU, intracranial injury and hospitalization period, those for hospital A were a use of the central nenous tube, intracranial injury and hospitalization period. CONCLUSION: It is expected that nosocomial bloodstream infection increases as aged group increases by the change of the population structure, as the usage of invasive instrument increases by development of new medical instrument as well as large scale hospitals. For these reasons, further studies developing countermeasures against nosocomial bloodstream infection are recommended.
Centers for Disease Control and Prevention (U.S.) ; Coagulase ; Cross Infection ; Enterococcus ; Epidemiologic Studies* ; Hospitalization ; Hospitals, University ; Humans ; Klebsiella pneumoniae ; Medical Records ; Pathology, Clinical ; Risk Factors ; Staphylococcus aureus ; Surveys and Questionnaires

Dystonia is a rare syndrome of sustained muscle contractions which frequently causes twisting and repetitive movements or abnormal postures. Dopa-responsive dystonia(DRD) is a slowly progressive dystonia with childhood on set. Initial gait disturbance with toe-walking, diurnal variation of symptoms, dramatic response to levodopa treatment and concurrent signs of parkinsonism are other characteristics of DRD. We report a 22 year old woman who showed the typical characteristics of DRD and was successfully managed with medical, surgical and rehabilitational methods. Better understanding of the disease entity and its treatment options are necessary for comprehensive rehabilitational management of DRD.
Dystonia* ; Female ; Gait ; Humans ; Levodopa ; Muscle Contraction ; Parkinsonian Disorders ; Posture ; Young Adult