• Journal of Periodontal and Implant Science
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• v.29 no.4
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• pp.995-1005
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• 1999

Alveolar bone destruction is a character-istic of periodontal disease. Treponema denticola are found in significantly increased numbers in the sites affected with periodontal disease. In order to clarify the role of T. denticola in destruction of alveo-lar bone in periodontal disease, this study was undertaken to determine the effect of sonicated extract of T. denticola on osteo-clast differentiation in co-culture system of mouse bone marrow cells and calvaria cells. The ability of osteoclast formation was estimated by counting the number of tar-tartrate resistant acid phosphatase(TRAP) positive cells. Sonicated extract of this bacteria stimulated osteoclast formation in a dose dependent manner(p<0.05). Indomathacin, an inhibitor of prostaglandin synthesis, decreased osteoclast formation induced by sonicated extract of this bacte-bacteria(p<0.05). Extract-induced osteoclast formation was decreased, when sonicated extract of bacteria was heated(p<0.05). These findings suggest that T. denticola induces osteoclast differentiation, and protein component of this bacteria and $PGE_2$ may play an important role in this process.

• Restorative Dentistry and Endodontics
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• v.29 no.5
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• pp.479-484
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• 2004

Immunoinhibitory protein extracted from sonicated Treponema denticola have been shown to suppress cell cycle progression of human lymphocytes. To study in detail about the effect of this microorganism on the function of lymphocytes. we investigated the levels of Interleukin-2 (IL-2) and Interleukin-4 (IL-4) production by T lymphocytes before and after the addition of $12.5{\;}\mu\textrm{g}/ml$ T. denticola sonicated extracts. In this study. levels of IL-2 and IL-4 produced from T cells pretreated with sonicated extracts were evaluated by using the quantitative sandwich enzyme immunoassay technique. In response to phytohemagglutinin (PHA) stimulation. T cell produced increased levels of IL-2 and IL-4. However. the expressions of both cytokines were significantly inhibited when PHA activated-T cells were pre-exposed to sonicated T. denticola extracts (p < 0.05). These findings suggest that the T. denticola sonicated extracts induced-immunosuppression in Th1 and Th2 cell functions could be a part of the pathogenic mechanism of the endodontic failure associated with this microorganism.

• Restorative Dentistry and Endodontics
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• v.27 no.5
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• pp.473-478
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• 2002

근관내 spirochetes의 존재유무가 명확하게 밝혀져 있지 않았으나 최근 PCR을 사용한 연구에서 Treponema denticola균주가 감염근관의 50% 이상의 경우에서 발견됨에 따라 이 세균이 치수 및 치근단 질환에 관여하는지에 대한 관심 이 높아졌다. 하지만 그 정확한 기전은 아직 밝혀져 있지 않다. 이와 관련하여 Shenker등이 T. denticola의 sonicated extract에서 순수분리된 단백질 (SIP)이 임파구 proliferation을 방해함을 보고한바 있다. 따라서 본 연구의 목적은 면역억제단백질 SIP이 어떤 기전에 의해서 임파구증식을 억제하는지를 밝히는 데 있다. 건강한 혈액 공여자로부터 추출해낸 T세포에 PHA (phytohemagglutinin)로 증식자극을 주게되는데 이 과정에서 SIP을 처리하거나 처리하지 않은 경우를 비교하여 세포주기 진행과정을 유세포분석기 (Becton-Dickinson FACS$^{tarplus}$) 를 통하여 평가하였다. 실험결과 세단계의 chromatography과정을 통해 순수정제된 SIP은 50kDa와 56kDa의 두가지 polypeptide로 구성되어 있고 0.25$\mu\textrm{g}$으로 처리된 T 임파구는 42.5%의 [$^3$H]thymidine incorporation 억제가 그리고, 0.5$\mu\textrm{g}$으로 처리한 경우는 75.1%의 억제가 일어나 dose-dependent한 양상이 나타났다. Propidium iodide와 유세포 분석기를 사용하여 세포주기를 분석한 결과 medium으로만 처리한 경우 97%이상의 임파구는 G$_0$/G$_1$ phase에 머물러 있었으나 PHA자극을 받은 경우 G$_0$/G$_1$ phase에서 58%, S phase에서 34.6%, G$_2$/M phase에서 7.4%로 분포되어 나타났다. SIP으로 전처리한 경우 세포 증식이 감소하여 0.25$\mu\textrm{g}$을 첨가한 경우 75.1%가 G$_0$/G$_1$ phase에 머물러 있었고 더 강한 농도의 0.5$\mu\textrm{g}$을 첨가한 경우는 87.7%가 G$_0$/G$_1$ phase에서 S phase로 진행되지 않고 머물러있었다. 따라서 SIP으로 전처리된 T 임파구는 그 증식이 G$_0$/G$_1$ phase에서 차단된 것으로 보인다. 이러한 면역억제현상이 in vitro 상태뿐 아니라 in vivo에서도 진행된다면 spirochete가 치수 및 치근단 질환의 병인론에 연관된 면역반응저하기전에 중요한 역할을 하는 것으로 추론할 수 있다.

• Journal of Periodontal and Implant Science
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• v.23 no.1
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• pp.1-15
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• 1993

P. gingivalis has been implicated as a strong pathogen in periodontal disease and known to have three serotypes of P. gingivalis. The purpose of this study is to investigate on the relationship between virulence, metabolic acids and genetic heterogeneity of P. gingivalis. P. gingivalis W50 standard strain and five strains of P. gingivalis serotype b Korean isolates were used in this study. For in vitro virulence test, lyophilized whole cell P. gingivalis were suspended, and sonicated with ultrasonic dismembranometer. Sonicated samples were applied to cultured cells derived from periodontal ligament, and cell activity was assayed with growth and survival assay. The metabolic acids were also extracted, and determined by High Performance Liquid Chromatography. Pst I-digested bacterial genomic DNA was electrophoresed, and densitometric analysis was performed to study the genetic heterogeneity. All of the P. gingivalis serotype b produced butyric acid. In cell activity study, butyric acid inhibited the cell activity irrespective of its concentration. Densitometric analysis showed restriction fragment length polymorphism. These results suggested that there existed heterogeneity of the metabolic acids and the virulence of P. gingivalis and such heterogeneity might be related to genetic heterogeneity.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.369-375
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• 2002

A sperm factor(s) for oocyte activation during fertilization has not been clearly identified. In this study to elucidate an oocyte activation factor(s), mouse sperm were sonicated and ultra-filtered with a 30 kilo-daltons (KD) cutoff membrane and the ultra-filtrate was then sequentially fractionated over Suporose 12 column and Superdex column, The recovered fractions were micro-injected into Mⅱmouse oocytes and second polar body formation (PBF) was examined. Suporose fraction RV2.10 prepared from sperm extract significantly increased PBF. Of Superdex fractions re-separated from Suporose fraction RV2.10, fraction RV2.12 also had the strongest PBF activity. By analyzing with micro-reverse phase column (URPC), the Superdex fraction RV2.12 appeared to be glutamic acid. In microinjection test, glutamic acid significantly increased PBF. This study suggests that glutamic acid should be a type of sperm factor for second polar body formation related to oocyte activation.

• Restorative Dentistry and Endodontics
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• v.30 no.1
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• pp.1-6
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• 2005

In order to examine the immunoresponse of host cells to Enterococcus faecalis, this in vitro study monitored the production of Interleukin-2 (IL-2), Interleukin-4 (IL-4) and Transforming growth factor-$\beta1\;(TGF-\beta1)$ in human lymphocytes. Lymphocytes were activated with PHA in the presence or abscence of sonicated extracts of E. Faecalis (SEF) and further incubated for 72 hours. The level of each cytokine was measured by ELISA. Data were analyzed with Kruskal-Wallis test and Mann-Whitney U test (P < 0.05). PHA-activated group did exhibit higher level of IL-2 and IL-4 than untreated control group. The levels of expression of both cytokines were significantly decreased following the treatment of high (25 ${\mu}g/ml$) and medium concentration (12.5 ${\mu}g/ml$)) of SEF (P > 0.05) than those of PHA activated group. But low concentration (5 ${\mu}g/ml$)) of SEF showed th similar level of IL-2 and IL-4 production as those of PHA activated group. $TGF-\beta1$ was unaffected by SEF treatment. These results suggested that E. faecalis may suppress IL-2 and IL-4 production by lymphocytes and this could be one of possible factors why E. faecalis are found frequently in the teeth with failed endodontic treatment.

• Natural Product Sciences
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• v.5 no.4
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• pp.181-185
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• 1999

Higenamine is a cardiotonic constituent of Aconite root, one of the most important oriental traditional medicine. Since Aconite root contains toxic aconitine alkaloids, variously processed roots have been often used. Much works have been done with the chemical significances concerning with the toxic aconitine alkaloids during the processing periods. However, effects of processing on higenamine have not yet been previously studied. In this paper, the extract pattern and the amounts of higenamine extracted with water from unprocessed and processed Aconite roots were compared. R-(+)-isomer was the only higenamine enantiomer detected although racemic higenamine was reported to be separated from Aconitum spp. Sonication for 1 hour resulted in higher higenamine extraction $(12.3\;{\mu}g/g)$ than boiling water extraction for 3 hours $(6.7\;{\mu}g/g)$ of unprocessed Aconite root. Extraction of not only higenamine but also most of the other components of unprocessed Aconite roots were reduced with boiling in water. Similarly, reduced extraction was observed with extracts of all three processed Aconite roots (Kyung-Po-Aconite root, Dang-Po-Aconite root and Huk-Peon-Aconite root) by either sonicated extraction or boiling water extraction.

• Restorative Dentistry and Endodontics
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• v.25 no.4
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• pp.606-618
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• 2000

Dental pulp infection is most commonly caused by extensive dental caries, and some bacterial species invade root canals; bacterial components and products are thought to be associated with the pathogenesis of periapical periodontitis. A principle driving force behind pulpal disease response appears to lie in the host immune system's to bacteria and their products. We examined the production of interleukin $1{\beta}$ (IL-$1{\beta}$) and tumor necrosis factor ${\alpha}$(TNF-${\alpha}$) from human peripheral mononuclear cells, lymphocytes and monocytes stimulated by heat-killed Acitnobacillus actinomycetemcomitans (ATCC 29523), Porphyromonas gingivalis (ATCC 33277) and Prevotella intermedia (ATCC 25611), and also by their sonicated bacterial extracts (SBE), respectively. The effects of three strains of heat-killed bacteria and their SBEs on the morphology of cultured blood cell lines HL-60 (KCLB 10240) and J774A.1 (KCLB 40067) were observed under the inverted microscope. Ultrastructural changes of J774A.1 exposed to heat-killed P. intermedia and its SBE were investigated using transmission electron microscopy. Production of IL-$1{\beta}$ was reduced in human peripheral mononuclear cells after stimulation by sonic bacterial extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. Heat-killed and sonic extract of P. gingivalis inhibited the production of TNF-${\alpha}$ in peripheral mononuclear cells. Production of TNF-${\alpha}$ was inhibited in peripheral monocytes after stimulation by sonic extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. HL-60 and J 774A.1 cells showed granular degeneration after treatment with heat-killed and sonic extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia Chromatin margination and shrinkage were observed in 774A.1 treated with heat-killed P. intermedia. Cell wall structure and organelles were destroyed and vacuoles were formed in cytoplasm in J774A.1 treated with P. intermedia sonic extract. These results suggest that A actinomycetemcomitans, P gingivalis and P intermedia may have an important role in the formation and progression of pulpal diseases via both modulation of production of IL-$1{\beta}$ and TNF-${\alpha}$ from blood mononuclear cells and cytopathic effects.

• Restorative Dentistry and Endodontics
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• v.20 no.1
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• pp.71-95
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• 1995

Bacteria have been regarded as a one of major etiologic factors in root canal infections. In endodontic treatment the effective removal of pathogenic microorganisms in the root canal is the key to successful outcome. Bacterial cell wall components may play an important role in the development of pulpal and periapical disease. The purpose of this study was to evaluate the effect of sonic extracts of Streptococcus spp. on cultured L929 cells and to characterize cell wall protein profiles of Streptococcus spp. Streptococcus spp. were isolated from infected root canals and identified with Vitek Systems(Biomeriux, USA). Five streptococci, namely S. sanguis, S. mitis, S uberis, S. mutans (ATCC 10449) and S. faecalis (ATCC 19433) weere enriched in brain heart infusion broth. Cell pellets were sonicated and cell wall extracts were dialyzed and membrane filtered. Prepared cell wall proteins were applied to cultured L929 cell. The cell reaction were evaluated by monitoring DNA synthesis, cell numbers and the change of cell morphology. The total cell wall protein profiles of microorganisms were characterized by sodium dodecyl sulfate polyacrylamide-gel eledruphoresis(SDS-PAGE). DNA synthesis of L929 cells were reduced by the increasing concentration of sonic extracts. DNA synthesis was significantly suppressed in more than $50{\mu}g$/ml of sonic extract conentration in five streptococci. S. nutans (ATCC 10449) showed stronger suppression on DNA synthesis than remaining four streptococci, which had the similar effect on DNA synthesis. Analysis of DNA synthesis measured by [$^3H$]-thymidine uptake was more sensitvie method than cell counting. Sonic extracts affected the microscopic findings of L929 cells. The protein profiles indicated that all five strains shared two major proteins with molecular masses of 70.8 and 57.5 kD respectively. S. uberis and S. mutans shared common minor proteins of which molecular weights were 147.9 and 112.2 kD respectively. However some minor proteins were unique for S. mitis, S. uberis and S. faecalis.

• Restorative Dentistry and Endodontics
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• v.20 no.1
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• pp.275-288
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• 1995

The inhibitory effect of cell wall extracts of streptococci, have been investigated to know host-parasite relationship or pathogenesis of abscess formation. Streptococci isolated from the infected root canals were sonicated to get cell wall extracts which have been known as one of the factors of pyogenesis. Lymphocytes separated by density gradient were stimulated with phytohemagglutinin and exposed to cell wall extracts of Streptococcus sanguis, S. mitis, S. uberis, S. mutans (ATCC 10449), and S. faecalis (ATCC 19433). [$^3H$]-thymidine uptake of lymphocytes was analyzed with scintillation counter and lactate dehyrogenase (LD) activity was measured with autochemistry analyzer. S. faeealis had the strongest inhibitory effect. beginning at $100\;{\mu}g/ml$ concentration of sonic extracts. S. sanguis and S. mitis had inhibitory effect at $300\;{\mu}g/ml$, while S. uberis and S. mutans showed no inhibitory, effect on DNA syntheis even at $300\;{\mu}g/ml$. Each streptococci showed different inhibitory effect on the DNA synthesis of lymphocytes, which finding indicated wide spectrum of susceptibility of lymphocytes according to streptococcus spp. There were no significant difference of LD activities between control and each streptococcal extracts. Streptococcal sonic extracts did not affect the morphological findings or number of colonies activated lymphocytes. These finding suggested the inhibitory effect of sonic extract of streptococci to lymphocytes could be detected by DNA synthesis inhibition, not by cellular membrane damage.