Simulation for improved collimation system of gamma-ray telescope system for boron neutron capture therapy at Kyoto University Reactor.

A gamma-ray telescope system has been used at Heavy Water Neutron Irradiation Facility at Kyoto University Reactor mainly for boron neutron capture therapy (BNCT) for multiple hepatic tumors. This system has been improved to accommodate BNCT for other sites, such as brain, head and neck, lung, breast, etc. Simulation for the collimation system was performed. It revealed that the effective telescope field-of-view could be expanded from approximately 3-21 cm.

Evaluation of PHITS for microdosimetry in BNCT to support radiobiological research.

The aim of this paper is to validate the microdosimetric functionality of the Monte Carlo code, PHITS, and verify its use for estimating dose and RBE for radiobiological studies performed at Kyoto University Institute for Integrated Radiation and Nuclear Science (KURNS). Lineal energy spectra produced by the KUR mixed irradiation mode were measured with a tissue equivalent proportional counter (TEPC) place in free air. The Monte Carlo calculation showed a good agreement with the measured data. In the second part of the study, a realistic set-up of a typical in-vivo radiobiological experiment was simulated with PHITS and the simulation results were compared against TLD and gold foil activation measurements. The Monte Carlo simulation results and the measured data showed an agreement within 3%. The calculated RBE also showed a close value to clinically utilised values. This study shows that PHITS can be utilised to evaluate thermal neutron fluxes and gamma ray absorbed dose rates inside a tumour like medium.

Control of protein function through oxidation and reduction of persulfidated states.

Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.

The impact of earlier intervention by an antimicrobial stewardship team for specific antimicrobials in a single weekly intervention.

OBJECTIVE: The objective of this study was to evaluate the effects of earlier intervention by an antimicrobial stewardship team (AST) on antimicrobial use, antimicrobial resistance rates, and the clinical outcomes, without changing the weekly intervention schedule. METHODS: A retrospective study was conducted at Fukuoka University Hospital between April 2013 and March 2016. The effects were compared among three study periods (SP): SP1 (patients receiving anti-methicillin-resistant Staphylococcus aureus agents and carbapenems for ≥14 days), SP2 (patients receiving specific antimicrobials for ≥14 days), and SP3 (patients receiving specific antimicrobials regardless of the duration of treatment). RESULTS: The timing of AST intervention was shortened from an average of 15.5days after administration in SP1 to 4.2 days in SP3. The antimicrobial use density (AUD) of carbapenems and piperacillin-tazobactam decreased significantly (SP2 vs. SP3, p<0.05), and the costs of specific antimicrobials decreased (SP1, US$ 1080000; SP2, US$ 944000; SP3, US$ 763000). The rates of carbapenem resistance among Pseudomonas aeruginosa isolates showed a significant reduction from 16.2% in SP2 to 8.7% in SP3 (p<0.05). The mortality rate and length of stay did not change during the study period. CONCLUSIONS: Earlier intervention by an AST could contribute to the proper use of antimicrobials without adversely affecting patient outcomes.

Tissue inhibitor of metalloproteinase-1 promotes cell proliferation through YAP/TAZ activation in cancer.

Tissue inhibitor of metalloproteinase-1 (TIMP-1), a member of the TIMP family (TIMP-1 to 4), is highly expressed in various types of cancer and forms a complex with its receptor CD63 and Integrin ß1. However, the precise oncogenic mechanism of TIMP-1 remains unclear. Yes-associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ) are transcription co-activators enhancing the transcription of specific genes related to cell proliferation. But the mechanism of aberrant YAP/TAZ activation in cancer is not fully understood. Here, we showed that TIMP-1 activates YAP/TAZ as novel downstream targets to promote cell proliferation. The TIMP-1-CD63-Integrin ß1 axis activates Src and promotes RhoA-mediated F-actin assembly, leading to LATS1/2 inactivation. This results in under-phosphorylation, protein stabilization and nuclear translocation of YAP/TAZ (YAP/TAZ activation); CTGF production; and cell proliferation. Furthermore, the TIMP-1-YAP/TAZ axis is aberrantly activated in various types of cancer cells or tissues. TIMP-1 knockdown inhibits cell proliferation through YAP/TAZ inactivation in cancer cells. This study found that TIMP-1 accelerates cell proliferation through YAP/TAZ activation in cancer, and suggests the TIMP-1-YAP/TAZ axis may be a novel potential drug target for cancer patients.

Note: Development of real-time epithermal neutron detector for boron neutron capture therapy.

The real-time detection of epithermal neutrons forms an important aspect of boron neutron capture therapy. In this context, we developed an epithermal neutron detector based on the combination of a small Eu:LiCaAlF6 scintillator and a quartz fiber in order to fulfill the irradiation-field requirements for boron neutron capture therapy. The irradiation test is performed with the use of a reactor-based neutron source. The thermal and epithermal neutron sensitivities of our epithermal neutron detector are estimated to be 9.52 × 10-8 ± 1.59 × 10-8 cm2 and 1.20 × 10-6 cm2 ± 8.96 × 10-9 cm2, respectively. We also subject the developed epithermal neutron detector to actual irradiation fields, and we confirm that the epithermal neutron flux can be measured in realtime.

The C-terminus of amelogenin enhances osteogenic differentiation of human cementoblast lineage cells.

BACKGROUND AND OBJECTIVES: Amelogenin proteins are the major constituent of developing extracellular enamel matrix and are believed to have an exclusively epithelial origin. Recent studies have suggested that amelogenins might induce the differentiation and maturation of various cells, including cementoblast lineage cells. However, the residues comprising the active site of amelogenin remain unclear. The purpose of this study was to identify the active site region of amelogenin by studying the effects of amelogenin fragments on the osteogenic differentiation of cementoblasts. MATERIAL AND METHODS: Amelogenin fragments lacking the C-terminus (rh163) and N-terminus (rh128) and a fragment consisting of the C-terminal region of rh174 (C11 peptide) were synthesized and purified. Human cementoblast lineage cells were cultured in osteogenic differentiation medium and treated with 0, 10, 100 or 1000 ng/mL of rh163, rh128 or C11 peptide. The mRNA levels of bone markers were examined by real-time polymerase chain reaction analysis. Alkaline phosphatase activity and calcium deposition were also determined. Mineralization was evaluated by alizarin red staining. RESULTS: The osteogenic differentiation of human cementoblast lineage cells was significantly enhanced by treatment with rh128 or C11 peptide, whereas rh163 had no significant effect as compared with untreated controls. CONCLUSIONS: The C-terminus of amelogenin promotes the osteogenic differentiation of human cementoblast lineage cells, indicating the possible utility of C11 peptide in periodontal tissue regeneration.

Improvement of depth dose distribution using multiple-field irradiation in boron neutron capture therapy.

It is important that improvements are made to depth dose distribution in boron neutron capture therapy, because the neutrons do not reach the innermost regions of the human body. Here, we evaluated the dose distribution obtained using multiple-field irradiation in simulation. From a dose volume histogram analysis, it was found that the mean and minimum tumor doses were increased using two-field irradiation, because of improved dose distribution for deeper-sited tumors.

Histopathologic impacts of everolimus introduction on kidney transplant recipients.

BACKGROUND: Introduction of everolimus (RAD) has been established as a new immunosuppressive medication for kidney transplant (KT) recipients. Administration of RAD is capable of reducing the dosage of coadministrated calcineurin inhibitors (CNI). However, histological investigations have rarely been performed. METHODS: To clarify histopathologic effects of RAD, a total of 9 adult KT recipients were enrolled (RAD group, n = 5; Mycophenolate mofetil (MMF) group, n = 4). Renal graft biopsies had been performed at 3 weeks and 1 year following KT. RESULTS: There were no differences in 1-, 3-, and 5-year graft survival rates (RAD group: 100%, 100%, and 80%, respectively; MMF group: 100%, 100%, and 75%, respectively), and patient survival between the 2 groups (no deaths during the 5 years post-transplantation). Interestingly, although 2 patients in the RAD group had developed CNI nephrotoxicity clinically, renal biopsies had proven no CNI-related lesions 1 year later, which might be due to the reduction in CNI. On the other hand, 1 patient, in the MMF group, had been diagnosed histologically with new-onset CNI nephrotoxicity 1 year following KT. Importantly, the frequency and mean arteriolar hyalinosis (ah) scores, which reflect CNI nephrotoxicity, were significantly higher in the MMF group at 1-year biopsy (P < .05, P < .0001). Two patients in the RAD group improved their ah scores between 3 weeks and 1 year. CONCLUSIONS: Pathological findings revealed that reversible CNI nephrotoxicity can be improved by RAD with reduced CNI maintenance therapy. It is reasonable to believe, therefore, that introduction of RAD is useful for patients who have been diagnosed with CNI nephrotoxicity.

Serum levels of toxic AGEs (TAGE) may be a promising novel biomarker in development and progression of NASH.

Nonalcoholic fatty liver disease (NAFLD) ranges from simple steatosis to nonalcoholic steatohepatitis (NASH), leads to fibrosis and potentially cirrhosis, liver failure, and hepatocellular carcinoma, and is one of the most common causes of liver disease worldwide. NAFLD has also been implicated in other medical conditions such as insulin resistance, obesity, metabolic syndrome, hyperlipemia, hypertension, cardiovascular disease, and diabetes. Continuous hyperglycemia has been implicated in the pathogenesis of diabetic micro- and macro-vascular complications via various metabolic pathways, and numerous hyperglycemia-induced metabolic and hemodynamic conditions exist, including the increased generation of various types of advanced glycation end-products (AGEs). We recently demonstrated that glyceraldehyde-derived AGEs (Glycer-AGEs), the predominant components of toxic AGEs (TAGE), played an important role in the pathogenesis of angiopathy in diabetic patients. Moreover, a growing body of evidence suggests that the interaction between TAGE and the receptor for AGEs may alter intracellular signaling, gene expression, and the release of pro-inflammatory molecules and also elicits the generation of oxidative stress in numerous types of cells including hepatocytes and hepatic stellate cells. Serum levels of TAGE were significantly higher in NASH patients than in those with simple steatosis and healthy controls. Moreover, serum levels of TAGE inversely correlated with adiponectin (adiponectin is produced by adipose tissue and is an anti-inflammatory adipokine that can increase insulin sensitivity). Furthermore, immunohistochemical staining of TAGE showed intense staining in the livers of patients with NASH. Serum levels of TAGE may be a useful biomarker for discriminating NASH from simple steatosis. The administration of atorvastatin (10 mg daily) for 12 months significantly improved NASH-related metabolic parameters and significantly decreased serum levels of TAGE. The steatosis grade and NAFLD activity score were also significantly improved. These results demonstrated that atorvastatin decreased the serum levels of TAGE in NASH patients with dyslipidemia and suggest the usefulness of TAGE as a biomarker for the attenuation of NASH. Serum levels of TAGE were significantly higher in non-B or non-C hepatocellular carcinoma (NBNC-HCC) patients than in NASH subjects without HCC or control subjects. TAGE may be involved in the pathogenesis of NBNC-HCC, and could, therefore, be a biomarker that could discriminate NBNC-HCC from NASH. We propose that serum levels of TAGE are promising novel targets for the diagnosis of and therapeutic interventions against NASH.

A Supramolecular Substance, [2] Rotaxane, Induces Apoptosis in Human Molt-3 Acute Lymphoblastic Leukemia Cells.

The antitumor effects of a supramolecular substance, the [2] rotaxane (TRO-A0001), and its molecular mechanisms were investigated. TRO-A0001 suppressed the proliferation of cultured human Molt-3 acute lymphoblastic leukemia cells for 12-72 h in a dose-dependent manner. Based on flow cytometry, TRO-A0001 clearly induced apoptosis after 24 h. The mitochondrial membrane potential disappeared after treatment with 1.0 µM of TRO-A0001. Expression of the cleaved forms of capase-9 and caspase-3 was significantly increased in cells exposed to TRO-A0001, whereas the expression of XIAP, a type of inhibitor of apoptosis family, was decreased. These results suggest that [2] rotaxane TRO-A0001 may be a highly promising new antitumor medicine.

Lactoferrin inhibits infection-related osteoclastogenesis without interrupting compressive force-related osteoclastogenesis.

BACKGROUND: Control of periodontal tissue inflammation during orthodontic treatment is very important in achieving a favourable therapeutic goal. We previously demonstrated that orally applied bovine lactoferrin (bLF) inhibited LPS-induced bone resorption but not orthodontic force-induced tooth movement in vivo. This study is designed to examine the underlying mechanism of it. METHODS: We examined the inhibitory effects of bLF on the expression of RANKL, OPG, TNF-α and COX-2 in osteoblasts loaded with compressive stress (CS) in comparison with LPS stimulated osteoblasts. Formation of osteoclasts was evaluated by co-culture system. RESULTS: Both CS- and LPS-applications upregulated COX-2 and RANKL but downregulated OPG. TNF-α was upregulated in LPS-stimulated osteoblasts but downregulated in CS-loaded osteoblasts. NS398 (a specific inhibitor of COX-2) significantly inhibited CS-induced RANKL-upregulation but not LPS-induced RANKL upregulation, indicating a critical role of COX-2/PGE2 pathway in CS-induced osteoclastogenesis. bLF significantly downregulated LPS-induced upregulation of RANKL and eliminated OPG suppression but not affected in CS-induced changes. Moreover, bLF significantly decreased LPS-induced osteoclast formation, whereas bLF had no effect on PGE2-induced osteoclast formation. CONCLUSIONS: bLF can effectively suppress harmful bone destruction associated with periodontitis without inhibiting bone remodelling by CS-loading. Therefore, oral administration of bLF may be highly beneficial for control of periodontitis in orthodontic patients.

The FGFR1 inhibitor PD173074 induces mesenchymal-epithelial transition through the transcription factor AP-1.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a crucial process in cancer progression that provides cancer cells with the ability to escape from the primary focus, invade stromal tissues and migrate to distant regions. Cell lines that lack E-cadherin show increased tumorigenesis and metastasis, and the expression levels of E-cadherin and Snail correlate inversely with the prognosis of patients suffering from breast cancer or oral squamous cell carcinoma (OSCC). Moreover, recent studies have shown that most EMT cases are regulated by soluble growth factors or cytokines. Among these factors, fibroblast growth factors (FGFs) execute diverse functions by binding to and activating members of the FGF receptor (FGFR) family, including FGFR1-4. Fibroblast growth factor receptor 1 is an oncoprotein that is involved in tumorigenesis, and PD173074 is known to be a selective inhibitor of FGFR1. However, the roles of FGFR1 and FGFR1 inhibitors have not yet been examined in detail. METHODS: Here, we investigated the expression of FGFR1 in head and neck squamous cell carcinoma (HNSCC) and the role of the FGFR1 inhibitor PD173074 in carcinogenesis and the EMT process. RESULTS: Fibroblast growth factor receptor 1 was highly expressed in 54% of HNSCC cases and was significantly correlated with malignant behaviours. Nuclear FGFR1 expression was also observed and correlated well with histological differentiation, the pattern of invasion and abundant nuclear polymorphism. Fibroblast growth factor receptor 1 was also overexpressed in EMT cell lines compared with non-EMT cell lines. Furthermore, treatment of HOC313 cells with PD173074 suppressed cellular proliferation and invasion and reduced ERK1/2 and p38 activation. These cells also demonstrated morphological changes, transforming from spindle- to cobble stone-like in shape. In addition, the expression levels of certain matrix metalloproteinases (MMPs), whose genes contain activator protein-1 (AP-1) promoter sites, as well as Snail1 and Snail2 were reduced following PD173074 treatment. CONCLUSION: Taken together, these data suggest that PD173074 inhibits the MAPK pathway, which regulates the activity of AP-1 and induces MET. Furthermore, this induction of MET likely suppresses cancer cell growth and invasion.

Orally administered liposomal lactoferrin inhibits inflammation-related bone breakdown without interrupting orthodontic tooth movement.

BACKGROUND: Bovine lactoferrin (bLF) modulates the production of tumor necrosis factor-alpha (TNF-α) and inhibits alveolar bone breakdown associated with periodontitis. This study is designed to examine the effects of orally administered liposomal bLF (LbLF) on orthodontic force (OF)-induced alveolar bone remodeling during experimental tooth movement. METHODS: Two groups of male Wistar rats were treated with either LbLF or control solution in drinking water 7 days before OF application. Lipopolysaccharide (LPS) was injected into the gingival sulcus in half the rats in each group. Thus, four groups: OF, OF+LbLF, OF+LPS, and OF+LPS+LbLF were established. RESULTS: Orally administered LbLF significantly reduced apical migration of junctional epithelium in the OF and OF+LPS groups. In OF+LPS, osteoclast number in the alveolar crestal area was increased by LPS treatment, whereas osteoclast number was significantly reduced in OF+LPS+LbLF through suppression of TNF-α production. Osteoclastic induction in the middle part, mainly from OF application, was not affected by LbLF administration. Inhibition of tooth movement was not induced by LbLF. CONCLUSIONS: Orally administered LbLF significantly inhibits LPS-induced alveolar bone resorption but not OF-induced bone remodeling. LbLF could be a potent therapeutic and preventive agent to control periodontal inflammation in patients undergoing orthodontic treatment.

Establishment of mesenchymal cell line derived from human developing odontoma.

OBJECTIVES: An odontoma, which shows proliferating odontogenic epithelium and mesenchymal tissue, is one of the most common odontogenic tumors encountered. These are commonly found in tooth-bearing regions, although the etiology remains unknown. There are no previous reports of an established line of immortalized human odontoma cells. METHODS: Using odontoma fragments obtained from a girl treated at our department, we established an immortalized human odontoma cell line and investigated cell morphology, dynamic proliferation, the presence of contamination, and karyotype. Moreover, cell characterization was examined using osteogenic and odontogenic markers. RESULTS: We successfully established a mesenchymal odontoma cell (mOd cells). The cells were found to be fibroblastic and had a high level of telomerase activity. Cell growth was confirmed after more than 200 population doublings without significant growth retardation. mOd cells expressed mRNA for differentiation markers, including collagen type I (COLI), alkaline phosphatase, bone sialoprotein, osteopontin, osteocalcin, cementum-derived protein (CP-23), dentin sialophosphoprotein (DSPP), and distal-less homeobox 3 (DLX3), as well as bone morphogenetic proteins (BMPs). In addition, they showed a high level of calcified nodule formation activity in vitro. CONCLUSIONS: We successfully established a cell line that may be useful for investigating the mechanisms of normal odontogenesis as well as characteristics of odontoma tumors.

Acceleration of tendon-bone healing in anterior cruciate ligament reconstruction using an enamel matrix derivative in a rat model.

We examined whether enamel matrix derivative (EMD) could improve healing of the tendon-bone interface following reconstruction of the anterior cruciate ligament (ACL) using a hamstring tendon in a rat model. ACL reconstruction was performed in both knees of 30 Sprague-Dawley rats using the flexor digitorum tendon. The effect of commercially available EMD (EMDOGAIN), a preparation of matrix proteins from developing porcine teeth, was evaluated. In the left knee joint the space around the tendon-bone interface was filled with 40 µl of EMD mixed with propylene glycol alginate (PGA). In the right knee joint PGA alone was used. The ligament reconstructions were evaluated histologically and biomechanically at four, eight and 12 weeks (n = 5 at each time point). At eight weeks, EMD had induced a significant increase in collagen fibres connecting to bone at the tendon-bone interface (p = 0.047), whereas the control group had few fibres and the tendon-bone interface was composed of cellular and vascular fibrous tissues. At both eight and 12 weeks, the mean load to failure in the treated specimens was higher than in the controls (p = 0.009). EMD improved histological tendon-bone healing at eight weeks and biomechanical healing at both eight and 12 weeks. EMD might therefore have a human application to enhance tendon-bone repair in ACL reconstruction.

SU-D-217BCD-02: Online Angular Tube Current Modulation in X-Ray Computed Tomography: Can Tube Current Be Modulated Appropriately?

PURPOSE: In X-ray computed tomography (CT), X-rays are significantly less attenuated in the anteroposterior direction and more in the lateral direction. Therefore, the tube current should be adjusted within one gantry rotation using angular tube current modulation (TCM). The aim of this study was to evaluate whether online angular TCM could reduce radiation dose appropriately. METHODS: A 128-detector dual-source CT (SOMATOM Definition Flash; Siemens Healthcare, Erlangen, Germany) and an online TCM system (CARE Dose 4D; Siemens Healthcare) were used. Dose profiles were acquired using the CT Dose Profiler (RTI Electronics, Molndal, Sweden) and an elliptical cylindrical phantom (MHT; Kyoto Kagaku, Kyoto, Japan) for helical CT scans with and without TCM. In addition, absorbed dose distributions within a single section were acquired using an anthropomorphic phantom (RANI 10; The Phantom Laboratory, Salem, NY) and radiophotoluminescent glass dosimeters (RPLDs) (GD- 302M; Chiyoda Technol, Tokyo, Japan) for helical CT scans with and without TCM after placing RPLDs within all holes of one section and pasting them around the section. A graph of each absorbed dose distribution was drawn using graphing software (ORIGIN 8.6; OriginLab, Northampton, MA). RESULTS: The acquired dose profiles suggested that online angular TCM could adjust the tube current in near-real time according to the attenuation measured from the previous projection. The profiles gradually stabilized because the tube current was adjusted properly. The absorbed doses were low and the distributions were stable with TCM compared with those without TCM. CONCLUSIONS: In X-ray CT, an online angular TCM can reduce radiation dose effectively by adjusting tube current within one gantry rotation in near-real time.

Ultrasound stimulation attenuates root resorption of rat replanted molars and impairs tumor necrosis factor-α signaling in vitro.

BACKGROUND AND OBJECTIVE: A therapeutic protocol to minimize root resorption induced by tooth replantation has not yet been universally established. In this context, noninvasive modality such as ultrasound therapy have been a focus of increased interest. This study aimed to evaluate the inhibitory effect of ultrasound therapy on root resorption of replanted rat molars. In addition, the study aimed to promote insights into the mechanism through which ultrasound mediates the metabolism of periodontal cells in vitro. MATERIAL AND METHODS: An experimental model of tooth replantation in rats, involving luxation and immediate replacement of the maxillary first molars, was used to assess the inhibitory effect of an ultrasound-therapy regimen (15 min of exposure to ultrasound, each day for 21 d) on root resorption. Moreover, the effect of ultrasound on osteoclastogenesis/cementoclastogenesis was examined in vitro using a mouse osteoblastic stromal cell line (ST2) and a mouse cementoblastic cell line (OCCM-30). RESULTS: The area of root resorption lacunae was statistically decreased (p < 0.01) in the ultrasound-treated sample. In addition, immunohistochemical staining, using murine TNF-α polyclonal antibody, failed to detect tumor necrosis factor-α (TNF-α) protein in the ultrasound-treated sample compared with the control. An in vitro study showed that the lipopolysaccharide (LPS)-induced expression of Tnfalpha mRNA was significantly reduced by ultrasound therapy in both osteoblastic and cementoblastic cells. Moreover, the TNF-α-induced up-regulation of Rankl mRNA was also inhibited by ultrasound. CONCLUSION: Ultrasound may contribute to the reduction of the trauma-induced inflammatory reaction through impairment of the TNF-α signaling pathway. It is therefore suggested that ultrasound shows potential as a therapeutic tool to optimize the regenerative potential of periodontal tissues on replanted teeth.

Topical application of Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces cell cycle arrest in the rat gingival epithelium in vivo.

BACKGROUND: Aggregatibacter actinomycetemcomitans is one of the etiological pathogens implicated in the onset of periodontal disease. This pathogen produces cytolethal distending toxin (CDT) that acts as a genotoxin to induce cell cycle arrest and cellular distension in cultured cell lines. Therefore, CDT is a possible virulence factor; however, the in vivo activity of CDT on periodontal tissue has not been explored. Here, CDT was topically applied into the rat molar gingival sulcus; and the periodontal tissue was histologically and immunohistochemically examined. MATERIALS AND METHODS: Recombinant purified A. actinomycetemcomitans CDT was applied to gingival sulcus of male Wistar rats and tissue samples were immunohistochemmically examined. RESULTS: One day after application, infiltration of neutrophils and dilation of blood vessels in the gingival connective tissue were found. At day three, desquamation and detachment of cells in the junctional epithelium was observed. This abrasion of junctional epithelium was not observed in rats treated with mutated CDT, in which a His274Ala mutation is present in the CdtB subunit. This indicates the tissue abrasion may be caused by the genotoxicity of CdtB. Expression of the proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, was significantly suppressed using CDT treatment in the junctional epithelium and gingival epithelium. CONCLUSION: Using the rat model, these data suggest CDT intoxication induces cell cycle arrest and damage in periodontal epithelial cells in vivo.